Modified epoxy resin immobilized enzyme, preparation method therefor and application thereof

This application is a U.S. national phase application filed under 35 U.S.C. § 371 claiming benefit to International Patent Application No. PCT/CN2020/072009, filed on Jan. 14, 2020, the disclosure of which is incorporated by reference herein in its entirety.

The present application contains a Sequence Listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy is named “206418_0014_00 US_Sequence_Listing_ST25.txt” and is 20,748 bytes in size. Except for changes to the bibliographic information and this Sequence Listing is identical to an ASCII formatted sequence listing of the international application No. PCT/CN2020/072009 filed on Jan. 14, 2020. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.

The present disclosure relates to the technical field of biocatalysis, in particular to a modified epoxy resin immobilized enzyme, a preparation method therefor and an application thereof.

Enzymes are biocatalysts widely used in different industrial processes because of their high activity, selectivity, and specificity compared to chemical catalysts. Enzymes are able to produce complex compounds under very mild reaction conditions, allowing to develop more sustainable processes and this attribute has made the enzymatic processes as emerging platform. Because of the biological origins of enzymes, they generally have operational characteristics that differ from those required for an industrial process.

The biocatalysts can be produced using whole living or dead cells or the crude enzyme or purified enzymes depending on the type and application of enzyme. The expanded pool of enzymes and advances in protein engineering has made it possible to produce economically viable biocatalyst on a commercial scale.

The increasing use of enzymes as catalysts in industrial processes has led to increasing demand of enzymes in immobilized form, as it offers unique process and cost advantages. The immobilized enzymes frequently termed as “biocatalysts” are widely used for industrial organic synthesis and biotransformation.

One of the most useful strategies to successfully use enzymes in biotechnological processes is their immobilization. A proper enzyme immobilization is a powerful tool to improve enzymatic properties, such as resistance to drastic reaction conditions (e.g., pH and temperature far from their physiological range), enhanced enzyme activity, multiple reusability or continuous use, and improvement of substrate specificity and enantiomer specificity, or product selectivity.

The emergence of novel immobilization platforms is enabling a seamless integration of immobilized enzymes in continuous flow bio-catalysis. The discovery and evolution of new and highly efficient enzymes, novel retrosynthetic approaches with an emphasis on biocatalysis, reduced cost of recombinant proteins and enzyme immobilization strategies all combine to augur well for flow biocatalysis.

The immobilization methods are based on the distinctive characteristics of functional groups of the amino acid side chains of the enzymes, which interact to (or react with) the support in several ways. The enzymes can be attached on the support surface by adsorption via reversible bonds, like van der Waals forces, hydrophobic, or ionic linkages, or by irreversible chemical bonds, such as covalent attachment (Sheldon et al 2013).

The covalent attachment provides an irreversible binding of the biocatalyst to the support and, for this reason, it prevents enzyme leakage and in effect enhancing the enzyme recyclability. Among covalent supports, Epoxy-activated supports are almost-ideal matrixes to perform a very easy immobilization of proteins on both a laboratory and industrial scale (Hannibal-Friedrich et al 1980; Hernaiz et al 2000; Calleri et al 2004; Podgornik, H. et al 2002). These supports are directly supplied in an activated form. Moreover, they are very stable during storage and transport even suspended in neutral aqueous media. Moreover, it is possible to carry out longterm immobilizations, permitting us to fully cover the support surface with the enzyme. Furthermore, epoxy-activated supports react with proteins under very mild experimental conditions (e.g. pH 7.0), promoting very small chemical modifications of the protein (secondary amine, thioether and ether).

Generally speaking, the soluble proteins are scarcely reactive with epoxy groups at neutral pH values. This low reactivity of epoxy supports causes the immobilization of enzymes on these supports to be produced via a two-step mechanism: first, a rapid and mild physical adsorption of the protein on the support is produced; secondly, a covalent reaction between the adsorbed protein and neighboring epoxide groups occurs [Wheatley et al 1999; Bauer-Arnaz et al 1998].

Due to this mechanism, commercial epoxy supports utilized to immobilize proteins are fairly hydrophobic, in order to adsorb proteins when they are incubated at high ionic strength (by a hydrophobic interaction). In some cases, the use of hydrophobic supports can promote the wrong folding of the protein structure caused by the stabilization of anomalous structures with internal hydrophobic amino acids located in the outer layer (Fitzpatrick P A et al 1993). Moreover, quite often the use of high salt concentrations can deactivate the activity of different enzymes especially of multimeric ones where the linkage among subunits is promoted by ionic forces (Fernandez-Lafuente et al 2009). Albeit the fact that epoxy supports are quite an important platform for immobilization, with the emerging new enzymes, especially more evolved multimeric enzymes, an overall improved and novel process of immobilization on epoxy supports are warranted.

Bolivar et al (2007) evaluated different immobilization strategies for the immobilization of Formate dehydrogenase from Candida boidinii using epoxy, amino-epoxy, glyoxyl (epoxy) or treatment of enzymes adsorbed on aminated supports with glutaraldehyde. The best results in terms of stability were achieved using amino-epoxy supports (by a 12-fold factor compared to soluble enzyme) and glyoxyl agarose supports (by a 150-fold factor). However, both cases activity recovery was just over 15% in addition to poor and identical stability with soluble enzyme.

Truppo et al (2012) evaluated the use of several polymer-based resins (SEPABEADS) from Mitsubishi for the immobilization of Januvia transaminase (CDX0117, Codexis) for the purpose of using the immobilized enzyme in organic solvents. The resins selected included three epoxide functionalized supports for covalent immobilization (EC-EP, EC-HFA/S, and EXE119), and two adsorption supports for immobilization through hydrophobic interaction (EXA252 and EXE120). Though many supports tested showed activity, SEPABEAD EXE120, (adsorption support) a highly hydrophobic octadecyl functionalized polymethacrylate resin, provided the highest specific activity

(FIG. 1). As much as 45% of the enzyme activity charged to the immobilization process was recovered on the EXE120 resin, resulting in a 4 wt % loading of transaminase on the resin (40 mg transaminase per 1 g solid support). Under the immobilized conditions, the epoxy support EC-EP showed poor enzyme binding and expression, which may be due to denaturation of enzyme by the epoxy support.

Hui Ren et al (2016) reported immobilization of thermophilic esterase AFEST from the archaeon Archaeoglobus fulgidus epoxy support Sepabeads EC-EP via covalent attachment, and the immobilized enzyme was then employed as a biocatalyst for poly(“-caprolactone) synthesis. The enzyme loading and recovered activity of immobilized enzyme was measured to be 72 mg/g and 10.4 U/mg using p-nitrophenyl caprylate as the substrate at 80° C., respectively. The immobilized enzyme good reusability, with monomer conversion values exceeding 75% during 15 batch reactions.

Ana I. Benitez-Mateos et al (2018) reported the use of porous carriers for the development of self-sufficient immobilized transaminase by binding along with the cofactor, PLP. In this work, w-transaminase from Halomonas elongata was co-immobilized with PLP onto porous methacrylate-based metal chelated carriers coated with polyethyleneimine. The packed-bed reactor continuously run up to 50 column volumes at 1.45 mL×min−1 in the enantioselective deamination of model amines (α-methylbenzyl amine), yielding >90% conversion in all cycles without exogenous addition of cofactor. Similar approach was made with transaminases from Chromobacterium violaceum and Pseudomonas fluorescens which showed similar activity. However, overall the maximum operating time the stability of enzyme reports is 133 minutes, despite the fact this approach seemed positive.

Despite several publications, there are following limitations on these methods, particularly in relation with the enzymes listed in Table 1.

Methods of the immobilized enzymes reported in an existing technology are not suitable for industrial applications of the enzymes in Table 1, and its stability needs to be further improved.

The present disclosure aims to provide a modified epoxy resin immobilized enzyme, a preparation method therefor and an application thereof, as to improve the recycling stability of enzymes.

In order to achieve the above purpose, according to one aspect of the present disclosure, a preparation method for a modified epoxy resin immobilized enzyme is provided. The preparation method includes the following steps: modifying an epoxy resin, adding a polyethyleneimine to a modified epoxy resin for further modification, and then adding an enzyme to be immobilized and a glutaraldehyde for immobilization, to obtain the modified epoxy resin immobilized enzyme.

Further, the step of modifying the epoxy resin includes using a sodium periodate to oxidize the epoxy resin or using an iminodiacetic acid to react with the epoxy resin.

Further, while the step of modifying the epoxy resin is to use the sodium periodate to oxidize the epoxy resin, before the sodium periodate is added, an acetic acid is firstly used to treat the epoxy resin, and after the enzyme to be immobilized and the glutaraldehyde are added for immobilization, it further includes a step of adding a cross-linking agent glutaraldehyde or dextran aldehyde for secondary cross-linking.

Further, while the step of modifying the epoxy resin is to use the iminodiacetic acid to react with the epoxy resin, after the polyethyleneimine is added to the modified epoxy resin for further modification, it further includes a step of adding metal ion solution for treatment, and the enzyme to be immobilized has a his tag; preferably, the metal ion solution is selected from one or more of a cobalt chloride, a cobalt sulfate, a nickel chloride, a copper sulfate, a ferrous chloride or a ferrous sulfate; preferably, the concentration of the metal ion solution is 5˜100 mmol/L, preferably 10˜50 mmol/L.

Further, the adding sequence of the enzyme to be immobilized and the glutaraldehyde is the enzyme to be immobilized and the glutaraldehyde, or the glutaraldehyde and the enzyme to be immobilized.

Further, in the step of adding the polyethyleneimine to the modified epoxy resin for further modification, it further includes adding a cofactor, and the cofactor is a nicotinamide adenine dinucleotide (NAD+), a nicotinamide adenine dinucleotide phosphate (NADP+) or a pyridoxal phosphate (PLP); preferably, the polyethyleneimine participates in the reaction in the form of polyethyleneimine aqueous solution, and the final concentration of the cofactor in the polyethyleneimine aqueous solution is 1˜10 mg/mL, preferably 3˜6 mg/mL; and preferably, before the cross-linking agent glutaraldehyde or dextran aldehyde is used, it further comprises a step of modifying the cross-linking agent with a polyethylene glycol (PEG), and the PEG modification of the cross-linking agent glutaraldehyde or dextran aldehyde includes dissolving the cross-linking agent with water, adding the PEG, and stirring at 20˜30° C. for 1˜6 h, herein the PEG is selected from PEG400˜PEG2000, and the mass ratio of PEG to the cross-linking agent is 1:1˜10:1, further preferably 2:1˜4:1.

Further, in the step of treating the epoxy resin with the acetic acid, the acetic acid used is acetic acid solution, and the concentration of the acetic acid in the acetic acid solution is 0.5˜3 M, preferably 1˜2 M; and the volume-to-mass ratio of the acetic acid solution to the epoxy resin is 5˜20:1, preferably 10˜15:1; preferably, in the step of oxidizing the epoxy resin with the sodium periodate, the concentration of the sodium periodate in sodium periodate solution used is 50˜500 mM, preferably 100˜200 mM; and the volume-to-mass ratio of the sodium periodate solution to the epoxy resin is 5˜20:1, preferably 5˜15:1; preferably, the molecular weight of the polyethyleneimine is 3 KDa˜70 KDa, and the concentration of the polyethyleneimine aqueous solution is 0.5%˜3%, preferably 1%˜2%; and pH of the polyethyleneimine aqueous solution is 6˜11, further preferably 7˜10; preferably, the volume/mass final concentration of the cross-linking agent glutaraldehyde or dextran aldehyde is 0.1%˜3%, preferably 0.3%˜2%; preferably, the mass ratio of the enzyme to the modified epoxy resin is 0.05˜0.3:1; and preferably, in the step of using the iminodiacetic acid to react with the epoxy resin, the iminodiacetic acid used is iminodiacetic acid aqueous solution, the concentration of the iminodiacetic acid aqueous solution is 0.5˜3 M, preferably 1˜2 M, and the volume-to-mass ratio of the iminodiacetic acid aqueous solution to the epoxy resin is 5˜20:1, preferably 10˜15:1; and pH of the iminodiacetic acid aqueous solution is 6.0˜10.0, preferably 7.0˜9.0.

Further, the treatment time after the acetic acid is mixed with the epoxy resin is 6˜24 h, preferably 10˜15 h; preferably, the reaction time after the sodium periodate solution is mixed with the epoxy resin is 1˜6 h, preferably 2˜3 h; preferably, the reaction time after the polyethyleneimine aqueous solution is mixed with the epoxy resin is 1˜20 h, preferably 3˜6 h; preferably, the reaction time after the enzyme is mixed with the modified epoxy resin is 2˜24 h, preferably 15˜20 h; preferably, after the cross-linking agent is added, the reaction time is 10˜120 min, preferably 20˜60 min; preferably, the action time after the iminodiacetic acid aqueous solution is mixed with the epoxy resin is 0.5˜6 h, preferably 1˜2 h; and preferably, the action time after the polyethyleneimine aqueous solution is mixed with a support is 1˜20 h, further, it is 3˜6 h; after the metal containing solution are added, the action time is 1˜6 h, further, it is 1˜3 h; and the action time after the enzyme solution is mixed with the support is 4˜48 h, and further, the action time is 15˜20 h.

Further, the epoxy resin is selected from one or more in a group consisting of Purolite®Lifetech™ECR8285, ECR8204, ECR8209, SEPLITE®LX1000EA, LX1000EP, LX103B, EP200, LX1000HFA, HFA001, LX107S, LX1000SW, LX1000SD, HECHENG®ES1, ES103, ES105, ES108 or ES109.

Further, the enzyme to be immobilized is selected from one or more in a group consisting of a transaminase derived from Chromobacterium violaceum DSM30191, a transaminase derived from Aspergillus fumigatus , a transaminase derived from Vibrio fluvialis strain JS17, a ketoreductase derived from Acetobacter sp. CCTCC M209061, a ketoreductase derived from Candida macedoniensis AKU4588, a cyclohexanone monooxygenase derived from Rhodococcus sp. Phi1, a cyclohexanone monooxygenase derived from Brachymonas petroleovorans , a monooxygenase derived from Rhodococcus ruber -SD1, an ammonia lyase derived from Photorhabdus luminescens , an ammonia lyase derived from Solenostemon scutellarioides , an Ene reductase derived from Saccharomyces cerevisiae , an Ene reductase derived from ChrySEQbacterium sp. CA49, an imine reductase derived from Streptomyces sp or Bacillus cereus , a leucine dehydrogenase derived from Bacillus cereus , a phenylalanine dehydrogenase derived from Bacillus sphaericus , a nitrilase derived from Aspergillus niger CBS 513.88 and a nitrilase derived from Neurospora crassa OR74A; and the transaminase derived from Chromobacterium violaceum DSM30191 is a mutant having a sequence of SEQ ID NO: 2 or SEQ ID NO: 3; the transaminase derived from Arthrobacter citreus is a mutant having a sequence of SEQ ID NO: 5 or SEQ ID NO: 6; the ketoreductase derived from Acetobacter sp. CCTCC M209061 is a mutant having a sequence of SEQ ID NO: 8 or SEQ ID NO: 9; the cyclohexanone monooxygenase derived from Rhodococcus sp. Phi1 is a mutant having a sequence of SEQ ID NO: 11 or SEQ ID NO: 12; and the cyclohexanone monooxygenase derived from Rhodococcus ruber -SD1 is a mutant having a sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

According to another aspect of the present disclosure, a modified epoxy resin immobilized enzyme is provided. The immobilized enzyme is prepared by any one of the above preparation methods.

According to another aspect of the present disclosure, an application of the modified epoxy resin immobilized enzyme in an aqueous buffer reaction system or an organic solvent reaction system is provided.

Further, the aqueous buffer reaction system or the organic solvent reaction system is reacted in a packed bed reactor or a continuous stirred tank reactor.

By applying the technical scheme of the present disclosure, the epoxy resin is modified, the polyethyleneimine is added to the modified epoxy resin for the further modification, and an aldehyde group in the resin and an amino group in the polyethyleneimine are covalently bound to each enzyme, then it is activated by the bifunctional reagent glutaraldehyde. In this way, a steric resin arm is increased, to form a network structure, it may be more easily bound to the enzyme by covalent binding, and the enzyme load may also be improved because the steric inhibition is reduced.

It should be noted that embodiments in the present application and features of the embodiments may be combined with each other in the case without conflicting. The present disclosure is described in detail below in combination with the embodiments.

IDA: Iminodiacetic acid disodium salt hydrate.

GA: Glutaraldehyde

PEI: Polyethyleneimine

PEG: Polyethylene glycol

In most cases, biocatalysis can rely on efficient biological catalysts. Enzymes are versatile biological catalysts with high stereoselectivity and regioselectivity and a high turnover rate. However, free enzymes are relatively sensitive and unstable, and they cannot be recovered and reused efficiently. To overcome these limitations and broaden their applicability, before use, free enzymes are usually attached to an inert, insoluble material via immobilization.

Epoxy resins have been used in immobilization of several kinds of enzymes like lipase, acylases, the process is very simple and easy to handle, but enzyme should be purified before immobilization to get positive activity recovery, and activity recovery is still not as good as other immobilization protocols even pure enzyme is used. In view of this, the present application proposes the following technical solution.

According to a typical embodiment of the present disclosure, a preparation method for a modified epoxy resin immobilized enzyme is provided. The preparation method includes the following steps: modifying an epoxy resin, adding a polyethyleneimine to a modified epoxy resin for further modification, and then adding an enzyme to be immobilized and a glutaraldehyde for immobilization, to obtain the modified epoxy resin immobilized enzyme.

By applying the technical scheme of the present disclosure, the epoxy resin is modified, the polyethyleneimine is added to the modified epoxy resin for the further modification, and an aldehyde group in the resin and an amino group in the polyethyleneimine are covalently bound to each enzyme, then it is activated by the bifunctional reagent glutaraldehyde. In this way, a steric resin arm is increased, to form a network structure, it may be more easily bound to the enzyme by covalent binding, and the enzyme load may also be improved because the steric inhibition is reduced.

In a typical embodiment of the present disclosure, the step of modifying the epoxy resin includes using a sodium periodate to oxidize the epoxy resin or using an iminodiacetic acid to react with the epoxy resin, as to obtain the epoxy resin with the improved performance. Preferably, while the step of modifying the epoxy resin is to use the sodium periodate to oxidize the epoxy resin, before the sodium periodate is added, an acetic acid is firstly used to treat the epoxy resin, and after the enzyme to be immobilized and the glutaraldehyde are added for immobilization, it further includes a step of adding a cross-linking agent glutaraldehyde or dextran aldehyde for secondary cross-linking, so that the immobilization is stronger.

In the present application, the modification of the epoxy resin with iminodiacetic acid and metal is modified. After the epoxy resin is reacted with the iminodiacetic acid, PEI is added and bound to the resin by ionic attachment, and then it is treated with the suitable metal. After that, a His-tagged enzyme is added, and then glutaraldehyde cross-linking is performed so that the attachment is stronger. PEI may bind to the metal stronger than the iminodiacetic acid, and may also cross-link with the glutaraldehyde, so that the enzyme leakage is much reduced. According to a typical embodiment of the present disclosure, while the step of modifying the epoxy resin is to use the iminodiacetic acid to react with the epoxy resin, after the polyethyleneimine is added to the modified epoxy resin for further modification, it further includes a step of adding metal ion solution for treatment, and the enzyme to be immobilized has a his tag; preferably, the metal ion solution is selected from one or more of a cobalt chloride, a cobalt sulfate, a nickel chloride, a copper sulfate, a ferrous chloride or a ferrous sulfate; and preferably, the concentration of the metal ion solution is 5˜100 mmol/L, preferably 10˜50 mmol/L.

Preferably, the adding sequence of the enzyme to be immobilized and the glutaraldehyde is the enzyme to be immobilized and the glutaraldehyde, or the glutaraldehyde and the enzyme to be immobilized successively.

According to a typical embodiment of the present disclosure, in the step of adding the polyethyleneimine to the modified epoxy resin for further modification, it further includes adding a cofactor, and the cofactor is NAD+, NADP+ or PLP; preferably, the polyethyleneimine participates in the reaction in the form of polyethyleneimine aqueous solution, and the final concentration of the cofactor in the polyethyleneimine aqueous solution is 1˜10 mg/mL, preferably 3˜6 mg/mL; and preferably, before the cross-linking agent glutaraldehyde or dextran aldehyde is used, it further comprises a step of modifying the cross-linking agent with PEG, and the PEG modification of the cross-linking agent glutaraldehyde or dextran aldehyde includes dissolving the cross-linking agent with water, adding the PEG, and stirring at 20˜30° C. for 1˜6 h, herein the PEG is selected from PEG400˜PEG2000, and the mass ratio of PEG to the cross-linking agent is 1:1˜10:1, the reusability is good, and further preferably it is 2:1˜4:1.

According to a typical embodiment of the present disclosure, in the step of treating the epoxy resin with the acetic acid, the acetic acid used is acetic acid solution, and the concentration of the acetic acid in the acetic acid solution is 0.5˜3 M, preferably 1˜2 M; and the volume-to-mass ratio of the acetic acid solution to the epoxy resin is 5˜20:1, preferably 10˜15:1; preferably, in the step of oxidizing the epoxy resin with the sodium periodate, the concentration of the sodium periodate in sodium periodate solution used is 50˜500 mM, preferably 100˜200 mM; and the volume-to-mass ratio of the sodium periodate solution to the epoxy resin is 5˜20:1, preferably 5˜15:1; preferably, the molecular weight of the polyethyleneimine is 3 KDa˜70 KDa, and the concentration of the polyethyleneimine aqueous solution is 0.5%˜3%, preferably 1%˜2%; and pH of the polyethyleneimine aqueous solution is 6˜11, further preferably 7˜10; preferably, the volume/mass final concentration of the cross-linking agent glutaraldehyde or dextran aldehyde is 0.1%˜3%, preferably 0.3%˜2%; preferably, the mass ratio of the enzyme to the modified epoxy resin is 0.05˜0.3:1; and preferably, in the step of using the iminodiacetic acid to react with the epoxy resin, the iminodiacetic acid used is iminodiacetic acid aqueous solution, the concentration of the iminodiacetic acid aqueous solution is 0.5˜3 M, preferably 1˜2 M, and the volume-to-mass ratio of the iminodiacetic acid aqueous solution to the epoxy resin is 5˜20:1, preferably 10˜15:1; and pH of the iminodiacetic acid aqueous solution is 6.0˜10.0, preferably 7.0˜9.0, the stability is best.

According to a typical embodiment of the present disclosure, the treatment time after the acetic acid is mixed with the epoxy resin is 6˜24 h, preferably 10˜15 h; preferably, the reaction time after the sodium periodate solution is mixed with the epoxy resin is 1˜6 h, preferably 2˜3 h; preferably, the reaction time after the polyethyleneimine aqueous solution is mixed with the epoxy resin is 1˜20 h, preferably 3˜6 h; preferably, the reaction time after the enzyme is mixed with the modified epoxy resin is 2˜24 h, preferably 15˜20 h; preferably, after the cross-linking agent is added, the reaction time is 10˜120 min, preferably 20˜60 min; preferably, the action time after the iminodiacetic acid aqueous solution is mixed with the epoxy resin is 0.5˜6 h, preferably 1˜2 h; and preferably, the action time after the polyethyleneimine aqueous solution is mixed with a support is 1˜20 h, further, it is 3˜6 h; after the metal containing solution are added, the action time is 1˜6 h, further, it is 1˜3 h; and the action time after the enzyme solution is mixed with the support is 4˜48 h, and further, the action time is 15˜20 h.

According to a typical embodiment of the present disclosure, the epoxy resin is selected from one or more in a group consisting of Purolite®Lifetech™ECR8285, ECR8204, ECR8209, SEPLITE®LX1000EA, LX1000EP, LX103B, EP200, LX1000HFA, HFA001, LX107S, LX1000SW, LX1000SD, HECHENG®ES1, ES103, ES105, ES108 or ES109.

According to a typical embodiment of the present disclosure, the enzyme to be immobilized is selected from one or more in a group consisting of a transaminase derived from Chromobacterium violaceum DSM30191, a transaminase derived from Aspergillus fumigatus , a transaminase derived from Vibrio fluvialis strain JSI7, a ketoreductase derived from Acetobacter sp. CCTCC M209061, a ketoreductase derived from Candida macedoniensis AKU4588, a cyclohexanone monooxygenase derived from Rhodococcus sp. Phi1, a cyclohexanone monooxygenase derived from Brachymonas petroleovorans , a monooxygenase derived from Rhodococcus ruber -SD1, an ammonia lyase derived from photorhabdus luminescens , an ammonia lyase derived from Solenostemon scutellarioides , an Ene reductase derived from Saccharomyces cerevisiae , an Ene reductase derived from ChrySEQbacterium sp. CA49, an imine reductase derived from Streptomyces sp or Bacillus cereus , a leucine dehydrogenase derived from Bacillus cereus , a phenylalanine dehydrogenase derived from Bacillus sphaericus , a nitrilase derived from Aspergillus niger CBS 513.88 and a nitrilase derived from Neurospora crassa OR74A; and the transaminase derived from Chromobacterium violaceum DSM30191 is a mutant having a sequence of SEQ ID NO: 2 or SEQ ID NO: 3; the transaminase derived from Arthrobacter citreus is a mutant having a sequence of SEQ ID NO: 5 or SEQ ID NO: 6; the ketoreductase derived from Acetobacter sp. CCTCC M209061 is a mutant having a sequence of SEQ ID NO: 8 or SEQ ID NO: 9; the cyclohexanone monooxygenase derived from Rhodococcus sp. Phi1 is a mutant having a sequence of SEQ ID NO: 11 or SEQ ID NO: 12; and the cyclohexanone monooxygenase derived from Rhodococcus ruber -SD1 is a mutant having a sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

The chemical processes involved in the reactions of the above enzymes is briefly described as follows:

R, R 1 and R 2 in the above reaction formulas may be each independently selected from H, a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted aralkyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted heterocycloalkyl, or a fused ring system formed by R 1 and its linked heterocycle.

According to a typical embodiment of the present disclosure, a modified epoxy resin immobilized enzyme is provided. The immobilized enzyme is prepared by any one of the above preparation methods.

According to a typical embodiment of the present disclosure, an application of the modified epoxy resin immobilized enzyme in an aqueous buffer reaction system or an organic solvent reaction system is provided. Preferably, the aqueous buffer reaction system or the organic solvent reaction system is reacted in a packed bed reactor or a continuous stirred tank reactor.

In a typical embodiment of the present disclosure, the epoxy resins (for example, the epoxy resins shown in Table 2, herein Epoxy is an epoxy group) are modified, thereby the reusability of the epoxy resin immobilized enzyme is improved.

Typically, the properties of the epoxy resins are changed by oxidizing the epoxy resins with the sodium periodate or modifying the epoxy resins with the iminodiacetic acid. The specific description is as follows:

I. Immobilization of Enzymes on Sodium Periodate Oxidized Epoxy Resins

After oxidation by sodium periodate, oxidized epoxy resins were further modified by PEI or PEI along with cofactors for each enzyme accordingly through covalent binding between aldehyde group in resin and amino group in PEI, followed by activation by bifunctional reagent glutaraldehyde, through this way, space arm of resin increased and net structure formed, and enzyme can be combined much easier by covalent binding because of reduced steric inhibition, and enzyme loading could also be improved. To make immobilization stronger, extra linker like glutaraldehyde or dextran aldehyde was added for secondary cross-linking.

Enzyme also can be combined on PEI modified oxidized epoxy resin by ionic adsorption primarily, followed by adding glutaraldehyde to perform crosslinking between enzyme, PEI and resin to form net structure and strong combination.

II. Immobilization of Enzymes on Iminodiacetic Modified Epoxy Resins

It is previously reported to immobilize the His-tagged enzyme on the metal-modified epoxy resin. The epoxy resin was first reacted with iminodiacetic acid to convert part of the epoxides on the surface of the beads and then treated with a suitable metal ion solution for the complexation on the resin. His-tagged enzyme was then added and a selective interaction between the poly-histidine tag and the metal allowed for a quick complexation, followed by a reaction between nucleophilic residues on the protein surface (Lys, Cys, or Ser) and the unreacted epoxy residues on the beads to give a successful along with covalent immobilization. The metal ion was then removed by washing with an EDTA solution. To ensure that no reactive epoxide remained, the beads were finally treated with glycine as capping agent. Main enzyme can be specifically bound onto resin, but stability was still not very good, after 6 cycles, less than 10% residual activity left. Complexation of metal and iminodiacetic acid modified resin was not strong enough, and enzyme can be easily leaked out.

In the present application, modification of epoxy by iminodiacetic acid and metal was revised. After reaction with iminodiacetic acid, PEI was added and combined with resin by ionic attachment, and then treated with suitable metal. Then His-tagged enzyme was added, and followed by glutaraldehyde crosslinking to make the attachment stronger. PEI can bind metal stronger than iminodiacetic acid, along with cross linking by glutaraldehyde, made enzyme leaking out reduce a lot.

To make the technical solution suitable for enzymes without His-tag, metal was not used after PEI modification, glutaraldehyde was added and form covalent binding with PEI and residual hydroxyl residual on surface of epoxy resin, then enzyme was added and bounded with covalent attachment.

In both methods, Enzyme also can be added before glutaraldehyde addition, and ionic adsorbed primarily with PEI and affinity adsorption, followed by adding glutaraldehyde to perform crosslinking between enzyme, PEI and resin to form net structure and strong combination.

The beneficial effects of the present disclosure are further described below in combination with the embodiments.

The enzymes used in the following embodiments and its sources are shown in Tables 3˜8 below.

Immobilization of Enzymes on Sodium Periodate Oxidized Epoxy Resins

5 g of an epoxy resin ECR8285 or LX1000EP is added to 20 mL of 1 M acetic acid respectively, it is mild stirred at a room temperature for 12 h, a support treated with the acetic acid is washed for 3 times with 20 mL of distilled water, and the treated support is suspended in 20 mL of 50 mM sodium periodate. It is mild stirred at the room temperature for 2 h, filtered and washed with the distilled water.

5 g of a modified epoxy resin is resuspended in 0.1 M phosphate buffer (PB), PEI is added, the final concentration is 1%, and pH is adjusted to pH 7.0. After being mild stirred for 2 h, 50-100 mM glutaraldehyde is added, and it is mild stirred at the room temperature for 1-2 h. Then the support resin is filtered and washed with 10 ml of water. The washed support is added to enzyme solution (10 ml of an enzyme, containing 5 mg/mL of a cofactor, dissolved in 40 mL of 100 mM PB, pH 7.0), it is stirred at 10-25° C. for 4 h, and overnights in a refrigerator. After standing overnight, the enzyme is filtered, and washed with 0.1 M PB (pH 7.0). Optionally, after standing overnight, excess glutaraldehyde (20-50 mM) or dextran aldehyde (10-50 mM) is added to solution, it is mild stirred for 1 h at 10-25° C., then filtered and washed with 0.1 M PB (pH 7.0).

Immobilization of enzymes on sodium periodate oxidized epoxy resins

5 g of epoxy beads are added to 50 mL of 1 M acetic acid, it is mild stirred at a room temperature for 12 h, a support treated with the acetic acid is washed for 3 times with 50 mL of distilled water, it is mild stirred for 10 minutes each time, and the treated support is suspended in 100 mL of a sodium periodate. It is mild stirred at the room temperature for 2 h, filtered and washed with the distilled water.

5 g of a modified epoxy resin is resuspended in 0.1 M PB, PEI is added, the final concentration is 1%, pH is adjusted to pH 7.0, and it is mild stirred for 2 h. Then the support is filtered and washed with 10 ml of water. The washed support is added to enzyme solution (10 ml of an enzyme, containing 5 mg/mL of a cofactor, dissolved in 40 mL of 100 mM PB, pH 7.0), it is stirred at the room temperature for 4 h, and overnights in a refrigerator. After standing overnight, 20-100 mM of a glutaraldehyde is added to solution, it is mild stirred at 10-25° C. for 1 h, then then filtered and washed with 0.1 M PB (pH 7.0).

Immobilization of Enzyme on Iminodiacetic Modified Epoxy Resin

4 g of a support is added to 8 mL of a support modification buffer (0.1 M sodium borate, 1 M iminodiacetic acid, pH 8.5), and it is shaken at a room temperature for 2 h. After 2 h, the support is filtered to remove the support modification buffer and washed with double distilled water, then resuspended with PB, and PEI is added so that the final concentration of PEI is 1%, pH is adjusted to 8.0-11.0, and it is mixed and mild stirred for 3 h. The support is filtered and washed for 3 times with 30 mL of water, then resuspended in 20 mL of PB, and metal containing solution are added, so that the concentration of the metal ions is 10-30 mM. The metal ions are selected from CoCl 2 or NiCl 2 .6H 2 O or CuSO 4 .5H 2 O or FeCl 2 or FeCl 2 .

It is shaken for 2 h at the room temperature. The resin is rinsed again with the double distilled water and washed with 0.1 M PB (pH 8.0). It is resuspended again (pretreated with a cofactor according to each enzyme) in a buffer containing 50 mM glutaraldehyde (0.1 M PB pH=8.0), and mild stirred for 1 h at the room temperature. It is filtered and washed for 3 times with 0.1M PB (pH=7.0).

The pretreated resin is resuspended with 16 mL of 0.1 M PB (pH=7.0), 4-8 mL of enzyme solution (50-100 mg/mL of the protein content, and 3-10 mg/mL of the cofactor) is added, it is mild stirred for 30 minutes, and filtered after overnight. It is washed twice with 20 ml of 0.05 M PB (pH 7.5, it contains 0.05 M EDTA and 0.5 M NaCl), and mild stirred for 10 minutes each time. Subsequently, it is washed for 3 times with 20 ml of water, and then washed with 0.1 M PB (pH 7.5).

Immobilization of Enzyme on Iminodiacetic Modified Epoxy Resin

4 g of a support is added to 8 mL of a support modification buffer (0.1 M sodium borate, 1 M iminodiacetic acid, pH 8.5), and it is shaken at a room temperature for 2 h. After 2 h, the support is filtered to remove the support modification buffer and washed with double distilled water, then resuspended with PB, and PEI is added so that the final concentration of PEI is 1%, pH is adjusted to 8.0-11.0, and it is mixed and mild stirred for 3 h. The support is filtered and washed for 3 times with 30 mL of water, then resuspended in 20 mL of PB, and metal containing solution are added, so that the concentration of the metal ions is 10-30 mM. The metal ions are selected from CoCl 2 or NiCl 2 .6H 2 O or CuSO 4 .5H 2 O or FeCl 2 or FeCl 2 .

It is shaken for 2 h at the room temperature. The resin is rinsed again with the double distilled water and washed with 0.1 M PB (pH 8.0). It is resuspended again with 16 mL of a buffer (0.1 M PB pH=8.0), 4-8 mL of enzyme solution (50-100 mg/mL of the protein content, and 3-10 mg/mL of the cofactor) is added, it is continuously mild stirred for 30 minutes, and filtered after overnight. It is washed twice with 20 ml of 0.05 M PB (pH 7.5, it contains 0.05 M EDTA and 0.5 M NaCl), and mild stirred for 10 minutes each time. Subsequently, it is washed for 3 times with 20 ml of water, and then washed with 0.1 M PB (pH 7.5).

Immobilization of Enzyme on Iminodiacetic Modified Epoxy Resin

4 g of a support is added to 8 mL of a support modification buffer (0.1 M sodium borate, 1 M iminodiacetic acid, pH 8.5), and it is shaken at a room temperature for 2 h. After 2 h, the support is filtered to remove the support modification buffer and washed with double distilled water, then resuspended with PB, and PEI is added so that the final concentration of PEI is 1%, pH is adjusted to 8.0-11.0, and it is suspended and mild stirred for 3 h. The support is filtered and washed for 3 times with 30 mL of water, then resuspended in 20 mL of PB, and metal containing solution are added, so that the concentration of the metal ions is 10-30 mM. The metal ions are selected from CoCl 2 or NiCl 2 .6H 2 O or CuSO 4 .5H 2 O or FeCl 2 or FeCl 2 .

The support is filtered and washed for 3 times with 30 ml of water, and then resuspended in 16 mL of PB (0.1 M pH=8.0), 4-8 mL of enzyme solution (50-100 mg/mL of the protein content, and 3-10 mg/mL of the cofactor) is added, it is continuously mild stirred for 30 minutes, and filtered after overnight. 16 mL of 0.1 M PB (pH=8.0, 5 mg/ml of the cofactor and 50 mM glutaraldehyde) is added, and it is shaken gently for 30 minutes at the room temperature. It is washed twice with 20 ml of 0.05 M PB (pH 7.5, it contains 0.05 M EDTA and 0.5 M NaCl), and mild stirred for 10 minutes each time. Subsequently, it is washed for 3 times with 20 ml of water, and then washed with 0.1 M PB (pH 7.5).

Immobilization of Enzyme on Iminodiacetic Modified Epoxy Resin

4 g of a support is added to 8 mL of a support modification buffer (0.1 M sodium borate, 1 M iminodiacetic acid, pH 8.5), and it is shaken at a room temperature for 2 h. After 2 h, the support is filtered to remove the support modification buffer and washed with double distilled water, then resuspended with PB, and PEI is added so that the final concentration of PEI is 1%, pH is adjusted to 8.0-11.0, and it is mild stirred for 3 h. The support is filtered and washed for 3 times with 30 mL of water, then resuspended in 20 mL of PB, and metal containing solution are added, so that the concentration of the metal ions is 10-30 mM. The metal ions are selected from CoCl 2 or NiCl 2 .6H 2 O Or CuSO 4 .5H 2 O or FeCl 2 or FeCl 2 .

The support is filtered and washed for 3 times with 30 ml of water, the pretreated support is resuspended in 16 mL of PB (0.1 M pH=8.0), 4-8 mL of enzyme solution (50-100 mg/mL of the protein content, and 3-10 mg/mL of the cofactor) is added, it is continuously mild stirred for 30 minutes, and filtered after overnight. 16 mL of 0.1 M PB (pH=8.0, 5 mg/ml of the cofactor and 50 mM glutaraldehyde) is added, and it is shaken gently for 30 minutes at the room temperature. It is washed twice with 20 ml of 0.05 M PB (pH 7.5, it contains 0.05 M EDTA and 0.5 M NaCl), and mild stirred for 10 minutes each time. Subsequently, it is washed for 3 times with 20 ml of water, and then washed with 0.1 M PB (pH 7.5).

Or the pretreated resin is resuspended with 16 mL of 0.1 M PB (pH=7.0), 4-8 mL of enzyme solution (50-100 mg/mL of the protein content, and 3-10 mg/mL of the cofactor) is added, it is mild stirred for 30 minutes, and filtered after overnight. It is washed twice with 20 ml of 0.05 M PB (pH 7.5, it contains 0.05 M EDTA and 0.5 M NaCl), and mild stirred for 10 minutes each time. Subsequently, it is washed for 3 times with 20 ml of water, and then washed with 0.1 M PB (pH 7.5).

As in Embodiment 2, the glutaraldehyde is changed to a PEI-modified glutaraldehyde, the molecular weight of PEG is 6000 Da, and the mass ratio of PEG to the glutaraldehyde is 1:1.

As in Embodiment 1, the glutaraldehyde is changed to aldehyde dextran.

As in Embodiment 5, the glutaraldehyde is changed to a PEI-modified glutaraldehyde, the molecular weight of PEG is 6000 Da, and the mass ratio of PEG to the glutaraldehyde is 1:1.

Conversion and reusability test of immobilized transaminase

In a 10 mL reaction bulb, 0.3 mL of MeOH is added, 0.1 g of a main raw material 1 or a main raw material 2 is dissolved, 4 eq of isopropylamine hydrochloride and 1.0 mg of pyridoxal-5′-phosphate (PLP) are added, and 0.1 M PB 7.0 is supplemented until the final volume of reaction solution is 1 mL, then 5 mg of enzyme powder or cross-linked enzyme aggregate wet enzyme or cross-linked enzyme aggregate lyophilized powder prepared from 20 mg of the enzyme powder is added, and it is stirred at 30° C. for 16-20 h. The conversion rate in the system is detected by a high performance liquid chromatography (HPLC), and reaction data is shown in Table 9 below:

Conversion and Reusability Test of Immobilized Ketoreductase

In a 10 mL reaction bulb, 0.5 mL of isopropanol (IPA) is added, 0.1 g of a main raw material 3 or 4 is dissolved, 0.5 mL of 0.1 M PB 7.0 and 1-10 mg of NAD+ are added, then 5 mg of enzyme powder or the immobilized enzyme prepared from 10 mg of the enzyme powder is added, it is stirred at 30° C. for 16-20 h. The conversion rate of the system is detected by a gas chromatography (GC), and reaction data is shown in Table 10 below:

Conversion and Reusability Test of Immobilized CHMOs

The activity of the CHMO epoxy support immobilized enzyme is detected by performing a reaction on the following substrate 5:

0.3 mL of isopropanol is loaded into a 10 mL reaction bulb, subsequently 100 mg of a substrate 5 is added, 3 mL of 0.1 M PB (pH 8.0) containing 5 mg of NADP+ is added, and then 2 mg of alcohol dehydrogenase ADH-Tb free enzyme and 20 mg of cyclohexanone monooxygenase free enzyme immobilized enzyme prepared from 50 mg of the free enzyme are added. It is reacted at 30° C. for 16-20 h to test the conversion rate. After each round of the reaction, the immobilized enzyme is separated and reused in the next round of the reaction, and the number of reuses is investigated. The conversion rate of the system is detected by GC, and reaction data is shown in Table 11 below:

Conversion and Reusability Test of Immobilized ERED

The activity of the ERED epoxy support immobilized enzyme is detected by performing a reaction on the following substrate 6:

3 mL of 0.1 M PB (pH 7.0-8.0) is loaded into a 10 mL reaction bulb, subsequently 100 mg of a substrate 6 is added, then 10 mg of NAD(P)+, 80 mg of an ammonium formate, 2 mg of FDH, and 10 mg of a ERED free enzyme or immobilized enzyme prepared from 30 mg of the free enzyme are added. It is reacted at 30° C. for 16-20 h to test the conversion rate. After each round of the reaction, the immobilized enzyme is separated and reused in the next round of the reaction, and the number of reuses is investigated. The conversion rate of the system is detected by GC, and reaction data is shown in Table 12 below:

Conversion and Reusability Test of Immobilized NITs

The activity of the NIT amino support immobilized enzyme is detected by performing a reaction on the following substrate 7:

2 mL of 0.1 M PB (pH 7.0-8.0) is added to a 10 mL reaction bulb, and 100 mg of the above substrate 9 is added, then 20 mg of NIT free enzyme or the immobilized enzyme prepared from 30 mg of the free enzyme is added. After 16 h of the reaction at 30° C., the conversion rate is detected. After each round of the reaction, the immobilized enzyme is separated and reused in the next round of the reaction, and the number of reuses is investigated. The conversion rate of the system is detected by GC, and reaction data is shown in Table 13 below:

Conversion and Reusability Test of Immobilized IREDs, which are Detected by the Following Substrate 8.

2 mL of 0.1 M PB (pH 7.0-8.0) is added to a 10 mL reaction ball, and then 100 mg of the substrate 8, 10 mg of NAD+, 60 mg of an ammonium formate, 10 mg of FDH, and 10 mg of IRED free enzyme or immobilized enzyme prepared from 30 mg of the free enzyme are added. After 20 h of the reaction at 30° C., the conversion rate is detected. After each round of the reaction, the immobilized enzyme is separated and reused in the next round of the reaction, and the number of reuses is investigated.

The conversion rate of the system is detected by HPLC, and reaction data is shown in Table 14 below:

Conversion and Reusability Test of Immobilized PAL

The activity and number of reuses of the immobilized enzyme are tested by performing a reaction on the following substrate 9:

8 mL of 4 M ammonium carbamate aqueous solution (pH 9.0˜9.5) is added into a 10 mL reaction bulb, and 100 mg of the above substrate 9 is added, then 10 mg of ammonia lyase free enzyme or immobilized enzyme prepared from 40 mg of the free enzyme is added. After 16-20 of the reaction at 30° C., the conversion rate is detected. After each round of the reaction, the immobilized enzyme is separated and reused in the next round of the reaction, and the number of reuses is investigated.

The conversion rate of the system is detected by HPLC, and reaction data is shown in Table 15 below:

Conversion and Reusability Test of Immobilized AADH

In a 10 mL reaction bulb, 5 mL of 0.1 M Tris-CI buffer (pH 8.0-9.0) is added, 100 mg of a main raw material 10, a main raw material 11 or a main raw material 12 is added, 108 mg of ammonium chloride is added, and pH is adjusted to 7.5-8.0, then 10-50 mg of NAD + , 150 mg of glucose, 5 mg of GDH, 10 mg of AADH or immobilized AADH prepared from 30 mg of the free enzyme are added. It is stirred at 30° C. for 16-20 h. The conversion rate of the system is detected by HPLC, and reaction data is shown in Table 16 below:

Conversion and Reusability Test of Immobilized FDH

In a 10 mL reaction bulb, 5 mL of 0.1 M Tris-CI buffer (pH 8.0-9.0) is added, 100 mg of a main raw material 12 is dissolved, and 108 mg of ammonium chloride and 80 mg of ammonium fomate are added, pH is adjusted to 7.5-8.0, then 10-50 mg of NAD + , 100 mg of AADH-Bc free enzyme, 5 mg of FDH or the immobilized FDH prepared from 10 mg of the free enzyme are added. It is stirred at 30° C. for 16-20 h. The conversion rate of the system is detected by HPLC, and reaction data is shown in Table 17 below:

Use of Transferase Amino Support Immobilized Enzyme in Packed Bed Continuous Reaction

The transaminase TA-Cv-V1 in the embodiment is immobilized on a support ECR8285, and immobilized by IDA 1 . The obtained immobilized enzyme is filled into a columnar reactor with a column volume of 120 mL, and the amount of the immobilized enzyme is 72 g.

500 g of a substrate 1 is dissolved in 1.5 L of methanol, and 4 eq of isopropylamine hydrochloride (1.8 L of 6 M isopropylamine hydrochloride aqueous solution) and 5 g of PLP are added without PB (0.1 M, pH 8.0), and the volume is fixed to 5 L.

The flow rate is set to 0.6 mL/min, namely the retention time is 200 min, and the continuous reaction is performed. Effluent at an outlet is detected for the conversion rate. The conversion rate is >98%. After 260 h of the continuous operation, the conversion rate is not decreased. After 280 h of the operation, the conversion rate is decreased to 90%. It is specifically shown in Table 18.

Use of transferase immobilized enzyme in continuous stirred tank reaction

The same immobilized enzyme TA-Ac-V1 in Embodiment 1 is used, the support is LX1000HFA, and it is immobilized by a mode of SP 2 . 50 g of the immobilized enzyme of the transaminase TA-Ac-V1 is added to a 200 mL reactor, and 150 mL of PB is added.

3.2 L of PB (0.1 M, pH 7.0), 1.8 L of isopropylamine hydrochloride aqueous solution (6 M) and 5 g of PLP are added to 500 g of a substrate 1, and it is prepared into a suspension by beating.

The substrate suspension is continuously added to a reaction bulb at a rate of 0.4 mL/min (namely the retention time is 500 min), and at the same time, the reaction system is extracted at an outlet at the same flow rate (a filter head is additionally installed at a tail end of a pipe, to prevent the immobilized enzyme from being extracted). Under this condition, the conversion rate may reach more than 90%, and the conversion rate is not decreased basically after 2000 h of the continuous operation. Results are shown in Table 19.

It is an ammonia lyase PAL-Ss immobilized enzyme, the support is HFA001, and it is immobilized by a mode of IDA 4 . 6 g of the obtained immobilized enzyme is filled into a 10 mL columnar reactor.

500 g of a substrate 9 is dissolved in 4.5 L of ammonium carbamate aqueous solution (4 M, pH 9.0˜9.5).

The flow rate is set to 0.03 mL/min, namely the retention time is 330 min, and the continuous reaction is performed. Effluent at an outlet is detected for the conversion rate, and the conversion rate is 80%. After 300 h of the continuous operation, the conversion rate is not decreased. After 310 h of the operation, the conversion rate is decreased to 70%. It is shown in Table 20.

It is the ketoreductase KRED-Ac-V1 immobilized enzyme prepared in the embodiment, the support is LX1000EP, and it is immobilized by a mode of IDA 4 . 6 g of the obtained immobilized enzyme is filled into a 10 mL columnar reactor.

100 g of the substrate 3 is dissolved in 0.3 L of isopropanol, 0.7 L of PB (0.1 M, pH 7.0) is added for dissolution, and then 0.1 g of NAD + is added.

The flow rate is set to 0.05 mL/min, namely the retention time is 200 min, and the continuous reaction is performed. Effluent at an outlet is detected for the conversion rate. The conversion rate is >90%. After 200 h of the continuous operation, the conversion rate is not decreased. After 210 h of the operation, the conversion rate is decreased to 80%. It is shown in Table 21.

Investigation of Various Parameters of Epoxy Resin Immobilization by Sodium Periodate Oxidation

The concentration and amount of an acetic acid, the concentration and amount of a sodium periodate, and the concentration of a cross-linking agent are investigated.

FDH is immobilized on the LX1000HFA support by the method in Embodiment 4, the concentration of IDA is set to 0.2˜3 mol/L, the volume/mass ratio of IDA solution and support is 2˜25:1, the concentration of the metal ions is set to 5˜100 mmol/L, and the range of the cross-linking agent dextran aldehyde (DA) is investigated. Herein, the IDA concentration of 1-2 mol/L is best, and it is best while the volume/mass ratio to the support is 10-20:1; the activity is better while the metal ion concentration is 10-100 mmol/L, in view of the cost, a concentration of 10˜50 mmol/L may be used; and the cross-linking agent DA is best in the range of 0.5%˜2%. The specific parameters and results are shown in Table 22.

The acetic acid concentration, the volume/mass ratio of the acetic acid solution to the support, the concentration of sodium periodate, the volume/mass ratio of the sodium periodate solution to the support, and the concentration of the cross-linking agent GA are investigated.

By the same method in Embodiment 3, FDH is immobilized to the support HFA, the different acetic acid concentrations, volume/mass ratios of acetic acid solution to the support, concentrations of sodium periodate, volume/mass ratios of sodium periodate solution to the support, and concentrations of the cross-linking GA are set. Results show that the optimal concentration of the acetic acid is 1-2 mol/L; the optimal volume/mass ratio of the acetic acid solution to the support is 10˜15:1; the optimal concentration of the sodium periodate is 0.1˜0.2 mol/L, and the effects are all better while the volume-to-mass ratio of the support is 5˜25, in view of cost saving, it may be 5˜15:1 preferably; and it is better while the concentration of the cross-linking agent is 0.5%˜2%. The specific parameters and results are shown in Table 23.

Investigation of Amount of PEI

By the same methods in Embodiment 2 (IDA4) and Embodiment 5 (SP3), FDH is immobilized to the support ECR8285, PEIs with different molecular weights are selected, and different PEI concentrations are set, to investigate the appropriate amount of PEI, and investigate the effects of different pH values on the immobilized enzyme. Results show that the PEIs have the similar effects while the molecular weight is from 3 KDa to 70 KDa, and the optimal concentration range of PEI is 1%˜2%; and while pH is 6.0˜10.0, the effect on the immobilized enzyme activity is not large, while pH is 7˜9, the stability is the best. The specific parameters and results are shown in Table 24.

Investigation of Proportion of PEG-Modified Cross-Linking Agent

By the same methods in Embodiment 7 (SP4) and Embodiment 9 (IDA4), FDH is immobilized on the support ECR8285, and the range of PEG and the ratio of PEG to GA in the methods of PEG-modified glutaraldehyde are investigated. Results show that PEG200, PEG2000 and PEG6000 may all modify the glutaraldehyde. While the ratio of PEG to GA is in the range of 1:1˜10:1, the reusability of the enzyme is better, and is best while the ratio is 2:1˜4:1. The specific parameters and results are shown in Table 25.

The above are only preferred embodiments of the present disclosure, and are not intended to limit the present disclosure. For those skilled in the art, the present disclosure may have various modifications and changes. Any modifications, equivalent replacements, improvements and the like made within the spirit and principle of the present disclosure shall be included within a scope of protection of the present disclosure.