Use of α-1,4-glucan lyase for preparation of 1,5-D-hydrofructose

This application is a Continuation Application of application ser. No. 08/633719 now abandoned filed Jul. 8, 1996, which is the U.S. national phase under 35 U.S.C. §371 of International Application No. PCT/EP94/03397, filed Oct. 15, 1994, which claims the benefit of priority under 35 U.S.C. §119 to Application No. 9321304.9, filed Oct. 15, 1993 in Great Britain, Application No. 9321305.6, filed Oct. 15, 1993 in Great Britain, Application No. 9321301.5, filed Oct. 15, 1993 in Great Britain, Application No. 9321302.5, filed Oct. 15, 1993 in Great Britain, and Application No. 9321303.1, filed Oct. 15, 1993, in Great Britain.

The present invention also relates to the use of an enzyme, in particular α-1,4-glucan lyase (“GL”), to prepare 1,5-D-anhydrofructose (“AF”) from substrates based on α-1,4-glucan.

The present invention also relates to the use of a sugar, in particular 1,5-D-anhydrofructose (“AF”), as an anti-oxidant, in particular as an anti-oxidant for food stuffs and beverages.

The present invention relates to the use of 1,5-D-anhydrofructose (“AF”) as a sweetener, in particular as a sweetener for foodstuffs and beverages, preferably human foodstuffs and beverages.

FR-A-2617502 and Baute et al in Phytochemistry [1988] vol. 27 No. 11 pp3401-3403 report on the production of AF in Morchella vulgaris by an apparent enzymatic reaction. The yield of production of AF is quite low. Despite a reference to a possible enzymatic reaction, neither of these two documents presents any amino acid sequence data for any enzyme let alone any nucleotide sequence information. These documents say that AF can be a precursor for the preparation of the antibiotic pyrone microthecin.

Yu et al in Biochimica et Biophysica Acta [1993] vol 1156 pp313-320 report on the preparation of GL from red seaweed and its use to degrade α-1,4-glucan to produce AF. The yield of production of AF is quite low. Despite a reference to the enzyme GL this document does not present any amino acid sequence data for that enzyme let alone any nucleotide sequence information coding for the same. This document also suggests that the source of GL is just algal.

A typical α-1,4-glucan based substrate is starch. Today, starches have found wide uses in industry mainly because they are cheap raw materials.

Starch degrading enzymes can be grouped into various categories. The starch hydrolases produce glucose or glucose-oligomers. A second group of starch degrading enzymes are phosphorylases that produce glucose-1-phosphate from starch in the presence of inorganic phosphate.

AF has also been chemically synthesised—see the work of Lichtenthaler in Tetrahedron Letters Vol 21 pp 1429-1432. However, this chemical synthesis involves a large number of steps and does not yield large quantities of AF.

The chemical synthetic route for producing AF is therefore very expensive.

There is therefore a need for a process that can prepare AF in a cheap and easy manner and also in a way that enables large quantities of AF to be made.

Furthermore, anti-oxidants are typically used to prevent oxygen having any deleterious effect on a substance such as a foodstuff. Two commonly used anti-oxidants are GRINDOX 142 and GRINDOX 1029. These anti-oxidants contain many components and are quite expensive to make.

There is therefore a need to have a simpler and cheaper form of anti-oxidant.

Furthermore, sweeteners are often used in the preparation of foodstuffs and beverages. However, many sweeteners are expensive and complex to prepare.

There is therefore a need to have a simpler and cheaper form of sweetener.

According to the present invention there is provided a method of preparing the sugar 1,5-D-anhydrofructose comprising treating an α-1,4-glucan with the enzyme α-1,4-glucan lyase characterised in that enzyme is used in substantially pure form.

Preferably if the glucan contains links other than and in addition to the α-1,4-links the α-1,4-glucan lyase is used in conjunction with a suitable reagent that can break the other links—such as a hydrolase—preferably glucanohydrolase.

Preferably the glucan is starch or a starch fraction prepared chemically or enzymatically. If prepared enzymatically the reaction can be performed before the addition of the α-1,4-glucan lyase or the reactions can be performed simultaneously. The suitable reagent can be an auxiliary enzyme. Preferred auxiliary enzymes are alpha- or beta-amylases. Preferably a debranching enzyme is used. More preferably the auxiliary enzyme is at least one of pullanase or isoamylase.

Preferably the α-1,4-glucan lyase either is bound to a support or, more preferably, is in a dissolved form.

Preferably the enzyme is isolated from either a fungus, preferably Morchella costata or Morchella vulgaris, or from a fungally infected algae, preferably Gracilariopsis lemaneiformis, or from algae lone, preferably Gracilariopsis lemaneiformis.

Preferably the enzyme is isolated and/or further purified from the fungus or from the fungally infected algae or algae alone using a gel that is not degraded by the enzyme.

Preferably the gel is based on dextrin or derivatives thereof.

Preferably the gel is a cyclodextrin—more preferably beta-cyclodextrin. Preferably the enzyme comprises the amino acid sequence SEQ. I.D. No. 1. or the amino acid sequence SEQ. I.D. No. 2 or the amino acid sequence SEQ. ID. No. 5 or the amino acid SEQ. I.D. No. 6, or any variant thereof.

In an alternative preferable embodiment, the enzyme comprises any one of the amino acid sequences shown in SEQ. I.D. No.s 9-11, or any variant thereof.

The term “any variant thereof” means any substitution of, variation of, modification of, replacement of, deletion of or addition of an amino acid from or to the sequence providing the resultant enzyme has lyase activity.

Preferably the enzyme is used in combination with amylopectin or dextrin.

Preferably, the enzyme is obtained from the expression of a nucleotide sequence coding for the enzyme.

Preferably the nucleotide sequence is a DNA sequence.

Preferably the DNA sequence comprises a sequence that is the same as, or is complementary to, or has substantial homology with, or contains any suitable codon substitutions for any of those of, SEQ. ID. No. 3 or SEQ. ID. No. 4 or SEQ. ID. No. 7 or SEQ. ID. No. 8.

In an alternative preferable embodiment, the DNA sequence comprises any one of the sequences that are the same as, or are complementary to, or have substantial homology with, or contain any suitable codon substitutions as shown as SEQ. ID. No.s 12-14.

The expression “substantial homology” covers homology with respect to structure and/or nucleotide components and/or biological activity.

The expression “contains any suitable codon substitutions” covers any codon replacement or substitution with another codon coding for the same amino acid or any addition or removal thereof providing the resultant enzyme has lyase activity.

In other words, the present invention also covers a modified DNA sequence in which at least one nucleotide has been deleted, substituted or modified or in which at least one additional nucleotide has been inserted so as to encode a polypeptide having the activity of a glucan lyase, preferably having an increased lyase activity.

Preferably the starch is used in high concentration—such as up to about 25% solution.

Preferably the substrate is treated with the enzyme in the presence of a buffer.

More preferably the substrate is treated with the enzyme in the presence of substantially pure water.

Preferably the substrate is treated with the enzyme in the absence of a co-factor.

According to the present invention there is also provided a method of preparing the sugar 1,5-D-anhydrofructose comprising treating an α-1,4-glucan with the enzyme α-1,4-glucan lyase characterised in that enzyme comprises the amino acid sequence SEQ. ID. No. 1. or the amino acid sequence SEQ. ID. No. 2 or the amino acid sequence SEQ. ID. No. 5. or the amino acid sequence SEQ. ID. No. 6, or any one of the amino acid sequences SEQ. I.D. No.s 9-11, or any variant thereof.

According to the present invention there is also provided the sugar 1,5-D-anhydrofructose when prepared by the method of the present invention.

AF prepared by the present method was confirmed and characterised by 13 C NMR.

One of key advantages of the present method is that the sugar 1,5-D-anhydrofructose can be prepared in much larger quantities than before and by a method that is relatively easier and cheaper than the known processes. For example the sugar can now be prepared in amounts of for example greater than 100 g—such as 500 g—compared to the prior art methods when only much smaller amounts were and could be produced—such as micro gram amounts.

Typical reactions that can be catalyzed by GL can be summarised as follows:

1). Amylopectin→AF+limit dextrin

2). Amylose→AF+limit dextrin

3). Dextrin→AF+glucose

In reaction 1), the ratio of the two products depend on the structure of amylopectin or the distribution of α-1,6glucosidic linkages in the amylopectin molecules.

In reaction 2) and 3), the ratio of the products depends on the degree of polymerisation (DP) number of the substrate. In reaction 3 the ratio between AF and glucose depends upon the DP. For example if the dextrin contains 10 glucose units the ratio AF:glucose would be 9:1.

Another advantage of the present invention is that glucans that contain links other than α1,4-links can be substantially degraded—whereas before only partial degradation was achieved. The substantial degradation of the 1,5-D-anhydrofructose precursor is one of the factors leading to the increased yields of 1,5-D-anhydrofructose.

Other advantages are AF is a naturally occurring substance and therefore it has a potential for human purposes. For example, it can be converted to the antibiotic microthecin by AF dehydrase. Antibiotics are known for their uses in food bio-preservation, which is an important area in food technology. However, to date, the preparation of AF and also microthecin has had a number of disadvantages. For example, only small quantities could be produced. Also, the process was costly.

The present invention overcomes these problems by providing a larger production of and much cheaper production of AF and so also other products such as microthecin. In this regard, it is possible to prepare gram to kilogram amounts of AF.

A further advantage is that the lyase is stable for at least one year at 4° C. and can be lyophilized without loss of activity.

Another advantage is that the lyase produces AF directly from starches and does not need the presence of any co-factors.

Another advantage is that the enzyme can be used in pure water. This result is very surprising.

Based on the simple properties of the present lyase, one can expect that the production cost of AF will be comparable to that of glucose. This is especially advantageous that the present lyase does not necessarily require the presence of any co-factors which are generally very expensive.

In general α-1,4-glucans can be used as substrate for the enzyme.

As a preferred substrate, starch is used.

In a preferred process, soluble or gelatinized starch or starch hydrolysate are used. The starch hydrolysates can be prepared either chemically or enzymatically.

If an enzyme is used for the partial starch degradation the enzyme can either be added before the addition of the lyase or any other additional starch degrading reagent (such as the enzyme glucanohydrolase) which may be added simultaneously.

The lyase will convert the glucan to AF. The enzyme will attach the substrate from the non reducing end and leave only the reducing sugar unconverted. The residual glucose can be removed by known methods some of which have been described here.

Using the reaction described here pure AF can be produced and also in large amounts.

In one embodiment, the α-1,4-glucan lyase is purified from the fungally infected algae—such as Gracilariopsis lemaneiformis —by affinity chromatography on β-cyclodextrin Sepharose, ion exchange chromatography on Mono Q HR 5/5 and gel filtration on Superose 12 columns. The purified enzyme produces 1,5-anhydro-D-fructose from α-1,4-glucans.

The fungal lyase isolated from fungal infected Gracilariopsis lemaneiformis is characterized as having a pH optimum at 3.5-7.5 when amylopectin is used, a temperature optimum at 50° C. and a pI of 3.9.

In another embodiment, the α-1,4-glucan lyase is purified from the fungus Morchella costata by affinity chromatography on β-cyclodextrin Sepharose, ion exchange chromatography on Mono Q HR 55 and gel filtration on Superose 12 columns. The purified enzyme produces 1,5-anhydro-D-fructose from α-1,4-glucans.

The fungal lyase shows a pI around 5.4 as determined by isoelectric focusing on gels with pH gradient of 3 to 9. The molecular weight determined by SDS-PAGE on 8-25% gradient gels was 110 kDa. The enzyme exhibited a pH optimum in the range pH 5-7. The temperature optimum was found to be between 30-45° C.

In another embodiment, the α-1,4-glucan lyase is purified from the fungus Morchella vulgaris by affinity chromatography on β-cyclodextrin Sepharose, ion exchange chromatography on Mono Q HR 5/5 and gel filtration on Superose 12 columns. The purified enzyme produces 1,5-anhydro-D-fructose from α-1,4-glucans.

In another embodiment, the α-1,4-glucan lyase is purified from algae—such as Gracilariopsis lemaneiformis —by affinity chromatography on β-cyclodextrin Sepharose, ion exchange chromatography on Mono Q HR 5/5 and gel filtration on Superose 12 columns. The purified enzyme produces 1,5-anhydro-D-fructose from α-1,4-glucans.

Typical pH and temperature optima for the lyase catalyzed reaction for some of the GL enzymes according to the present invention are as follows:

The enzymes of the present invention convert amylose and amylopectin to 1,5-anhydrofructose.

Among the maltosaccharides tested, we found that the lyase showed low activity towards maltose, and lower activity to maltotriose and maltoheptaose with the highest activity to maltotetraose and maltopentaose. The enzyme showed no substrate inhibition up to a concentration 10 mg ml −1 among these maltosaccharides.

The enzymes from each of the preferred sources has been sequenced and the amino acid sequences are presented later. Also presented later are the DNA sequences coding for the enzymes.

The present invention therefore describes a new starch degrading enzyme—namely a new α-1,4-glucan lyase. This is an enzyme that has been purified and characterized for the first time.

As mentioned above, the present invention also relates to some specific uses of AF.

In particular, the present invention relates to the use of 1,5-D-anhydrofructose (“AF”), as an anti-oxidant, in particular as an anti-oxidant for food stuffs and beverages.

Therefore according to the present invention there is provided the use of 1,5-D-anhydrofructose (AF) as an anti-oxidant.

Preferably AF is or is used in an edible substance.

Preferably AP is used in or as a foodstuff or beverage.

Preferably, AF is used in combination with another anti-oxidant.

Preferably the AF is prepared by the method according to the present invention.

The main advantages of using AF as an anti-oxidant are that it is a natural product, it is non-metabolisable, it is easy to manufacture, it is water-soluble, and it is generally non-toxic.

In a preferred embodiment the present invention therefore relates to the enzymatic preparation of pure AF which can be used as an attractive water soluble antioxidant for food and non-food purposes. In the application examples are given for the use of AF as an antioxidant in food formulations.

In the accompanying examples it is seen that AF is comparable with known high quality commercial available food antioxidants. Non-food examples include use in polymer chemistry as oxygen scavengers during the synthesis of polymers. Also, AF could be used for the synthesis of bio-degradable plastic.

Experiments have shown that AF can be an efficient reducing agent (antioxidant), as it can easily reduce 3,5-dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid.

AF is a naturally occurring substance and therefore it has a tremendous potential for use as an acceptable antioxidant. AF can also be converted into the antibiotic microthecin by AF dehydrase. Antibiotics are known for their uses in food biopreservation, an important area in food biotechnology.

In another aspect, the present invention also relates to the use of 1,5-D-anhydrofructose as a sweetener, in particular as a sweetener for foodstuffs and beverages, preferably human foodstuffs and beverages.

Thus according to this aspect of the present invention there is provided the use of 1,5-D-anhydrofructose as a sweetener.

Preferably the AF is used as or in a human foodstuff or beverage.

The AF may be used in any desired amount such as a 5% solution or 100 mg/kg to 500 mg/kg.

The advantages of using AF as a sweetener are that it is a natural product, it is generally non-toxic, it is water soluble, it is non-metabolisable and it is easy to manufacture.

The present invention therefore also relates to a novel application of AF as a sweetener.

Preferably the AF is prepared by the method according to the present invention.

Further aspects of the present invention include:

a method of preparing the enzyme α-1,4-glucan lyase (GL) comprising isolating the enzyme from a fungally infected algae, fungus or algae alone;

an enzyme comprising the amino acid sequence SEQ. ID. No. 1. or SEQ. ID. No. 2 or SEQ. ID. No. 5. or SEQ. ID. No. 6, or any variant thereof;

an enzyme comprising the amino acid sequence SEQ. ID. No. 9. or SEQ. ID. No. 10 or SEQ. ID. No. 11, or any variant thereof;

a nucleotide sequence coding for the enzyme α-1,4-glucan lyase, preferably wherein the sequence is not in its natural environment (i.e. it does not form part of the natural genome of a cellular organism capable of expressing the enzyme, preferably wherein the nucleotide sequence is a DNA sequence;

a nucleotide sequence wherein the DNA sequence comprises at least a sequence that is the same as, or is complementary to, or has substantial homology with, or contains any suitable codon substitutions for any of those of, SEQ. ID. No. 3 or SEQ. ID. No. 4 or SEQ. ID. No. 7 or SEQ. ID. No. 8, preferably wherein the sequence is in isolated form;

a nucleotide sequence wherein the DNA sequence comprises at least a sequence that is the same as, or is complementary to, or has substantial homology with, or contains any suitable codon substitutions for any of those of, SEQ. ID. No. 12 or SEQ. ID. No. 13 or SEQ. ID. No. 14, preferably wherein the sequence is in isolated form; and

the use of beta-cyclodextrin to purify an enzyme, preferably GL.

Other preferred embodiments of the present invention include any one of the following: A transformed host organism having the capability of producing AF as a consequence of the introduction of a DNA sequence as herein described; such a transformed host organism which is a microorganism—preferably wherein the host

organism is selected from the group consisting of bacteria, moulds, fungi and yeast; preferably the host organism is selected from the group consisting of Saccharomyces, Kluyveromyces, Aspergillus, Trichoderma Hansenula, Pichia, Bacillus Streptomyces, Eschericia such as Aspergillus oryzae, Saccharomyces cerevisiae, bacillus sublilis, Bacillus amyloliquefascien, Eschericia coli.; A method for preparing the sugar 1,5-D-anhydrofructose comprising the use of a transformed host organism expressing a nucleotide sequence encoding the enzyme α-1,4-glucan lyase, preferably wherein the nucleotide sequence is a DNA sequence, preferably wherein the DNA sequence is one of the sequences hereinbefore described; A vector incorporating a nucleotide sequence as hereinbefore described, preferably wherein the vector is a replication vector, preferably wherein the vector is an expression vector containing the nucleotide sequence downstream from a promoter sequence, preferably the vector includes a marker (such as a resistance marker); Cellular organisms, or cell line, transformed with such a vector; A method of producing the product α-1,4-glucan lyase or any nucleotide sequence or part thereof coding for same, which comprises culturing such an organism (or cells from a cell line) transfected with such a vector and recovering the product.

In particular, in the expression systems, the enzyme should preferably be secreted to ease its purification. To do so the DNA encoding the mature enzyme is fused to a signal sequence, a promoter and a terminator from the chosen host.

For expression in Aspergillus niger the gpdA (from the Glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans ) promoter and signal sequence is fused to the 5′ end of the DNA encoding the mature lyase. The terminator sequence from the A. niger trpC gene is placed 3′ to the gene (Punt, P. J. et al 1991—(1991): 3. Biotech. 17, 19-34). This construction is inserted into a vector containing a replication origin and selection origin for E. coli and a selection marker for A. niger. Examples of selection markers for A. niger are the amdS gene, the argB gene, the pyrG gene, the hygB gene, the BmlR gene which all have been used for selection of transformants. This plasmid can be transformed into A. niger and the mature lyase can be recovered from the culture medium of the transformants. Eventually the construction could be transformed into a protease deficient strain to reduce the proteolytic degradation of the lyase in the culture medium (Archer D. B. et al 992—Biotechnol. Lett. 14, 357-362).

Instead of Aspergillus niger as host, other industrial important microorganisms for which good expression systems are known could be used such as: Aspergillus oryzae, Aspergillus sp., Trichoderma sp., Saccharomyces cerevisiae, Kluyveromyces sp., Hansenula sp., Pichia sp., Bacillus subtilis, B. amyloliquefaciens, Bacillus sp., Streptomyces sp. or E. coli.

The following samples were deposited in accordance with the Budapest Treaty at the recognised depositary The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St. Machar Drive, Aberdeen, Scotland, United Kingdom, AB21 RY on Jun 20, 1994:

E. Coli containing plasmid pGL1 (NCIMB 40652)—[ref. DH5alpha-pGL1]; and

E. Coli containing plasmid pGL2 (NCIMB 40653)—[ref. DH5alpha-pGL2].

The following sample was accepted as a deposit in accordance with the Budapest Treaty at the recognised depositary The Culture Collection of Algae and Protozoa (CCAP) at Dunstaffnage Marine Laboratory PO Box 3, Oban, Argyll, Scotland, United Kingdom, PA34 4AD on Oct. 11, 1994:

Fungally infected Gracilariopsis lemaneiformis (CCAP 1373/1)—[ref. GLQ-1 (Qingdao)].

Thus highly preferred embodiments of the present invention include a GL enzyme obtainable from the expression of the GL coding sequences present in plasmids that are the subject of either deposit NCIMB 40652 or deposit NCIMB 40653; and a GL enzyme obtainable from the fungally infected algae that is the subject of deposit CCAP 1373/1.

The following samples were deposited in accordance with the Budapest Treaty at the recognised depositary The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St. Machar Drive, Aberdeen, Scotland, United Kingdom, AB2 1 RY on Oct. 3, 1994:

E. Coli containing plasmid pMC (NCIMB 40687)—[ref. DH5alpha-pMC];

E. Coli containing plasmid pMV1 (NCIMB 40688)—[ref. DH5alpha-pMVI]; and.

E. Coli containing plasmid pMV2 (NCIMB 40689)—[ref. DH15alpha-pMV2].

Plasmid pMC is a pBluescript II KS containing a 4.1 kb fragment isolated from a genomic library constructed from Morchella costata. The fragment contains a gene coding for α-1,4-glucan lyase.

Plasmid pMV1 is a pBluescript II KS containing a 2.45 kb fragment isolated from a genomic library constructed from Morchella vulgaris. The fragment contains the 5′ end of a gene coding for α-1,4-glucan lyase.

Plasmid MV2 is a pPUC19 containing a 3.1 kb fragment isolated from a genomic library constructed from Morchella vulgaris. The fragment contains the 3′ end of a gene coding for α-1,4-glucan lyase.

In the following discussions, MC represents Morchella costata and MV represents Morchella vulgaris.

As mentioned, the GL coding sequence from Morchella vulgaris was contained in two plasmids. With reference to FIG. 15 pMV1 contains the nucleotides from position 454 to position 2902; and pMV2 contains the nucleotides downstream from (and including) position 2897. With reference to FIGS. 12 and 13, to ligate the coding sequences one can digest pMV2 with restriction enzymes EcoRI and BamHI and then insert the relevant fragment into pMV1 digested with restriction enzymes EcoRI and

Thus highly preferred embodiments of the present invention include a GL enzyme obtainable from the expression of the GL coding sequences present in plasmids that are the subject of either deposit NCIMB 40687 or deposit NCIMB 40688 and deposit NCIMB 40689.

The following sample was also accepted as a deposit in accordance with the Budapest Treaty at the recognised depositary The Culture Collection of Algae and Protozoa (CCAP) at Dunstaffnage Marine Laboratory PO Box 3, Oban, Argyll, Scotland, United Kingdom, PA34 4AD on Oct. 11, 1994:

Fungally infected Gracilariopsis lemaneiformis (CCAP 1373/2)—[ref. GLSC-1 (California)].

Thus a highly preferred embodiment of the present invention includes a GL enzyme obtainable from the algae that is the subject of deposit CCAP 1373/2.

The present invention will now be described only by way of example.

In the following Examples reference is made to the accompanying figures in which:

FIG. 1 shows stained fungally infected algae;

FIG. 2 shows stained fungally infected algae;

FIG. 3 shows sections of fungal hypha;

FIG. 4 shows sections of fungally infected algae;

FIG. 5 shows a section of fungally infected algae;

FIG. 6 shows a plasmid map of pGL1;

FIG. 7 shows a plasmid map of pGL2;

FIG. 8 shows the amino acid sequence represented as SEQ. I.D. No.3 showing positions of the peptide fragments that were sequenced;

FIGS. 9A and 9B show the alignment of SEQ. I.D. No. 1 with SEQ. I.D. No.2;

FIG. 11 shows a plasmid map of pMC;

FIG. 12 shows a plasmid map of pMV1;

FIG. 13 shows a plasmid map of pMV2;

FIGS. 14A, 14 B and 14 C show the GL coding sequence and part of the 5′ and 3′ non-translated regions for genomic DNA obtained from Morchella costata;

FIGS. 15A, 15 B and 15 C show the GL coding sequence and part of the 5′ and 3′ non-translated regions for genomic DNA obtained from Morchella vulgaris;

FIGS. 16A and 16B show a comparison of the GL coding sequences and non-translated regions from Morchella costata and Morchella vulgaris;

FIG. 17 shows the amino acid sequence represented as SEQ. I.D. No. 5 showing positions of the peptide fragments that were sequenced;

FIG. 18 shows the amino acid sequence represented as SEQ. I.D. No. 6 showing positions of the peptide fragments that were sequenced;

FIG. 19 shows a graph of oxygen consumption with and without the presence of AF; and

FIG. 20 shows a TLC plate.

In more detail, FIG. 1 shows Calcoflour White stainings revealing fungi in upper part and lower part of Gracilariopsis lemaneiformis (108×and 294×).

FIG. 2 shows PAS/Anilinblue Black staining of Gracilariopsis lemaneiformis with fungi. The fungi have a significant higher content of carbohydrates.

FIG. 3 shows a micrograph showing longitudinal and grazing sections of two thin-walled fungal hypha (f) growing between thick walls (w) of algal cells. Note thylacoid membranes in the algal chloroplast (arrows).

FIG. 4 shows the antisense detections with clone 2 probe (upper row) appear to be restricted to the fungi illustrated by Calcoflour White staining of the succeeding section (lower row) (46x and 108x).

FIG. 5 shows intense antisense detections with clone 2 probe are found over the fungi in Gracilariopsis lemaneiformis (294x).

FIG. 6 shows a map of plasmid pGL1—which is a pBluescript II KS containing a 3.8 kb fragment isolated from a genomic library constructed from fungal infected Gracilariopsis lemaneiformis. The fragment contains a gene coding for alpha-1,4-glucan lyase.

FIG. 7 shows a map of plasmid pGL2—which is a pBluescript II SK containing a 3.6 kb fragment isolated from a genomic library constructed from fungal infected Gracilariopsis lemaneiformis. The fragment contains a gene coding for alpha-1,4-glucan lyase.

FIG. 9 shows the alignment of SEQ. I.D. No. 1 (GL1) with SEQ. I.D. No.2 (GL2). The total number of residues for GL1 is 1088; and the total number of residues for GL2 is 1091. In making the comparison, a structure-genetic matrix was used (Open gap cost: 10; Unit gap cost: 2). In FIG. 9 the character to show that two aligned residues are identical is ‘;’; and the character to show that two aligned residues are similar is ‘:’. Amino acids said to be ‘similar’ are: A,S,T; D,E; N,Q; R,K; I,L,M,V; F,Y,W. Overall there is an identity of 845 amino acids (i.e. 77.67%); a similarity of 60 amino acids (5.51%). The number of gaps inserted in GL1 are 3 and the number of gaps inserted in GL2 are 2.

FIG. 10 is a microphotograph of a fungal hypha (f) growing between the algal walls (w). Note grains of floridean starch (s) and thylakoids (arrows) in the algal cell.

In FIG. 14, the total number of bases is 4726—and the DNA sequence composition is: 1336 A; 1070 C; 1051 G; 1269 T. The ATG start codon is shown in bold. The introns are underlined. The stop codon is shown in italics.

In FIG. 15, the total number of bases is 4670—and the DNA sequence composition is: 1253 A; 1072 C; 1080 G; 1265 T. The ATG start codon is shown in bold. The introns are underlined. The stop codon is shown in italics.

In FIG. 16, the two aligned sequences are those obtained from MC (total number of residues: 1066) and MV (total number of residues: 1070). The comparison matrix used was a structure-genetic matrix (Open gap cost: 10; Unit gap cost : 2). In this Figure, the character to show that two aligned residues are identical is ‘:’. The character to show that two aligned residues are similar is ‘.’. The amino acids said to be similar are: A,S,T; D,E; N,Q; R,K; I,L,M,V; F,Y,W. Overall there is: Identity: 920 (86.30%); Similarity: 51 (4.78%). The number of gaps inserted in MC is 1 and the number of gaps inserted in MV is 1.

In the attached sequence listings: SEQ. I.D. No. 5 is the amino-acid sequence for GL obtained from Morchella costata; SEQ. I.D. No. 6 is the amino-acid sequence for GL obtained from Morchella vulgaris; SEQ. I.D. No. 7 is the nucleotide coding sequence for GL obtained from Morchella costata; and SEQ. I.D. No. 8 is the nucleotide coding sequence for GL obtained from Morchella vulgaris.

In SEQ. I.D. No. 5 the total number of residues is 1066. The GL enzyme has an amino acid composition of:

In SEQ.I.D. No. 6 the total number of residues is 1070. The GL enzyme has an amino acid composition of:

Experiments

1 The Soluble Enzyme System

1.1. Effect of pH on the Stability and Activity of the Lyase Isolated from Fugal Infected Gracilariopsis lemaneiformis.

Two buffer systems, namely HOAc and NaOAc and sodium citrate—citric acid in a concentration of 5 mM—were tested at 37° C. The pH range tested was from pH 3 to pH 5.2. The lyase showed maximum activity in a pH range between 3.6 to 4.2. At pH 3, the stability and activity of the enzyme decreased by about 90%. At pH 5.2, the activity decreased by about 64%. However, the enzyme was considerably more stable at this pH than at pH 3, as the AF yield obtained at pH 5.2 was 75% of the AF yield obtained at pH 3.8. Slightly higher AF yield was obtained in the HOAc and NaOAc buffer than in citrate buffer. This is not due to any differential effect of the two buffers (final conc. is 125 μM in the AF assay mixture) in the AF assay method.

1.2. Effect of Temperature on the Activity and Stability of the Lyase

This experiment was conducted at optimal pH range. At 25° C. the production of AF was linear up to at least 9 days. This indicates that no loss of activity and stability of the lyase occurred within 9 days. With increasing temperature, the stability of the enzyme decreased.

The half life of the enzyme activity at the following temperature was:

30° C. 5 days

37° C. 2.5 days

40° C. less than 1 day

500C. less than 1 day

1.3. Effect of Substrate Concentration on the Stability of the Lyase and AF Yield

It was observed that amylopectin and dextrins have a stabilizing effect on the lyase while the smallest substrate maltose does not. This was verified for both the soluble enzyme system and the immobilized enzyme system.

AF yield increases with the increase in amylopectin concentration up to 25%. In the case of dextrin, the AF yield decreases when the concentration exceeds 30% (30%, 40% and 50% were tested).

1.4 Activation and Inactivation of Lyase

No metal ions are found necessary for the activity and the enzyme catalysed reaction can surprisingly proceed in pure water. The fact that the addition of EDTA in the reaction mixture up to 20 mM had little effect on the activity clearly demonstrates that metal ions are not essential for the activity of the lyase enzyme according to the present invention.

This means that in the AF purification step, the ion exchange chromatography step that takes away salts from the reaction system can be omitted, if water is used as reaction medium. However, inclusion of NaCl in the reaction mixture in a concentration of 0.85% (0.145 M) can increase the AF yield up to 1-fold.

1.5. Substrate Specificity

Upon cooling solubilized starch will tend to form rigid gels when the starch concentration becomes to high. Therefore it is an advantage to utilize partly degraded starch as substrate for the 1,4-glucan lyase.

The specificity of α-1,4-glucan lyase isolated from M. costata for different oligosaccharides was tested. The oligosaccharides were maltose (G2), maltotriose (G3), maltotetraose (G4), maltopentaose (G5), maltohexaose (G6) and maltoheptaose (G7). The oligosaccharides were dissolved in H 2 O at a concentration of 8 mg/ml. The enzyme assay contained 150 μl substrate G2/G3/G4/G5/G6/G7, 120 μl 0.1M MBS pH 6.3 and 30 μl purified enzyme. The reaction mixture was incubated for 60 min at 30° C. Afterwards the reaction was stopped by boiling for 3 min and 900 μl absolute ethanol was added for precipitation. After centrifugation at 20.000×g for 5 min at 4° C. the supernatant was transferred to a new eppendorf tube and lyophilized.

The freeze-dried samples were dissolved in 1000 μl H 2 O and were filtrated through a 0.22 μm Millipore filter before 25 μl of the sample was loaded on the Dionex HPLC.

1.7 HPLC

Analytical Procedures

Analyses were performed on a Dionex 4500i chromatography system consisting of a GPM-2 pump and a PED detector which was used in pulse-amperometric detection mode.

The anion exchange columns were a CarboPac PA-100 (4×250 mm) and a CarboPac PA-100 guard column (3×25 mm) from Dionex.

The eluent were 200 mM sodium hydroxide (A), 500 mM sodium acetate (B) and 18 M ohm de-ionized water (C). The pump was programmed in 2 different ways, method no. 1 and method no. 2:

Standards

Glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose (all from Sigma) and 1,5-anhydrofructose were used as standards. All compounds were dissolved in 18 M ohm de-ionized water which was filtered through a 0.22 μm Millipore filter before use.

1.7 Results

The analyses show that the purified enzyme which was isolated from M. costata indeed was able to use maltooligosaccharides as substrate 1 for 1,5-anhydrofructose formation.

When maltose was used as substrate, almost no 1,5-anhydrofructose was formed but when the other maltooligosaccharides (G3-G7) were used, high amounts of this compound were produced.

It is clear that higher amounts of 1,5-anhydrofructose were obtained when a longer maltooligosaccharide was used.

This observation corresponds perfectly well with the theory of the lyase forming 1,5-anhydrofructose from the non-reducing end of the substrate, leaving only the terminal glucose molecule unchanged.

1.8 Formation of AF

α-1,4-glucan lyase from M.costata hydrolyses starch to the end-product 1,5-anhydrofructose. The end-product was shown by HPLC, method 2. The enzyme assay contained 500 μl amylopectin (20 mg/ml, dissolved in H 2 O), 400 μl 0.1M MES pH 6.3 and 100 μl purified enzyme. The reaction mixture was incubated at 30° C. and the reaction was stopped by boiling after 30 or 120 min incubation. High-molecular oligosaccharides were precipitated by addition of 3 vol abs. ethanol and the sample was centrifuged and freeze-dried as described above. The samples were dissolved in 125 μl H 2 O and 25 μl were applied on the HPLC column.

The HPLC elution profile clearly shows that α-1,4-glucan lyase from M.costata produces 1,5-anhydrofructose by hydrolysis of starch. Equal amounts of 1,5-anhydrofructose were found after 30 and 120 min. incubation which indicate that the enzyme activity is not inhibited by the endproduct 1,5-anhydrofructose.

13 C NMR spectra (water) of AF prepared in this way shows that it adopts one major form giving rise to the following signals: δ93.5 (quart, C-2), 81.5 (CH, C-5), 77.7 (CH, C-3), 72.6 (CH 2 , C-1), 69,8 (CH, C-4), 62.0 (CH 2 , C-6). Assignments are based on H—H C—H and C—H 2D correlation spectra.

1.6. The Cooperative Effect of Lyase with Pullulanase and Isoamylase

As it can be seen from Table 1, the inclusion of pullulanase in the reaction mixture will obviously increase the AF yield by about 15-23%, depending on whether soluble starch or amylopectin is used as substrate.

The reaction mixture contained 0.3 ml 2% potato amylopectin (Sigma) in water or 0. 3 ml 2% soluble starch (Merck), 2 μl lyase and 0.36 units pullulanase (BM) as indicated.

The reaction was carried out at 30° C. for 1 day. At the end of the reaction, samples were taken for AF and Glc analysis.

In the case of isoamylase, the advantage is that the optimal pH of the lyase overlaps with that of Pseudomonas isoamylase (pH 3.0-4.5). The problem, however, is that isoamylase will produce an excess amount of long chain amylose that precipitates from the solution, and therefore is no longer suitable as a substrate for the lyase. It can be expected that the cooperation of the lyase with isoamylase will be efficient, if the chain of amylose is not too long.

2. The Immobilized Enzyme System

Immobilization of the lyase was achieved by using succinimide-activated Sepharose (Affigel 15 gel, Bio-Rad) and glutaradehye-activated Silica gel (BM). The recovery of lyase activity after immobilization on Affigel 15 gel was between 40% to 50%. There may be some lyase that is still active after immobilization, but is inaccessible to the substrate because of the steric hindrance, especially in the case of macromolecules like starches. Immobilized enzymes used in the industry usually have an activity recovery of around 50%.

The most interesting thing of the Affigel 15 gel immobilized lyase is that its stability has been greatly improved at pH 5.5. When the column was operated at this pH, the stability was at least 16 days long. The pH shift in the stability is very important considering the optimal pH of pullulanase which is around pH 5.5. This is the prerequisite for the lyase and pullulanase to cooperate efficiently in the same reactor with the same physico-chemical environment. The soluble lyase has an optimal pH between 3.6 and 4.2, and at this pH range pullulanase shows little or no activity.

With the silica gel immobilized lyase, the activity recovery is very high, around 80-100%. However, the silica gel immobilized enzyme was not stable when the column was operated neither at pH 3.8 nor pH 5.5. It is possible that some lyase was adsorbed on the surface of the silica gel beads and was slowly released from the silica gel after each washing of the column. It may therefore be the adsorbed lyase that contributes to the high recovery rate and the decrease in column activity.

3. Purification of AF

3.1. The Lyase-Amylopectin/Soluble Starch System

In this system, the reaction system contained AF, limit dextrin, the lyase, and buffer salts at the end of the reaction. AF was separated from the macromolecules limit dextrin and the lyase) by ethanol (final conc. 50%) precipitation. Unprecipitated low-molecular-weight amylopectin was separated by ultrafiltration using Amicon YM3 membranes (cut-off 3,000). Ethanol was removed by evaporation at 40° C. in a rotary evaporator. Buffer salts were removed from AF by mixed ion exchangers. Purified solid AF was obtained by freeze-drying.

3.2. The Lyase-Pullulanase/Amylopectin/Soluble Starch System

In this system the final products are AF and glucose. If at least a substantially pure sample of AF is to be prepared, the by-product glucose must be removed. This can be achieved by enzymatic methods. First the glucose is converted into gluconic acid and hydrogen peroxide by glucose oxidase.

Catalase is needed to dispel H 2 O 2 formed. H 2 O 2 will oxidize AF into two new compounds which are at present of unknown structure. The other impurities in the AF preparation are the oxidation products of AF. It was observed that AF can slowly be oxidized by air-level of oxygen, especially at high temperature, high AF concentration and long time of exposure.

Gluconic acid was removed together with the buffer salts by ion exchange chromatography.

In this system, the low-molecular-weight amylopectin molecules may alternatively be hydrolysed by amyloglucosidase instead of using ultrafiltration.

3.3. The Purity Checking of AF

The purity of the AF preparations were confirmed by TLC, Dionex and NMR.

3.4 Analysis of the Antioxidative Activity of Anhydro Fructose

Electrochemical Oxygen Consumption

Method

The activity of AF was investigated in a methyl linoleate emulsion as described by Jorgensen and Skibsted (Z. Lebensm. Unters. Forsch. (1993) 196: 423-429) with minor modifications: To 5.00 ml of a 1.33 mM methyl linoleate emulsion in 5.0 mM aqueous phosphate buffer with pH=5.8 and 0.2 w/w % Tween 20 as emulsifier was added AF in the following concentrations: 0, 15, 146 and 680μM. The oxidation in the system was initiated by addition of 50 μl 0.26 M metmyoglobin (MMb) final concentration 0.26 mM. Immediately after initiating the reaction the sample was injected to a thermostated (25.0±0.1° C.) 70 μl closed cell, effectively excluding diffusion of oxygen into the system. The oxygen consumption was measured by a Clark electrode, which was connected to a PC data collection program. The relative oxygen concentration (%) was registered every 30s.

Results

Curves corresponding to oxygen consumption for the different samples are illustrated in FIG. 19 . For samples without addition of AF a relative decrease in oxygen concentration is seen immediately after injection of the sample. For samples containing AF a lag-phase is observed before the curve breaks off and the oxygen concentration is reduced. After the lag-phase only a minor reduction in the oxygen consumption rate is observed compared to samples without AF added. A tendency for samples having the highest amount of AF to have the longest lag-phase is observed. As well the rate for oxygen consumption is lower for these samples, which is seen by a smaller slope of the curves compared to the slope for the references (0 μM).

ESR Analysis

Method

Hydroxyl radicals were generated by a Fenton reaction with H 2 O 2 (0.17 mM) and FeSO 4 (4.8 μM). The generated radicals were trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO, 9.7 mM). AF was added in concentrations of 1.3 mM and 6.3 mM. A water soluble extract of rosemary ( Rosmarinus officinalis L.) was analyzed in a concentration of 0.25 mg/ml (in grams equivalent to 1.26 mM AF). Measurements were carried out at room temperature (20±1° C.) after 120 s and repeated for the same reaction mixture after 300 s with the following spectrometer settings: Center field 3475.60 G; sweep width 55 G; microwave power 20 mW; modulation frequency 100 kHz; modulation amplitude 1.01 G; receiver gain 1.00·10 5 ; conversion time 81.92 ms time constant 163.84 ms and sweep time 83.89 s.

Results

The generated hydroxyl radicals were trapped by DMPO. The spin adduct gives rise to a characteristic 1:2:2:1 ESR spectrum. The peak height of the spectrum is proportional to the quantitative amount of generated spin adduct. Addition of both DMPO and AF will set up a competition between the spin trap and AF. A reduction of peak height will indicate a good scavenging activity of AF.

At a concentration of 1.3 mM AF no scavenging activity of hydroxyl radicals is seen, at 6.3 mM Af the peak height is reduced, indicating that a part of the generated hydroxyl radicals is scavenged by AF.

4. Use of AF as an Anti-oxidant

Use of AF as an Anti-oxidant in a 50% Mayonnaise

50% mayonnaise is used for salads, open sandwiches, etc. in both the catering and the retail trades. The low oil content of 50% mayonnaise makes it suitable for low-calorie applications.

A typical mayonnaise composition is as follows:

MAYODAN 602 ensures a fine, stable oil dispersion and the required viscosity, thereby providing 50% mayonnaise with a long shelf life.

Flavouring 10251 is a natural lemon flavouring which provides mayonnaise with the fresh taste of lemon.

Typically the mayonnaise is prepared by the following method:

1) Dry mix the MAYODAN 602, sugar and salt. Disperse in oil in a ratio of 1 part powder to 2 parts oil.

2) Add flavouring and potassium sorbate to the water and pour into the Koruma mixer. Add 1).

3) Add the egg yolk.

4) Add the oil continuously in a vacuum.

5) After ⅔ of the oil has been added (slowly), blend the tarragon vinegar with the remaining ⅓ of the oil, and add.

The following data show that when AF is added to the mayonnaise as an anti-oxidant the results are comparable to the known food anti-oxididants GRINDOX 142 and GRINDOX 1029.

In the test procedure the anti-oxidants were added to the mayonnaise to provide an anti-oxidant concentration in the order of about 500 ppm. The mayonnaise was then placed in a bomb calorimeter at temperature 80° C. containing pure O 2 . An induction period to the onset of substantial oxidation of the product is then measured.

The results were as follows.

These results show that AF is an excellent food anti-oxidant and is comparable with the known foodstuffs anti-oxidants GRINDOX 142 or GRINDOX 1029.

Use of AF as an Anti-oxidant in a Salad Dressing

Yogurt Salad Dressing with 50% Oil

Yogurt salad dressing with 50% oil is used for salads, potatoes, raw vegetable salad, meat, fish and boiled vegetables.

MAYODAN 525 provides unique emulsion stability, prevents syneresis, ensures uniform oil dispersion and viscosity, improves tolerance to production processes and ensures a long shelf life.

Flavouring 2072 is a nature-identical, acid masking flavouring reducing the acidulated taste of dressing without affecting its pH value.

Process

1. Dry mix MAYODAN 525, sugar and salt. Disperse in oil in a ratio of 1 part powder to 2 parts oil.

2. Fill flavouring, potassium sorbate and yogurt into the Koruma mixer. Add 1).

3. Add the egg yolk.

4. Add the oil continuously in a vacuum.

5. After ⅔ of the oil has been added (slowly), blend the vinegar with the remaining ⅓ of the oil, and add.

Add spices if required.

Protection Factor (PF)

For each temperature defined as PF=IP of the oil with added antioxidant/IP of the same oil without added antioxidant

Life Extension (LE) %

LE=(PF−1.0)×100

6. Preparations of α-1.4-glucan Lyase

Introduction

With regard to a further embodiments of the present invention the enzyme α-1,4-glucan lyase for use in preparing the AF may be isolated from a fungally infected algae, preferably fungally infected Gracilariopsis lemaneiformis more preferably fungally infected Gracilariopsis lemaneiformis from Qingdao (China).

Alternatively the enzyme may be obtained from a fungus. For example, the fungus can be any one of Discina perlata, Discina parma, Gyromitra gigas, Gyromitra influa, Mitrophora hybrida, Morchella conica, Morchella costata, Morchella elata, Morchella hortensis, Morchella rotunda, Morchella vulgaris, Peziza badia, Sarcosphaera eximia, Disciotis venosa, Gyromitra esculenta, Helvella crispa, Helvella lacunosa, Leptopodia elastica, Verpa digitaliformis, and other forms of Morchella. Preferably the fungus is Morchella costata or Morchella vulgaris.

With regard to a further embodiment of the present invention the enzyme α-1,4-glucan lyase for use in preparing the AF may be isolated from algae alone, preferably Gracilariopsis lemaneiformis more preferably Gracilariopsis lemaneiformis from Santa Cruz (Calif.).

The initial enzyme purification can be performed by the method as described by Yu et al (ibid). However, preferably, the initial enzyme purification includes an optimized procedure in which a solid support is used that does not decompose under the purification step. This gel support further has the advantage that it is compatible with standard laboratory protein purification equipment. The details of this optimized purification strategy are given later on. The purification is terminated by known standard techniques for protein purification.

The purity of the enzyme can be readily established using complementary electrophoretic techniques.

A. Source=Fungally Infected Algae

The following sequence information was used to generate primers for the PCR reactions mentioned below and to check the amino acid sequence generated by the respective nucleotide sequences.

Amino Acid Sequence Assembled from Peptides from Fungus Infected Gracilariopsis lemaneiformis

Tyr Arg Trp Gln Glu Val Leu Tyr Thr Ala Met Tyr Gln Asn Ala

Ala Phe Gly Lys Pro Ile Ile Lys Ala Ala Ser Met Tyr Asn Asn

Asp Ser Asn Val Arg Arg Ala Gln Asn Asp His Phe Leu Leu Gly

Gly His Asp Gly Tyr Arg Ile Leu Cys Ala Pro Val Val Trp Glu

Asn Ser Thr Glu Arg Glu Leu Tyr Leu Pro Val Leu Thr Gln Trp

Tyr Lys Phe Gly Pro Asp Phe Asp Thr Lys Pro Leu Glu Gly Ala

The Amino Acid Sequence (27-34) Used to Generate Primer A and B (Met Tyr Asn Asn Asp Ser Asn Val)

Primer A

ATG TA(TC) AA(CT) AA(CT) GA(CT) TC(GATC) AA(CT) GT 128 mix

Primer B

ATG TA(TC) AA(CT) AA(CT) GA(CT) AG(CT) AA(CT) GT 64 mix

The Amino Acid Sequence (45-50) Used to Generate Primer C (Gly Gly His Asp Gly Tyr)

Primer C

TA (GATC)CC (GA)TC (GA)TG (GATC)CC (GATC)CC 256 mix

[The Sequence Corresponds to the Complementary Strand.]

The Amino Acid Sequence (74-79) Used to Generate Primer E (Gln Trp Tyr Lys Phe Gly)

Primer E

GG(GATC) CC(GA) AA(CT) TF(GA) TAC CA(CT) TG 64 mix

[The Sequence Corresponds to the Complementary Strand.]

The Amino Acid Sequence (1-6) Used to Generate Primer F1 and F2 (Tyr Arg Trp Gln Glu Val)

Primer F1

TA(TC) CG(GATC) TGG CA(GA) GA(GA) GT 32 mix

Primer F2

TA(TC) AG(GA) TGG CA(GA) GA(GA) GT 16 mix

The Sequence Obtained from the First PCR Amplification (Clone 1)

Met Tyr Asn Asn Asp Ser Asn Val Arg Arg Ala Gln Asn Asp His Phe Leu Leu Gly Gly His Asp Gly

The Sequence Obtained from the Second PCR Amplification (Clone 1)

Met Tyr Asn Asn Asp Ser Asn Val Arg Arg Ala Gln Asn Asp His Phe Leu Leu Gly

Gly His Asp Gly Tyr Arg Ile Leu Cys Ala Pro Val Val Trp Glu Asn Ser Thr Glu

Arg Glu Leu Tyr Leu Pro Val Leu Thr Gln Trp Tyr Lys Phe Gly Pro

The Sequence Obtained from the Third PCR Amplification (Clone2)

Tyr Arg Trp Gln Glu Val Leu Tyr Thr Ala Met Tyr Gln Asn Ala Ala Phe Gly Lys

Pro Ile Ile Lys Ala Ala Ser Met Tyr Asp Asn Asp Arg Asn Val Arg Gly Ala Gln Asp

Asp His Phe Leu Leu Gly Gly His Asp Gly Tyr Arg Ile Leu Cys Ala Pro Val Val

Trp Glu Asn Thr Thr Ser Arg Asp Leu Tyr Leu Pro Val Leu Thr Lys Trp Tyr Lys

Phe Gly

A.1. Cytological Investigations of Gracilariopsis lemaneiformis

A.1.1.1 Detection of Fungal Infection in Gracilariopsis lemaneiformis

Sections of Gracilariopsis lemaneiformis collected in China were either hand cut or cut from paraffin embedded material. Sectioned material was carefully investigated by light microscopy. Fungal hyphae were clearly detected in Gracilariopsis lemaneiformis.

The thalli of the Gracilariopsis lemaneiformis are composed of cells appearing in a highly ordered and almost symmetric manner. The tubular thallus of G. lemaneiformis is composed of large, colorless central cells surrounded by elongated, slender, ellyptical cells and small, round, red pigmented peripherial cells. All algal cell types are characterized by thick cell walls. Most of the fungal hyphae are found at the interphase between the central layer of large cells and the peripherial layer. These cells can clearly be distinguished from the algae cells as they are long and cylindrical. The growth of the hyphae is observed as irregularities between the highly ordered algae cells. The most frequent orientation of the hypha is along the main axis of the algal thallus. Side branches toward the central and periphery are detected in some cases. The hypha can not be confused with the endo/epiphytic 2nd generation of the algae.

Calcofluor White is known to stain chitin and cellulose containing tissue. The reaction with chitin requires four covalently linked terminal n-acetyl glucosamine residues. It is generally accepted that cellulose is almost restricted to higher plants although it might occur in trace amounts in some algae. It is further known that chitin is absent in Gracilaria.

Calcofluor White was found to stain domains corresponding to fungi hyfa cell walls in sectioned Gracilariopsis lemaneiformis material.

The hypha appear clear white against a faint blue background of Gracilaria tissue when observed under u.v. light—see FIG. 1 . Chitin is the major cell wall component in most fungi but absent in Gracilaria. Based upon these observations we conclude that the investigated algae is infected by a fungi. 40% of the lower parts of the investigated Gracilariopsis lemaneiformis sections were found to be infected with fungal hyphae. In the algae tips 25% of the investigated Gracilariopsis lemaneiformis sections were found to be infected.

Staining of sectioned Gracilariopsis lemaneiformis with Periodic acid Schiff (PAS) and Aniline blue black revealed a significantly higher content of carbohydrates within the fungal cells as compared with the algae cells—see FIG. 2 . Safranin 0 and Malachit Green showed the same colour reaction of fungi cells as found in higher plants infected with fungi.

An Acridin Orange reaction with sectioned Gracilariopsis lemaneiformis showed clearly the irregularly growth of the fungus.

A. 1.1.2 Electron Microscopy

Slides with 15 μm thick sections, where the fungus was detected with Calcofluor White were fixed in 2% OsO 4 , washed in water and dehydrated in dimethoxypropane and absolute alcohol. A drop of a 1:1 mixture of acetone and Spurr resin was placed over each section on the glass slide, and after one hour replaced by a drop of pure resin. A gelatin embedding capsule filled with resin was placed face down over the section and left over night at 4° C. After the polymerization at 55° C. for 8 hrs, the thick sections adhering to the resin blocks could can be separated from the slide by immersion in liquid nitrogen.

Blocks were trimmed and 100 nm thick sections were cut using a diamond knife on a microtome. The sections were stained in aqueous uranyl acetate and in lead citrate. The sections were examined in an electron microscope at 80 kV.

The investigation confirmed the ligth microscopical observations and provided further evidence that the lyase producing, chinese strain of G. lamneiformis is infected by a fungal parasite or symbiont.

Fungal hyphae are build of tubular cells 50 to 100 μm long and only few microns in diameter. The cells are serially arranged with septate walls between the adjacent cells. Ocasional branches are also seen. The hyphae grow between the thick cell walls of algal thallus without penetrating the wall or damaging the cell. Such a symbiotic association, called mycophycobiosis, is known to occur between some filamentous marine fungi and large marine algae (Donk and Bruning, 1992—Ecology of aquatic fungi in and on algae. In Reisser, W.(ed.): Algae and Symbioses: Plants, Animals, Fungi, Viruses, Interactions Explored. Biopress Ltd., Bristol.)

Examining the microphotograph in FIG. 10, several differences between algal and fungal cells can be noticed. In contrast to several μm thick walls of the alga, the fungal walls are only 100-200 nm thick. Plant typical organells as chloroplasts with thyllacoid membranes as well as floridean starch grains can be seen in algal cells, but not in the fungus.

Intercellular connections of red algae are characterized by specific structures termed pit plugs, or pit connections The structures are prominent, electron dense cores and they are important features in algal taxonomy (Pueschel, C. M.: An expanded survey of the ultrastructure of Red algal pit plugs. J. Phycol. 25, 625, (1989)). In our material, such connections were frequently observed in the algal thallus, but never between the cells of the fungus.

A. 1.2 In situ Hybridization Experiments

In situ hybridization technique is based upon the principle of hybridization of an antisense ribonucotide sequence to the mRNA. The technique is used to visualize areas in microscopic sections where said mRNA is present. In this particular case the technique is used to localize the enzyme α-1,4-glucan lyase in sections of Gracilariopsis lemaneiformis.

A. 1.2. 1 Preparation of 35 S Labelled Probes for In situ Hybridization

A 238 bp PCR fragment from a third PCR amplification—called clone 2 (see above)—was cloned into the pGEM-3Zf(+) Vector (Promega). The transcription of the antisense RNA was driven by the SP6 promotor, and the sense RNA by the T7 promotor. The Ribonuclease protection assay kit (Ambion) was used with the following modifications. The transcripts were run on a 6% sequencing gel to remove the unincorporated nucleotide and eluted with the elution buffer supplied with the T7RNA polymerase in vitro Transcription Kit (Ambion). The antisense transcript contained 23 non-coding nucleotides while the sense contained 39. For hybridization 10 7 cpm/ml of the 35 S labelled probe was used.

In situ hybridisation was performed essentially as described by Langedale et.al.(1988). The hybridization temperature was found to be optimal at 45° C. After washing at 45° C. the sections were covered with KodaK K-5 photographic emulsion and left for 3 days at 5° C. in dark (Ref: Langedale, J. A., Rothermel, B. A. and Nelson, T. (1988). Genes and development 2: 106-115. Cold Spring Harbour Laboratory).

The in situ hybridization experiments with riboprobes against the mRNA of α-1,4-glucan lyase, show strong hybridizations over and around the hypha of the fungus detected in Gracilariopsis lemaneiformis —see FIGS. 4 and 5. This is considered a strong indication that the α-1,4-glucan lyase is produced. A weak random background reactions were detected in the algae tissue of both Gracilariopsis lemaneiformis. This reaction was observed both with the sense and the antisense probes. Intense staining over the fungi hypha was only obtained with antisense probes.

These results were obtained with standard hybridisation conditions at 45° C. in hybridization and washing steps. At 50° C. no staining over the fungi was observed, whereas the background staining remained the same. Raising the temperature to 55° C. reduced the background staining with both sense and antisense probes significantly and equally.

Based upon the cytological investigations using complementary staining procedures it is concluded that Gracilariopsis lemaneiformis is fungus infected. The infections are most pronounced in the lower parts of the algal tissue.

In sectioned Gracilariopsis lemaneiformis material in situ hybridization results clearly indicate that hybridization is restricted to areas where fungal infections are found—see FIG. 4 . The results indicate that α-1,4-glucan lyase mRNA appears to be restricted to fungus infected areas in Gracilariopsis lemaneiformis. Based upon these observations we conclude that α-1,4-glucan lyase activity is detected in fungally infected Gracilariopsis lemaneiformis.

A.2. Enzyme Purification and Characterization

Purification of α-1,4-glucan lyase from fungal infected Gracilariopsis lemaneiformis material was performed as follows.

A.2. 1 Materials and Methods

The algae were harvested by filtration and washed with 0.9% NaCl. The cells were broken by homogenization followed by sonication on ice for 6×3 min in 50 mM citrate-NaOH pH 6.2 (Buffer A). Cell debris were removed by centrifugation at 25,000×g for 40 min. The supernatant obtained at this procedure was regarded as cell-free extract and was used for activity staining and Western blotting after separation on 8-25% gradient gels.

A.2.2 Separation by β-cyclodextrin Sepharose Gel

The cell-free extract was applied directly to a β-cyclodextrin Sepharose gel 4B column (2.6×18 cm) pre equilibrated with Buffer A. The column was washed with 3 volumes of Buffer A and 2 volumes of Buffer A containing 1 M NaCl. α-1,4-glucan lyase was eluted with 2% dextrins in Buffer A. Active fractions were pooled and the buffer changed to 20 mM Bis-tris propane-HCl (pH 7.0, Buffer B).

Active fractions were applied onto a Mono Q HR 5/5 column pre-equilibrated with Buffer B. The fungal lyase was eluted with Buffer B in a linear gradient of 0.3 M NaCl.

The lyase preparation obtained after β-cyclodextrin Sepharose chromatography was alternatively concentrated to 150 μl and applied on a Superose 12 column operated under FPLC conditions.

A.2.3 Assay for α-1,4-glucan Lyase Activity and Conditions for Determination of Substrate Specificity, pH and Temperature Optimum

The reaction mixture for the assay of the α-1,4-glucan lyase activity contained 10 mg ml− 1 amylopectin and 25 mM Mes-NaOH (pH 6.0). The reaction was carried out at 30° C. for 30 min and stopped by the addition of 3,5-dinitrosalicylic acid reagent. Optical density at 550 nm was measured after standing at room temperature for 10 min.

A.3. Amino Acid Sequencing of the α-1.4-glucan Lyase from Fungus Infected Gracilariopsis lemaneiformis

A.3.1 Amino Acid Sequencing of the Lyases

The lyases were digested with either endoproteinase Arg-C from Clostridium histolyticum or endoproteinase Lys-C from Lysobacter enzymogenes, both sequencing grade purchased from Boehringer Mannheim, Germany. For digestion with endoproteinase Arg-C, freeze dried lyase (0.1 mg) was dissolved in 50μl 10 M urea, mM methylamine, 0.1 M Tris-HCl, pH 7.6. After overlay with N 2 and addition of 10 μl of 50 mM DTr and 5 mM EDTA the protein was denatured and reduced for min at 50° C. under N 2 . Subsequently, 1 μg of endoproteinase Arg-C in 10μl of 50 mM Tris-HCl, pH 8.0 was added, N 2 was overlayed and the digestion was carried out for 6h at 37° C. For subsequent cysteine derivatization, 12.5 μl 100 mM iodoacetamide was added and the solution was incubated for 15 min at RT in the dark under N 2 .

For digestion with endoproteinase Lys-C, freeze dried lyase (0.1 mg) was dissolved in 50 μl of 8 M urea, 0.4 M NH 4 HCO 3 , pH 8.4. After overlay with N 2 and addition of 5 μl of 45 mM DTT, the protein was denatured and reduced for 15 min at 50° C. under N 2 . After cooling to RT, 5 μl of 100 mM iodoacetamide was added for the cysteines to be derivatized for 15 min at RT in the dark under N 2 .

Subsequently, 90 μl of water and 5 μg of endoproteinase Lys-C in 50 μl of 50 mM tricine and 10 mM EDTA, pH 8.0, was added and the digestion was carried out for 24 h at 37° C. under N 2 .

The resulting peptides were separated by reversed phase HPLC on a VYDAC C18 column (0.46×15 cm; 10 μm; The Separations Group; California) using solvent A: 0.1% TEA in water and solvent B: 0.1% TFA in acetonitrile. Selected peptides were rechromatographed on a Develosil C18 column (0.46×10 cm; 3 μm; Dr. Ole Schou, Novo Nordisk, Denmark) using the same solvent system prior to sequencing on an Applied Biosystems 476A sequencer using pulsed-liquid fast cycles.

The amino acid sequence information from the enzyme derived from fungus infected Gracilariopsis lemaneiformis is shown below, in particular SEQ. ID. No. 1. and SEQ. ID. No. 2.

SEQ. I.D. No. 1 has

Number of residues: 1088.

Amino acid composition (including the signal sequence)

SEQ. I.D. No. 2 has

Number of residues: 1091.

Amino acid composition (including the signal sequence)

A.3.2 N-terminal Analysis

Studies showed that the N-terminal sequence of native glucan lyase 1 was blocked. Deblocking was achieved by treating glucan lyase 1 blotted onto a PVDF membrane with anhydrous TFA for 30 min at 40° C. essentially as described by LeGendre et al. (1993) [Purification of proteins and peptides by SDS-PAGE; In: Matsudaira, P. (ed.) A practical guide to protein and peptide purification for microsequencing, 2nd edition; Academic Press Inc., San Diego; pp. 74-101.]. The sequence obtained was TALSDKQTA, which matches the sequence (sequence position from 51 to 59 of SEQ. I.D. No.1) derived from the clone for glucan lyase 1 and indicates N-acetylthreonine as N-terminal residue of glucan lyase 1. Sequence position 1 to 50 of SEQ. I.D. No. 1 represents a signal sequence.

A.4. DNA Sequencing of Genes Coding for the α-1.4-glucan Lyase from Fungus Infected Gracilariopsis lemaneiformis

A.4.1 Methods for Molecular Biology

DNA was isolated as described by Saunders (1993) with the following modification: The polysaccharides were removed from the DNA by ELUTIP-d (Schleicher & Schuell) purification instead of gel purification. (Ref:Saunders, G. W. (1993). Gel purification of red algal genomic DNA: An inexpensive and rapid method for the isolation of PCR-friendly DNA. Journal of phycology 29(2): 251-254 and Schleicher & Schuell: ELUTIP-d. Rapid Method for Purification and Concentration of DNA.)

A.4.2 PCR

The preparation of the relevant DNA molecule was done by use of the Gene Amp DNA Amplification Kit (Perkin Elmer Cetus, USA) and in accordance with the manufactures instructions except that the Taq polymerase was added later (see PCR cycles) and the temperature cycling was changed to the following:

A.4.3 Cloning of PCR Fragments

PCR fragments were cloned into pT7Blue (from Novagen) following the instructions of the supplier.

A.4.4 DNA Sequencing

Double stranded DNA was sequenced essentially according to the dideoxy method of Sanger et al. (1979) using the Auto Read Sequencing Kit (Pharmacia) and the Pharmacia LKB A.L.F.DNA sequencer. (Ref.: Sanger, F., Nicklen, S. and Coulson, A. R.(1979). DNA sequencing with chain-determinating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5467.)

The sequences are shown as SEQ. I.D. No.s 1 and 2. In brief:

SEQ. I.D. No. 3 has

Total number of bases: 3267.

DNA sequence composition: 850 A; 761 C; 871 G; 785 T

SEQ. I.D. No. 4 has

Total number of bases: 3276.

DNA sequence composition: 889 A; 702 C; 856 G; 829 T

A.4.5 Screening of the Library

Screening of the Lambda Zap library obtained from Stratagene, was performed in accordance with the manufacturer's instructions except that the prehybridization and hybridization was performed in 2×SSC, 0.1% SDS, 10×Denhardt's and 100g/ml denatured salmon sperm DNA. To the hybridization solution a 32P-labeled denatured probe was added. Hybridization was performed over night at 55° C. The filters were washed twice in 2×SSC, 0.1% SDS and twice in 1×SSC, 0.1% SDS.

A.4.6 Probe

The cloned PCR fragments were isolated from the pT7 blue vector by digestion with appropriate restriction enzymes. The fragments were separated from the vector by agarose gel electrophoresis and the fragments were purified from the agarose by Agarase (Boehringer Mannheim). As the fragments were only 90-240 bp long the isolated fragments were exposed to a ligation reaction before labelling with 32P-dCTP using either Prime-It random primer kit (Stratagene) or Ready to Go DNA labelling kit (Pharmacia).

A.4.7 Results

A.4.7.1 Generation of PCR DNA Fragments Coding for α-1,4-glucan Lyase

The amino acid sequences of three overlapping tryptic peptides from α-1,4-glucan lyase were used to generate mixed oligonucleotides, which could be used as PCR primers (see the sequences given above).

In the first PCR amplification primers A/B (see above) were used as upstream primers and primer C (see above) was used as downstream primer. The size of the expected PCR product was 71 base pairs.

In the second PCR amplification primers A/B were used as upstream primers and E was used as downstream primer. The size of the expected PCR product was 161 base pairs.

In the third PCR amplification primers F1 (see above) and F2 (see above) were used as upstream primers and E was used as downstream primer. The size of the expected PCR product was 238 base pairs.

The PCR products were analysed on a 2% LMT agarose gel and fragments of the expected sizes were cut out from the gel and treated with Agarase (Boehringer Manheim) and cloned into the pT7blue Vector (Novagen) and sequenced.

The cloned fragments from the first and second PCR amplification coded for amino acids corresponding to the sequenced peptides (see above). The clone from the third amplification (see above) was only about 87% homologous to the sequenced peptides.

A.4.7.2 Screening of the Genomic Library with the Cloned PCR Fragments

Screening of the library with the above-mentioned clones gave two clones. One clone contained the nucleotide sequence of SEQ I.D. No. 4 (gene 2). The other clone contained some of the sequence of SEQ I.D. No.3 (from base pair 1065 downwards) (gene 1).

The 5′ end of SEQ. I.D. No. 3 (i.e. from base pair 1064 upwards) was obtained by the RACE (rapid amplification of cDNA ends) procedure (Michael, A. F., Michael, K. D. & Martin, G. R.(1988). Proc.Natl.Acad.Sci.USA 85:8998-99002.) using the 5′ race system from Gibco BRL. Total RNA was isolated according to Collinge et al.(Collinge, D. B., Milligan D. E:, Dow, J. M., Scofield, G. & Daniels, M. J.(1987). Plant Mol Biol 8: 405-414). The 5′ race was done according to the protocol of the manufacturer, using 1 μg of total RNA. The PCR product from the second amplification was cloned into pT7blue vector from Novagen according to the protocol of the manufacturer. Three independent PCR clones were sequenced to compensate for PCR errors.

An additional PCR was performed to supplement the clone just described with XbaI and NdeI restriction sites immediately in front of the ATG start codon using the following oligonucleotide as an upstream primer:

and a primer containing the complement sequence of bp 1573-1593 in sequence GL1 (i.e. SEQ. I.D. No. 3) was used-as a downstream primer.

The complete sequence for gene 1 (i.e. SEQ. I.D. No. 3) was generated by cloning the 3′ end of the gene as a BamHI-HindIII fragment from the genomic clone into the pBluescript II KS+ vector from Stratagene and additionally cloning the PCR generated 5′ end of the gene as a XbaI-BamHI fragment in front of the 3′ end.

Gene 2 was cloned as a HindIII blunt ended fragment into the EcoRV site of pbluescript II SK+ vector from Stratagene. A part of the 3′ untranslated sequence was removed by a SacI digestion, followed by religation. HindIII and HpaI restriction sites were introduced immediately in front of the start ATG by digestion with HindIII and NarI and religation in the presence of the following annealed oligonucleotides

No introns were found in the clones sequenced.

The clone 1 type (SEQ. ID. No.3) can be aligned with all ten peptide sequences (see FIG. 8) showing 100% identity. Alignment of the two protein sequences encoded by the genes isolated from the fungal infected algae Gracilariopsis lemaneiformis shows about 78% identity, indicating that both genes are coding for a α-1.4-glucan lyase.

Expression of the GL Gene in Micro-organisms

(E.G. Analyses of Pichia Lyase Transformants and Aspergillus Lyase Transformants)

The DNA sequence encoding the GL was introduced into microorganisms to produce an enzyme with high specific activity and in large quantities.

In this regard, gene 1 (i.e. SEQ. I.D. No. 3) was cloned as a NotI-HindIII blunt ended (using the DNA blunting kit from Amersham International) fragment into the Pichia expression vector pHIL-D2 (containing the AOX1 promoter) digested with EcoRI and blunt ended (using the DNA blunting kit from Amersham International) for expression in Pichia pastoris (according to the protocol stated in the Pichia Expression Kit supplied by Invitrogen).

In another embodiment, the gene I (i.e. SEQ. I.D. No. 3) was cloned as a NotI-HindIII blunt ended fragment (using the DNA blunting kit from Amersham International) into the Aspergillus expression vector pBARMTE1 (containing the methyl tryptophan resistance promoter from Neuropera crassa ) digested with SmaI for expression in Aspergillus niger (Pall et al (1993) Fungal Genet Newsletter vol 40 pages 59-62). The protoplasts were prepared according to Daboussi et al (Curr Genet (1989) vol 15 pp 453-456) using lysing enzymes Sigma L-2773 and the lyticase Sigma L-8012. The transformation of the protoplasts was followed according to the protocol stated by Buxton et al (Gene (1985) vol 37 pp 207-214) except that for plating the transformed protoplasts the protocol laid out in Punt et al (Methods in Enzymology (1992) vol 216 pp 447-457) was followed but with the use of 0.6% osmotic stabilised top agarose.

The results showed that lyase activity was observed in the transformed Pichia pastoris and Aspergillus niger.

A.5.1 General Methods

Preparation of cell-free extracts.

The cells were harvested by centrifugation at 9000 rpm for 5 min and washed with 0.9% NaCl and resuspended in the breaking buffer (50 mM K-phosphate, pH 7.5 containing 1 mM of EDTA, and 5% glycerol). Cells were broken using glass beads and vortex treatment. The breaking buffer contained 1 mM PMSF (protease inhibitor). The lyase extract (supernatant) was obtained after centrifugation at 9000 rpm for 5 min followed by centrifugation at 20,000×g for 5min.

Assay of lyase activity by alkaline 3,5-dinitrosalicylic acid reagent (DNS)

One volume of lyase extract was mixed with an equal volume of 4% amylopectin solution. The reaction mixture was then incubated at a controlled temperature and samples were removed at specified intervals and analyzed for AF.

The lyase activity was also analyzed using a radioactive method.

The reaction mixture contained 10 μl 14 C-starch solution (1 μCi; Sigma Chemicals Co.) and 10 μl of the lyase extract. The reaction mixture was left at 25° C. overnight and was then analyzed in the usual TLC system. The radioactive AF produced was detected using an Instant Imager (Pachard Instrument Co., Inc., Meriden, Conn.).

Electrophoresis and Western Blotting

SDS-PAGE was performed using 8-25% gradient gels and the PhastSystem (Pharmacia). Western blottings was also run on a Semidry transfer unit of the PhastSystem.

Primary antibodies raised against the lyase purified from the red seaweed collected at Qingdao (China) were used in a dilution of 1:100. Pig antirabbit IgG conjugated to alkaline phosphatase (Dako A/S, Glostrup, Denmark) were used as secondary antibodies and used in a dilution of 1:1000.

Results

1. Lyase activity was determined 5 days after induction (according to the manual) and proved the activity to be intracellular for all samples in the B series.

A time course of sample B27 is as follows. The data are also presented in FIG. 1

2. Western-blotting Analysis

The CFE of all samples showed bands with a molecular weight corresponding to the native lyase.

Results

I. Lyase Activity was Determined after 5 days Incubation (Minimal Medium Containing 0.2% Casein Enzymatic Hydrolysate Analysis by the Alkaline 3,5-dinitrosalicylic Acid Reagent

1). Lyase Activity Analysis of the Culture Medium

Among 35 cultures grown with 0.2% amylopectin included in the culture medium, AF was only detectable in two cultures. The culture medium of 5.4+ and 5.9+ contained 0.13 g AF/liter and 0.44 g/liter, respectively. The result indicated that active lyase had been secreted from the cells. Lyase activity was also measurable in the cell-free extract.

2). Lyase Activity Analysis in Cell-free Extracts

The results show that Gene 1 of GL was expressed intracellular in A. niger.

Experiments with transformed E.coli (using cloning vectors pQE30 from the Qia express vector kit from Qiagen) showed expression of enzyme that was recognised by anti-body to the enzyme purified from fungally infected Gracilariopsis lemaneiformis.

B. Source=Fungus

B.1. Enzyme Purification and Characterization of the α-1,4-glucan Lyase from the Fungus Morchella costata

B.1.1 Materials and Methods

The fungus Morchella costata was obtained from American Type Culture Collection (ATCC). The fungus was grown at 25° C. on a shaker using the culture medium recommended by ATCC. The mycelia were harvested by filtration and washed with 0.9% NaCl.

The fungal cells were broken by homogenization followed by sonication on ice for 6×3 min in 50 mM citrate-NaOH pH 6.2 (Buffer A). Cell debris were removed by centrifugation at 25,000×g for 40 min. The supernatant obtained at this procedure was regarded as cell-free extract and was used for activity staining and Western blotting after separation on 8-25% gradient gels.

B. 1.2 Separation by β-cyclodextrin Sepharose Gel

The cell-free extract was applied directly to a β-cyclodextrin Sepharose gel 4B column ( 2.6×18 cm) pre equilibrated with Buffer A. The column was washed with 3 volumes of Buffer A and 2 volumes of Buffer A containing 1 M NaCl. α-1,4-glucan lyase was eluted with 2% dextrins in Buffer A. Active fractions were pooled and the buffer changed to 20 mM Bis-tris propane-HCl (pH 7.0, Buffer B).

Active fractions were applied onto a Mono Q HR 5/5 column pre-equilibrated with Buffer B. The fungal lyase was eluted with Buffer B in a linear gradient of 0.3 M NaCl. The lyase preparation obtained after β-cyclodextrin Sepharose chromatography was alternatively concentrated to 150 μl and applied on a Superose 12 column operated under FPLC conditions.

B.1.3 Assay for α-1,4-glucan lyase activity and conditions for determination of substrate specificity, pH and temperature optimum

The reaction mixture for the assay of the α-1,4-glucan lyase activity contained 10 mg ml −1 amylopectin and 25 mM Mes-NaOH (pH 6.0).

The reaction was carried out at 30 ° C. for 30 min and stopped by the addition of 3,5-dinitrosalicylic acid reagent. Optical density at 550 nm was measured after standing at room temperature for 10 min. 10 mM EDTA was added to the assay mixture when cell-free extracts were used.

The substrate amylopectin in the assay mixture may be replaced with other substrates and the reaction temperature may vary as specified in the text.

In the pH optimum investigations, the reaction mixture contained amylopection or maltotetraose 10 mg ml −1 in a 40 mM buffer. The buffers used were glycine-NaOH (pH 2.0-3.5), HoAc-NaoAc (pH 3.5-5.5), Mes-NaOH (pH 5.5-6.7), Mops-NaOH (6.0-8.0) and bicine-NaOH (7.6-9.0). The reactions were carried out at 30° C. for 30 min. The reaction conditions in the temperature optimum investigations was the same as above except that the buffer Mops-NaOH (pH 6.0) was used in all experiments. The reaction temperature was varied as indicated in the text.

SDS-PAGE, Native-PAGE and isoelectrofocusing were performed on PhastSystem (Pharmacia, Sweden) using 8-25% gradient gels and gels with a pH gradient of 3-9, respectively. Following electrophoresis, the gels were stained by silver staining according to the procedures recommended by the manufacturer (Pharmacia). The glycoproteins were stained by PAS adapted to the PhastSystem. For activity staining, the electrophoresis was performed under native conditions at 6° C.

Following the electrophoresis, the gel was incubated in the presence of 1% soluble starch at 30° C. overnight. Activity band of the fungal lyase was revealed by staining with I 2 /KI solution.

B. 1.4 Results

B. 1.4.1 Purification, Molecular Mass and Isoelectric Point of the α-1,4-glucan Lyase

The fungal lyase was found to adsorb on columns packed with β-cyclodextrin Sepharose, starches and Red Sepharose. Columns packed with β-cyclodextrin Sepharose 4B gel and starches were used for purification purposes.

The lyase preparation obtained by this step contained only minor contaminating proteins having a molecular mass higher than the fungal lyase. The impurity was either removed by ion exchange chromatography on Mono Q HR 5/5 or more efficiently by gel filtration on Superose 12.

The purified enzyme appeared colourless and showed no absorbance in the visible light region. The molecular mass was determined to 110 kDa as estimated on SDS-PAGE.

The purified fungal lyase showed a isoelectric point of pI 5.4 determined by isoelectric focusing on gels with a pH gradient of 3 to 9. In the native electrophoresis gels, the enzyme appeared as one single band. This band showed starch-degrading activity as detected by activity staining. Depending the age of the culture from which the enzyme is extracted, the enzyme on the native and isoelectric focusing gels showed either as a sharp band or a more diffused band with the same migration rate and pI.

B. 1.4.2 The pH and Temperature Optimum of the Fungal Lyase Catalayzed Reaction

The pH optimum pH range for the fungal lyase catalyzed reaction was found to be between pH 5 and pH 7.

B. 1.4.3 Substrate Specificity

The purified fungal lyase degraded maltosaccharides from maltose to maltoheptaose. However, the degradation rates varied. The highest activity achieved was with maltotetraose (activity as 100%), followed by maltohexaose (97%), maltoheptaose (76%), maltotriose (56%) and the lowest activity was observed with maltose (2%).

Amylopectin, amylose and glycogen were also degraded by the fungal lyase (% will be determined). The fungal lyase was an exo-lyase, not a endolyase as it degraded p-nitrophenyl α-D-maltoheptaose but failed to degrade reducing end blocked p-nitrophenyl α-D-maltoheptaose.

B. 1.5 Morchella Vulgaris

The protocols for the enzyme purification and charaterisation of alpha α-1,4-glucal lyase obtained from Morchella Vulgaris were the same as those above for Morchella Costata (with similar results).

B. 2. Amino Acid Sequencing of the α-1,4-glucan Lyase from Fungus

B. 2.1 Amino Acid Sequencing of the Lyases

The lyases were digested with either endoproteinase Arg-C from Clostridium histolyticum or endoproteinase Lys-C from Lysobacter enzymogenes, both sequencing grade purchased from Boehringer Mannheim, Germany. For digestion with endoproteinase Arg-C, freezedried lyase (0.1 mg) was dissolved in 50 μl 10 M urea, mM methylamine, 0.1 M Tris-HCl, pH 7.6. After overlay with N 2 and addition of 10 μl of 50 mM DTT and 5 mM EDTA the protein was denatured and reduced for min at 50° C. under N 2 . Subsequently, 1 μg of endoproteinase Arg-C in 10 μl of 50 mM Tris-HCl, pH 8.0 was added, N 2 was overlayed and the digestion was carried out for 6 h at 37° C.

For subsequent cysteine derivatization, 12.5 μl 100 mM iodoacetamide was added and the solution was incubated for 15 min at RT in the dark under N 2 .

For digestion with endoproteinase Lys-C, freeze dried lyase (0.1 mg) was dissolved in 50 μl of 8 M urea, 0.4 M NH 4 HCO 3 , pH 8.4. After overlay with N 2 and addition of 5 μl of 45 mM DIT, the protein was denatured and reduced for 15 min at 50° C. under N 2 . After cooling to RT, 5 μl of 100 mM iodoacetamide was added for the cysteines to be derivatized for 15 min at RT in the dark under N 2 . Subsequently, 90 μl of water and 5 μg of endoproteinase Lys-C in 50 μl of 50 mM tricine and 10 mM EDTA, pH 8.0, was added and the digestion was carried out for 24 h at 37° C. under N 2 .

The resulting peptides were separated by reversed phase HPLC on a VYDAC C18 column (0.46×15 cm; 10 μm; The Separations Group; California) using solvent A: 0.1% TFA in water and solvent B: 0.1% TFA in acetonitrile. Selected peptides were rechromatographed on a Develosil C18 column (0.46×10 cm; 3 μm; Dr. Ole Schou, Novo Nordisk, Denmark) using the same solvent system prior to sequencing on an Applied Biosystems 476A sequencer using pulsed-liquid fast cycles.

The amino acid sequence information from the enzyme derived from the fungus Morchella costata is shown FIG. 17 .

The amino acid sequence information from the enzyme derived from the fungus Morchella vulgaris is shown FIG. 18 .

B.3. DNA Sequencing of Genes Coding for the α-1,4glucan Lyase from Fungus

B.3.1 Methods for Molecular Biology

DNA was isolated as described by Dellaporte et al (1983—Plant Mol Biol Rep vol 1 pp19-21).

B.3.2 PCR

The preparation of the relevant DNA molecule was done by use of the Gene Amp DNA Amplification Kit (Perkin Elmer Cetus, USA) and in accordance with the manufactures instructions except that the Taq polymerase was added later (see PCR cycles) and the temperature cycling was changed to the following:

B.3.3 Cloning of PCR Fragments

PCR fragments were cloned into pT7Blue (from Novagen) following the instructions of the supplier.

B.3.4 DNA Sequencing

Double stranded DNA was sequenced essentially according to the dideoxy method of Sanger et al. (1979) using the Auto Read Sequencing Kit (Pharmacia) and the Pharmacia LKB A.L.F.DNA sequencer. (Ref: Sanger, F., Nicklen, S. and Coulson, A. R.(1979). DNA sequencing with chain-determinating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5467.)

B.3.5 Screening of the Libraries

Screening of the Lambda Zap libraries obtained from Stratagene, was performed in accordance with the manufacturer's instructions except that the prehybridization and hybridization was performed in 2×SSC, 0.1% SDS, 10×Denhardt's and 100μg/ml denatured salmon sperm DNA.

To the hybridization solution a 32P-labeled denatured probe was added. Hybridization was performed over night at 55° C. The filters were washed twice in 2×SSC, 0.1% SDS and twice in 1×SSC, 0.1% SDS.

B.3.6 Probe

The cloned PCR fragments were isolated from the p77 blue vector by digestion with appropriate restriction enzymes. The fragments were separated from the vector by agarose gel electrophoresis and the fragments were purified from the agarose by Agarase (Boehringer Mannheim). As the fragments were only 90-240 bp long the isolated fragments were exposed to a ligation reaction before labelling with 32P-dCTP using either Prime-It random primer kit (Stratagene) or Ready to Go DNA labelling kit (Pharmacia).

B.3.7 Results

B.3.7.1 Generation of PCR DNA Fragments Coding for α-1,4-glucan Lyase

The amino acid sequences (shown below) of three overlapping tryptic peptides from α-1,4-glucan lyase were used to generate mixed oligonucleotides, which could be used as PCR primers for amplification of DNA isolated from both MC and MV.

Lys Asn Leu His Pro Gln His Lys Met Leu Lys Asp Thr Val Leu Asp Ile Val Lys

Pro Gly His Gly Glu Tyr Val Gly Trp Gly Glu Met Gly Gly Ile Gln Phe Met Lys

Glu Pro Thr Phe Met Asn Tyr Phe Asn Phe Asp Asn Met Gln Tyr Gln Gln Val Tyr

Ala Gln Gly Ala Leu Asp Ser Arg Glu Pro Leu Tyr His Ser Asp Pro Phe Tyr

In the first PCR amplification primers A1/A2 (see below) were used as upstream primers and primers B1/B2 (see below) were used as downstream primer.

The PCR products were analysed on a 2% LMT agarose gel and fragments of the expected sizes were cut out from the gel and treated with Agarase (Boehringer Manheim) and cloned into the pT7blue Vector (Novagen) and sequenced.

The cloned fragments from the PCR amplification coded for amino acids corresponding to the sequenced peptides (see above) and in each case in addition to two intron sequences. For MC the PCR amplified DNA sequence corresponds to the sequence shown as from position 1202 to position 1522 with reference to FIG. 14 . For MV the PCR amplified DNA sequence corresponds to the sequence shown as from position 1218 to position 1535 with reference to FIG. 15 .

B.3.7.2 Screening of the Genomic Libraries with the Cloned PCR Fragments

Screening of the libraries with the above-mentioned clone gave two clones for each source. For MC the two clones were combined to form the sequence shown in FIG. 14 (see below). For MV the two clones could be combined to form the sequence shown in FIG. 15 in the manner described above.

An additional PCR was performed to supplement the MC clone with PstI, PvuII, AscI and NcoI restriction sites immediately in front of the ATG start codon using the following oligonucleotide as an upstream primer:

and a primer containing the complement sequence of bp 1297-1318 in FIG. 4 was used as a downstream primer.

The complete sequence for MC was generated by cloning the 5′ end of the gene as a BglII-EcoRI fragment from one of the genomic clone (first clone) into the BamHI-EcoRI sites of pBluescript II KS+ vector from Stratagene. The 3′ end of the gene was then cloned into the modified pBluescript II KS+ vector by ligating an NspV (blunt ended, using the DNA blunting kit from Amersham International)-EcoRI fragment from the other genomic clone (second clone) after the modified pBluescript II KS+ vector had been digested with EcoRI and EcoRV. Then the intermediate part of the gene was cloned in to the further modified pBluescript II KS+ vector as an EcoRI fragment from the first clone by ligating that fragment into the further modified pBluescript II KS+ vector digested with EcoRI.

B.4. Expression of the GL Gene in Micro-organisms

The DNA sequence encoding the GL can be introduced into microorganisms to produce the enzyme with high specific activity and in large quantities.

In this regard, the MC gene (FIG. 14) was cloned as a XbaI-XhoI blunt ended (using the DNA blunting kit from Amersham International) fragment into the Pichia expression vector pHIL-D2 (containing the AOX1 promoter) digested with EcoRI and blunt ended (using the DNA blunting kit from Amersham International) for expression in Pichia pastoris (according to the protocol stated in the Pichia Expression Kit supplied by Invitrogen).

In another embodiment, the MC gene 1 (same as FIG. 14 except that it was modified by PCR to introduce restriction sites as described above) was cloned as a PvuII-XhoI blunt ended fragment (using the DNA blunting kit from Amersham International) into the Aspergillus expression vector pBARMTE1 (containing the methyl tryptophan resistance promoter from Neuropera crassa ) digested with SmaI for expression in Aspergillus niger (Pall et al (1993) Fungal Genet Newslett. vol 40 pages 59-62). The protoplasts were prepared according to Daboussi et al (Curr Genet (1989) vol 15 pp 453-456) using lysing enzymes Sigma L-2773 and the lyticase Sigma L-8012. The transformation of the protoplasts was followed according to the protocol stated by Buxton et al (Gene (1985) vol 37 pp 207-214) except that for plating the transformed protoplasts the protocol laid out in Punt et al (Methods in Enzymology (1992) vol 216 pp 447-457) was followed but with the use of 0.6% osmotic stabilised top agarose.

The results showed that lyase activity was observed in the transformed Pichia pastoris and Aspergillus niger.

Analyses of Pichia Lyase Transportations and Aspergillus Lyase Transformants

General Methods

Preparation of Cell-free Extracts

The cells were harvested by centrifugation at 9000 rpm for 5 min and washed with 0.9% NaCl and resuspended in the breaking buffer (50 mM K-phosphate, pH 7.5 containing 1 mM of EDTA, and 5% glycerol). Cells were broken using glass beads and vortex treatment. The breaking buffer contained 1 mM PMSF (protease inhibitor). The lyase extract (supernatant) was obtained after centrifugation at 9000 rpm for 5 min followed by centrifugation at 20,000×g for 5 min.

Assay of Lyase Activity by Alkaline 3,5-dinitrosalicylic Acid Reagent (DNS)

One volume of lyase extract was mixed with an equal volume of 4% amylopectin solution. The reaction mixture was then incubated at a controlled temperature and samples were removed at specified intervals and analyzed for AF.

The lyase activity was also analyzed using a radioactive method.

The reaction mixture contained 10 μl 14 C-starch solution (1 μCi; Sigma Chemicals Co.) and 10 μl of the lyase extract. The reaction mixture was left at 25° C. overnight and was then analyzed in the usual TLC system. The radioactive AF produced was detected using an Instant Imager (Pachard Instrument Co., Inc., Meriden, Conn.).

Electrophoresis and Western Blotting

SDS-PAGE was performed using 8-25% gradient gels and the PhastSystem (Pharmacia). Western blottings was also run on a Semidry transfer unit of the PhastSystem. Primary antibodies raised against the lyase purified from the red seaweed collected at Qingdao (China) were used in a dilution of 1:100. Pig antirabbit IgG conjugated to alkaline phosphatase (Dako A/S, Glostrup, Denmark) were used as secondary antibodies and used in a dilution of 1:1000.

Results

I. Lyase Activity was Determined after 5 days Incubation (Minimal Medium Containing 0.2% Casein Enzymatic Hydrolysate Analysis by the Alkaline 3,5-dinitrosalicylic Acid Reagent

The results show that the MC-lyase was expressed intracellular in A. niger.

II. Lyase Activity Test by Radioactive Method

The cell-free extracts of the following cultures contained 14 C labelled AF

51+, 54+, 55+, 59+, 512, 513, 514, 515, 516, 518, 519.

The TLC of the degradation products of the α-1,4-glucan lyase reaction using 14 C-starch as substrate are shown in FIG. 20 . The reaction mixture was applied on the TLC. The lane number corresponds to the name of the culture: 1, 512; 2, 513; 3, 514; 4, 515; 5, 516; 6, 517; 7, 518; 8, 519; 9, 520. The fast moving spots are AF.

C. Source=Algae Alone

The protocols for the enzyme purification and charaterisation of alpha 1,4-glucal lyase obtained from Gracilarioposis lemaneiformis (as obtained from Santa Cruz) were essentially the same as those described above for, for example, Morchella Costata (with similar results).

1. Characterization of α-1,4-glucan lyase from the parasite-free red seaweed Gracilariopsis lemaneiformis collected in California.

The amino acid composition of the lyase is given in the following table.

2. Sequence Analsis

Comparison of the peptide sequences from the Californian algae with the amino acid sequence from the fungally infected algae from China showed a high degree of homology (78 to 80% identity between the amino acid sequence generated from the PCR fragments and the corresponding sequences in the GL obtained from the algae from China) between the two protein sequences.

Three Oligonucleotides was generated from these two sequences from the Californian algae to generate a PCR fragment of app. 970 bp.

Primer 1 was used as the upstream primer and primer 2 was used as the downstream primer in the first PCR amplification. In the second PCR amplification primer 1 was used as the upstream primer and primer 3 was used as the downstream primer. A PCR fragment of the expected size was generated and cloned into the pT7blue vector from Novagen. Three independent plasmids containing a PCR fragment were sequenced and it was seen that these three cloned PCR fragments contained the codons for peptide sequences originating from three different proteins. This indicates that there are at least three different genes coding for α-1,4-glucan lyase in the Californian algae.

3. The substrate concentration at which half of the maximal velocity rate was reached is 3.76 mg/ml for amylopectin and 3.37 mg/ml for glycogen.

4. The degradation rates of the lyase on various substrates are given below.

Reaction conditions: The reaction mixture contained 10 mM of HOAc-NaOAc (pH 3.8). The substrate concentration was 10 mg/ml. The final volume was 100 ul after the addition of lyase and water. The reaction time was 40 min at 45° C.

The lyase was not able to degrade pullulan, nigeran tetrasaccharide, trehalose, isomaltose, glucose, α-, β- and r-cyclodextrins. The lyase degraded panose and nigerose though at a slow rate.

5. The temperature optimum for the lyase was 48° C. when amylopectin was used as substrate and 50° C. when glycogen was used as substrate. At 50° C., the reactivity of glycogen was similar to that of amylopectin; below 50° C., amylopectin was a better substrate than glycogen.

6. The pH optimum range for the lyase was between pH 3.5 and pH 7.0; the optimal pH was 3.8. The buffers used in the pH tests were glycine-HCl (pH 2.2-3.6); NaOAc-HOAc (pH 3.5-5.5); Mes-NaOH (pH 5.5-6.7); Mops-NaOH (pH 6.0-8.0) and bicine-NaOH (pH 7.6-9.0). All buffers used were 40 mM.

7. At a final concentration of 2 mM, p-chloromercuribenzoic acid (PCMB) inhibited the lyase activity by 96%, indicating the —SH group(s) is essential for the enzymatic activity.

7. Further Studies

7.1 Effect of Alcohols in Increasing the Activity and Stability of the Lyase Purified from the Fungal Infected Algae

1-propanol, 2-propanol and 1-butanol were tested at the following concentrations (0%, 1%, 5% and 10%). The optimal concentration of l-propanol was 5% which increased the AF yield by 34% after 6 days of incubation; the optimal concentration for 2-propanol was 1% which increased the AF yield by 20% after 10 days incubation; the optimal concentration for 1-butanol was 5% which increased the AF yield by 52% after 3-day incubation.

Ethanol was tested at the following concentrations (0, 1, 3, 5, 7, 9, 11, 13, 15%). The optimal concentration for 7 days incubation was 5% which increased the AF yield by 12%. For 10 days incubation the optimal concentration was 3% which increased AF yield by 16%.

7.2 Effect of Different Reaction Media Upon the Production of AP by the Lyase Purified from the Fungal Infected Algae and the Fugnal Lyase from M. costata and M. vulgaris

2.1. The Lyase from the Fungal Infected Algae

The results (see table below) indicate that the best reaction medium is 5 mM of HOAc-NaOAc (pH 3.9) (BACE for short) and containing mM concentrations of Na 2 -EDTA. The production of AF using either pure water or 0.85% NaCl as reaction medium decreased the yield. Inclusion of 0.85% of NaCl in BACE also decreased the AF yield.

2.2. The Following Buffers: Mes-NaOH, Mops-NaOH, Hepes-NaOH, and Bicine-NaOH were the Optimal Reaction Media for the Lyase from M. costata and M. vulgaris. In the HOAc-NaOAc Buffer, the Lyase was Unstable and Therefore Use of this Buffer System caused a Decrease in AF Yield

7.3. The Effect of Endoamylases and Debranching Enzymes Upon the AF Production

3.1. The Effect of Endoamylase

The starch used for AF production may first be liquified either by endoamylases, or by acid hydrolysis.

Endoamylase degraded starch is more suitable as substrate for the lyase as compared to native starch. Starch has a limited solubility at the temperature used for the lyase-catalyzed reaction. Treatment of starch with endoamylases led to increased glucose yield. It was found that a reducing matter of around 10-15% (on a dry mater basis) was most suitable as substrate for the lyase with respect to AF yield and further treatment with the endoamylase to a reducing matter of 19% was no longer suitable for the lyase.

3.2. The Effect of Pullulanase and Isoamylase

As seen from the results below, both the isoamylase and the pullulanase increased AF yield by up to 50% at pH 4.5 and 5.0. The reaction system consisted of the lyase from the fungal affected red algae with or without the addition of isoamylase or pullulanase (MegaZyme Ltd.). Amylopectin was used as substrate. The AF produced in the presence of only the lyase was expressed as 100%.

4. The Relative Degrading Rates of the Fungal Lyase Towards Various Substrates

4.1. The Lyase from M. costata.

The activity observed with maltotetraose is expressed as 100%.

4.2. The Lyase from M. vulgaris Lyase

The activity observed for maltotetraose is treated as 100%. The final concentration of all substrates was 10 mg ml −1 .

The lyase from M. costata and M. vulgaris was unable to degrade the following sugars.

Trehalose, panose, nigerose, nigerotetraose, glucose, isomaltose, alpha-, beta and gama-cyclodextrins, pullulalans and non-reducing end blocked p-nitrophenyl α-D-maltoheptaoside as there was no AF detectable on a TLC plates after these substrates had been incubated for 48 h with the fungal lyase.

7.5. pH and Temperature Optimum for the Lyase Catalyzed Reaction.

7.6. The Stabilizing Effect of Glycogen on the Lyase from the Fungal Infected Gracilariopsis lemaneiformis

The results indicate that at higher temperatures the reaction rates were higher when glycogen was used as substrate instead of amylopectin.

7.7. The Molecular Masses and pI Values of the Lyases

The molecular masses of the lyases from the fungal infected G. lemaneiformis, both forms of lyase from apparent fungal free G. lemaneiformis, from M. costata and M. vulgans were estimated to 110,000±10,000 daltons used SDS-PAGE on a gradient gel (8-25%).

The pI of the lyase from the fungal infected G. lemaneiformis was around 3.9. For the lyase from M. vuglaris, the pI was around pH 4.6 and the pI for the lyase from M. costata was around 5.0. These values were obtained by isoelectric focusing on a gel with a pH gradient from 3 to 9.

The pI values deduced from the amino acid compositions are: The lyase from the fungal infected G. lemaneiformis: 4.58 and for the lyase from M. costata: 6.30.

7.8. Immunological Test of the Lyase by Western Blotting

The results showed that the antibodies to the algal lyase could recognize the fungal lyase both in cell-free extracts and in purified form, as revealed by Western blottings. The antibodies to the algal lyase purified form the algae collected from China also recognized the lyase from the algae collected from Sant Cruz, Calif.

7.9. Reversible and Irreversible Inhibitors of the Fungal Lyase

9.1. The Reversible Inhibitors, Glucose and Maltose

At a substrate concentration of 10 mg/ml, the activity for the M. costata lyase decreased by 19.3% in the presence of 0.1 M glucose when amylopectin was used as substrate; the activity was not affected when glycogen was used as substrate. In the presence of 0.1 M of maltose the activity decreased by 48.8% and 73.4%, respectively for glycogen and amylopectin.

It seems that the inhibition by 0.1 M glucose is competitive as increasing the substrate from 1% to 2% decreased the inhibition from 19.3 to 7%, whereas the inhibition by 0.1 M maltose is non-competitive as the increase of substrate did not significantly affect the inhibition degree.

For the M. vulgaris lyase, 0.1 M glucose and maltose did also inhibit the reaction when either amylopectin or glycogen was used as substrate.

9.2. The Reversible Inhibitor Deoxyjirimycin

At a final substrate concentration of 2%, the activity was decreased to 10.4% for the algal lyase and the M.costata lyase in the presence of 25 μM of deoxyjirimycin, using amylopectin as substrate. At 100 μM, the activity of both lyases was completely lost.

9.3. Irreversible Inhibitor: PCMB

Under the same assay conditions and in the presence of 2 mM PCMB, the activity decreased by 60% for the M. costata lyase and 98% for the lyase from the fungal infected red algae. This means that the fungal lyase was much less sensitive to heavy metal inhibition.

7.10. Examples of Laboratory Scale Production of AF

10.1. Production of AF Using Dextrin as Substrate

The reactor contained 1000 g dextrins (obtained by treatment of starch with Termamyl to a final reducing matter of 10%) in a final volume of 4.6 liter (HOAC-NaOAC, pH 3.9, containing 5 mM Na 2 -EDTA). The reaction was initiated by adding 3 mg lyase purified from fungal infected algae. The reaction was performed at room temperature. At day 19, another batch of lyase (4 mg) was added.

10.2. Using 4 C-Starch for the Production of 14 C-AF

The uniformly labelled 14 C-starch (340 μCi obtained from Sigma) was vaccum-dried to remove the ethanol it contained and then dissolved in 2 ml water. The reaction was initiated by adding 20 μl lyase purified from the fungal infected algae and 20 μl pullulanase (MegaZyme Ltd.) The reaction was performed overnight at 30 ° C. At the end of the reaction, the reaction mixture was filtered using a filter with a molecular mass cut off of 10,000 to remove the enzymes and unreacted starch molecules.

The filtrate was applied on a Ca 2 carbohydrate column (Chrompack) using a Waters HPLC. Water was used as eluent. The flow rate was 0.5 ml/min. AF was efficiently separated from glucose and maltosaccharides. The pooled AF fractions were freeze-dried and totally 140 μCi 14 C-AF was obtained.

These findings relate to an even further aspect of the present invention, namely the use of a reagent that can increase the hydrophobicity of the reaction medium (preferably an alcohol) to increase the stability and activity of the lyase according to the present invention. This increased stability leads to a increased AF yield.

Other modifications of the present invention will be apparent to those skilled in the art without departing from the scope of the invention.

(1) GENERAL INFORMATION:

(iii) NUMBER OF SEQUENCES: 39

(2) INFORMATION FOR SEQ ID NO: 1:

(A) LENGTH: 1088 amino

#acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

#1:

Met Phe Ser Thr Leu Ala Phe Val Ala Pro Se

#r Ala Leu Gly Ala Ser

1               5

#                10

#                15

Thr Phe Val Gly Ala Glu Val Arg Ser Asn Va

#l Arg Ile His Ser Ala

20

#            25

#            30

Phe Pro Ala Val His Thr Ala Thr Arg Lys Th

#r Asn Arg Leu Asn Val

35

#        40

#        45

Ser Met Thr Ala Leu Ser Asp Lys Gln Thr Al

#a Thr Ala Gly Ser Thr

50

#    55

#    60

Asp Asn Pro Asp Gly Ile Asp Tyr Lys Thr Ty

#r Asp Tyr Val Gly Val

65

#70

#75

#80

Trp Gly Phe Ser Pro Leu Ser Asn Thr Asn Tr

#p Phe Ala Ala Gly Ser

85

#                90

#                95

Ser Thr Pro Gly Gly Ile Thr Asp Trp Thr Al

#a Thr Met Asn Val Asn

100

#           105

#           110

Phe Asp Arg Ile Asp Asn Pro Ser Ile Thr Va

#l Gln His Pro Val Gln

115

#       120

#       125

Val Gln Val Thr Ser Tyr Asn Asn Asn Ser Ty

#r Arg Val Arg Phe Asn

130

#   135

#   140

Pro Asp Gly Pro Ile Arg Asp Val Thr Arg Gl

#y Pro Ile Leu Lys Gln

145                 1

#50                 1

#55                 1

#60

Gln Leu Asp Trp Ile Arg Thr Gln Glu Leu Se

#r Glu Gly Cys Asp Pro

165

#               170

#               175

Gly Met Thr Phe Thr Ser Glu Gly Phe Leu Th

#r Phe Glu Thr Lys Asp

180

#           185

#           190

Leu Ser Val Ile Ile Tyr Gly Asn Phe Lys Th

#r Arg Val Thr Arg Lys

195

#       200

#       205

Ser Asp Gly Lys Val Ile Met Glu Asn Asp Gl

#u Val Gly Thr Ala Ser

210

#   215

#   220

Ser Gly Asn Lys Cys Arg Gly Leu Met Phe Va

#l Asp Arg Leu Tyr Gly

225                 2

#30                 2

#35                 2

#40

Asn Ala Ile Ala Ser Val Asn Lys Asn Phe Ar

#g Asn Asp Ala Val Lys

245

#               250

#               255

Gln Glu Gly Phe Tyr Gly Ala Gly Glu Val As

#n Cys Lys Tyr Gln Asp

260

#           265

#           270

Thr Tyr Ile Leu Glu Arg Thr Gly Ile Ala Me

#t Thr Asn Tyr Asn Tyr

275

#       280

#       285

Asp Asn Leu Asn Tyr Asn Gln Trp Asp Leu Ar

#g Pro Pro His His Asp

290

#   295

#   300

Gly Ala Leu Asn Pro Asp Tyr Tyr Ile Pro Me

#t Tyr Tyr Ala Ala Pro

305                 3

#10                 3

#15                 3

#20

Trp Leu Ile Val Asn Gly Cys Ala Gly Thr Se

#r Glu Gln Tyr Ser Tyr

325

#               330

#               335

Gly Trp Phe Met Asp Asn Val Ser Gln Ser Ty

#r Met Asn Thr Gly Asp

340

#           345

#           350

Thr Thr Trp Asn Ser Gly Gln Glu Asp Leu Al

#a Tyr Met Gly Ala Gln

355

#       360

#       365

Tyr Gly Pro Phe Asp Gln His Phe Val Tyr Gl

#y Ala Gly Gly Gly Met

370

#   375

#   380

Glu Cys Val Val Thr Ala Phe Ser Leu Leu Gl

#n Gly Lys Glu Phe Glu

385                 3

#90                 3

#95                 4

#00

Asn Gln Val Leu Asn Lys Arg Ser Val Met Pr

#o Pro Lys Tyr Val Phe

405

#               410

#               415

Gly Phe Phe Gln Gly Val Phe Gly Thr Ser Se

#r Leu Leu Arg Ala His

420

#           425

#           430

Met Pro Ala Gly Glu Asn Asn Ile Ser Val Gl

#u Glu Ile Val Glu Gly

435

#       440

#       445

Tyr Gln Asn Asn Asn Phe Pro Phe Glu Gly Le

#u Ala Val Asp Val Asp

450

#   455

#   460

Met Gln Asp Asn Leu Arg Val Phe Thr Thr Ly

#s Gly Glu Phe Trp Thr

465                 4

#70                 4

#75                 4

#80

Ala Asn Arg Val Gly Thr Gly Gly Asp Pro As

#n Asn Arg Ser Val Phe

485

#               490

#               495

Glu Trp Ala His Asp Lys Gly Leu Val Cys Gl

#n Thr Asn Ile Thr Cys

500

#           505

#           510

Phe Leu Arg Asn Asp Asn Glu Gly Gln Asp Ty

#r Glu Val Asn Gln Thr

515

#       520

#       525

Leu Arg Glu Arg Gln Leu Tyr Thr Lys Asn As

#p Ser Leu Thr Gly Thr

530

#   535

#   540

Asp Phe Gly Met Thr Asp Asp Gly Pro Ser As

#p Ala Tyr Ile Gly His

545                 5

#50                 5

#55                 5

#60

Leu Asp Tyr Gly Gly Gly Val Glu Cys Asp Al

#a Leu Phe Pro Asp Trp

565

#               570

#               575

Gly Arg Pro Asp Val Ala Glu Trp Trp Gly As

#n Asn Tyr Lys Lys Leu

580

#           585

#           590

Phe Ser Ile Gly Leu Asp Phe Val Trp Gln As

#p Met Thr Val Pro Ala

595

#       600

#       605

Met Met Pro His Lys Ile Gly Asp Asp Ile As

#n Val Lys Pro Asp Gly

610

#   615

#   620

Asn Trp Pro Asn Ala Asp Asp Pro Ser Asn Gl

#y Gln Tyr Asn Trp Lys

625                 6

#30                 6

#35                 6

#40

Thr Tyr His Pro Gln Val Leu Val Thr Asp Me

#t Arg Tyr Glu Asn His

645

#               650

#               655

Gly Arg Glu Pro Met Val Thr Gln Arg Asn Il

#e His Ala Tyr Thr Leu

660

#           665

#           670

Cys Glu Ser Thr Arg Lys Glu Gly Ile Val Gl

#u Asn Ala Asp Thr Leu

675

#       680

#       685

Thr Lys Phe Arg Arg Ser Tyr Ile Ile Ser Ar

#g Gly Gly Tyr Ile Gly

690

#   695

#   700

Asn Gln His Phe Gly Gly Met Trp Val Gly As

#p Asn Ser Thr Thr Ser

705                 7

#10                 7

#15                 7

#20

Asn Tyr Ile Gln Met Met Ile Ala Asn Asn Il

#e Asn Met Asn Met Ser

725

#               730

#               735

Cys Leu Pro Leu Val Gly Ser Asp Ile Gly Gl

#y Phe Thr Ser Tyr Asp

740

#           745

#           750

Asn Glu Asn Gln Arg Thr Pro Cys Thr Gly As

#p Leu Met Val Arg Tyr

755

#       760

#       765

Val Gln Ala Gly Cys Leu Leu Pro Trp Phe Ar

#g Asn His Tyr Asp Arg

770

#   775

#   780

Trp Ile Glu Ser Lys Asp His Gly Lys Asp Ty

#r Gln Glu Leu Tyr Met

785                 7

#90                 7

#95                 8

#00

Tyr Pro Asn Glu Met Asp Thr Leu Arg Lys Ph

#e Val Glu Phe Arg Tyr

805

#               810

#               815

Arg Trp Gln Glu Val Leu Tyr Thr Ala Met Ty

#r Gln Asn Ala Ala Phe

820

#           825

#           830

Gly Lys Pro Ile Ile Lys Ala Ala Ser Met Ty

#r Asn Asn Asp Ser Asn

835

#       840

#       845

Val Arg Arg Ala Gln Asn Asp His Phe Leu Le

#u Gly Gly His Asp Gly

850

#   855

#   860

Tyr Arg Ile Leu Cys Ala Pro Val Val Trp Gl

#u Asn Ser Thr Glu Arg

865                 8

#70                 8

#75                 8

#80

Glu Leu Tyr Leu Pro Val Leu Thr Gln Trp Ty

#r Lys Phe Gly Pro Asp

885

#               890

#               895

Phe Asp Thr Lys Pro Leu Glu Gly Ala Met As

#n Gly Gly Asp Arg Ile

900

#           905

#           910

Tyr Asn Tyr Pro Val Pro Gln Ser Glu Ser Pr

#o Ile Phe Val Arg Glu

915

#       920

#       925

Gly Ala Ile Leu Pro Thr Arg Tyr Thr Leu As

#n Gly Glu Asn Lys Ser

930

#   935

#   940

Leu Asn Thr Tyr Thr Asp Glu Asp Pro Leu Va

#l Phe Glu Val Phe Pro

945                 9

#50                 9

#55                 9

#60

Leu Gly Asn Asn Arg Ala Asp Gly Met Cys Ty

#r Leu Asp Asp Gly Gly

965

#               970

#               975

Val Thr Thr Asn Ala Glu Asp Asn Gly Lys Ph

#e Ser Val Val Lys Val

980

#           985

#           990

Ala Ala Glu Gln Asp Gly Gly Thr Glu Thr Il

#e Thr Phe Thr Asn Asp

995

#       1000

#      1005

Cys Tyr Glu Tyr Val Phe Gly Gly Pro Phe Ty

#r Val Arg Val Arg Gly

1010

#   1015

#  1020

Ala Gln Ser Pro Ser Asn Ile His Val Ser Se

#r Gly Ala Gly Ser Gln

1025                1030

#                1035

#               1040

Asp Met Lys Val Ser Ser Ala Thr Ser Arg Al

#a Ala Leu Phe Asn Asp

1045

#               1050

#              1055

Gly Glu Asn Gly Asp Phe Trp Val Asp Gln Gl

#u Thr Asp Ser Leu Trp

1060

#           1065

#          1070

Leu Lys Leu Pro Asn Val Val Leu Pro Asp Al

#a Val Ile Thr Ile Thr

1075

#       1080

#      1085

(2) INFORMATION FOR SEQ ID NO: 2:

(A) LENGTH: 1091 amino

#acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

#2:

Met Tyr Pro Thr Leu Thr Phe Val Ala Pro Se

#r Ala Leu Gly Ala Arg

1               5

#                10

#                15

Thr Phe Thr Cys Val Gly Ile Phe Arg Ser Hi

#s Ile Leu Ile His Ser

20

#            25

#            30

Val Val Pro Ala Val Arg Leu Ala Val Arg Ly

#s Ser Asn Arg Leu Asn

35

#        40

#        45

Val Ser Met Ser Ala Leu Phe Asp Lys Pro Th

#r Ala Val Thr Gly Gly

50

#    55

#    60

Lys Asp Asn Pro Asp Asn Ile Asn Tyr Thr Th

#r Tyr Asp Tyr Val Pro

65

#70

#75

#80

Val Trp Arg Phe Asp Pro Leu Ser Asn Thr As

#n Trp Phe Ala Ala Gly

85

#                90

#                95

Ser Ser Thr Pro Gly Asp Ile Asp Asp Trp Th

#r Ala Thr Met Asn Val

100

#           105

#           110

Asn Phe Asp Arg Ile Asp Asn Pro Ser Phe Th

#r Leu Glu Lys Pro Val

115

#       120

#       125

Gln Val Gln Val Thr Ser Tyr Lys Asn Asn Cy

#s Phe Arg Val Arg Phe

130

#   135

#   140

Asn Pro Asp Gly Pro Ile Arg Asp Val Asp Ar

#g Gly Pro Ile Leu Gln

145                 1

#50                 1

#55                 1

#60

Gln Gln Leu Asn Trp Ile Arg Lys Gln Glu Gl

#n Ser Lys Gly Phe Asp

165

#               170

#               175

Pro Lys Met Gly Phe Thr Lys Glu Gly Phe Le

#u Lys Phe Glu Thr Lys

180

#           185

#           190

Asp Leu Asn Val Ile Ile Tyr Gly Asn Phe Ly

#s Thr Arg Val Thr Arg

195

#       200

#       205

Lys Arg Asp Gly Lys Gly Ile Met Glu Asn As

#n Glu Val Pro Ala Gly

210

#   215

#   220

Ser Leu Gly Asn Lys Cys Arg Gly Leu Met Ph

#e Val Asp Arg Leu Tyr

225                 2

#30                 2

#35                 2

#40

Gly Thr Ala Ile Ala Ser Val Asn Glu Asn Ty

#r Arg Asn Asp Pro Asp

245

#               250

#               255

Arg Lys Glu Gly Phe Tyr Gly Ala Gly Glu Va

#l Asn Cys Glu Phe Trp

260

#           265

#           270

Asp Ser Glu Gln Asn Arg Asn Lys Tyr Ile Le

#u Glu Arg Thr Gly Ile

275

#       280

#       285

Ala Met Thr Asn Tyr Asn Tyr Asp Asn Tyr As

#n Tyr Asn Gln Ser Asp

290

#   295

#   300

Leu Ile Ala Pro Gly Tyr Pro Ser Asp Pro As

#n Phe Tyr Ile Pro Met

305                 3

#10                 3

#15                 3

#20

Tyr Phe Ala Ala Pro Trp Val Val Val Lys Gl

#y Cys Ser Gly Asn Ser

325

#               330

#               335

Asp Glu Gln Tyr Ser Tyr Gly Trp Phe Met As

#p Asn Val Ser Gln Thr

340

#           345

#           350

Tyr Met Asn Thr Gly Gly Thr Ser Trp Asn Cy

#s Gly Glu Glu Asn Leu

355

#       360

#       365

Ala Tyr Met Gly Ala Gln Cys Gly Pro Phe As

#p Gln His Phe Val Tyr

370

#   375

#   380

Gly Asp Gly Asp Gly Leu Glu Asp Val Val Gl

#n Ala Phe Ser Leu Leu

385                 3

#90                 3

#95                 4

#00

Gln Gly Lys Glu Phe Glu Asn Gln Val Leu As

#n Lys Arg Ala Val Met

405

#               410

#               415

Pro Pro Lys Tyr Val Phe Gly Tyr Phe Gln Gl

#y Val Phe Gly Ile Ala

420

#           425

#           430

Ser Leu Leu Arg Glu Gln Arg Pro Glu Gly Gl

#y Asn Asn Ile Ser Val

435

#       440

#       445

Gln Glu Ile Val Glu Gly Tyr Gln Ser Asn As

#n Phe Pro Leu Glu Gly

450

#   455

#   460

Leu Ala Val Asp Val Asp Met Gln Gln Asp Le

#u Arg Val Phe Thr Thr

465                 4

#70                 4

#75                 4

#80

Lys Ile Glu Phe Trp Thr Ala Asn Lys Val Gl

#y Thr Gly Gly Asp Ser

485

#               490

#               495

Asn Asn Lys Ser Val Phe Glu Trp Ala His As

#p Lys Gly Leu Val Cys

500

#           505

#           510

Gln Thr Asn Val Thr Cys Phe Leu Arg Asn As

#p Asn Gly Gly Ala Asp

515

#       520

#       525

Tyr Glu Val Asn Gln Thr Leu Arg Glu Lys Gl

#y Leu Tyr Thr Lys Asn

530

#   535

#   540

Asp Ser Leu Thr Asn Thr Asn Phe Gly Thr Th

#r Asn Asp Gly Pro Ser

545                 5

#50                 5

#55                 5

#60

Asp Ala Tyr Ile Gly His Leu Asp Tyr Gly Gl

#y Gly Gly Asn Cys Asp

565

#               570

#               575

Ala Leu Phe Pro Asp Trp Gly Arg Pro Gly Va

#l Ala Glu Trp Trp Gly

580

#           585

#           590

Asp Asn Tyr Ser Lys Leu Phe Lys Ile Gly Le

#u Asp Phe Val Trp Gln

595

#       600

#       605

Asp Met Thr Val Pro Ala Met Met Pro His Ly

#s Val Gly Asp Ala Val

610

#   615

#   620

Asp Thr Arg Ser Pro Tyr Gly Trp Pro Asn Gl

#u Asn Asp Pro Ser Asn

625                 6

#30                 6

#35                 6

#40

Gly Arg Tyr Asn Trp Lys Ser Tyr His Pro Gl

#n Val Leu Val Thr Asp

645

#               650

#               655

Met Arg Tyr Glu Asn His Gly Arg Glu Pro Me

#t Phe Thr Gln Arg Asn

660

#           665

#           670

Met His Ala Tyr Thr Leu Cys Glu Ser Thr Ar

#g Lys Glu Gly Ile Val

675

#       680

#       685

Ala Asn Ala Asp Thr Leu Thr Lys Phe Arg Ar

#g Ser Tyr Ile Ile Ser

690

#   695

#   700

Arg Gly Gly Tyr Ile Gly Asn Gln His Phe Gl

#y Gly Met Trp Val Gly

705                 7

#10                 7

#15                 7

#20

Asp Asn Ser Ser Ser Gln Arg Tyr Leu Gln Me

#t Met Ile Ala Asn Ile

725

#               730

#               735

Val Asn Met Asn Met Ser Cys Leu Pro Leu Va

#l Gly Ser Asp Ile Gly

740

#           745

#           750

Gly Phe Thr Ser Tyr Asp Gly Arg Asn Val Cy

#s Pro Gly Asp Leu Met

755

#       760

#       765

Val Arg Phe Val Gln Ala Gly Cys Leu Leu Pr

#o Trp Phe Arg Asn His

770

#   775

#   780

Tyr Gly Arg Leu Val Glu Gly Lys Gln Glu Gl

#y Lys Tyr Tyr Gln Glu

785                 7

#90                 7

#95                 8

#00

Leu Tyr Met Tyr Lys Asp Glu Met Ala Thr Le

#u Arg Lys Phe Ile Glu

805

#               810

#               815

Phe Arg Tyr Arg Trp Gln Glu Val Leu Tyr Th

#r Ala Met Tyr Gln Asn

820

#           825

#           830

Ala Ala Phe Gly Lys Pro Ile Ile Lys Ala Al

#a Ser Met Tyr Asp Asn

835

#       840

#       845

Asp Arg Asn Val Arg Gly Ala Gln Asp Asp Hi

#s Phe Leu Leu Gly Gly

850

#   855

#   860

His Asp Gly Tyr Arg Ile Leu Cys Ala Pro Va

#l Val Trp Glu Asn Thr

865                 8

#70                 8

#75                 8

#80

Thr Ser Arg Asp Leu Tyr Leu Pro Val Leu Th

#r Lys Trp Tyr Lys Phe

885

#               890

#               895

Gly Pro Asp Tyr Asp Thr Lys Arg Leu Asp Se

#r Ala Leu Asp Gly Gly

900

#           905

#           910

Gln Met Ile Lys Asn Tyr Ser Val Pro Gln Se

#r Asp Ser Pro Ile Phe

915

#       920

#       925

Val Arg Glu Gly Ala Ile Leu Pro Thr Arg Ty

#r Thr Leu Asp Gly Ser

930

#   935

#   940

Asn Lys Ser Met Asn Thr Tyr Thr Asp Lys As

#p Pro Leu Val Phe Glu

945                 9

#50                 9

#55                 9

#60

Val Phe Pro Leu Gly Asn Asn Arg Ala Asp Gl

#y Met Cys Tyr Leu Asp

965

#               970

#               975

Asp Gly Gly Ile Thr Thr Asp Ala Glu Asp Hi

#s Gly Lys Phe Ser Val

980

#           985

#           990

Ile Asn Val Glu Ala Leu Arg Lys Gly Val Th

#r Thr Thr Ile Lys Phe

995

#       1000

#      1005

Ala Tyr Asp Thr Tyr Gln Tyr Val Phe Asp Gl

#y Pro Phe Tyr Val Arg

1010

#   1015

#  1020

Ile Arg Asn Leu Thr Thr Ala Ser Lys Ile As

#n Val Ser Ser Gly Ala

1025                1030

#                1035

#               1040

Gly Glu Glu Asp Met Thr Pro Thr Ser Ala As

#n Ser Arg Ala Ala Leu

1045

#               1050

#              1055

Phe Ser Asp Gly Gly Val Gly Glu Tyr Trp Al

#a Asp Asn Asp Thr Ser

1060

#           1065

#          1070

Ser Leu Trp Met Lys Leu Pro Asn Leu Val Le

#u Gln Asp Ala Val Ile

1075

#       1080

#      1085

Thr Ile Thr

1090

(2) INFORMATION FOR SEQ ID NO: 3:

(A) LENGTH: 3267 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

#3:

#TCGTAGGG     60

#CAGCTACT    120

#CGGCTACT    180

#TCGGAGTA    240

#CCCCGGGT    300

#ATCCGTCC    360

#GCTACAGG    420

#TCAAGCAG    480

#TGACTTTC    540

#ACGGAAAT    600

#ATGAAGTT    660

#TATACGGT    720

#AGGGATTC    780

#GCACTGGA    840

#TTAGACCT    900

#CAGCACCT    960

#GGTTCATG   1020

#GACAAGAG   1080

#ACGGTGCT   1140

#AGTTCGAG   1200

#TCTTCCAG   1260

#ACAACATC   1320

#GGCTCGCT   1380

#TTTGGACC   1440

#GGGCACAT   1500

#ACGAGGGG   1560

#ACGACTCC   1620

#TCGGTCAT   1680

#GGCCTGAC   1740

#ACTTCGTC   1800

#TCAATGTG   1860

#ACTGGAAG   1920

#GGGAACCG   1980

#AGGAAGGG   2040

#GTCGTGGT   2100

#CTACATCA   2160

#TGCCTCTC   2220

#CGCCGTGT   2280

#TCAGGAAC   2340

#TGTACATG   2400

#GGCAGGAA   2460

#AGGCTGCT   2520

#TTCTTGGT   2580

#CCGAACGC   2640

#ACACCAAG   2700

#CGCAAAGT   2760

#TGAACGGT   2820

#TATTCCCC   2880

#CCACCAAT   2940

#GTGGTACG   3000

#TCTACGTT   3060

#GTTCTCAG   3120

#AGAACGGT   3180

#TTGTTCTC   3240

#

#           3267

(2) INFORMATION FOR SEQ ID NO: 4:

(A) LENGTH: 3276 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

#4:

#TCACGTGT     60

#GTCTAGCT    120

#CGACTGCT    180

#ACGTCCCT    240

#CCACTCCC    300

#ACAATCCA    360

#ATTGTTTC    420

#TCCTCCAG    480

#AGATGGGC    540

#TATATGGC    600

#ATAATGAA    660

#GGTTGTAC    720

#AAGAGGGG    780

#GGAACAAG    840

#ATAACTAC    900

#TTCCCATG    960

#AACAGTAC   1020

#GTACTTCC   1080

#TTGACCAA   1140

#CTCTTCTG   1200

#CGAAATAT   1260

#AAAGACCA   1320

#ATAACTTC   1380

#TCACCACG   1440

#ACAAGTCG   1500

#GCTTCTTG   1560

#AGGGTTTG   1620

#GGCCGAGC   1680

#TTTTCCCA   1740

#TCTTCAAA   1800

#ACAAAGTT   1860

#CTTCGAAC   1920

#GATATGAG   1980

#TCTGTGAA   2040

#GCCGCAGT   2100

#GGGTTGGA   2160

#ACATGAAC   2220

#ATGGACGA   2280

#TACCGTGG   2340

#ATCAAGAA   2400

#GTTACCGC   2460

#CGATTATC   2520

#ACCACTTC   2580

#AGAATACA   2640

#CTGACTAT   2700

#ATTCTGTG   2760

#GCTACACG   2820

#TGTTTGAG   2880

#GCGGTATT   2940

#TACGGAAA   3000

#ATGGTCCA   3060

#CTGGAGCG   3120

#GTGATGGA   3180

#TGCCAAAC   3240

#

#     3276

(2) INFORMATION FOR SEQ ID NO: 5:

(A) LENGTH: 1066 amino

#acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

#5:

Met Ala Gly Phe Ser Asp Pro Leu Asn Phe Cy

#s Lys Ala Glu Asp Tyr

1               5

#                10

#                15

Tyr Ser Val Ala Leu Asp Trp Lys Gly Pro Gl

#n Lys Ile Ile Gly Val

20

#            25

#            30

Asp Thr Thr Pro Pro Lys Ser Thr Lys Phe Pr

#o Lys Asn Trp His Gly

35

#        40

#        45

Val Asn Leu Arg Phe Asp Asp Gly Thr Leu Gl

#y Val Val Gln Phe Ile

50

#    55

#    60

Arg Pro Cys Val Trp Arg Val Arg Tyr Asp Pr

#o Gly Phe Lys Thr Ser

65

#70

#75

#80

Asp Glu Tyr Gly Asp Glu Asn Thr Arg Thr Il

#e Val Gln Asp Tyr Met

85

#                90

#                95

Ser Thr Leu Ser Asn Lys Leu Asp Thr Tyr Ar

#g Gly Leu Thr Trp Glu

100

#           105

#           110

Thr Lys Cys Glu Asp Ser Gly Asp Phe Phe Th

#r Phe Ser Ser Lys Val

115

#       120

#       125

Thr Ala Val Glu Lys Ser Glu Arg Thr Arg As

#n Lys Val Gly Asp Gly

130

#   135

#   140

Leu Arg Ile His Leu Trp Lys Ser Pro Phe Ar

#g Ile Gln Val Val Arg

145                 1

#50                 1

#55                 1

#60

Thr Leu Thr Pro Leu Lys Asp Pro Tyr Pro Il

#e Pro Asn Val Ala Ala

165

#               170

#               175

Ala Glu Ala Arg Val Ser Asp Lys Val Val Tr

#p Gln Thr Ser Pro Lys

180

#           185

#           190

Thr Phe Arg Lys Asn Leu His Pro Gln His Ly

#s Met Leu Lys Asp Thr

195

#       200

#       205

Val Leu Asp Ile Val Lys Pro Gly His Gly Gl

#u Tyr Val Gly Trp Gly

210

#   215

#   220

Glu Met Gly Gly Ile Gln Phe Met Lys Glu Pr

#o Thr Phe Met Asn Tyr

225                 2

#30                 2

#35                 2

#40

Phe Asn Phe Asp Asn Met Gln Tyr Gln Gln Va

#l Tyr Ala Gln Gly Ala

245

#               250

#               255

Leu Asp Ser Arg Glu Pro Leu Tyr His Ser As

#p Pro Phe Tyr Leu Asp

260

#           265

#           270

Val Asn Ser Asn Pro Glu His Lys Asn Ile Th

#r Ala Thr Phe Ile Asp

275

#       280

#       285

Asn Tyr Ser Gln Ile Ala Ile Asp Phe Gly Ly

#s Thr Asn Ser Gly Tyr

290

#   295

#   300

Ile Lys Leu Gly Thr Arg Tyr Gly Gly Ile As

#p Cys Tyr Gly Ile Ser

305                 3

#10                 3

#15                 3

#20

Ala Asp Thr Val Pro Glu Ile Val Arg Leu Ty

#r Thr Gly Leu Val Gly

325

#               330

#               335

Arg Ser Lys Leu Lys Pro Arg Tyr Ile Leu Gl

#y Ala His Gln Ala Cys

340

#           345

#           350

Tyr Gly Tyr Gln Gln Glu Ser Asp Leu Tyr Se

#r Val Val Gln Gln Tyr

355

#       360

#       365

Arg Asp Cys Lys Phe Pro Leu Asp Gly Ile Hi

#s Val Asp Val Asp Val

370

#   375

#   380

Gln Asp Gly Phe Arg Thr Phe Thr Thr Asn Pr

#o His Thr Phe Pro Asn

385                 3

#90                 3

#95                 4

#00

Pro Lys Glu Met Phe Thr Asn Leu Arg Asn As

#n Gly Ile Lys Cys Ser

405

#               410

#               415

Thr Asn Ile Thr Pro Val Ile Ser Ile Asn As

#n Arg Glu Gly Gly Tyr

420

#           425

#           430

Ser Thr Leu Leu Glu Gly Val Asp Lys Lys Ty

#r Phe Ile Met Asp Asp

435

#       440

#       445

Arg Tyr Thr Glu Gly Thr Ser Gly Asn Ala Ly

#s Asp Val Arg Tyr Met

450

#   455

#   460

Tyr Tyr Gly Gly Gly Asn Lys Val Glu Val As

#p Pro Asn Asp Val Asn

465                 4

#70                 4

#75                 4

#80

Gly Arg Pro Asp Phe Lys Asp Asn Tyr Asp Ph

#e Pro Ala Asn Phe Asn

485

#               490

#               495

Ser Lys Gln Tyr Pro Tyr His Gly Gly Val Se

#r Tyr Gly Tyr Gly Asn

500

#           505

#           510

Gly Ser Ala Gly Phe Tyr Pro Asp Leu Asn Ar

#g Lys Glu Val Arg Ile

515

#       520

#       525

Trp Trp Gly Met Gln Tyr Lys Tyr Leu Phe As

#p Met Gly Leu Glu Phe

530

#   535

#   540

Val Trp Gln Asp Met Thr Thr Pro Ala Ile Hi

#s Thr Ser Tyr Gly Asp

545                 5

#50                 5

#55                 5

#60

Met Lys Gly Leu Pro Thr Arg Leu Leu Val Th

#r Ser Asp Ser Val Thr

565

#               570

#               575

Asn Ala Ser Glu Lys Lys Leu Ala Ile Glu Th

#r Trp Ala Leu Tyr Ser

580

#           585

#           590

Tyr Asn Leu His Lys Ala Thr Trp His Gly Le

#u Ser Arg Leu Glu Ser

595

#       600

#       605

Arg Lys Asn Lys Arg Asn Phe Ile Leu Gly Ar

#g Gly Ser Tyr Ala Gly

610

#   615

#   620

Ala Tyr Arg Phe Ala Gly Leu Trp Thr Gly As

#p Asn Ala Ser Asn Trp

625                 6

#30                 6

#35                 6

#40

Glu Phe Trp Lys Ile Ser Val Ser Gln Val Le

#u Ser Leu Gly Leu Asn

645

#               650

#               655

Gly Val Cys Ile Ala Gly Ser Asp Thr Gly Gl

#y Phe Glu Pro Tyr Arg

660

#           665

#           670

Asp Ala Asn Gly Val Glu Glu Lys Tyr Cys Se

#r Pro Glu Leu Leu Ile

675

#       680

#       685

Arg Trp Tyr Thr Gly Ser Phe Leu Leu Pro Tr

#p Leu Arg Asn His Tyr

690

#   695

#   700

Val Lys Lys Asp Arg Lys Trp Phe Gln Glu Pr

#o Tyr Ser Tyr Pro Lys

705                 7

#10                 7

#15                 7

#20

His Leu Glu Thr His Pro Glu Leu Ala Asp Gl

#n Ala Trp Leu Tyr Lys

725

#               730

#               735

Ser Val Leu Glu Ile Cys Arg Tyr Tyr Val Gl

#u Leu Arg Tyr Ser Leu

740

#           745

#           750

Ile Gln Leu Leu Tyr Asp Cys Met Phe Gln As

#n Val Val Asp Gly Met

755

#       760

#       765

Pro Ile Thr Arg Ser Met Leu Leu Thr Asp Th

#r Glu Asp Thr Thr Phe

770

#   775

#   780

Phe Asn Glu Ser Gln Lys Phe Leu Asp Asn Gl

#n Tyr Met Ala Gly Asp

785                 7

#90                 7

#95                 8

#00

Asp Ile Leu Val Ala Pro Ile Leu His Ser Ar

#g Lys Glu Ile Pro Gly

805

#               810

#               815

Glu Asn Arg Asp Val Tyr Leu Pro Leu Tyr Hi

#s Thr Trp Tyr Pro Ser

820

#           825

#           830

Asn Leu Arg Pro Trp Asp Asp Gln Gly Val Al

#a Leu Gly Asn Pro Val

835

#       840

#       845

Glu Gly Gly Ser Val Ile Asn Tyr Thr Ala Ar

#g Ile Val Ala Pro Glu

850

#   855

#   860

Asp Tyr Asn Leu Phe His Ser Val Val Pro Va

#l Tyr Val Arg Glu Gly

865                 8

#70                 8

#75                 8

#80

Ala Ile Ile Pro Gln Ile Glu Val Arg Gln Tr

#p Thr Gly Gln Gly Gly

885

#               890

#               895

Ala Asn Arg Ile Lys Phe Asn Ile Tyr Pro Gl

#y Lys Asp Lys Glu Tyr

900

#           905

#           910

Cys Thr Tyr Leu Asp Asp Gly Val Ser Arg As

#p Ser Ala Pro Glu Asp

915

#       920

#       925

Leu Pro Gln Tyr Lys Glu Thr His Glu Gln Se

#r Lys Val Glu Gly Ala

930

#   935

#   940

Glu Ile Ala Lys Gln Ile Gly Lys Lys Thr Gl

#y Tyr Asn Ile Ser Gly

945                 9

#50                 9

#55                 9

#60

Thr Asp Pro Glu Ala Lys Gly Tyr His Arg Ly

#s Val Ala Val Thr Gln

965

#               970

#               975

Thr Ser Lys Asp Lys Thr Arg Thr Val Thr Il

#e Glu Pro Lys His Asn

980

#           985

#           990

Gly Tyr Asp Pro Ser Lys Glu Val Gly Asp Ty

#r Tyr Thr Ile Ile Leu

995

#       1000

#      1005

Trp Tyr Ala Pro Gly Phe Asp Gly Ser Ile Va

#l Asp Val Ser Lys Thr

1010

#   1015

#  1020

Thr Val Asn Val Glu Gly Gly Val Glu His Gl

#n Val Tyr Lys Asn Ser

1025                1030

#                1035

#               1040

Asp Leu His Thr Val Val Ile Asp Val Lys Gl

#u Val Ile Gly Thr Thr

1045

#               1050

#              1055

Lys Ser Val Lys Ile Thr Cys Thr Ala Ala

1060

#           1065

(2) INFORMATION FOR SEQ ID NO: 6:

(A) LENGTH: 1070 amino

#acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

#6:

Met Ala Gly Leu Ser Asp Pro Leu Asn Phe Cy

#s Lys Ala Glu Asp Tyr

1               5

#                10

#                15

Tyr Ala Ala Ala Lys Gly Trp Ser Gly Pro Gl

#n Lys Ile Ile Arg Tyr

20

#            25

#            30

Asp Gln Thr Pro Pro Gln Gly Thr Lys Asp Pr

#o Lys Ser Trp His Ala

35

#        40

#        45

Val Asn Leu Pro Phe Asp Asp Gly Thr Met Cy

#s Val Val Gln Phe Val

50

#    55

#    60

Arg Pro Cys Val Trp Arg Val Arg Tyr Asp Pr

#o Ser Val Lys Thr Ser

65

#70

#75

#80

Asp Glu Tyr Gly Asp Glu Asn Thr Arg Thr Il

#e Val Gln Asp Tyr Met

85

#                90

#                95

Thr Thr Leu Val Gly Asn Leu Asp Ile Phe Ar

#g Gly Leu Thr Trp Val

100

#           105

#           110

Ser Thr Leu Glu Asp Ser Gly Glu Tyr Tyr Th

#r Phe Lys Ser Glu Val

115

#       120

#       125

Thr Ala Val Asp Glu Thr Glu Arg Thr Arg As

#n Lys Val Gly Asp Gly

130

#   135

#   140

Leu Lys Ile Tyr Leu Trp Lys Asn Pro Phe Ar

#g Ile Gln Val Val Arg

145                 1

#50                 1

#55                 1

#60

Leu Leu Thr Pro Leu Val Asp Pro Phe Pro Il

#e Pro Asn Val Ala Asn

165

#               170

#               175

Ala Thr Ala Arg Val Ala Asp Lys Val Val Tr

#p Gln Thr Ser Pro Lys

180

#           185

#           190

Thr Phe Arg Lys Asn Leu His Pro Gln His Ly

#s Met Leu Lys Asp Thr

195

#       200

#       205

Val Leu Asp Ile Ile Lys Pro Gly His Gly Gl

#u Tyr Val Gly Trp Gly

210

#   215

#   220

Glu Met Gly Gly Ile Glu Phe Met Lys Glu Pr

#o Thr Phe Met Asn Tyr

225                 2

#30                 2

#35                 2

#40

Phe Asn Phe Asp Asn Met Gln Tyr Gln Gln Va

#l Tyr Ala Gln Gly Ala

245

#               250

#               255

Leu Asp Ser Arg Glu Pro Leu Tyr His Ser As

#p Pro Phe Tyr Leu Asp

260

#           265

#           270

Val Asn Ser Asn Pro Glu His Lys Asn Ile Th

#r Ala Thr Phe Ile Asp

275

#       280

#       285

Asn Tyr Ser Gln Ile Ala Ile Asp Phe Gly Ly

#s Thr Asn Ser Gly Tyr

290

#   295

#   300

Ile Lys Leu Gly Thr Arg Tyr Gly Gly Ile As

#p Cys Tyr Gly Ile Ser

305                 3

#10                 3

#15                 3

#20

Ala Asp Thr Val Pro Glu Ile Val Arg Leu Ty

#r Thr Gly Leu Val Gly

325

#               330

#               335

Arg Ser Lys Leu Lys Pro Arg Tyr Ile Leu Gl

#y Ala His Gln Ala Cys

340

#           345

#           350

Tyr Gly Tyr Gln Gln Glu Ser Asp Leu His Al

#a Val Val Gln Gln Tyr

355

#       360

#       365

Arg Asp Thr Lys Phe Pro Leu Asp Gly Leu Hi

#s Val Asp Val Asp Phe

370

#   375

#   380

Gln Asp Asn Phe Arg Thr Phe Thr Thr Asn Pr

#o Ile Thr Phe Pro Asn

385                 3

#90                 3

#95                 4

#00

Pro Lys Glu Met Phe Thr Asn Leu Arg Asn As

#n Gly Ile Lys Cys Ser

405

#               410

#               415

Thr Asn Ile Thr Pro Val Ile Ser Ile Arg As

#p Arg Pro Asn Gly Tyr

420

#           425

#           430

Ser Thr Leu Asn Glu Gly Tyr Asp Lys Lys Ty

#r Phe Ile Met Asp Asp

435

#       440

#       445

Arg Tyr Thr Glu Gly Thr Ser Gly Asp Pro Gl

#n Asn Val Arg Tyr Ser

450

#   455

#   460

Phe Tyr Gly Gly Gly Asn Pro Val Glu Val As

#n Pro Asn Asp Val Trp

465                 4

#70                 4

#75                 4

#80

Ala Arg Pro Asp Phe Gly Asp Asn Tyr Asp Ph

#e Pro Thr Asn Phe Asn

485

#               490

#               495

Cys Lys Asp Tyr Pro Tyr His Gly Gly Val Se

#r Tyr Gly Tyr Gly Asn

500

#           505

#           510

Gly Thr Pro Gly Tyr Tyr Pro Asp Leu Asn Ar

#g Glu Glu Val Arg Ile

515

#       520

#       525

Trp Trp Gly Leu Gln Tyr Glu Tyr Leu Phe As

#n Met Gly Leu Glu Phe

530

#   535

#   540

Val Trp Gln Asp Met Thr Thr Pro Ala Ile Hi

#s Ser Ser Tyr Gly Asp

545                 5

#50                 5

#55                 5

#60

Met Lys Gly Leu Pro Thr Arg Leu Leu Val Th

#r Ala Asp Ser Val Thr

565

#               570

#               575

Asn Ala Ser Glu Lys Lys Leu Ala Ile Glu Se

#r Trp Ala Leu Tyr Ser

580

#           585

#           590

Tyr Asn Leu His Lys Ala Thr Phe His Gly Le

#u Gly Arg Leu Glu Ser

595

#       600

#       605

Arg Lys Asn Lys Arg Asn Phe Ile Leu Gly Ar

#g Gly Ser Tyr Ala Gly

610

#   615

#   620

Ala Tyr Arg Phe Ala Gly Leu Trp Thr Gly As

#p Asn Ala Ser Thr Trp

625                 6

#30                 6

#35                 6

#40

Glu Phe Trp Lys Ile Ser Val Ser Gln Val Le

#u Ser Leu Gly Leu Asn

645

#               650

#               655

Gly Val Cys Ile Ala Gly Ser Asp Thr Gly Gl

#y Phe Glu Pro Ala Arg

660

#           665

#           670

Thr Glu Ile Gly Glu Glu Lys Tyr Cys Ser Pr

#o Glu Leu Leu Ile Arg

675

#       680

#       685

Trp Tyr Thr Gly Ser Phe Leu Leu Pro Trp Le

#u Arg Asn His Tyr Val

690

#   695

#   700

Lys Lys Asp Arg Lys Trp Phe Gln Glu Pro Ty

#r Ala Tyr Pro Lys His

705                 7

#10                 7

#15                 7

#20

Leu Glu Thr His Pro Glu Leu Ala Asp Gln Al

#a Trp Leu Tyr Lys Ser

725

#               730

#               735

Val Leu Glu Ile Cys Arg Tyr Trp Val Glu Le

#u Arg Tyr Ser Leu Ile

740

#           745

#           750

Gln Leu Leu Tyr Asp Cys Met Phe Gln Asn Va

#l Val Asp Gly Met Pro

755

#       760

#       765

Leu Ala Arg Ser Met Leu Leu Thr Asp Thr Gl

#u Asp Thr Thr Phe Phe

770

#   775

#   780

Asn Glu Ser Gln Lys Phe Leu Asp Asn Gln Ty

#r Met Ala Gly Asp Asp

785                 7

#90                 7

#95                 8

#00

Ile Leu Val Ala Pro Ile Leu His Ser Arg As

#n Glu Val Pro Gly Glu

805

#               810

#               815

Asn Arg Asp Val Tyr Leu Pro Leu Phe His Th

#r Trp Tyr Pro Ser Asn

820

#           825

#           830

Leu Arg Pro Trp Asp Asp Gln Gly Val Ala Le

#u Gly Asn Pro Val Glu

835

#       840

#       845

Gly Gly Ser Val Ile Asn Tyr Thr Ala Arg Il

#e Val Ala Pro Glu Asp

850

#   855

#   860

Tyr Asn Leu Phe His Asn Val Val Pro Val Ty

#r Ile Arg Glu Gly Ala

865                 8

#70                 8

#75                 8

#80

Ile Ile Pro Gln Ile Gln Val Arg Gln Trp Il

#e Gly Glu Gly Gly Pro

885

#               890

#               895

Asn Pro Ile Lys Phe Asn Ile Tyr Pro Gly Ly

#s Asp Lys Glu Tyr Val

900

#           905

#           910

Thr Tyr Leu Asp Asp Gly Val Ser Arg Asp Se

#r Ala Pro Asp Asp Leu

915

#       920

#       925

Pro Gln Tyr Arg Glu Ala Tyr Glu Gln Ala Ly

#s Val Glu Gly Lys Asp

930

#   935

#   940

Val Gln Lys Gln Leu Ala Val Ile Gln Gly As

#n Lys Thr Asn Asp Phe

945                 9

#50                 9

#55                 9

#60

Ser Ala Ser Gly Ile Asp Lys Glu Ala Lys Gl

#y Tyr His Arg Lys Val

965

#               970

#               975

Ser Ile Lys Gln Glu Ser Lys Asp Lys Thr Ar

#g Thr Val Thr Ile Glu

980

#           985

#           990

Pro Lys His Asn Gly Tyr Asp Pro Ser Lys Gl

#u Val Gly Asn Tyr Tyr

995

#       1000

#      1005

Thr Ile Ile Leu Trp Tyr Ala Pro Gly Phe As

#p Gly Ser Ile Val Asp

1010

#   1015

#  1020

Val Ser Gln Ala Thr Val Asn Ile Glu Gly Gl

#y Val Glu Cys Glu Ile

1025                1030

#                1035

#               1040

Phe Lys Asn Thr Gly Leu His Thr Val Val Va

#l Asn Val Lys Glu Val

1045

#               1050

#              1055

Ile Gly Thr Thr Lys Ser Val Lys Ile Thr Cy

#s Thr Thr Ala

1060

#           1065

#          1070

(2) INFORMATION FOR SEQ ID NO: 7:

(A) LENGTH: 3201 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

#7:

#GTGTTGCG     60

#AGAGCACC    120

#TAGGTGTG    180

#AGACCTCT    240

#CTCTGAGT    300

#CGGGAGAT    360

#GCAACAAG    420

#TAGTGCGC    480

#AAGCCCGT    540

#TGCATCCG    600

#GCGAGTAT    660

#TGAACTAT    720

#ATTCTCGC    780

#AGCACAAG    840

#GAAAGACC    900

#GTATCAGT    960

#CAAAGTTG   1020

#AAAGTGAC   1080

#TTCACGTC   1140

#TCCCTAAC   1200

#ATATCACT   1260

#GAGTTGAC   1320

#CGAAGGAT   1380

#ATGTTAAT   1440

#AACAATAC   1500

#ACCCGGAC   1560

#TCGATATG   1620

#ATGGAGAC   1680

#CCTCTGAG   1740

#CAACTTGG   1800

#GGCGTGGA   1860

#GTAACTGG   1920

#TGTGCATC   1980

#AGGAGAAA   2040

#CGTGGCTC   2100

#ACCCCAAG   2160

#TTTTGGAG   2220

#ACTGCATG   2280

#ATACTGAG   2340

#CTGGTGAC   2400

#ACAGAGAT   2460

#ACGATCAA   2520

#CTAGGATT   2580

#GAGAGGGT   2640

#ACCGCATC   2700

#ATGGTGTT   2760

#AGTCGAAG   2820

#TCTCAGGA   2880

#CAAAAGAC   2940

#AAGAGGTG   3000

#TCGTCGAT   3060

#AGAACTCC   3120

#GCGTCAAG   3180

#

#                3201

(2) INFORMATION FOR SEQ ID NO: 8:

(A) LENGTH: 3213 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

#8:

#CTGCTGCC     60

#AGGGTACA    120

#TGTGTGTA    180

#AGACTTCT    240

#CTCTGGTT    300

#CGGGCGAG    360

#GAAACAAG    420

#TAGTGCGT    480

#CAGCCCGT    540

#TGCATCCG    600

#GAGAGTAT    660

#TGAATTAT    720

#ATAGTCGT    780

#AGCACAAG    840

#GGAAGACC    900

#GTATCAGC    960

#CGAAGTTG   1020

#AAAGTGAC   1080

#TGCATGTC   1140

#TCCCTAAT   1200

#ACATCACC   1260

#GATATGAT   1320

#CGCAAAAT   1380

#ATGTTTGG   1440

#AAGACTAC   1500

#ACCCTGAC   1560

#TCAATATG   1620

#ATGGAGAC   1680

#CCTCTGAG   1740

#CAACCTTC   1800

#GACGTGGT   1860

#GTACGTGG   1920

#TGTGTATA   1980

#AGAAATAT   2040

#GGCTTAGA   2100

#CCAAGCAT   2160

#TAGAAATT   2220

#GCATGTTC   2280

#CTGAGGAT   2340

#GTGACGAC   2400

#GAGATGTC   2460

#ATCAGGGA   2520

#GGATTGTT   2580

#AGGGTGCC   2640

#CCATCAAG   2700

#GTGTTAGC   2760

#CGAAGGTC   2820

#ATGACTTC   2880

#TCAAACAG   2940

#ACGACCCC   3000

#TTGACGGC   3060

#GCGAAATT   3120

#GTACCACA   3180

#

#       3213

(2) INFORMATION FOR SEQ ID NO: 9:

(A) LENGTH: 317 amino

#acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-sit

#e

(B) LOCATION: 201

#/note= “X denotes a misc. amino

acid”

#9:

Met Thr Asn Tyr Asn Tyr Asp Asn Leu Asn Ty

#r Asn Gln Pro Asp Leu

1               5

#                10

#                15

Ile Pro Pro Gly His Asp Ser Asp Pro Asp Ty

#r Tyr Ile Pro Met Tyr

20

#            25

#            30

Phe Ala Ala Pro Trp Val Ile Ala His Gly Ty

#r Arg Gly Thr Ser Asp

35

#        40

#        45

Gln Tyr Ser Tyr Gly Trp Phe Leu Asp Asn Va

#l Ser Gln Ser Tyr Thr

50

#    55

#    60

Asn Thr Gly Asp Asp Ala Trp Ala Gly Gln Ly

#s Asp Leu Ala Tyr Met

65

#70

#75

#80

Gly Ala Gln Cys Gly Pro Phe Asp Gln His Ph

#e Val Tyr Glu Ala Gly

85

#                90

#                95

Asp Gly Leu Glu Asp Val Val Thr Ala Phe Se

#r Tyr Leu Gln Gly Lys

100

#           105

#           110

Glu Tyr Glu Asn Gln Gly Leu Asn Ile Arg Se

#r Ala Met Pro Pro Lys

115

#       120

#       125

Tyr Val Phe Gly Phe Phe Gln Gly Val Phe Gl

#y Ala Thr Ser Leu Leu

130

#   135

#   140

Arg Asp Asn Leu Pro Ala Gly Glu Asn Asn Va

#l Ser Leu Glu Glu Ile

145                 1

#50                 1

#55                 1

#60

Val Glu Gly Tyr Gln Asn Gln Asn Val Pro Ph

#e Glu Gly Leu Ala Val

165

#               170

#               175

Asp Val Asp Met Gln Asp Asp Leu Arg Val Ph

#e Thr Thr Arg Pro Ala

180

#           185

#           190

Phe Trp Thr Ala Asn Lys Val Gly Xaa Gly Gl

#y Asp Pro Asn Asn Lys

195

#       200

#       205

Ser Val Phe Glu Trp Ala His Asp Arg Gly Le

#u Val Cys Gln Thr Asn

210

#   215

#   220

Val Thr Cys Phe Leu Lys Asn Glu Lys Asn Pr

#o Tyr Glu Val Asn Gln

225                 2

#30                 2

#35                 2

#40

Ser Leu Arg Glu Lys Gln Leu Tyr Thr Lys Se

#r Asp Ser Leu Asp Asn

245

#               250

#               255

Ile Asp Phe Gly Thr Thr Pro Asp Gly Pro Se

#r Asp Ala Tyr Ile Gly

260

#           265

#           270

His Leu Asp Tyr Gly Gly Gly Val Glu Cys As

#p Ala Leu Phe Pro Asp

275

#       280

#       285

Trp Gly Arg Pro Asp Val Ala Gln Trp Trp Gl

#y Asp Asn Tyr Lys Lys

290

#   295

#   300

Leu Phe Ser Ile Gly Leu Asp Phe Val Trp Gl

#n Asp Met

305                 3

#10                 3

#15

(2) INFORMATION FOR SEQ ID NO: 10:

(A) LENGTH: 323 amino

#acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-sit

#e

(B) LOCATION: 272

#/note= “X is a misc. amino acid”

(ix) FEATURE:

(A) NAME/KEY: Modified-sit

#e

(B) LOCATION: 273

#/note= “X is a misc. amino acids”

(ix) FEATURE:

(A) NAME/KEY: Modified-sit

#e

(B) LOCATION: 274

#/note= “X is a misc. amino acid”

#10:

Met Thr Asn Tyr Asn Tyr Asp Asn Tyr Asn Ty

#r Asn Gln Ser Asp Leu

1               5

#                10

#                15

Ile Ala Pro Gly Tyr Pro Ser Asp Pro Asn Ph

#e Tyr Ile Pro Met Tyr

20

#            25

#            30

Phe Ala Ala Pro Trp Val Val Val Lys Gly Cy

#s Ser Gly Asn Ser Asp

35

#        40

#        45

Glu Gln Tyr Ser Tyr Gly Trp Phe Met Asp As

#n Val Ser Gln Thr Tyr

50

#    55

#    60

Met Asn Thr Gly Gly Thr Ser Trp Asn Cys Gl

#y Glu Glu Asn Leu Ala

65

#70

#75

#80

Tyr Met Gly Ala Gln Cys Gly Pro Phe Asp Gl

#n His Phe Val Tyr Gly

85

#                90

#                95

Asp Gly Asp Gly Leu Glu Asp Val Val Gln Al

#a Phe Ser Leu Leu Gln

100

#           105

#           110

Gly Lys Glu Phe Glu Asn Gln Val Leu Asn Ly

#s Arg Ala Val Met Pro

115

#       120

#       125

Pro Lys Tyr Val Phe Gly Tyr Phe Gln Gly Va

#l Phe Gly Ile Ala Ser

130

#   135

#   140

Leu Leu Arg Glu Gln Arg Pro Glu Gly Gly As

#n Asn Ile Ser Val Ser

145                 1

#50                 1

#55                 1

#60

Glu Ile Val Glu Gly Tyr Gln Ser Asn Asn Ph

#e Pro Leu Glu Gly Leu

165

#               170

#               175

Ala Val Asp Val Asp Met Gln Gln Asp Leu Ar

#g Cys Ser Ser Pro Leu

180

#           185

#           190

Lys Ile Glu Phe Trp Thr Ala Asn Lys Val Gl

#y Thr Gly Gly Asp Ser

195

#       200

#       205

Asn Asn Lys Ser Val Phe Glu Trp Ala His As

#p Lys Gly Leu Val Cys

210

#   215

#   220

Gln Thr Asn Val Thr Cys Phe Leu Arg Asn As

#p Asn Gly Gly Ala Asp

225                 2

#30                 2

#35                 2

#40

Tyr Glu Val Asn Gln Thr Leu Arg Glu Lys Gl

#y Leu Tyr Thr Lys Asn

245

#               250

#               255

Asp Ser Leu Thr Asn Thr Asn Phe Gly Thr Th

#r Asn Asp Gly Pro Xaa

260

#           265

#           270

Xaa Xaa Tyr Ile Gly His Leu Asp Tyr Gly Gl

#y Gly Gly Asn Cys Asp

275

#       280

#       285

Ala Leu Phe Pro Asp Trp Gly Arg Pro Gly Va

#l Ala Glu Trp Trp Gly

290

#   295

#   300

Asp Asn Tyr Ser Lys Leu Phe Lys Ile Gly Le

#u Asp Phe Val Trp Gln

305                 3

#10                 3

#15                 3

#20

Asp Met Thr

(2) INFORMATION FOR SEQ ID NO: 11:

(A) LENGTH: 202 amino

#acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-sit

#e

(B) LOCATION: 43

#/note= “X is a misc. amino acid”

(ix) FEATURE:

(A) NAME/KEY: Modified-sit

#e

(B) LOCATION: 176

#/note= “X is a misc. amino acid”

#11:

Met Thr Asn Tyr Asn Tyr Asp Asn Leu Asn Ty

#r Asn Gln Pro Asp Val

1               5

#                10

#                15

Val Pro Pro Gly Tyr His Asp His Pro Asn Ty

#r Tyr Ile Pro Met Tyr

20

#            25

#            30

Tyr Ala Ala Pro Trp Leu Val Val Gln Gly Xa

#a Ala Gly Thr Ser Lys

35

#        40

#        45

Gln Tyr Ser Tyr Gly Trp Phe Met Asp Asn Va

#l Ser Gln Ser Tyr Met

50

#    55

#    60

Asn Thr Gly Asp Thr Ala Trp Asn Cys Gly Gl

#n Glu Asn Leu Ala Tyr

65

#70

#75

#80

Met Gly Ala Gln Tyr Gly Pro Phe Asp Gln Hi

#s Phe Val Tyr Gly Asp

85

#                90

#                95

Gly Asp Gly Leu Glu Asp Val Val Lys Ala Ph

#e Ser Phe Leu Gln Gly

100

#           105

#           110

Lys Glu Phe Glu Asp Lys Lys Leu Asn Lys Ar

#g Ser Val Met Pro Pro

115

#       120

#       125

Lys Tyr Val Phe Gly Phe Phe Gln Gly Val Ph

#e Gly Ala Leu Ser Leu

130

#   135

#   140

Leu Lys Gln Asn Leu Pro Ala Gly Glu Asn As

#n Ile Ser Val Gln Glu

145                 1

#50                 1

#55                 1

#60

Ile Val Glu Gly Tyr Gln Asp Asn Asp Tyr Pr

#o Phe Glu Gly Leu Xaa

165

#               170

#               175

Val Asp Val Asp Met Gln Asp Asp Leu Arg Va

#l Phe Thr Thr Lys Pro

180

#           185

#           190

Glu Tyr Trp Ser Ala Asn Met Val Gly Glu

195

#       200

(2) INFORMATION FOR SEQ ID NO: 12:

(A) LENGTH: 953 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(573,

# “”)

#/note= “g is a misc nucleic acid”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(601,

# “”)

#/note= “g is a misc. nucleic acid”

#12:

#CACCTGGC     60

#TGATCGCA    120

#ATGTATCC    180

#CGTACATG    240

#GACTTGAA    300

#GACTGAAT    360

#TCGGAGCC    420

#AAGAAATT    480

#TTGATATG    540

#AGGTGGGG    600

#GCCTTGTC    660

#TGAATCAG    720

#ATTTTGGA    780

#GTGGTGTG    840

#GGGGCGAT    900

#C           953

(2) INFORMATION FOR SEQ ID NO: 13:

(A) LENGTH: 969 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(814.

#.821, “”)

#/note= “Each g between (and

including)

#814 and 821 is a misc. nucleic acid.”

#13:

#CTCCAGGA     60

#TAGTTGTT    120

#ATAATGTC    180

#ACTTGGCA    240

#GAGATGGT    300

#ACCAAGTT    360

#GAGTCTTT    420

#CTGTTTCA    480

#TAGATGTG    540

#CGGCAAAT    600

#ATGACAAA    660

#GGGCAGAT    720

#CACTGACG    780

#ATCTGGAC    840

#GTGTGGCT    900

#TGTGGCAA    960

#

#

#        969

(2) INFORMATION FOR SEQ ID NO: 14:

(A) LENGTH: 607 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(128,

# “”)

#/note= “g is a misc. nucleic acid”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(232,

# “”)

#/note= “g is a misc. nucleic acid”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(249,

# “”)

#/note= “g is a misc. nucleic acid”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(526,

# “”)

#/note= “g is a misc. nucleic acid”

#14:

#CTCCAGGT     60

#TGGTCGTT    120

#ATGTCTCT    180

#TGGCATAC    240

#ATGGCCTT    300

#AAAAACTC    360

#TTTTCGGT    420

#TGCAAGAG    480

#ATGTTGAT    540

#ACATGGTA    600

#

#

#         607

(2) INFORMATION FOR SEQ ID NO: 15:

(A) LENGTH: 90 amino

#acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

#15:

Tyr Arg Trp Gln Glu Val Leu Tyr Thr Ala Me

#t Tyr Gln Asn Ala Ala

1               5

#                10

#                15

Phe Gly Lys Pro Ile Ile Lys Ala Ala Ser Me

#t Tyr Asn Asn Asp Ser

20

#            25

#            30

Asn Val Arg Arg Ala Gln Asn Asp His Phe Le

#u Leu Gly Gly His Asp

35

#        40

#        45

Gly Tyr Arg Ile Leu Cys Ala Pro Val Val Tr

#p Glu Asn Ser Thr Glu

50

#    55

#    60

Arg Glu Leu Tyr Leu Pro Val Leu Thr Gln Tr

#p Tyr Lys Phe Gly Pro

65

#70

#75

#80

Asp Phe Asp Thr Lys Pro Leu Glu Gly Ala

85

#                90

(2) INFORMATION FOR SEQ ID NO: 16:

(A) LENGTH: 23 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(6,

#“”)

#/note= “N is T OR C”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(9,

#“”)

#/note= “N IS C OR T”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(12,

#“”)

#/note= “N IS C OR T”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(15,

#“”)

#/note= “N IS C OR T”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(18,

#“”)

#/note= “N IS G OR A OR T OR C”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(21,

#“”)

#/note= “N IS C OR T”

#16:

#

#                23

(2) INFORMATION FOR SEQ ID NO: 17:

(A) LENGTH: 23 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(6,

#“”)

#/note= “N IS T OR C”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(9,

#“”)

#/note= “N IS C OR T”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(12,

#“”)

#/note= “N IS C OR T”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(15,

#“”)

#/note= “N IS C OR T”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(18,

#“”)

#/note= “N IS C OR T”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(21,

#“”)

#/note= “N IS C OR T”

#17:

#

#                23

(2) INFORMATION FOR SEQ ID NO: 18:

(A) LENGTH: 17 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(3,

#“”)

#/note= “N IS G OR A OR T OR C”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(6,

#“”)

#/note= “N IS G OR A”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(9,

#“”)

#/note= “N IS G OR A”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(12,

#“”)

#/note= “N IS G OR A OR T OR C”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(15,

#“”)

#/note= “N IS G OR A OR T OR C”

#18:

#

#

#   17

(2) INFORMATION FOR SEQ ID NO: 19:

(A) LENGTH: 20 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(3,

#“”)

#/note= “N IS G OR A OR T OR C”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(6,

#“”)

#/note= “N IS G OR A”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(9,

#“”)

#/note= “N IS C OR T”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(12,

#“”)

#/note= “N IS G OR A”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(18,

#“”)

#/note= “N IS C OR T”

#19:

#

#

# 20

(2) INFORMATION FOR SEQ ID NO: 20:

(A) LENGTH: 17 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(3,

#“”)

#/note= “N IS T OR C”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(6,

#“”)

#/note= “N IS G OR A OR T OR C”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(12,

#“”)

#/note= “N IS G OR A”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(15,

#“”)

#/note= “N IS G OR A”

#20:

#

#

#   17

(2) INFORMATION FOR SEQ ID NO: 21:

(A) LENGTH: 17 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(3,

#“”)

#/note= “N IS T OR C”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(6,

#“”)

#/note= “N IS G OR A”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(12,

#“”)

#/note= “N IS G OR A”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(15,

#“”)

#/note= “N IS G OR A”

#21:

#

#

#   17

(2) INFORMATION FOR SEQ ID NO: 22:

(A) LENGTH: 71 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

#22:

#TTGGCGGC     60

#

#

#       71

(2) INFORMATION FOR SEQ ID NO: 23:

(A) LENGTH: 23 amino

#acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

#23:

Met Tyr Asn Asn Asp Ser Asn Val Arg Arg Al

#a Gln Asn Asp His Phe

1               5

#                10

#                15

Leu Leu Gly Gly His Asp Gly

20

(2) INFORMATION FOR SEQ ID NO: 24:

(A) LENGTH: 160 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

#24:

#TTGGTGGA     60

#AACGGAAT    120

#

#   160

(2) INFORMATION FOR SEQ ID NO: 25:

(A) LENGTH: 54 amino

#acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

#25:

Met Tyr Asn Asn Asp Ser Asn Val Arg Arg Al

#a Gln Asn Asp His Phe

1               5

#                10

#                15

Leu Leu Gly Gly His Asp Gly Tyr Arg Ile Le

#u Cys Ala Pro Val Val

20

#            25

#            30

Trp Glu Asn Ser Thr Glu Arg Glu Leu Tyr Le

#u Pro Val Leu Thr Gln

35

#        40

#        45

Trp Tyr Lys Phe Gly Pro

50

(2) INFORMATION FOR SEQ ID NO: 26:

(A) LENGTH: 238 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

#26:

#GGAAACCG     60

#AGGATGAC    120

#TGTGGGAG    180

#CGGCCC      238

(2) INFORMATION FOR SEQ ID NO: 27:

(A) LENGTH: 79 amino

#acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

#27:

Tyr Arg Trp Gln Glu Val Leu Tyr Thr Ala Me

#t Tyr Gln Asn Ala Ala

1               5

#                10

#                15

Phe Gly Lys Pro Ile Ile Lys Ala Ala Ser Me

#t Tyr Asp Asn Asp Arg

20

#            25

#            30

Asn Val Arg Gly Ala Gln Asp Asp His Phe Le

#u Leu Gly Gly His Asp

35

#        40

#        45

Gly Tyr Arg Ile Leu Cys Ala Pro Val Val Tr

#p Glu Asn Thr Thr Ser

50

#    55

#    60

Arg Asp Leu Tyr Leu Pro Val Leu Thr Lys Tr

#p Tyr Lys Phe Gly

65

#70

#75

(2) INFORMATION FOR SEQ ID NO: 28:

(A) LENGTH: 28 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

#28:

#

#             28

(2) INFORMATION FOR SEQ ID NO: 29:

(A) LENGTH: 36 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

#29:

#

#       36

(2) INFORMATION FOR SEQ ID NO: 30:

(A) LENGTH: 34 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

#30:

#

#        34

(2) INFORMATION FOR SEQ ID NO: 31:

(A) LENGTH: 75 amino

#acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

#31:

Lys Asn Leu His Pro Gln His Lys Met Leu Ly

#s Asp Thr Val Leu Asp

1               5

#                10

#                15

Ile Val Lys Pro Gly His Gly Glu Tyr Val Gl

#y Trp Gly Glu Met Gly

20

#            25

#            30

Gly Ile Gln Phe Met Lys Glu Pro Thr Phe Me

#t Asn Tyr Phe Asn Phe

35

#        40

#        45

Asp Asn Met Gln Tyr Gln Gln Val Tyr Ala Gl

#n Gly Ala Leu Asp Ser

50

#    55

#    60

Arg Glu Pro Leu Tyr His Ser Asp Pro Phe Ty

#r

65

#70

#75

(2) INFORMATION FOR SEQ ID NO: 32:

(A) LENGTH: 23 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(3,

#“”)

#/note= “N IS G OR A”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(6,

#“”)

#/note= “N IS C OR T”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(9,

#“”)

#/note= “N IS G OR A”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(15,

#“”)

#/note= “N IS G OR A OT T OR C”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(18,

#“”)

#/note= “N IS G OR A”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(21,

#“”)

#/note= “N IS C OR T”

#32:

#

#                23

(2) INFORMATION FOR SEQ ID NO: 33:

(A) LENGTH: 23 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(3,

#“”)

#/note= “N IS G OR A”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(6,

#“”)

#/note= “N IS C OR T”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(9,

#“”)

#/note= “N IS G OR A”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(15,

#“”)

#/note= “N IS G OR A”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(18,

#“”)

#/note= “N IS G OR A”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(21,

#“”)

#/note= “N IS C OR T”

#33:

#

#                23

(2) INFORMATION FOR SEQ ID NO: 34:

(A) LENGTH: 20 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(3,

#“”)

#/note= “N IS G OR A”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(6,

#“”)

#/note= “N IS G OR A OR T OR C”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(9,

#“”)

#/note= “N IS G OR A”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(12,

#“”)

#/note= “N IS G OR A”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(15,

#“”)

#/note= “N IS G OR A”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(18,

#“”)

#/note= “N IS G OR A”

#34:

#

#

# 20

(2) INFORMATION FOR SEQ ID NO: 35:

(A) LENGTH: 20 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(3,

#“”)

#/note= “N IS G OR A”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(6,

#“”)

#/note= “N IS G OR A OR T OR C”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(9,

#“”)

#/note= “N IS G OR A”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(12,

#“”)

#/note= “N IS G OR A OR T OR C”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(15,

#“”)

#/note= “N IS G OR A”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(18,

#“”)

#/note= “N IS G OR A”

#35:

#

#

# 20

(2) INFORMATION FOR SEQ ID NO: 36:

(A) LENGTH: 37 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

#36:

#

#      37

(2) INFORMATION FOR SEQ ID NO: 37:

(A) LENGTH: 23 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(6,

#“”)

#/note= “N IS G OR A OR T OR C”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(9,

#“”)

#/note= “N IS C OR T”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(12,

#“”)

#/note= “N IS C OR T”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(15,

#“”)

#/note= “N IS C OR T”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(18,

#“”)

#/note= “N IS C OR T”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(21,

#“”)

#/note= “N IS C OR T”

#37:

#

#                23

(2) INFORMATION FOR SEQ ID NO: 38:

(A) LENGTH: 21 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(1,

#“”)

#/note= “N IS A OR G”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(4,

#“”)

#/note= “N IS G OR A OR T OR C”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(13,

#“”)

#/note= “N IS G OR A OR T OR C”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(16,

#“”)

#/note= “N IS G OR A OR T OR C”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(19,

#“”)

#/note= “N IS G OR A OR T OR C”

#38:

#

#

#21

(2) INFORMATION FOR SEQ ID NO: 39:

(A) LENGTH: 23 base

#pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(6,

#“”)

#/note= “N IS G OR A”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(9,

#“”)

#/note= “N IS C OR T”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(15,

#“”)

#/note= “N IS G OR A OR T OR C”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(18,

#“”)

#/note= “N IS G OR A”

(ix) FEATURE:

(A) NAME/KEY: misc_

#difference

(B) LOCATION: replace(21,

#“”)

#/note= “N IS G OR A”

#39:

#

#                23