Enzymes mixture obtained from Penicillium funiculosum

The present invention relates to a novel micro-organism, new enzymes and new enzymes mixture. In addition, the present invention relates to the composition of the enzymes mixture, its preparation and its use in feed, food and other industries including but not limited to the paper industry and the textile industry.

Enzymes have been used for a long time for a variety of different industrial applications. Examples are known in the baking industry, in the wine and fruit juices industry (where enzymes are used to breakdown pectins and β-glucans), in the textile industry (where cellulases are used to obtain soft and smooth cellulosic fabrics) and also, which is not the least application, for animal feed. In this case the enzymes improve the digestibility of vegetable sources.

This last use enables the livestock to digest feed more efficiently. The value of a feed can be measured by the FCR (Feed Conversion Ratio), a nutritive ratio of the amount of feed consumed relative to the weight gain of the animal. A decrease in FCR, for a feed, indicates the animal gains proportionately more weight for a given quantity of feed ; i.e. the animal is able to utilize the feed more efficiently.

Poor digestibility of the feed components (starch, fat, protein/amino acids) is a noted feature of cereal-based feeds and, for example, particularly those containing a high barley or wheat content. In these cases it may be necessary to formulate the teed to contain higher levels of energy from other sources and other supplements such as amino acids. These enzymes increase the Apparent Metabolizable Energy value of the cereals incorporated into the Feed.

Another approach to resolve this problem has been to add enzyme supplements, cellulases, endo-1,3(4)-β-glucanases (β-glucanases), endo-1,4-β-xylanases (xylanases) etc., or mixtures of enzyme activities, to these cereal-based feeds. Enzyme supplements may have a specific use to hydrolyze the β-glucans, or to hydrolyze the arabinoxylans, found in the cereals (typically barley and wheat). The addition of enzymes has differents goals. One advantage which clearly proves the efficacy of feed enzyme supplements is the reduction in viscosity of materials in the gut of the animals which receive cereal-based feed containing the appropriate enzyme supplement. The higher viscosity is due, in part, to β-glucans and arabinoxylans found in barley and wheat. The lower viscosity, resulting from enzyme action, permits an easier absorption of nutritional components in the animal's gut. The other advantage is the release of nutrients entrapped by the cell walls of the cereals decreasing the requirement for other costly feed supplements. Overall the result is a significant reduction in the cost of the feed with a similar or beneficial effect as measured by the FCR.

Enzymes preparations originating from a range of different micro-organisms have been described to improve feed digestibility.

If we consider prior art related to the use of enzymes in the animal feed we can mention the European Patent No 0.699.762 which describes use of a phytase issued from Schwanniomyces occidentalis . This phytase is a phytase obtained from genetically modified organism obtained by including cloned gene that we would like to avoid in the present invention.

If we consider the WO 95/26398 patent application, again a modified cellulase is obtained by inclusion of foreign DNA sequence in an host cell which modifies the nature of the original strain which is chosen in the following list of micro-organisms: Bacillus, Streptomyces, Saccharomyces, Schizosaccharomyces, Aspergillus. In the present invention our main aim was to avoid foreign gene inclusion in the micro-organism which is the producer of the enzyme.

In the WO 96/05739 patent application, a mixture of enzymes (xylanase, protease and, optionally, β-glucanase) is obtained from different micro-organisms. The authors give example (page 5) of enzymes mixture with a ratio of xylanase activity to β-glucanase activity of the order of 1:5. It has been found that when a xylanase is included in a cereal-based diet at or around its optimum dosage level, the co-presence of enzymes possessing β-glucanase activity increase the FCR of the feed which is of course disadvantageous. Consequently the authors advise against the presence of β-glucanase, they recommend a maximum ratio of xylanase activity to β-glucanase activity of 1:0-0.25.

In some cases, in order to ensure all the enzyme activities relevant to the feed application are present, preparations are made up from preparations from more than one micro-organism. In a number of cases the enzyme preparations have been obtained from microorganisms subjected to genetic modification using recombinant DNA techniques.

We have discovered and developed a new micro-organism belonging to the class of Penicillium funiculosum , that contains new enzymes and a mixture of enzyme activities which can be used successfully to increase mainly the digestibility of cereal-based animal feeds.

Accordingly, the present invention relates to a new micro-organism derived from Penicillium funiculosum and a method for cultivating this micro-organism and for recovering the enzymes produced by this micro-organism.

In addition, in accordance with this invention, there are provided new enzymes issued from this micro-organism, nucleic acid sequences therefrom and new compositions containing those enzymes.

Further, in accordance with this invention, there is provided a method for improving the digestibility of aminoacids and cereal-based animal feeds and amino acids.

Another subject of the present invention is the reduction of phosphorus excretion and ammonia excretion from the battery where animals are fed.

A. The New Strain Penicillium funiculosum

This new strain of the fungus Penicillium funiculosum is deposited under the number IMI 378536 in a recognized International Depositary Authority under the Budapest Treaty (1977), the International Mycological Institute (IMI), Bakeham Lane, Englefield Green, Egham, Surrey, TW20 9TY, UK.

Filiation

The new strain has been obtained from Penicillium funiculosum IMI 134756 after successive UV and β-irradiations treatment of spores, including screening on selective medium. No genetic modification has been obtained by recombinant DNA techniques using inclusion of foreign DNA or RNA.

Identification and Typing

Penicillium funiculosum IMI 378536 has been characterised by growth on Czapek Dox agar at 25° C. Colony characteristics and micro-morphology are typical for Penicillium funiculosum . The identification of the micro-organism as a Penicillium funiculosum has been confirmed at the International Mycological Institute, Bakeham Lane, Englefield Green, Egham, Surrey, TW20 9TY, UK. Growth is as a tough basal felt, with aerial growth, as ropes or bundles of hyphae (funiculose), mycelium is white with underlying red colouration in the substrate, margins are reverse pale but coloured red towards centres and may become deep red. This penicillium is typical, it shows conidiophores short mostly arising from funicles, biverticillate, acerose conidiogenous cells, conidia are elliptical and smooth.

The micro-organism used for the production of the enzyme preparation of this invention is grown under aerobic conditions in a medium which contains cellulose, corn steep liquor, calcium carbonate and ammonium sulphate.

B. Process of Fermentation

This new fungus is manufactured by fermentation of the deposited strain first on a seed medium preferably constituted of (in weight):

The production medium has preferably the following constitution (in weight):

For the fermentation, charge the fermenter with sufficient water, add the ingredients to water in suitable agitated container, stir until the ingredients have dissolved. Sterilise by sealing the fermentor and raising the content to typically 121° C. The fermentation vessel is inoculated with the seed fermenter.

The main source of carbon which is added during the process of fermentation is cellulose ; amongst different cellulose sources we prefer to use ARBOCEL, SOLKAFLOC, CLAROCEL, ALPHACEL, FIBRACEL with different grades.

The pH during the fermentation is preferably controlled by the addition of sulphuric acid, or another acid, and ammonia in gas or liquid form, or another base.

At the end of the fermentation time, eliminate solids by solid-liquid separation such as filtration or centrifugation, collect the liquid phase and concentrate for example by ultrafiltration on organic or mineral membrane.

These enzymes may also be manufactured from means of recombinant DNA technology and thus be produced by recombinant homologous species or heterologous species. The host for the transfer of the gene coding for the enzyme may be selected from a fungal species, a bacterial cell or a plant cell. Any conventional technique may be used to insert the gene encoding the enzyme of interest in the host cell such as plasmids (integrative or not), phage vectors and viral vectors. The Penicillium funiculosum comprising heterologous genes inclusion or modification of the genome with homologous genes by inclusion, deletion or modification of said homologous gene are also part of this invention.

In accordance with the invention the enzyme may be provided as an isolated pure enzyme preparation or as a crude preparation such as the cultivation medium in which Penicillium funiculosum has been grown.

It may be also possible to include this or those enzymes in compositions containing one further enzyme, the type of which depending on the intended use of the composition. The added enzymes may be selected from for example carbohydrases, lipases and proteases.

C. Compositions of the <<Mixture of Enzyme Activities>>

1. Liquid Composition

For liquid composition, after addition of antimicrobial agents measurement of the concentration of enzymes and correct dilution to product strength is carried out.

The preferred composition of the liquid solution by weight is the following:

Antimicrobial are chosen from products such as sorbic acid and salts, benzoic acid and salts, methyl 4-hydroxybenzoate and n-propyl 4-hydroxybenzoate, fumaric acid, salts and esters. Salts such as sodium chloride or potassium chloride could also be used.

The most preferred anti-freezing agents are 1,2-propandiol, ethylene glycol, glycerol.

2. Powder Composition

For powder preparations, the concentrated solution obtained is dried with eventually the presence of a carrier. The powder obtained after drying of the concentrated solution in absence of a carrier can be further mixed with a suitable carrier.

The preferred composition of the powder form is the following:

Preferred carriers are chosen from wheat flour, starch, gypsum, maltodextrin, corn solids, by-products from cereal processing such as maize grits, wheat middlings, wheat bran, rye tailings, minerals mixture. F r = T substrate - T water T test - T water     F r = T substrate - T water T test - T water D .    Enzyme     characteristics

We obtain a new enzymes mixture produced by Penicillium funiculosum . This enzymes mixture contains new enzymes such as cellulases, β-glucanases, xylanases, xylanase accessory enzymes such as arabinofuranosidase and feruloyl esterases.

1. Procedure

The enzyme preparation is characterised by assays that include assays for cellulase, cellobiohydrolase, β-glucosidase, endo-1,3(4)-β-glucanase, laminarinase endo-1,4-β-xylanase (using different substrates), β-xylosidase, arabinofuranosidase and feruloyl esterase (using different substrates) activities.

1.1. Cellulase by DNS CMC Method

The assay for cellulase activity is based on the enzymatic hydrolysis of the glycosidic bonds in carboxymethylcellulose (CMC), a β-1,4 glucan. The products of the reaction, β-1,4 glucan oligosaccharides, are determined by the resulting increase in reducing value (as glucose).

A solution containing 1 ml of a 1%(w/v) CMC solution in 0.1M sodium acetate buffer, pH 5.0 (or at different pH values); 1 ml of appropriately diluted enzyme solution was incubated at 50° C. for 10 minutes. The enzyme reaction is stopped by the addition of 2 ml of a DNS solution (1%(w/v) 3,5-dinitrosalicylic acid, 1.6%(w/v) sodium hydroxide, 30%(w/v) potassium sodium (+)-tartrate in distilled water). The solution is mixed and placed into a boiling water bath, 95° C. minimum, for 5 minutes then cooled to 25° C. 10 ml distilled water is added to the solution and the absorbance is measured at 540 nm using a 2 cm path length glass cell.

The result is converted to μmoles reducing sugar (as glucose) by comparison with a standard curve for 2 ml of 0.00-0.04%(w/v) glucose solutions treated with DNS solution in an equivalent manner.

The observed enzyme reaction absorbance is corrected for non-specific absorbance by carrying out a reaction in which the DNS solution is added to the mixture before the enzyme solution. One unit of cellulase activity is defined as the amount of enzyme which produces 1 μmole glucose equivalents.min −1 under the conditions of the assay (50° C. and pH 5.0 or other pH).

1.2 Cellobiohydrolase by the p-Nitrophenyl β-D-Cellobiopyranoside Method

The assay of cellobiohydrolase is based on the enzymatic hydrolysis of p-nitrophenyl β-D-cellobiopyranoside. A product of the reaction, p-nitrophenol is determined colorimetrically.

A solution containing 1 ml of a 0.1%(w/v) p-nitrophenyl β-D-cellobiopyranoside in distilled water: 1 ml distilled water; 1 ml 0.2M sodium acetate buffer, pH 5.0; 1 ml of appropriately diluted enzyme solution was incubated at 50° C. for 30 minutes. The enzyme reaction is stopped by the addition of 4 ml of 0.4M glycine solution. The solution is mixed and cooled to 20° C. The absorbance is measured at 400 nm using a 1 cm path length glass cell.

The result is converted to μmoles p-nitrophenol by comparison with the molar extenction coefficient of p-nitrophenol under these conditions.

The observed enzyme reaction absorbance is corrected for non-specific absorbance by carrying out a reaction in which the glycine solution is added to the mixture before the enzyme solution. One unit of cellobiohydrolase activity is defined as the amount of enzyme which produces 1 μmole p-nitrophenol from p-nitrophenyl β-D-cellobiopyranoside per minute under the conditions of the assay (50° C. and pH 5.0).

1.3 β-Glucosidase by the p-Nitrophenyl β-D-Glucopyranoside Method

The assay of β-glucosidase is based on the enzymatic hydrolysis of p-nitrophenyl β-D-glucopyranoside. A product of the reaction, p-nitrophenol is determined colorimetrically.

A solution containing 1 ml of a 0.1%(w/v) p-nitrophenyl β-D-glucopyranoside in distilled water; 1 ml distilled water; 1 ml 0.2M sodium acetate buffer, pH 5.0; 1 ml of appropriately diluted enzyme solution was incubated at 50° C. for 30 minutes. The enzyme reaction is stopped by the addition of 4 ml of 0.4M glycine solution. The solution is mixed and cooled to 20° C. The absorbance is measured at 400 nm using a 1 cm path length glass cell.

The result is converted to μmoles p-nitrophenol by comparison with the molar extenction coefficient of p-nitrophenol under these conditions.

The observed enzyme reaction absorbance is corrected for non-specific absorbance by carrying out a reaction in which the glycine solution is added to the mixture before the enzyme solution. One unit of β-glucosidase activity is defined as the amount of enzyme which produces 1 μmole p-nitrophenol from p-nitrophenyl β-D-glucopyranoside per minute under the conditions of the assay (50° C. and pH 5.0).

1.4. endo-1,3(4)-β-Glucanase by the DNS Barley β-Glucan Method

An assay for endo-1,3(4)-β-glucanase activity is based on the enzymatic hydrolysis of the glycosidic bonds in barley β-glucan, a β-1,3(4)-glucan. The products of the reaction, β-1,3(4)-glucan oligosaccharides, are determined by the resulting increase in reducing value (as glucose).

A solution containing 1 ml of a 1%(w/v) barley β-glucan solution in 0.1M sodium acetate buffer, pH 5.0 (or at different pH values); 1 ml of appropriately diluted enzyme solution was incubated at 50° C. for 10 minutes. The enzyme reaction is stopped by the addition of 2 ml of a DNS solution (1%(w/v) 3,5-dinitrosalicylic acid, 1.6%(w/v) sodium hydroxide, 30%(w/v) potassium sodium (+)-tartrate in distilled water). The solution is mixed and placed into a boiling water bath, 95° C. minimum, for 5 minutes then cooled to 25° C. 10 ml distilled water is added to the solution and the absorbance is measured at 540 nm using a 2 cm path length glass cell.

The result is converted to μmoles reducing sugar (as glucose) by comparison with a standard curve for 2 ml of 0.00-0.04%(w/v) glucose solutions treated with DNS solution in an equivalent manner.

The observed enzyme reaction absorbance is corrected for non-specific absorbance by carrying out a reaction in which the DNS solution is added to the mixture before the enzyme solution. One unit of endo-1,3(4)-β-glucanase activity is defined as the amount of enzyme which produces 1 μmole glucose equivalents.min −1 under the conditions of the assay (50° C. and pH 5.0 or other pH).

1.5. endo-1,3(4)-β-glucanase by the azo Barley β-Glucan Method

An assay for endo-1,3(4)-β-glucanase activity is based on the enzymatic hydrolysis of a barley β-glucan which has a bound chromophore (azo-barley β-glucan). The products of the reaction, oligomers that are soluble after ethanol precipitation, are determined by the resulting increase in absorbance at 590 nm.

A solution containing 0.5 ml of azo barley β-glucan substrate (ready-to-use form) and 0.2 ml of enzyme dilution (containing between 0.15 to 0.60 units.ml −1 in 0.01M sodium acetate buffer, pH 4.6) was incubated at 30° C. for 20 minutes exactly. The enzyme reaction is stopped by the addition of 2.5 ml of Precipitation Solution (containing 18.1 g sodium acetate and 3.0 g zinc mixed in 300 ml of glass distilled water, pH adjusted to pH 5.0 with hydrochloric acid, transfer contents to a 1 l volumetric flask and make up to volume with 96% v/v ethanol). The solution is mixed and allowed to stand at room temperature for 10 minutes. The solution is transfered in centrifuge tube and centrifuged at 1000 g for 10 minutes in a benchtop centrifuge. The absorbance of the supernatant is measured at 590 nm using a 1 cm path length glass cell.

The observed enzyme reaction absorbance is corrected for non-specific absorbance by carrying out a reaction in which the Precipitation Solution is added to the mixture before the enzyme solution. One unit of endo-1,3(4)-β-glucanase activity is defined as the amount of enzyme which hydrolyses the substrate to give an absorbance of 0.820 units at 590 nm, using a standard substrate, under the conditions of the assay (30° C. and pH 4.6).

1.6. Laminarinase (endo-1,3-β-Glucanase) by the DNS Laminarin Method

The assay for laminarinase (endo-1,3(4)-β-glucanase) activity is based on the enzymatic hydrolysis of the glycosidic bonds in laminarin, a β-1,3-glucan. The products of the reaction, β-1,3-glucan oligosaccharides, are determined by the resulting increase in reducing value (as glucose).

A solution containing 1 ml of a 1%(w/v) laminarin solution in 0.1M sodium acetate buffer, pH 5.0; 1 ml of appropriately diluted enzyme solution was incubated at 50° C. for 10 minutes. The enzyme reaction is stopped by the addition of 2 ml of a DNS solution (1%(w/v) 3,5-dinitrosalicylic acid, 1.6%(w/v) sodium hydroxide, 30%(w/v) potassium sodium (+)-tartrate in distilled water). The solution is mixed and placed into a boiling water bath. 95° C. minimum, for 5 minutes then cooled to 25° C. 10 ml distilled water is added to the solution and the absorbance is measured at 540 nm using a 2 cm path length glass cell.

The result is converted to μmoles reducing sugar (as glucose) by comparison with a standard curve for 2 ml of 0.00-0.04%(w/v) glucose solutions treated with DNS solution in an equivalent manner.

The observed enzyme reaction absorbance is corrected for non-specific absorbance by carrying out a reaction in which the DNS solution is added to the mixture before the enzyme solution. One unit of laminarinase activity is defined as the amount of enzyme which produces 1 μmole glucose equivalents.min −1 under the conditions of the assay (50° C. and pH 5.0).

1.7. endo-1,4-β-Xylanase by the DNS Birchwood Xylan Method

An assay for endo-1,4-β-xylanase activity is based on the enzymatic hydrolysis of the xylosidic bonds in birchwood xylan, a β-1,4-xylan. The products of the reaction, β-1,4-xylan oligosaccharides are determined by the resulting increase in reducing value (as xylose).

A solution containing 1 ml of a 1%(w/v) birchwood xylan solution in 0.1M sodium acetate buffer, pH 5.0 (or at different pH values); 1 ml of appropriately diluted enzyme solution was incubated at 50° C. for 10 minutes. The enzyme reaction is stopped by the addition of 2 ml of a DNS solution (1%(w/v) 3,5-dinitrosalicylic acid, 1.6%(w/v) sodium hydroxide, 30%(w/v) potassium sodium (+)-tartrate in distilled water). The solution is mixed and placed into a boiling water bath, 95° C. minimum, for 5 minutes then cooled to 25° C. 10 ml distilled water is added to the solution and the absorbance is measured at 540 nm using a 2 cm path length glass cell.

The result is converted to μmoles reducing sugar (as xylose) by comparison with a standard curve for 2 ml of 0.00-0.03%(w/v) xylose solutions treated with DNS solution in an equivalent manner.

The observed enzyme reaction absorbance is corrected for non-specific absorbance by carrying out a reaction in which the DNS solution is added to the mixture before the enzyme solution. One unit of endo-1,4-β-xylanase activity is defined as the amount of enzyme which produces 1 μmole xylose equivalents.min −1 under the conditions of the assay (50° C. and pH 5.0 or other pH).

1.8. endo-1,4-β-Xylanase by the DNS Wheat Arabinoxylan Method

An assay for endo-1,4-β-xylanase activity is based on the enzymatic hydrolysis of the xylosidic bonds in wheat arabinoxylan, an arabinose substituted β-1,4-xylan. The products of the reaction, arabino-β-1,4-xylan oligosaccharides are determined by the resulting increase in reducing value (as xylose).

A solution containing 1 ml of a 1%(w/v) wheat arabinoxylan solution in 0.1M sodium acetate buffer, pH 5.0 (or at different pH values); 1 ml of appropriately diluted enzyme solution was incubated at 50° C. for 10 minutes. The enzyme reaction is stopped by the addition of 2 ml of a DNS solution (1%(w/v) 3,5-dinitrosalicylic acid, 1.6%(w/v) sodium hydroxide, 30%(w/v) potassium sodium (+)-tartrate in distilled water). The solution is mixed and placed into a boiling water bath, 95° C. minimum, for 5 minutes then cooled to 25° C. 10 ml distilled water is added to the solution and the absorbance is measured at 540 nm using a 2 cm path length glass cell.

The result is converted to μmoles reducing sugar (as xylose) by comparison with a standard curve for 2 ml of 0.00-0.03%(w/v) xylose solutions treated with DNS solution in an equivalent manner.

The observed enzyme reaction absorbance is corrected for non-specific absorbance by carrying out a reaction in which the DNS solution is added to the mixture before the enzyme solution. One unit of endo-1,4-β-xylanase activity is defined as the amount of enzyme which produces 1 μmole xylose equivalents.min −1 under the conditions of the assay (50° C. and pH 5.0 or other pH).

1.9. endo-1,4-β-Xylanase by the Viscometric Wheat Araboxylan Method

An assay for endo-1,4-β-xylanase activity is based on the enzymatic hydrolysis of a standard wheat arabinoxylan solution, the activity being determined by the reduction in relative viscosity against time.

A solution containing 1 ml of a 1%(w/v) wheat arabinoxylan solution in 0.1M sodium acetate buffer, pH 5.5 (or at different pH values); 3 ml distilled water and 1 ml of appropriately diluted enzyme solution is injected into a Haake microviscometer (using a gold ball calibrated to 0.1-2.0 mPa·s) and the ball drop time (T test ) measured (in ms over the defined drop length) every 30 seconds over a period of 15-20 minutes at 30° C. Mean ball drop times are measured for water (5 ml distilled water) and substrate (1 ml of a 1%(w/v) wheat arabinoxylan solution in 0.1M sodium acetate buffer, pH 5.5 and 4 ml distilled water) as T water and T substrate respectively. Controls are measured in an equivalent manner. The relative fluidity (F r ) is calculated for each value of T test as follows: F r = T substrate - T water T test - T water

The slope of a plot of F r against time (the elapsed time at which each measurement of T test is made) is calculated in relative fluidity change per minute (ΔFr.min −1 ) and is proportional to the enzyme concentration. One unit of endo-1,4-β-xylanase activity is defined as the amount of enzyme which will hydrolyse the substrate, reducing the viscosity of the solution, to give a change in relative fluidity of 1 (dimensionless unit).min −1 under the conditions of the assay (30° C. and pH 5.5 or other pH).

1.10 β-Xylosidase by the p-Nitrophenyl β-D-Xylopyranoside Method

The assay of β-xylosidase is based on the enzymatic hydrolysis of p-nitrophenyl β-D-xylopyranoside. A product of the reaction, p-nitrophenol is determined colorimetrically.

A solution containing 1 ml of a 0.1%(w/v) p-nitrophenyl β-D-xylopyranoside in distilled water; 1 ml distilled water; 1 ml 0.2M sodium acetate buffer, pH 5.0; 1 ml of appropriately diluted enzyme solution was incubated at 50° C. for 30 minutes. The enzyme reaction is stopped by the addition of 4 ml of 0.4M glycine solution. The solution is mixed and cooled to 20° C. The absorbance is measured at 400 nm using a 1 cm path length glass cell.

The result is converted to μmoles p-nitrophenol by comparison with the molar extenction coefficient of p-nitrophenol under these conditions.

The observed enzyme reaction absorbance is corrected for non-specific absorbance by carrying out a reaction in which the glycine solution is added to the mixture before the enzyme solution. One unit of xylosidase activity is defined as the amount of enzyme which produces 1 μmole p-nitrophenol from p-nitrophenyl β-D-xylopyranoside per minute under the conditions of the assay (50° C. and pH 5.0).

1.11 α-N-Arabinofuranosidase by the p-Nitrophenyl α-L-Arabinofuranoside Method

The assay of α-N-arabinofuranosidase (arabinofuranosidase) is based on the enzymatic hydrolysis of p-nitrophenyl α-L-arabinofuranoside. A product of the reaction, p-nitrophenol is determined colorimetrically.

A solution containing 1 ml of a 0.1%(w/v) p-nitrophenyl α-L-arabinofuranoside in distilled water; 1 ml distilled water; 1 ml 0.2M sodium acetate buffer, pH 5.0; 1 ml of appropriately diluted enzyme solution was incubated at 50° C. for 30 minutes. The enzyme reaction is stopped by the addition of 4 ml of 0.4M glycine solution. The solution is mixed and cooled to 20° C. The absorbance is measured at 400 nm using a 1 cm path length glass cell.

The result is converted to μmoles p-nitrophenol by comparison with the molar extenction coefficient of p-nitrophenol under these conditions.

The observed enzyme reaction absorbance is corrected for non-specific absorbance by carrying out a reaction in which the glycine solution is added to the mixture before the enzyme solution. One unit of arabinofuranosidase activity is defined as the amount of enzyme which produces 1 μmole p-nitrophenol from p-nitrophenyl α-L-arabinofuranoside per minute under the conditions of the assay (50° C. and pH 5.0).

1.12 Feruloyl Esterase by the FAXX Method

An assay of feruloyl esterase (ferulic acid esterase) is based on the enzymatic hydrolysis of O-[5-O-(trans-feruloyl)-α-L-arabinofuranosyl]-(1→3)-O-β-D-xylopyranosyl-(1→4)-D-xyfopyranose (FAXX). FAXX is prepared from enzyme-hydrolysed wheat bran, purified and characterised by NMR. FAXX hydrolysis is measured spectophotometrically.

The enzyme reaction is followed at 325 nm, using a 1 cm path length cell, in solution containing 0.050 mM FAXX in 0.1M MOPS buffer, pH 6.0 at 37° C.

One unit of feruloyl esterase activity on FAXX is defined as the amount of enzyme which converts 1 μmole substrate to product per minute under the conditions of the assay (37° C. and pH 6.0).

1.13 Feruloyl Esterase by the Ara 2 F Method

An assay of feruloyl esterase (ferulic acid esterase) is based on the enzymatic hydrolysis of Ara 2 F (ferulic acid linked 1,2 to arabinose). Ara 2 F is prepared from enzyme-hydrolysed sugar beet pulp, purified and characterised by NMR. Ara 2 F hydrolysis is measured spectophotometrically.

The enzyme reaction is followed at 325 nm, using a 1 cm path length cell, in solution containing 0.050 mM Ara 2 F in 0.1M MOPS buffer, pH 6.0 at 37° C.

One unit of feruloyl esterase activity on Ara 2 F is defined as the amount of enzyme which converts 1 μmole substrate to product per minute under the conditions of the assay (37° C. and pH 6.0).

1.14 Feruloyl Esterase by the Hydrolysis of Methyl Esters: Methyl Ferulic Acid (MFA); Methyl Caffeic Acid (MCA); Methyl Sinapic Acid (MSA); Methyl p-Coumaric Acid (MPCA) Methods

An assay of feruloyl esterase (ferulic acid esterase) is based on the enzymatic hydrolysis of methyl esters of ferulic acid (MFA), caffeic acid (MCA), sinapic acid (MSA) and p-coumaric acid (MpCA). Methyl ester hydrolysis is measured in 0.1M MOPS buffer, pH 6.0 at 37° C. Assays are based on two different techniques.

In the spectrophotometric method the methyl ester substrate concentration is 0.10 mM and ester hydrolysis is followed at 325 nm using a 1 cm path length cell. In this method the initial substrate concentration is limited.

In the HPLC method, the methyl ester substrate concentration is 1.0 mM and ester hydrolysis is followed at by measuring the release of free acid by HPLC after 10-30 minute intervals. In this method there is no limit over substrate concentration and activities measured are considerably higher than those for the spectrophotometric method.

One unit of feruloyl esterase activity is defined as the amount of enzyme which converts 1 μmole substrate to product per minute under the conditions of the assay (37° C. and pH 6.0).

1.15 Protein Concentration by Modified Bradford Coomassie Blue-binding Protein Assay

The assay of protein concentration is based on the modified Bradford Coomassie blue-binding protein assay using Brilliant Blue G (Coomassie blue) measured in a spectrophotometer at 595 nm using 1 cm light path glass cuvettes. The method (Sigma B 6916) is standardised using Bovine Serum Albumin (Sigma P 0914).

1.16 Isoelectric Point by Isoelectric Focusing

Isoelectric points of proteins are determined by standard methods using pre-cast vertical 5% polyacrylamide gels such as gels from NOVEX® for pH 3-10 (pl performance range 3.5-8.5) or pH 3-7 (pl performance range 3.0-6.5) in the NOVEX® XCell II™ Mini-Cell. NOVEX® cathode, anode and IEF sample buffers for pH 3-10 or pH 3-7 are used. NOVEX® standard protocol for isoelectric focusing, fixing, staining with Coomassie R-250 Blue Stain, and destaining are used.

1.17 SDS-PAGE (Sodium Dodecylsulphate Polyacrylamide Gel Elctrophoresis)

Analytical separation and molecular weight determination of proteins are carried out standard SDS-PAGE methods. Pre-cast NOVEX® NuPAGE™ gels (NuPAGE™ Bis-Tris gels or NuPAGE™ Tris-Acetate gels with NOVEX® recommended running buffers) are used in the NOVEX® XCell II™ Mini-Cell. NOVEX® sample preparation and running buffers, and molecular weight standards are used. NOVEX® standard protocol for SDS-PAGE, fixing, staining with Coomassie R-250 Blue Stain, and destaining are used.

2. Results on the Enzymes Mixture

2.1. Optimal pH

2.1.1. Activity endo-1,3(4)-β-Glucanase

The assay of endo-1,3(4)-β-glucanase from Penicillium funiculosum was carried out under standard conditions at 50° C. using the DNS barley β-glucan method. Enzyme activity was measured between pH 3.0 and pH 7.0. The optimal pH for enzyme activity is pH 4.0-5.0.

2.1.2. Activity endo-1,4-β-Xylanase

The assay of endo-1,4-β-xylanase from Penicillium funiculosum was carried out under standard conditions at 50° C. using the DNS birchwood xylan method.

2.2. Optimal Temperature

2.2.1. Activity endo-1,3( 4 )-β-Glucanase

The assay of endo-1,3(4)-β-glucanase from Penicillium funiculosum was carried out under standard conditions at pH 5.0 (the optimal pH for this enzyme) using the DNS barley β-glucan method. Enzyme activity was measured between 30 and 70° C. The optimal temperature lies between 50 and 60° C. with the greatest activity being measured at 60° C. The results in detail, in the form of a table vs. temperature are given.

2.2.2. Activity endo-1,4-β-Xylanase

The assay of endo-1,4-β-xylanase from Penicillium funiculosum was carried out under standard conditions at pH 5.5 and pH 3.5 using the DNS birchwood xylan method. Enzyme activity was measured between 30 and 70° C. The optimal temperature lies between 50 and 60° C. with the greatest activity being measured at 50° C. for pH 5.5 and at 60° C. for pH 3.5. The results in detail, in the form of a table vs. temperature are given.

Enzymes produced by Penicillium funiculosum have high levels of cellulase, endo-1,3(4)-β-glucanase and other glycanolytic activities. In addition, they are also characterised by high levels of endo-1,4-β-xylanase and xylanase accessory enzyme activities. The broad spectrum of hemicellulolytic enzymes is a characteristic of enzyme preparations from this micro-organism.

Each activity measured can reported as a ratio to a major activity for that preparation. An example of obtained results is shown in table A. These ratios may change in preparations from different fermentation batches.

3—Properties of Components in the Enzyme Mixture

3.1. Purification Methods

Hydrophobic Interaction Chromatography

The preparation obtained after filtration and concentration of the fermentation medium, to 112.6 mg/ml protein concentration, was diluted 1/1 with Hydrophobic Interaction Chromatogaphy (HIC) buffer (50 mM phosphate buffer, pH 7.0/1.7 M (NH 4 ) 2 SO 4 /0.04% sodium azide), exchanged into HIC buffer (PD-10 columns; Phamacia). Portions (10 ml) were applied to a column (10×5 cm diameter, 200 ml) of PhenylSepharose™ high performance HIC gel (Pharmacia) and separated using a gradient of reducing ammonium sulphate (NH 4 ) 2 SO 4 )concentration (1.7-0.0 M) at 10 ml/min. Fractions (10 ml) were collected and assayed for xylanase activity.

HIC gave two major peaks of xylanase activity. The first, named A, eluted from the column when the NH 4 ) 2 SO 4 concentration was reduced to about 0.6 M, while the second, named B, eluted at about 0.25 M NH 4 ) 2 SO 4 concentration. Fractions comprising peaks A and B from each injection were pooled separately. In total fraction A corresponded to 2.8% of the total xylanase activity whilst fraction B corresponded to 97.2% of the total xylanase activity. The yield was 77%.

Ion-Exchange Chromatography

Pooled fractions for peak A and B from HIC were precipitated by increasing the NH 4 ) 2 SO 4 concentration to 100% saturation followed by centrifugation (10 000×g for 30 minutes). Pellets were redissolved in 20 mM Tris-HCl buffer, pH 8.0/0.04% sodium azide and desalted to the same buffer using PD-10 columns. Samples (5 ml) were applied to a MonoQ™ HR 10/10 anion-exchange column (Pharmacia) previously equilibrated with 20 mM Tris-HCl buffer, pH 8.0/0.04% sodium azide and eluted at 2 ml/min with increasing concentration of sodium chloride (NaCl (0-1 M) in the same buffer. Fractions (2 ml) were collected and assayed for xylanase activity.

Peak A:

Separation of peak A by anion-exchange chromatography gave a single peak of xylanase activity which eluted at about 0.3M NaCl. The most active fractions were pooled and analysed by SDS-PAGE (sodium dodecylsulphate polyacrylamide gel electrophoresis). This showed a single major band of molecular weight 48 kDa. Recovery of xylanase activity after IEF (isoelectric focusing) confirmed that this major Coomassie-stained band was a xylanase.

Peak B:

Separation of peak B by anion-exchange chromatography gave two major peaks of xylanase activity, one of which eluted in the void (unbound material; peak B-I) and the other at 0.1 M NaCl (peak B-II). There were also two minor peaks eluting at 0.13 M and 0.19 M NaCl. The active fractions corresponding to each peak were pooled and analysed by SDS-PAGE, but none of the samples were pure.

Gel Filtration Chromatography

Pooled fractions comprising B-I and B-II were freeze dried, redissolved in water, and desalted (using PD-10 columns). Samples (0.2 ml) were applied to a Superdex™ 75 HR column (Pharmacia) and eluted at 0.4 ml/minute with 20 mM Bis-Tris buffer, pH 6.0/0.2 M NaCl/0.04% sodium azide. Fractions (0.4 ml) were collected and assayed for xylanase activity.

3.2. Properties of Xylanases

3.2.1 Isoelectric Point by Isoelectric Focusing

Isoelectric points of proteins are determined by standard methods using pre-cast vertical 5% polyacrylamide gets from NOVEX® for pH 3-10 and pH 3-7. NOVEX® cathode, anode and IEF sample buffers, and standard protocol for isoelectric focusing, fixing, staining with Coomassie R-250 Blue Stain, and destaining are used.

For xylanase A, a sample following MonoQ was used. For xylanases B-I and B-II, a sample following HIC, xylanase B, was used. For each of A and B, a small sample (10 μl) was loaded into a single well and a large sample (50 μl) was loaded into a triple well. After focussing the samples, the gel was cut in half such that one half contained the two small samples (A+B) and the molecular weight markers (this half was stained with Coomassie), while the other half contained the two large samples. The gel half containing the large samples was cut to separate the two samples lanes, and subsequently each lane was fractionated into 2 mm pieces. Each 2 mm piece was soaked separately overnight in 100 mM MOPS buffer, pH 6.0/0.04% sodium azide. Fractions were assayed for xylanase activity.

For xylanase sample A, the stained IEF gel showed a single major band of pl 3.55 marker and a few minor contaminating bands. Xylanase activity was found only in the fraction corresponding to this band, confirming the xylanase major band.

For xylanase sample B, the stained IEF gel indicates several bands over a range of pl values. Xylanase activity occured in two separated fractions of the unstained gel, and corresponding to proteins of pl 4.2 and 4.8.

3.2.2 Molecular Weight by SDS-PAGE

To confirm the molecular weights of xylanases in peak B from HIC, the fractions with xylanase activity eluted from the IEF gel were deslated, freeze-dried, and separated by SDS-PAGE. Denaturing PAGE was performed using 10% Tris-glycine gels (NOVEX®) with dithiothreitol (DTT 50 mM) included in the sample buffer as a reducing agent.

The stained gel indicated that both xylanases were pure, with molecular weights of 36 kDa and 15 kDa for xylanase B-I and xylanase B-II respectively.

All three purified xylanases were subjected to SDS-PGE analysis, xylanase A fraction after anion-exchange chromatography, xyalanase B-I and B-II fractions after gel filtration chromatography. Xylanase A gave a single band of molecular weight 48 kDa. Xylanase B-I gave one major and four minor bands after Coomassie staining. The major band was confirmed as the xylanase since it was of molecular weight 36 kDa. The purity is estimated at 90%. Xylanase B-II gave a major band of molecular weight 15 kDa and 2-3 minor bands. This xylanase is approximately 95% pure.

3.2.3 Enzyme Activity

The tests for enzyme activity measurement are described previously.

3.2.3.1 Analysis of Xylanase A

Xylanase Activity on Birchwood Xylan vs pH

3.2.3.2 Analysis of Xylanase B-I

Xylanase Activity on Birchwood Xylan vs pH

3.2.3.3 Analysis of Xylanase BII

Xylanase Activity on Birchwood Xylan vs pH

3.2.4 Sequences

One embodiment of the present invention is related to the amino acid and the nucleic acid sequences of the above described proteins or their variants.

For this purpose, the sequences for xylanases were identified from amino acid sequences of purified proteins (xylanase A, xylanase B-I and xylanase B-II) and from comparisons of amino acid and nucleotide sequences of known fungal xylanases.

It is understood for the invention that variants refers to any polypeptide or any protein analog, protein fragment, derived or mutated protein from the native protein or polypeptide and having the same biological fuctions as the said native protein or polypeptide. Different variants may be exist at natural state. Those variants may be for example allelic variations characterized by differencies into the sequence of genes encoding for the said protein or may result from differential splicing or from post-traductional modifications. Variants are obtainable by substitution, deletion, addition and/or modification of one or more amino acids. The all modifications are well known and can be performed by any method known of one skilled in the art.

Variants are molecules having for example more affinity for their substrate or having new biological properties.

Another object of the present invention is also the use of the sequences for the expression of the disclosed proteins or polypeptides in host cells of uni- or pluricellular organisms. For this purpose, the said sequences may be comprised into the genome of a vector. The said vector may be a plasmid, a phage or a virus. In hence, another embodiment of the invention is a host isolated cell from uni- or pluricellular organism, transfected or infected by a vector as above described. In a preferred embodiment the host cell is a bacteria.

The use of said vectors comprising the nucleic acid sequence of the disclosed proteins for the expression of said protein in any host cell is another embodiment of the present invention.

3.2.4.1 Sequences of Xylanase C

The production of probes was based on comparisons of amino acid and nucleotide sequences of known fungal xylanases. Conserved regions were identified and used to design PCR primers, whose products would be used to screen a genomic library of Penicillium funiculosum.

Two pairs of degenerate primers were made. The first pair were designed to amplify a 200 pb (approximate) product from a xylanase type B (or type 2) gene. The second pair were designed to amplify a 250 bp product from a xylanase type A (or type 1) gene.

A 258 bp band was produced with primers 3 and 4. After cloning into pGEMT and sequencing this was found to have significant sequence similarity to fungal xylanase type A/1. The plasmid containing the cloned product has been named pPFXYLA.

The complete sequence of xylanase C is shown in FIG. 1 and in SEQ ID NO 1.

3.2.4.2 sequences of Xylanase BI

The internal amino acid sequence, together with sequence alignments of other fungal cellobiohydrolases were used to design degenerate PCR primers (SEQ ID NO 3 and NO 4). A 290 bp product (SEQ ID NO 5) was amplified and cloned into pGEMT(Promega) to create pGEMTCB2 and sequenced. As shown in FIG. 2, the primer sequences are underlined. This PCR product is currently being used as a probe to screen a Penicillium funiculosum IMI134756 genomic library.

3.2.4.3 Sequences of Xylanase BII

The all sequence of the xylanase BII gene includes 1.3 kb of 5′ untranslated and upstream region and 0.85 kb of 3′ untranslated, a 54 bp intron and 669 bp encoding a 223 amino acid protein

Reverse transcription-PCR (RT-PCR) was used to prove the existence of the 54 bp intron. Total RNA was isolated from mycelia of Penicillium funiculosum IMI-134756 cultures, harvested after 4 days growth on 1% (w/v) oat spelt xylan. Primers were designed to amplify up to 195 bp fragment from messenger RNA (249 bp from genomic DNA) and to 433 bp (487 bp with genomic DNA).

Sequencing of 3 kb at the 3′ end of the plasmid (pPFXYNC2, revealed a gene (designated per A) that contains two putative introns and encodes a polypeptide of aproximatively 570 amino acids. The polypeptide showed significant sequence similarity to fungal amino acid permeases.

3.2.4.4 Sequence of Xylanase A

The internal sequence of Xylanase A was obtained and is represented by the following amino acid sequence:

AEAINYNQDY (SEQ ID NO 10)

3.3 Properties of Feruloyl Esterases

3.3.1 Purification

It is carried out following the same process as for xylanases.

The enzymes mixture contains at least two distinct feruloyl esterases. One of these (FaeB) has a molecular weight of 38,945-41,051 Da by mass spectrometry (35.450 Da from the primary amino acid sequence and 37 kDa by SDS-PAGE). FaeB has a pl of 4.2, it is a type B feruloyl esterase and is specific for MpCA and Ara 2 F substrates (activity against MpCA, MCA, MFA and Ara 2 F; but not against MSA and FAXX).

The other feruloyl esterase (FaeA) has a molecular weight of 29 kDa (by SDS-PAGE). FaeA has a pl of 4.65, it is a type A feruloyl esterase and is specific for FAXX and MSA substrates (activity against MSA, MCA, MFA and FAXX but not MpCA Ara 2 F).

3.3.2. Isoelectric Point by Isoelectric Focusing

Isoelectric points of proteins are determined by the standard methods. The enzymes mixture was applied as a wide strip (about 20 mm) to an IEF gel and electrophoresed at reduced temperature (5° C.). After focusing and band sharpening, the gel was cut down the middle of the sample lane. One half of the sample lane and the IEF standards were fixed, stained and destained using the standard protocol. The other half lane was cut into 2 mm wide sections and each section soaked overnight in 1 ml of 100 mM MOPS buffer, pH 6.0. Feruloly esterase activity was determined for each section of the gel using MFA, MPCA and MSA as substrates.

The stained IEF gel indicates the presence of very many proteins in cellulase with pl's ranging from very acidic (pl 2.4) to about pl 7. Most of the proteins are acidic (pl range 2.4-5). Two peaks of feruloyl esterase activity were detected in fractions cut from the gel. One, corresponding to FaeB, had a pl of 4.2 and activity only against MFA and MpCA (not MSA). The other, corresponding to FaeA, had a pl of 4.65 and activity against all three substrates tested .

3.3.3 Molecular Weight by SDS-PAGE

Molecular weights were analysed by SDS-PAGE using 10% Tris-Glycine gels. SDS-PAGE gels were run, fixed, stained with Coomassie Blue Stain and destained using the standard protocol.

The enzymes mixture contains at least two distinct feruloyl esterases. One corresponding to FaeB (pl 4.2) has a molecular weight of 37 kDa. The other, corresponding to FaeA (pl 4.65) has a molecular weight of 29 kDa.

The molecular weight of FaeB is estimated at 34,450 Da from the primary amino acid sequence, and at 38,945-41,051 Da by mass spectrometry.

3.3.4 Feruloyl Estease Activity

Assays for feruloyl esterase activity performed on the enzymes mixture using the spectrophotometric method

The enzymes mixture contains activity against all the substrates tested. With the methyl esters, activity is highest against MpCA and lowest against MSA. The activities against Ara 2 F and FAXX are higher than against the methyl esters which is indicative that the esterase activities are due to true feruloyl esterases and not general esterases or side activities of other cell wall-degrading esterases (e.g. acetyl xylan esterase, pectin esterase).

3.3.5 Sequences

3.3.5.1 Sequence of FEA-A

According to trypsin digests of the purified protein internal amino acid sequences were obtained as shown as following:

Sequence 1 (SEQ ID NO 11)

Sequence 2 (SEQ ID NO 12)

Sequence 3 (SEQ ID NO 13)

Sequence 4 (SEQ ID NO 14)

Several degenate PCR primers were designed from amino acid sequences obtained from the purified protein. Many products were cloned into pGEMT(Promega) and sequenced.

A plasmid named pGEMTD19 (180 bp) (FIG. 3) was found by PCR to contain sequence that was recognisable as peptide sequence 4 shown above. As shown in FIG. 3, the primer sequences have been double underlined previously known sequence, singly underlined.

The nucleic acid and amino acid sequences of FAE-A are disclosed in SEQ ID NO 7.

3.3.5.2 Sequence of FEA-B

Primers designed from peptide sequence of FAE-B were used to amplify up a probe, that was subsequently used to screen a Penicillium funiculosum genomic library. A 2291 bp clone was isolated and has been sequenced (SEQ ID ). The gene encoding for a 304 amino acid polypeptide and has one putative intron. The predicted amino acid sequence is shown in FIG. 4 SEQ ID NO. 9 wherein the mature protein (mature protein length=338) is in Bold. This protein comprises two distinct domains separated by a highly glycosylated linker. As shown in FIG. 4, the catalytic domain is in Bold, whereas the binding domain is in Bold double underlined and the linker is represented in dotted Bold line.

The protein is also featured by a putative active site motif (serine=nucleophile) as shown underlined in FIG. 4 with the following Putative catalytic triads:

(1) S1361/D174/H216

(2) S136/D2201/H276.

The FAE-B protein comprises also a secretion sequence (353) and 10 cysteines.

3.4 Properties of Glucanases

The enzymes mixture was subjected to 2D gel electrophoresis. IEF was carried out using pre-cast vertical 5% polyacrylamide gels from NOVEX® for pH 3-7 (pl performance range 3.0-6.5) in the NOVEX® XCell II™ Mini-Cell. NOVEX® cathode, anode and IEF sample buffers for pH 3-7 and the NOVEX® standard protocol for isoelectric focusing are used. One lane was cut off and electrophoresed in the second dimension using a 10% Laemmli SDS-PAGE gel. A second lane was separated from the gel, cut into 35 fractions, the gel strips soaked in buffer, and fractions assayed for enzyme activity. The third lane was left on the gel, fixed, stained with Coomassie R-250 Blue Stain and destained using the NOVEX® standard protocol.

Significant endo-1,3(4)-β-glucanase (DNS barley β-glucan method) and cellulase (DNS CMC method) activities were found in fractions corresponding to proteins with pl 4.2, M.W. 36 kDa and pl 5.4, M.W. 27 kDa. To eliminate Xylanase B-I as being in one of the fractions, the fractions were tested for activity using the DNS birchwood xylan method. No xylanase activity was detected in the fractions corresponding to β-glucanase or cellulase activities.

FIG. 1 : Penicillium funiculosum xylanase C protein amino acid sequence (SEQ ID NO:2).

FIG. 2 : Nucleotide and amino acid sequences of xylanase BI (XynBI) PCR product (SEQ ID NO:6).

FIG. 3 : Nucleotide and amino acid sequences of Feruloyl estease A (faeA) PCR product ((SEQ ID NO:9).

FIG. 4 : Penicillium funiculosum Feruloyl estease B (faeB) protein (FAE-V or FAE-I) amino acid sequence (SEQ ID NO:9).

<160> NUMBER OF SEQ ID NOS: 14

<210> SEQ ID NO 1

<211> LENGTH: 2898

<212> TYPE: DNA

<213> ORGANISM: Pennicilium funiculosum

<220> FEATURE:

<221> NAME/KEY: RBS

<222> LOCATION: (570)...(576)

<221> NAME/KEY: RBS

<222> LOCATION: (724)...(730)

<221> NAME/KEY: CDS

<222> LOCATION: (1317)...(1589)

<221> NAME/KEY: intron

<222> LOCATION: (1590)...(1642)

<221> NAME/KEY: CDS

<222> LOCATION: (1644)...(2042)

<400> SEQUENCE: 1

catccataca ggtttctcct gtatggaatt tgtaattact tatcactaat tgtcacaatt 60

ctcaggtttt ctgaacatgc tttcttttcc gtccggcatt gttaagcttg atcatagcct 120

gtgtttctta aaggcggttc cggcggtaat ctcagtacta cgtcctacgt atgtaggtag 180

ttatatctat cctacccttt gactgtatgt tccctacttt gcagtactta gacaacgatt 240

ttctaggcag gttcctagaa tgctctcgtt ttcgtttact atcattgttt ttatgcgcgt 300

aatgtggagc tattgacgtg tatgtcactt tacatgccta taactaactt aatctaaacg 360

tccattaggg gttcaacata tgtattcccc gcctccgcat gttcactcca atgtggaaat 420

tatctccacc gtgcccaaca cttccctgat aatgagcaat gtcgatgagt atttcaatgc 480

aagaatctga gccgaatcaa gttttacaag gttagtcatt ggttgatcct gttgtcgaaa 540

gctagtgcag gttatgctcc gccgatgaag gctaaatata taacgggaga taaccctaac 600

tacctaggta tcaacccaac gcgacattga aaaatcttca aaaacatcct tactagcgga 660

tatcaatagc gagaactgcg attaaacatt gtcgatcttc ggcactttag cagcatgact 720

tcaggctaaa cacattgatg caaaccgctt tctcatttta cctgaaactt gggatgacga 780

atcctccaac cagttgtcga aacgaacagg ctccacatct cgcaacaaat caaagcaacg 840

agtctaatat atgtattcgt catctagtaa agatcaaacg atttcgtttc agcggtggta 900

cataccccaa gcactccgac aagtcggtta tccagacctg tcgatcttga atatcgccca 960

tacagagctc ttctttaaag accacgaccg aacatcaagg agaatttagc cagaaatttt 1020

ccggtatgcg agaatgatcc cagatttgca acagaaaagt gctctattgc gaatatcctc 1080

aagcatattc cccgaaactc cgcgatggag accttgaggg gggtcatatg gatgcgaggc 1140

atgatgaaac aaacaacccg ttactgttaa atgagaaatg agaatgcggg tatataaaca 1200

tgcggattgt cctcgccaga aaatcccccc cccccccccc caccaaaaca aaagacgtac 1260

cactcatttt ctggatatcc acacttggcg agaatcaaag aaccatttca atcaag atg 1319

Met

1

aag ctc ttc cta gct gca att gtc ctt tgc gca act gcc gcg aca gcc 1367

Lys Leu Phe Leu Ala Ala Ile Val Leu Cys Ala Thr Ala Ala Thr Ala

5 10 15

ttc cca tca gaa ctt gct caa cgc gct gcg gga gac ctt agc aag cgt 1415

Phe Pro Ser Glu Leu Ala Gln Arg Ala Ala Gly Asp Leu Ser Lys Arg

20 25 30

caa tca atc acg acc agc cag act ggg acg aac aac ggc tac tac tac 1463

Gln Ser Ile Thr Thr Ser Gln Thr Gly Thr Asn Asn Gly Tyr Tyr Tyr

35 40 45

tcg ttc tgg acc aac ggc gga gga gaa gtc act tac aca aat ggc gac 1511

Ser Phe Trp Thr Asn Gly Gly Gly Glu Val Thr Tyr Thr Asn Gly Asp

50 55 60 65

aat ggc gag tac agc gtg aca tgg gtc gat tgt ggt gac ttt aca tct 1559

Asn Gly Glu Tyr Ser Val Thr Trp Val Asp Cys Gly Asp Phe Thr Ser

70 75 80

ggc aag ggc tgg aat cca gcc aat gca cag taagtttccc tctttccttc 1609

Gly Lys Gly Trp Asn Pro Ala Asn Ala Gln

85 90

taagcttata ttgtacgtac tcacaatttg cagg act gtc acg tac tca gga gaa 1664

Thr Val Thr Tyr Ser Gly Glu

95

ttt aac ccc tct gga aac gct tat ttg gct gtc tac ggg tgg aca aca 1712

Phe Asn Pro Ser Gly Asn Ala Tyr Leu Ala Val Tyr Gly Trp Thr Thr

100 105 110

gat cct ctt gtc gaa tac tac atc ctg gag tcc tac ggc acc tat aac 1760

Asp Pro Leu Val Glu Tyr Tyr Ile Leu Glu Ser Tyr Gly Thr Tyr Asn

115 120 125 130

cca tca tct gga ctt act tca ctt ggc cag gtc act agc gat ggt ggc 1808

Pro Ser Ser Gly Leu Thr Ser Leu Gly Gln Val Thr Ser Asp Gly Gly

135 140 145

acc tac gat atc tac tca acc cag cgt gtc aac cag cct tcc att gag 1856

Thr Tyr Asp Ile Tyr Ser Thr Gln Arg Val Asn Gln Pro Ser Ile Glu

150 155 160

gga act tcc acc ttc aac cag tac tgg tca gtt cgc acc gag aag cga 1904

Gly Thr Ser Thr Phe Asn Gln Tyr Trp Ser Val Arg Thr Glu Lys Arg

165 170 175

gtc ggc gga act gtc acc acg gcc aac cac ttt gca gca tgg aag gca 1952

Val Gly Gly Thr Val Thr Thr Ala Asn His Phe Ala Ala Trp Lys Ala

180 185 190

ctt gga ctt gaa atg ggc act tat aac tat atg att gtg tcc acc gaa 2000

Leu Gly Leu Glu Met Gly Thr Tyr Asn Tyr Met Ile Val Ser Thr Glu

195 200 205 210

ggc tac gag agc agt ggc tct agt acc atc aca gtg tcc tag 2042

Gly Tyr Glu Ser Ser Gly Ser Ser Thr Ile Thr Val Ser *

215 220

acatgtctca atgacgcttg ttacacagct gtccctttat tgacacttat aaatgactta 2102

tggaagggag tcggcaaaat ttttatgttc gaagtttcat atgtctattg tggaaatcgg 2162

cccatatttt cagggctagt cactctttga ttgcatctta agttacttga tcaaattaag 2222

ccctaacacc aagatctgga atgcgagcaa tatcaagtat ttattcattt attttaaacc 2282

ccggagtggg ctgtctttga tagtatagta atgatgcaca tttgttgtgg cagccttacc 2342

tgtttttcca ttggcattcg agatatctac cgacatgttc cttcagcaag cagtatttat 2402

cgcgtctcga tcaagcatcg acggcctttt ggggaaacca agaaaaatat tttggcctcc 2462

atatctctgt cgcacattcc ctccttctct gaaacctttg cttttgggaa cgttcgaaaa 2522

aacagagcgg ttgcaagcag tagctccatc caggcaagat gcataccgat gcatactagt 2582

gagtaggcca gttagcgaat tgtttgttct cagtgccgat gatgaaatta tgcaattaaa 2642

gacttactgc gagacccgcc accaaagggc atgaaaacat gcttcatctc ttttgtggga 2702

ttctcccatc tgcttggatc aaagctatat cccggacatc aatagttagc gatattgaat 2762

cgaacatctg ccatgccttg taggcgggaa agtgacaccg aataggctat aggaaccact 2822

cacgcatgag gattgggaaa cacatcaggg tcgcgatgta aagtatatgc ctgagtagat 2882

actgtgacac ctccag 2898

<210> SEQ ID NO 2

<211> LENGTH: 223

<212> TYPE: PRT

<213> ORGANISM: Penicillium funiculosum

<400> SEQUENCE: 2

Met Lys Leu Phe Leu Ala Ala Ile Val Leu Cys Ala Thr Ala Ala Thr

1 5 10 15

Ala Phe Pro Ser Glu Leu Ala Gln Arg Ala Ala Gly Asp Leu Ser Lys

20 25 30

Arg Gln Ser Ile Thr Thr Ser Gln Thr Gly Thr Asn Asn Gly Tyr Tyr

35 40 45

Tyr Ser Phe Trp Thr Asn Gly Gly Gly Glu Val Thr Tyr Thr Asn Gly

50 55 60

Asp Asn Gly Glu Tyr Ser Val Thr Trp Val Asp Cys Gly Asp Phe Thr

65 70 75 80

Ser Gly Lys Gly Trp Asn Pro Ala Asn Ala Gln Thr Val Thr Tyr Ser

85 90 95

Gly Glu Phe Asn Pro Ser Gly Asn Ala Tyr Leu Ala Val Tyr Gly Trp

100 105 110

Thr Thr Asp Pro Leu Val Glu Tyr Tyr Ile Leu Glu Ser Tyr Gly Thr

115 120 125

Tyr Asn Pro Ser Ser Gly Leu Thr Ser Leu Gly Gln Val Thr Ser Asp

130 135 140

Gly Gly Thr Tyr Asp Ile Tyr Ser Thr Gln Arg Val Asn Gln Pro Ser

145 150 155 160

Ile Glu Gly Thr Ser Thr Phe Asn Gln Tyr Trp Ser Val Arg Thr Glu

165 170 175

Lys Arg Val Gly Gly Thr Val Thr Thr Ala Asn His Phe Ala Ala Trp

180 185 190

Lys Ala Leu Gly Leu Glu Met Gly Thr Tyr Asn Tyr Met Ile Val Ser

195 200 205

Thr Glu Gly Tyr Glu Ser Ser Gly Ser Ser Thr Ile Thr Val Ser

210 215 220

<210> SEQ ID NO 3

<211> LENGTH: 8

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Basis for the design of PCR primer sequences

based on internal sequence

<400> SEQUENCE: 3

Tyr Ala Gly Thr Cys Asp Pro Asp

1 5

<210> SEQ ID NO 4

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Penicillium funiculosum

<400> SEQUENCE: 4

Ala Lys Tyr Gly Thr Gly Tyr Cys Asp

1 5

<210> SEQ ID NO 5

<211> LENGTH: 285

<212> TYPE: DNA

<213> ORGANISM: Pennicium funiculosum

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (1)...(285)

<221> NAME/KEY: primer_bind

<222> LOCATION: (1)...(27)

<221> NAME/KEY: primer_bind

<222> LOCATION: (264)...(285)

<400> SEQUENCE: 5

gcc aag tac ggt acg ggt tat tgt gac tct caa tgc cct cgt gac ttg 48

Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp Leu

1 5 10 15

aag ttc atc gct ggt cag gcc aac gtc gag ggc tgg acg cct tcc acc 96

Lys Phe Ile Ala Gly Gln Ala Asn Val Glu Gly Trp Thr Pro Ser Thr

20 25 30

aac aac tcg aac act gga atc ggc aac cac gga tct tgc tgc gcg gag 144

Asn Asn Ser Asn Thr Gly Ile Gly Asn His Gly Ser Cys Cys Ala Glu

35 40 45

ctt gat atc tgg gaa gca aac agt atc tca gag gcc ttg act cct cac 192

Leu Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu Ala Leu Thr Pro His

50 55 60

cct tgc gat aca ccc ggc cta act gtt tgc act gct gat gac tgt ggt 240

Pro Cys Asp Thr Pro Gly Leu Thr Val Cys Thr Ala Asp Asp Cys Gly

65 70 75 80

ggt acc tac agc tcc aat cgt tat gcc ggt act tgc gac ccc gat 285

Gly Thr Tyr Ser Ser Asn Arg Tyr Ala Gly Thr Cys Asp Pro Asp

85 90 95

<210> SEQ ID NO 6

<211> LENGTH: 95

<212> TYPE: PRT

<213> ORGANISM: Penicillium funiculosum

<400> SEQUENCE: 6

Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp Leu

1 5 10 15

Lys Phe Ile Ala Gly Gln Ala Asn Val Glu Gly Trp Thr Pro Ser Thr

20 25 30

Asn Asn Ser Asn Thr Gly Ile Gly Asn His Gly Ser Cys Cys Ala Glu

35 40 45

Leu Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu Ala Leu Thr Pro His

50 55 60

Pro Cys Asp Thr Pro Gly Leu Thr Val Cys Thr Ala Asp Asp Cys Gly

65 70 75 80

Gly Thr Tyr Ser Ser Asn Arg Tyr Ala Gly Thr Cys Asp Pro Asp

85 90 95

<210> SEQ ID NO 7

<211> LENGTH: 168

<212> TYPE: DNA

<213> ORGANISM: Penicillium funiculosum

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (1)...(168)

<221> NAME/KEY: primer_bind

<222> LOCATION: (1)...(18)

<221> NAME/KEY: primer_bind

<222> LOCATION: (148)...(168)

<400> SEQUENCE: 7

ccg ggt act caa cct gtg gca tac tac gga cag cat ggt gtg agt gat 48

Pro Gly Thr Gln Pro Val Ala Tyr Tyr Gly Gln His Gly Val Ser Asp

1 5 10 15

acg gta ctg cct ttc tca ttg gga gaa ggg att agg gat acg ttt gtc 96

Thr Val Leu Pro Phe Ser Leu Gly Glu Gly Ile Arg Asp Thr Phe Val

20 25 30

aag gat gat cat tgt aca ccg aca aac ccg ccc gcc cct gct gct gga 144

Lys Asp Asp His Cys Thr Pro Thr Asn Pro Pro Ala Pro Ala Ala Gly

35 40 45

agt gga acc cac atc aag tat gta 168

Ser Gly Thr His Ile Lys Tyr Val

50 55

<210> SEQ ID NO 8

<211> LENGTH: 56

<212> TYPE: PRT

<213> ORGANISM: Penicillium funiculosum

<400> SEQUENCE: 8

Pro Gly Thr Gln Pro Val Ala Tyr Tyr Gly Gln His Gly Val Ser Asp

1 5 10 15

Thr Val Leu Pro Phe Ser Leu Gly Glu Gly Ile Arg Asp Thr Phe Val

20 25 30

Lys Asp Asp His Cys Thr Pro Thr Asn Pro Pro Ala Pro Ala Ala Gly

35 40 45

Ser Gly Thr His Ile Lys Tyr Val

50 55

<210> SEQ ID NO 9

<211> LENGTH: 349

<212> TYPE: PRT

<213> ORGANISM: Penicillium funiculosum

<220> FEATURE:

<221> NAME/KEY: SIGNAL

<222> LOCATION: (1)...(18)

<221> NAME/KEY: DOMAIN

<222> LOCATION: (19)...(294)

<223> OTHER INFORMATION: Catalytic

<221> NAME/KEY: ACT_SITE

<222> LOCATION: (134)...(138)

<223> OTHER INFORMATION: Putative

<221> NAME/KEY: CHAIN

<222> LOCATION: (295)...(314)

<221> NAME/KEY: BINDING

<222> LOCATION: (315)...(353)

<400> SEQUENCE: 9

Met Ala Ile Pro Leu Val Leu Val Leu Ala Trp Leu Leu Pro Val Val

-15 -10 -5

Leu Ser Leu Thr Gln Val Asn Asn Phe Gly Asp Asn Pro Gly Ser Leu

1 5 10

Gln Met Tyr Ile Tyr Val Pro Asn Lys Leu Ala Ser Lys Pro Ala Ile

15 20 25 30

Ile Val His Pro Cys Gly Gly Ser Ala Thr Glu Tyr Tyr Gly Met Tyr

35 40 45

Asp Tyr His Ser Pro Ala Asp Gln Tyr Gly Tyr Ile Leu Ile Tyr Pro

50 55 60

Ser Ala Thr Arg Asp Tyr Asn Cys Phe Asp Ala Tyr Ser Ser Ala Ser

65 70 75

Leu Thr His Asn Gly Gly Ser Asp Ser Leu Ser Ile Val Asn Met Val

80 85 90

Lys Tyr Val Ile Ser Thr Tyr Gly Ala Asp Ser Ser Lys Val Tyr Met

95 100 105 110

Thr Gly Ser Ser Ser Gly Ala Ile Met Thr Asn Val Leu Ala Gly Ala

115 120 125

Tyr Pro Asp Val Phe Ala Ala Gly Ser Ala Phe Ser Gly Met Pro Tyr

130 135 140

Ala Cys Leu Tyr Gly Ala Gly Ala Ala Asp Pro Ile Met Ser Asn Gln

145 150 155

Thr Cys Ser Gln Gly Gln Ile Gln His Thr Gly Gln Gln Trp Ala Ala

160 165 170

Tyr Val His Asn Gly Tyr Pro Gly Tyr Thr Gly Gln Tyr Pro Arg Leu

175 180 185 190

Gln Met Trp His Gly Thr Ala Asp Asn Val Ile Ser Tyr Ala Asp Leu

195 200 205

Gly Gln Glu Ile Ser Gln Trp Thr Thr Ile Met Gly Leu Ser Phe Thr

210 215 220

Gly Asn Gln Thr Asn Thr Pro Leu Ser Gly Tyr Thr Lys Met Val Tyr

225 230 235

Gly Asp Gly Ser Lys Phe Gln Ala Tyr Ser Ala Ala Gly Val Gly His

240 245 250

Phe Val Pro Thr Asp Val Ser Val Val Leu Asp Trp Phe Gly Ile Thr

255 260 265 270

Ser Gly Thr Thr Thr Thr Thr Thr Pro Thr Thr Thr Pro Thr Thr Ser

275 280 285

Thr Ser Pro Ser Ser Thr Gly Gly Cys Thr Ala Ala His Trp Ala Gln

290 295 300

Cys Gly Gly Ile Gly Tyr Ser Gly Cys Thr Ala Cys Ala Ser Pro Tyr

305 310 315

Thr Cys Gln Lys Ala Asn Asp Tyr Tyr Ser Gln Cys Leu

320 325 330

<210> SEQ ID NO 10

<211> LENGTH: 10

<212> TYPE: PRT

<213> ORGANISM: Penicillium funiculosum

<400> SEQUENCE: 10

Ala Glu Ala Ile Asn Tyr Asn Gln Asp Tyr

1 5 10

<210> SEQ ID NO 11

<211> LENGTH: 14

<212> TYPE: PRT

<213> ORGANISM: Penicillium funiculosum

<400> SEQUENCE: 11

Gln Tyr Thr Leu Thr Leu Pro Ser Asn Tyr Asn Pro Asn Lys

1 5 10

<210> SEQ ID NO 12

<211> LENGTH: 10

<212> TYPE: PRT

<213> ORGANISM: Penicillium funiculosum

<400> SEQUENCE: 12

Ala Val Ala Val Met Ser Gly Ala Asn Leu

1 5 10

<210> SEQ ID NO 13

<211> LENGTH: 22

<212> TYPE: PRT

<213> ORGANISM: Penicillium funiculosum

<220> FEATURE:

<221> NAME/KEY: UNSURE

<222> LOCATION: (6)...(6)

<223> OTHER INFORMATION: Cys or Ala

<400> SEQUENCE: 13

Thr Glu Tyr Ser Gly Xaa Ala Ala Asp Ser Glu His Pro Val Trp Trp

1 5 10 15

Ile Ala Phe Asp Gly Pro

20

<210> SEQ ID NO 14

<211> LENGTH: 28

<212> TYPE: PRT

<213> ORGANISM: Pennicilium funiculosum

<400> SEQUENCE: 14

Asp Thr Phe Val Lys Asp Asp His Cys Thr Pro Thr Asn Pro Pro Ala

1 5 10 15

Pro Ala Ala Gly Ser Gly Thr His Ile Lys Tyr Val

20 25