阅读说明：本技术 Pei固定化酶、其制备方法及其应用 (PEI immobilized enzyme, preparation method and application thereof ) 是由 洪浩 詹姆斯·盖吉 肖毅 张娜 罗杰斯卡·维亚撒·威廉姆斯 崔瑜霞 赵佳东 高妍妍 于 2020-04-29 设计创作，主要内容包括：本发明提供了一种固定化酶、其制备方法及其应用。该固定化酶包括活化的PEI以及共价结合在活化的PEI上的酶,酶选自如下任意一种：转氨酶、酮还原酶、单加氧酶、氨裂解酶、烯还原酶、亚胺还原酶、氨基酸脱氢酶及腈水解酶。通过将酶与PEI固定,将酶固定于PEI形成的多聚网状结构之间,提高了机械稳定性,实现了酶的固定化,而且避免了在固定化过程中酶直接与戊二醛等交联剂的直接共价结合而降低活性。(The invention provides an immobilized enzyme, a preparation method and application thereof. The immobilized enzyme comprises activated PEI and an enzyme covalently bonded on the activated PEI, wherein the enzyme is selected from any one of the following enzymes: transaminase, ketoreductase, monooxygenase, ammonia lyase, alkene reductase, imine reductase, amino acid dehydrogenase, and nitrilase. By fixing the enzyme and the PEI, the enzyme is fixed between the poly-reticular structures formed by the PEI, the mechanical stability is improved, the immobilization of the enzyme is realized, and the reduction of the activity caused by the direct covalent bonding of the enzyme and crosslinking agents such as glutaraldehyde and the like in the immobilization process is avoided.)

1. An immobilized enzyme comprising an activated cationic polymer and an enzyme covalently bound to the activated cationic polymer,

wherein the activated cationic polymer is cofactor-activated PEI;

the enzyme is selected from any one of the following: transaminase, ketoreductase, monooxygenase, ammonia lyase, alkene reductase, imine reductase, amino acid dehydrogenase, and nitrilase.

2. The immobilized enzyme according to claim 1, wherein the transaminase is derived from B.thuringiensis, Arthrobacter citreus or Chromobacterium violacea DSM 30191;

the ketoreductase is derived from Acetobacter sp.CCTCC M209061 or Candidamacedoniensis.AKU4588;

the monooxygenase is cyclohexanone monooxygenase from Brachymonas petroleovarans or Rhodococcus ruber-SD 1;

the ammonia lyase is derived from phenylalanine ammonia lyase of phosphatohydus luminescens or Solenostemon cutellarioides;

the alkene reductase is derived from Saccharomyces cerevisiae or Chryseobacterium sp.CA49;

the imine reductase is derived from Streptomyces sp or Bacillus cereus;

the amino acid dehydrogenase is leucine dehydrogenase derived from Bacillus cereus or phenylalanine dehydrogenase derived from Bacillus sphaericus;

the nitrilase is derived from Aspergillus niger CBS 513.88 OR Neurospora crassa OR 74A.

3. The immobilized enzyme according to claim 1 or 2, wherein the molecular weight of the PEI is from 3kDa to 600 kDa;

preferably, the mass ratio of the enzyme to the PEI in the immobilized enzyme is 0.1-1.5: 1, more preferably, the mass ratio is 0.2-1: 1.

4. the immobilized enzyme according to claim 1 or 2, wherein the cofactor is PLP, NAD or NADP.

5. The immobilized enzyme according to claim 4, wherein the enzyme is one or more, the cofactor is one or more, one or more of the cofactors corresponds to one or more of the enzymes,

preferably, the mass ratio of the cofactor to the PEI in the immobilized enzyme is 1-80: 120, more preferably 1-60: 100.

6. A preparation method of an immobilized enzyme, which is characterized by comprising the following steps:

activating the cationic polymer by using an activating agent to obtain an activated cationic polymer;

immobilizing an enzyme with the activated cationic polymer to obtain the immobilized enzyme;

wherein the activator is a cofactor;

the enzyme is selected from any one of the following: transaminase, ketoreductase, monooxygenase, ammonia lyase, alkene reductase, imine reductase, amino acid dehydrogenase, and nitrilase.

7. The method of claim 6, wherein said method of making further comprises the step of pretreating said PEI prior to activating said PEI with said activator,

preferably, the pre-treatment comprises:

diluting PEI with pure water to a final concentration of 1 g/mL-8 g/mL, more preferably 1 g/mL-5 g/mL, to obtain diluted PEI;

and adjusting the pH value of the diluted PEI to be 5-11, and more preferably 7.0-10.0 to obtain the pretreated PEI.

8. The process according to claim 7, wherein the enzyme is a free enzyme, the activator is a cofactor, and the cofactor is PLP, NAD or NADP;

preferably, in the step of pretreating, purified water is used for adjusting the concentration of the diluted PEI to be 1 g/mL-8 g/mL, preferably 1 g/mL-3 g/mL, and the pH value is 6-11, more preferably 8.0-11.0, so as to obtain the pretreated PEI;

more preferably, the step of activating PEI with the activator to obtain activated PEI comprises:

adding the cofactor into the pretreated PEI, controlling the final concentration of the cofactor to be 0.3-20 mg/mL, further preferably controlling the final concentration of the cofactor to be 1-10 mg/mL, and then stirring for 10-600 min, more preferably stirring for 30-180 min to obtain the activated PEI.

9. The method according to claim 8, wherein the free enzyme is an enzyme solution of one enzyme or a mixture of a plurality of enzymes, and one or more of the cofactors corresponds to one or more of the enzymes.

10. The method of claim 8 or 9, wherein the step of binding the enzyme to the activated PEI to obtain the immobilized enzyme comprises:

dropwise adding the activated PEI into the enzyme solution of the free enzyme at the temperature of 4-25 ℃ to obtain PEI adsorptive enzyme;

dripping a cross-linking agent into the PEI adsorbent enzyme for immobilization to obtain the PEI immobilized enzyme;

preferably, the concentration of the free enzyme is 0.05-0.3 g/mL;

preferably, the mass ratio of the PEI to the free enzyme is 0.2-1: 1.

11. the method of claim 10, wherein the step of adding the cross-linking agent dropwise to the PEI immobilization enzyme to perform immobilization to obtain the PEI immobilized enzyme comprises:

dropwise adding the cross-linking agent into the PEI adsorbent enzyme, and controlling the final concentration of the cross-linking agent to be 0.2g/100 mL-1 g/100mL to obtain a substance to be immobilized;

standing, centrifuging and drying the object to be immobilized in sequence to obtain a dry enzyme;

cleaning the dry enzyme to obtain the immobilized enzyme;

preferably, the standing time is 10-300 min, more preferably 30-120 min, the drying is natural drying, the cleaning is performed by sequentially adopting a phosphate buffer solution for cleaning 3-5 times, water for cleaning 3-5 times and the phosphate buffer solution for cleaning 3-5 times, the phosphate buffer solution contains NaCl, and the concentration of the NaCl is 0.5-1M.

12. The method of claim 6, wherein the transaminase is derived from B.thuringiensis, Arthrobacter citreus, or Chromobacterium violacea DSM 30191;

the ketoreductase is derived from Acetobacter sp.CCTCC M209061 or Candidamacedoniensis.AKU4588;

the monooxygenase is cyclohexanone monooxygenase from Brachymonas petroleovarans or Rhodococcus ruber-SD 1;

the ammonia lyase is derived from phenylalanine ammonia lyase of phosphatohydus luminescens or Solenostemon cutellarioides;

the alkene reductase is derived from Saccharomyces cerevisiae or Chryseobacterium sp.CA49;

the imine reductase is derived from Streptomyces sp or Bacillus cereus;

the amino acid dehydrogenase is leucine dehydrogenase derived from Bacillus cereus or phenylalanine dehydrogenase derived from Bacillus sphaericus;

the nitrilase is derived from Aspergillus niger CBS 513.88 OR Neurospora crassa OR 74A.

13. Use of the immobilized enzyme of any one of claims 1 to 5 or prepared by the preparation method of any one of claims 6 to 12 in biocatalytic reactions.

14. Use according to claim 13, wherein the biocatalytic reaction is a batch or continuous reaction.

Technical Field

The invention relates to the field of immobilized enzymes, and particularly relates to an immobilized enzyme, and a preparation method and application thereof.

Background

The use of microbial cells or isolated or engineered enzymes has led to significant advances in biocatalysis and to a shift in the manner of manufacture. Many classes of enzymes such as acyltransferases, amidases, transaminases, ketoreductases, oxidases, monooxygenases, hydrolases and the like are used in reactions involving antibiotics, herbicides, pharmaceutical intermediates and new-generation therapeutics.

When free enzyme is used as a biocatalyst, there is a great waste of enzyme, since it is very difficult to recover water-soluble enzyme. The water-insoluble immobilized enzyme can be easily recovered by very simple filtration after each cycle.

It is well known to use a support or carrier to immobilize free enzymes. However, solid phase carriers have disadvantages of high cost and limited mass transfer, and the preparation of carriers or supports, especially polymer resins, causes serious environmental pollution. Most solid-phase carrier immobilized enzyme methods have inherent drawbacks: less enzyme loading and low enzyme specificity. One way to overcome this drawback is to avoid contaminating the protein or enzyme load by purifying the enzyme, but the cost of enzyme purification is also high, further increasing the cost of immobilized biocatalysts.

Carrier-free immobilization methods for enzymes such as Lipase (Lipase), penicillin acylase (penicillin acylase), protease (protease), and aminoacylase (aminoacylase) have been reported. However, the non-supported, non-supported immobilized enzymes cannot sufficiently satisfy mass production because of their low mechanical stability against shearing force and stirring conditions and also cause filtration problems.

Furthermore, the carrier-free immobilized enzyme method is also not suitable for certain sensitive enzymes such as Transaminase (TA), Ketoreductase (KRED), monooxygenase (CHMO), ammonia lyase (PAL), alkene reductase (ERED), Imine Reductase (IRED), Amino Acid Dehydrogenase (AADH) and Nitrilase (Nitrilase).

Therefore, it is necessary to develop a method of immobilized enzyme without carrier to improve the activity of sensitive enzyme and its recycling rate.

Disclosure of Invention

The invention mainly aims to provide an immobilized enzyme, a preparation method and application thereof, and aims to solve the problem that the activity and the recycling of a sensitive enzyme are difficult to realize in the prior art.

In order to achieve the above object, according to one aspect of the present invention, there is provided an immobilized enzyme comprising an activated cationic polymer and an enzyme covalently bound to the activated cationic polymer, the activated cationic polymer being a cofactor-activated PEI; the enzyme is selected from any one of the following: transaminase, ketoreductase, monooxygenase, ammonia lyase, alkene reductase, imine reductase, amino acid dehydrogenase, and nitrilase.

Further, the transaminase is derived from b.thuringiensis, artrobacter citreus or Chromobacterium violacea DSM 30191; preferably, the ketoreductase is derived from Acetobacter sp.CCTCCMM 209061 or Candida macedonienensis.AKU4588; preferably, the monooxygenase enzyme is cyclohexanone monooxygenase from Brachymonas petrolea or Rhodococcus ruber-SD 1; preferably, the ammonia lyase is derived from phenylalanine ammonia lyase from phosphatorhaddus luminescenses or solenostemmon cutellarioides; preferably, the alkene reductase is derived from Saccharomyces cerevisiae or Chryseobacterium sp.CA49; preferably, the imine reductase is derived from Streptomyces sp. or Bacillus cereus; preferably, the amino acid dehydrogenase is a leucine dehydrogenase derived from Bacillus cereus or a phenylalanine dehydrogenase derived from Bacillus sphaericus; preferably, the nitrilase is derived from Aspergillus niger CBS 513.88 OR Neurospora crassa OR 74A.

Furthermore, the molecular weight of PEI is 3 KDa-600 KDa; preferably, the mass ratio of the enzyme to the PEI in the immobilized enzyme is 0.1-1.5: 1, more preferably, the mass ratio is 0.2-1: 1.

further, the cofactor is PLP, NAD or NADP

Further, the immobilized enzyme also comprises a cofactor of the enzyme, the cofactor is covalently bound to PEI, and preferably the cofactor is PLP, NAD or NADP.

Further, the enzyme is one or more, the cofactor is one or more, the one or more cofactor corresponds to the one or more enzyme, preferably, the mass ratio of the cofactor to the PEI in the immobilized enzyme is 1-80: 120, more preferably 1-60: 100.

According to a second aspect of the present application, there is provided a method for producing an immobilized enzyme, the method comprising: activating the cationic polymer by using an activating agent to obtain an activated cationic polymer; fixing the enzyme and the activated cationic polymer to obtain an immobilized enzyme; wherein the activator is a cofactor; the enzyme is selected from any one of the following: transaminase, ketoreductase, monooxygenase, ammonia lyase, alkene reductase, imine reductase, amino acid dehydrogenase, and nitrilase.

Further, the preparation method further comprises the step of pretreating the PEI before activating the PEI with an activating agent, preferably the pretreatment comprises: diluting PEI to a final concentration of 1g/100 mL-8 g/100mL, more preferably 1g/100 mL-5 g/100mL, with pure water to obtain diluted PEI; and adjusting the pH value of the diluted PEI to be 5-11, and more preferably 7.0-10.0 to obtain the pretreated PEI.

Further, the enzyme is free enzyme, the activator is a cofactor, and the cofactor is PLP, NAD, or NADP; preferably, in the step of pretreatment, purified water is used for adjusting the concentration of diluted PEI to be 1g/100 mL-8 g/100mL, preferably 1g/100 mL-3 g/100mL, and the pH value is 6-11, more preferably 8.0-11.0, so as to obtain pretreated PEI; more preferably, the step of activating the PEI with an activator to obtain activated PEI comprises: adding a cofactor into the pretreated PEI, controlling the final concentration of the cofactor to be 0.3-20 mg/mL, further preferably controlling the final concentration of the cofactor to be 1-10 mg/mL, and then stirring for 10-600 min, more preferably stirring for 30-180 min to obtain the activated PEI.

Furthermore, the free enzyme is enzyme solution of one enzyme or mixed solution of a plurality of enzymes, and the cofactor is corresponding cofactor of the plurality of enzymes.

Further, the step of immobilizing the enzyme with activated PEI to obtain an immobilized enzyme comprises: dropwise adding activated PEI into the free enzyme solution at the temperature of 4-25 ℃ to obtain PEI adsorbed enzyme; dripping a cross-linking agent into the PEI adsorbent enzyme for immobilization to obtain a PEI immobilized enzyme; preferably, the concentration of the free enzyme is 0.05-0.3 g/mL; preferably, the mass ratio of the PEI to the free enzyme is 0.2-1: 1.

further, dripping a cross-linking agent into the PEI immobilized enzyme for immobilization to obtain the PEI immobilized enzyme, wherein the PEI immobilized enzyme comprises: dripping a cross-linking agent into PEI (polyetherimide) adsorbent enzyme, and controlling the final concentration of the cross-linking agent to be 0.2g/100 mL-1 g/100mL to obtain a substance to be immobilized; standing, centrifuging and drying the object to be immobilized in sequence to obtain dry enzyme; cleaning the dried enzyme to obtain immobilized enzyme; preferably, the standing time is 10-300 min, more preferably 30-120 min, the drying is natural drying, the cleaning is sequentially cleaning for 3-5 times by using a phosphate buffer solution, cleaning for 3-5 times by using water and cleaning for 3-5 times by using the phosphate buffer solution, the phosphate buffer solution contains NaCl, and the concentration of the NaCl is 0.5-1M.

Further, the transaminase is derived from b.thuringiensis, artrobacter citreus or Chromobacterium violacea DSM 30191; preferably, the ketoreductase is derived from Acetobacter sp.CCTCCMM 209061 or Candida macedonienensis.AKU4588; preferably, the monooxygenase enzyme is cyclohexanone monooxygenase from Brachymonas petrolea or Rhodococcus ruber-SD 1; preferably, the ammonia lyase is derived from phenylalanine ammonia lyase from phosphatorhaddus luminescenses or solenostemmon cutellarioides; preferably, the alkene reductase is derived from Saccharomyces cerevisiae or Chryseobacterium sp.CA49; preferably, the imine reductase is derived from Streptomyces sp. or Bacillus cereus; preferably, the amino acid dehydrogenase is a leucine dehydrogenase derived from Bacillus cereus or a phenylalanine dehydrogenase derived from Bacillus sphaericus; preferably, the nitrilase is derived from Aspergillus niger CBS 513.88 OR Neurospora crassa OR 74A.

According to a third aspect of the present application, there is provided a use of any of the immobilized enzymes described above or an immobilized enzyme prepared by any of the preparation methods described above in a biocatalytic reaction.

Further, the biocatalytic reaction is a batch reaction or a continuous reaction.

By applying the technical scheme of the invention, the enzyme and the PEI are fixed, and the enzyme is fixed between the poly-reticular structures formed by the PEI, so that the mechanical stability is improved, the immobilization of the enzyme is realized, and the reduction of the activity caused by the direct covalent bonding of the enzyme and crosslinking agents such as glutaraldehyde and the like in the immobilization process is avoided.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.

As mentioned in the background art, although some immobilized enzymes without carrier support have been reported in the prior art, they are not suitable for sensitive enzymes, and therefore, there is still a need to develop a new immobilization mode to improve the activity and recycling efficiency of sensitive enzymes. In a typical embodiment of the present application, there is provided an immobilized enzyme comprising an activated cationic polymer and an enzyme covalently bound to the activated cationic polymer, the activated cationic polymer being PEI activated with a cofactor or a crosslinking agent, the crosslinking agent being a polyol-treated or untreated crosslinking agent, the enzyme being selected from any one of: transaminase, ketoreductase, monooxygenase, ammonia lyase, alkene reductase, imine reductase, amino acid dehydrogenase, and nitrilase.

In the immobilized enzyme, the enzymes and the PEI form net-shaped combination, so that the mechanical stability is improved, the immobilization of the enzymes is realized, and the activity, the stability and the recycling times of the sensitive enzymes can be improved.

Among the above immobilized enzymes, PEI, i.e., Polyethylene imine, abbreviated as PEI, and polyethyleneimine, also called polyazepine, are water-soluble high-molecular polymers, which are soluble in water and ethanol and insoluble in benzene. The commercial products are usually 20% to 50% strength aqueous solutions. PEI is originally reported to be used as a polycation non-viral vector, and has the advantages of low price, good transfection effect in vivo and in vitro, convenient use, large-scale production, no immunogenicity and the like, so PEI has great attention on the aspect of the non-viral vector as a medicament. The PEI polymer contains a plurality of amino groups, and can be subjected to cross-linking reaction with glutaraldehyde to generate various poly-reticular nanostructures. The network structure is dispersed with various functional groups such as aldehyde group, amino group and the like, and can be combined with enzyme protein through various modes such as covalent interaction, hydrogen bond interaction, ion interaction, hydrophobic interaction and the like, but not only covalent binding with single glutaraldehyde, and the activity of the enzyme is extremely easily destroyed through the covalent bond interaction.

Therefore, the immobilized enzyme realizes the immobilization of the enzyme by immobilizing the enzyme with the PEI and immobilizing the enzyme between the poly-reticular structures formed by the PEI, and avoids the direct covalent bonding of the enzyme with crosslinking agents such as glutaraldehyde and the like to reduce the activity in the immobilization process. The specific molecular weight of the PEI is not particularly limited as long as it can immobilize an enzyme. In a preferred embodiment of the present application, the molecular weight of PEI ranges from 3kDa to 600 kDa. The specific molecular weight of PEI can be reasonably optimized and selected according to different types of immobilized enzymes, and the immobilization effect on the existing enzymes is relatively better within the molecular weight range.

In the immobilized enzyme, the mass ratio of the enzyme to the PEI can be reasonably adjusted according to different specific enzyme types. In a preferred embodiment, the mass ratio of the enzyme to the PEI in the immobilized enzyme is 0.1-1.5: 1, preferably, the mass ratio is 0.2-1: 1. in the range of 0.1-1.5: 1, so that the recycling frequency of the immobilized enzyme is higher, and the mass ratio is in a range of 0.2-1: within 1 range, the recycling frequency of the immobilized enzyme is higher.

The cofactor is PLP, NAD or NADP, and the crosslinking agent is glutaraldehyde; preferably, the polyalcohol is PEG, and more preferably, the PEG is PEG 400-6000.

The immobilized enzyme may be a single enzyme or a plurality of enzymes. When a plurality of enzymes are immobilized, co-immobilization of the main enzyme and the coenzyme is possible. In a preferred embodiment of the present application, the immobilized enzyme further comprises a cofactor for the enzyme, which is covalently bound to the PEI, preferably the cofactor is PLP, NAD or NADP. The co-immobilized enzyme of the main enzyme and the coenzyme has the effects of stable catalytic activity and high recycling frequency.

As described above, the enzyme in the immobilized enzyme of the present application may be one or more, and accordingly, the cofactor may be one or more, and the cofactor corresponds to one or more enzymes. Preferably, the mass ratio of the cofactor to the PEI in the immobilized enzyme is 1-80: 120, more preferably 1-60: 100.

When two or more enzymes have different cofactors, the immobilized enzyme contains different cofactors of the respective enzymes; when two or more enzymes have the same cofactor, the amount of the cofactor contained in the immobilized enzyme is reasonably proportioned according to the amount of each different main enzyme. The amount of PEI varies depending on the amount of the particular cofactor employed.

In a second exemplary embodiment of the present application, there is provided a method for preparing an immobilized enzyme, the method comprising: activating the cationic polymer by using an activating agent to obtain an activated cationic polymer; fixing the enzyme and the activated cationic polymer to obtain an immobilized enzyme; wherein the activator is a polyol unmodified crosslinking agent, a polyol modified crosslinking agent or a cofactor, and the enzyme is selected from any one of the following: transaminase, ketoreductase, monooxygenase, ammonia lyase, alkene reductase, imine reductase, amino acid dehydrogenase, and nitrilase.

According to the preparation method of the immobilized enzyme, the PEI is activated by adopting glutaraldehyde or a cofactor, and then the PEI is combined with the enzyme to obtain the immobilized enzyme. The method is simple, and the activity of the enzyme is not easily influenced in the fixing process, so that the immobilized enzyme has high activity, good stability and more recycling times.

In order to more effectively activate PEI, in a preferred embodiment of the present application, the preparation method further comprises the step of pre-treating PEI before activating PEI with an activator, more preferably the pre-treating comprises: diluting PEI to a final concentration of 1g/100 mL-8 g/100mL, more preferably 1g/100 mL-5 g/100mL, with pure water to obtain diluted PEI; and adjusting the pH value of the diluted PEI to be 5-11, and more preferably 7.0-10.0 to obtain the pretreated PEI. The reason for the dilution is: the viscosity and concentration of PEI are reduced, the activation process is facilitated, and the formation of precipitates due to overhigh concentration is prevented. The purpose of adjusting the pH is: too high or too low a pH can cause denaturing inactivation of the enzyme and can also affect the degree of binding of various levels of amino functionality on PEI to glutaraldehyde, and too high a pH can also easily lead to the formation of precipitates during activation.

In the above-mentioned preparation method, the activating agent differs depending on whether the enzyme is a precipitated enzyme or a free enzyme, and the specific preparation steps are slightly different. In a preferred embodiment, the enzyme is a precipitating enzyme, the cross-linking agent is glutaraldehyde, and the activating agent is glutaraldehyde or pegylated glutaraldehyde. More preferably, in the step of pretreating, the pH of the diluted PEI is adjusted to be 5.0-11.0, preferably 7.0-10.0, so as to obtain the pretreated PEI. In a more preferred embodiment, the step of activating PEI with an activator to obtain activated PEI comprises: and (3) dripping glutaraldehyde or PEGylated glutaraldehyde into the pretreated PEI, and controlling the final concentration of the glutaraldehyde or PEGylated glutaraldehyde in the system to be 0.1-4 g/100mL, preferably 0.3-2 g/100mL, so as to obtain the activated PEI.

When the enzyme is a precipitating enzyme, PEI with better activation performance can be obtained by activating PEI with glutaraldehyde or PEGylated glutaraldehyde and controlling the concentration of glutaraldehyde or PEGylated glutaraldehyde within the preferred range, thereby being more favorable for effective combination with the enzyme.

The step of activating PEI by glutaraldehyde or PEGylated glutaraldehyde has no special requirement, as long as the activity efficiency of glutaraldehyde or PEGylated glutaraldehyde on PEI is controlled to be optimal. In order to activate the PEI more fully and uniformly, in a preferred embodiment, the dropping speed of the glutaraldehyde or the PEGylated glutaraldehyde is controlled to be 10-50 mL/min in the process of dropping the glutaraldehyde or the PEGylated glutaraldehyde into the pretreated PEI, and more preferably, the step of stirring is further included in the dropping process, the stirring temperature is preferably 4-25 ℃, the stirring speed is preferably 50-400 rpm, further preferably 100-300 rpm, and the stirring time is 1-12 hours, further preferably 2-10 hours.

The precipitating enzyme can be formed by the existing method, and is preferably obtained by precipitating the enzyme by using a precipitating agent in the application, and the precipitating agent is selected from organic solvents (such as acetonitrile), ammonium sulfate, PEG 400-6000 or PEI with the molecular weight of 3-70 KDa. More preferably, PEG 400-6000 or PEI with the molecular weight of 3 KDa-70 KDa is adopted to obtain the precipitating enzyme. It should be noted that, when the precipitant is PEI of 3 KDa-70 KDa, the PEI is contained in the precipitating enzyme, and the PEI and the enzyme are physically adsorbed in the precipitating process, so that the PEI and the enzyme can be covalently bound to form the immobilized enzyme directly under the crosslinking action of glutaraldehyde. Of course, if the reaction is now with glutaraldehyde-activated PEI, the precipitated enzyme aggregates are also covalently bound to the PEI through the arm action of the crosslinker, forming a network of enzyme-to-enzyme and enzyme-to-PEI bonds.

When the precipitating enzyme is formed by organic solvent, ammonium sulfate, PEG 400-6000 and the like, the PEI activated by glutaraldehyde needs to be combined. In a preferred embodiment, the step of binding the enzyme to the activated PEI to obtain an immobilized enzyme comprises: dropwise adding activated PEI into the precipitating enzyme, and controlling the pH value to be 5-11, preferably 7.0-9.0 in the dropwise adding process to obtain a mixture; standing, centrifuging, sieving and drying the mixture in sequence to obtain immobilized enzyme; more preferably, the mass ratio of the enzyme to the activated PEI is 0.2-1: 1.

when the pH value is within the range of 5-11, the method has the beneficial effect of improving the firmer combination of the PEI and the activating agent on the basis of ensuring the enzyme activity, and when the pH value is controlled within the range of 7.0-9.0 in the dropwise adding process, the method has the most beneficial effect on the enzyme activity, the best combination degree of the PEI and the activating agent and more excellent effect of easier operation. The mass ratio of the enzyme to the activated PEI is 0.2-1: 1, the technical effect of obtaining the immobilized enzyme with the highest activity on the basis of not wasting the enzyme can be obtained.

In the preferred embodiment described above, the effect of the standing is to further enhance the formation of an immobilized enzyme of higher mechanical strength by internal interaction between the enzyme molecule and the activated PEI. Sieving to form immobilized enzyme with uniform particle size. In a preferred embodiment, the standing time is 1 to 5 hours; more preferably, the sieving is 20-50 mesh sieving; more preferably, the drying is natural air drying, more preferably overnight air drying; more preferably, after the mixture is dried and before the PEI immobilized enzyme is obtained, the preparation method further comprises: and cleaning the dried mixture by using a phosphate buffer solution, water and the phosphate buffer solution in sequence, wherein each cleaning is repeated for 3-5 times, the pH value of the phosphate buffer solution is 7.0-8.0, the phosphate buffer solution contains NaCl, and the concentration of the NaCl is 0.5-1M.

In the preferred embodiment, the mixture is washed with a phosphate buffer solution to prevent the washing solution from adversely affecting the activity of the enzyme. The buffer at the above concentrations and pH ranges does not affect the activity of the enzyme.

When the enzyme is a free enzyme, in a preferred embodiment, the enzyme is a free enzyme, the activator is a cofactor, and the cofactor is PLP, NAD, or NADP. Under the action of the cofactor, PEI is activated, the activated PEI can effectively adsorb free enzyme, and then the PEI and the enzyme can be fixed in a crosslinking way under the action of a crosslinking agent.

In a preferred embodiment, the step of pretreating comprises adjusting the concentration of diluted PEI to 1g/100 mL-8 g/100mL, preferably 1g/100 mL-3 g/100mL, with purified water to a pH of 6-11, preferably 8.0-11.0, to obtain pretreated PEI. The concentration and pH of diluted PEI are in the above range aiming at free enzyme, so that the distribution of effective functional groups of the activated PEI is more favorable for the combination with enzyme molecules, and the immobilized enzyme has good precipitation state and is more favorable for being separated from the solution.

For immobilization of free enzymes, in a preferred embodiment, the step of activating PEI with an activating agent to obtain activated PEI comprises: adding a cofactor into the pretreated PEI, controlling the final concentration of the cofactor to be 0.3-20 mg/mL, further preferably controlling the final concentration of the cofactor to be 1-10 mg/mL, and then stirring for 10-600 min, more preferably stirring for 30-180 min to obtain the activated PEI.

The final concentration of the cofactor is controlled within the above range, and the activation degree of the PEI is optimally and more favorably combined with enzyme molecules in the later period on the basis of no cofactor waste. And stirring the added cofactor, so as to be beneficial to fully realizing the activation of the cofactor on the PEI.

The free enzyme is enzyme solution of one enzyme or mixed solution of a plurality of enzymes, and correspondingly, the cofactor is a cofactor corresponding to one enzyme or a plurality of enzymes.

In a preferred embodiment, the step of immobilizing the enzyme with activated PEI to obtain an immobilized enzyme comprises: dropwise adding activated PEI into the free enzyme solution at the temperature of 4-25 ℃ to obtain PEI adsorbed enzyme; dripping a cross-linking agent into the PEI adsorbent enzyme for immobilization to obtain a PEI immobilized enzyme; more preferably, the concentration of the free enzyme is 0.05-0.3 g/mL; more preferably, the mass ratio of the activated PEI to the free enzyme is 0.2-1: 1.

the concentration of free enzyme is controlled within the range of 0.05-0.3 g/mL, so that immobilized enzyme with proper particle size and uniform distribution is facilitated, and the immobilized enzyme is separated from the solution conveniently. The mass ratio of the activated PEI to the free enzyme is controlled to be 0.2-1: 1, the immobilized enzyme has the highest profitability and recovery without wasting the free enzyme.

In a preferred embodiment, the step of adding a cross-linking agent dropwise into the PEI immobilized enzyme for immobilization to obtain the PEI immobilized enzyme comprises the following steps: dripping a cross-linking agent into PEI (polyetherimide) adsorbent enzyme, and controlling the final concentration of the cross-linking agent to be 0.2g/100 mL-1 g/100mL to obtain a substance to be immobilized; standing, centrifuging and drying the object to be immobilized in sequence to obtain dry enzyme; cleaning the dried enzyme to obtain immobilized enzyme; preferably, the standing time is 10-300 min, more preferably 30-120 min, the drying is natural drying, the cleaning is sequentially cleaning for 3-5 times by using a phosphate buffer solution, cleaning for 3-5 times by using water and cleaning for 3-5 times by using the phosphate buffer solution, the phosphate buffer solution contains NaCl, and the concentration of the NaCl is 0.5-1M.

The final concentration of the cross-linking agent is controlled within the range of 0.2g/100 mL-1 g/100mL, so that the immobilized enzyme with firm combination can be formed, and the highest enzyme activity recovery effect can be achieved. Here, the actions of standing, washing and the like are the same as those described above.

In a third exemplary embodiment of the present application, there is further provided an application of any one of the immobilized enzymes described above, or an immobilized enzyme prepared by any one of the preparation methods described above, in a biocatalytic reaction. The immobilized enzyme has the advantages of high stability and high recycling efficiency, so that the immobilized enzyme can be repeatedly used in biocatalytic reactions.

In a more preferred embodiment, the biocatalytic reaction used for the immobilized enzyme is a batch reaction or a continuous reaction, and more preferably a continuous reaction. The immobilized enzyme has high recycling efficiency, so the method is suitable for continuous biocatalytic reaction and improves the reaction efficiency.

The advantageous effects of the present application will be further described with reference to specific examples.

Table 1:

table 2: partial enzyme parent and mutant sequences

Table 3:

table 4:

table 5:

table 6:

the chemical process of the reaction in which the above enzyme participates is briefly described as follows:

r, R1, and R2 in the above reaction formulae may each independently be selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aralkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocycloalkyl, or R1 and the heterocycle to which it is attached form a fused ring system.

PB in the following examples means a phosphate buffer.