Immobilized enzyme as well as preparation method and application thereof

Abstract

The invention provides an immobilized enzyme as well as a preparation method and application thereof. The immobilized enzyme comprises an enzyme and a carrier for loading the enzyme, wherein the enzyme is alpha-amino acid fatty acyl transferase or polypeptide ligase; the carrier is a polymethacrylate matrix carrier; the polymethacrylate matrix carrier comprises an amino carrier or an epoxy carrier; wherein the amino carrier is an amino carrier modified by a long-chain polymer; the epoxy carrier is an epoxy carrier modified by a long-chain polymer; wherein the long-chain polymer comprises polyethylene glycol (Mw) of 2-8 KDa, polyvinyl alcohol (Mw) of 1.7-31 KDa, polyethyleneimine (Mw) of 1.8-70 KDa, polyacrylamide (Mw) of 50-300 KDa or polylysine (Mw) of 1.6-41 KDa. The immobilized enzyme disclosed by the invention is good in intermolecular dispersity and relatively large in spacing, can efficiently catalyze a substrate to synthesize oligopeptide or polypeptide, and has relatively high substrate conversion rate.

Description

Technical Field

[0001] The present invention relates to the field of oligopeptide and polypeptide synthesis, and in particular to an immobilized enzyme, a preparation method and application thereof. Background Art

[0002] In the literature, peptides composed of 2 to 10 amino acids are usually called oligopeptides (small molecule peptides), and peptides composed of 10 to 100 amino acids are called polypeptides, with a molecular weight of less than 10,000 Da. Many scientific studies have shown that oligopeptides play an important role in nutritional metabolism, and they have the functions of promoting physical development, digestive system development, neurobehavioral development, bone development, and immune system development. In recent years, people have become more and more interested in polypeptides. Currently, there are more than 60 approved polypeptide drugs on the market, and there are about more than 100 candidate drugs in different stages of clinical development. The importance of polypeptides has been recognized in the nutrition and cosmetics industries.

[0003] At present, the synthesis methods of oligopeptides and polypeptides are mainly divided into chemical method, solid phase synthesis and enzymatic synthesis. Compared with the chemical route, enzymatic synthesis has the advantages of simple steps, low production cost, no risk of product racemization, and more environmentally friendly. In the process of solid phase synthesis, the amino acid substrate needs to go through the steps of protection and deprotection, which leads to complex procedures, high production costs, and the generation of a large amount of organic waste.

[0004] The enzymatic synthesis of oligopeptides mainly includes the use of amino acid ester acyltransferase to catalyze the synthesis of oligopeptides through the reaction of amino acid methyl ester with another amino acid or dipeptide and tripeptide, or the use of amino acid ligase to catalyze the synthesis of oligopeptides through two amino acids or dipeptides and tripeptides. However, when using amino acid ester acyltransferase to synthesize oligopeptides, its hydrolysis cannot be avoided, and the product yield will be reduced. The conversion rate of amino acid ligase to synthesize oligopeptides is relatively low, the selectivity is poor, and there are a large number of self-ligation products.

[0005] The enzymatic synthesis of peptides mainly includes the chemical-enzymatic combination method, in which the chemically synthesized peptides are further synthesized into peptides by the catalysis of proteases. Since the synthesis of peptide bonds in the presence of water is thermodynamically unfavorable, the use of pure organic solvents with very low water activity can be beneficial to the coupling of peptides. However, proteases show very low activity in pure organic solvents, and peptide substrates are often difficult to dissolve when the side chains are (partially) protected. Another method is to carry out the coupling reaction in water, applying some effective ligases known in nature, but they require specific recognition sequences. Another method is to use substrate mimetics and proteases, however, the synthesis of the required highly active peptide esters is challenging, and some amino acids may undergo side reactions, such as Cys, Met, Trp, His, and Tyr.

[0006] At present, α-amino acid ester acyltransferase and polypeptide ligase are mainly used to synthesize oligopeptides and polypeptides. They not only have a synthetic effect, but also a hydrolytic effect. If the product is not separated from the enzyme in time, the product will be hydrolyzed, resulting in a decrease in yield. The free enzyme reaction cannot achieve timely separation of the product and the enzyme, and the reaction can only be terminated by adjusting the acid and alkali. During the post-treatment process, due to the presence of the free enzyme, there will also be a certain degree of emulsification, which makes the post-treatment difficult. The continuous reaction of the immobilized enzyme can achieve instant separation of the enzyme and the reaction substrate, which has the advantages of saving labor, increasing production capacity, and simplifying the production process. Therefore, it is crucial to develop an immobilized α-amino acid ester acyltransferase and polypeptide ligase for efficient synthesis of oligopeptides or polypeptides. Summary of the invention

[0007] The main purpose of the present invention is to provide an immobilized enzyme, a preparation method and application thereof, so as to solve the problems of low conversion rate of immobilized enzymes for synthesizing oligopeptides and lack of immobilized enzymes for synthesizing polypeptides in the prior art.

[0008] In order to achieve the above-mentioned object, according to the first aspect of the present invention, an immobilized enzyme is provided, which comprises an enzyme and a carrier for loading the enzyme; wherein the enzyme is α-amino acid acyltransferase or polypeptide ligase; the carrier is a polymethacrylate matrix carrier modified with a long-chain polymer; the polymethacrylate matrix carrier comprises an amino-type carrier or an epoxy-type carrier; wherein the long-chain polymer comprises polyethylene glycol Mw=2~8 KDa, polyvinyl alcohol Mw=1.7~31 KDa, polyethyleneimine Mw=1.8~70 KDa, polyacrylamide Mw=50~300 KDa or polylysine Mw=1.6~41 KDa.

[0009] Furthermore, the α-amino acid acyltransferase is derived from Pedobacter or Sphingobacterium; and the polypeptide ligase is derived from Bacillus subtilis.

[0010] Furthermore, the amino acid sequence of the α-amino acid acyltransferase is selected from any one of the following: SEQ ID NOs: 1-10; the amino acid sequence of the polypeptide ligase is selected from any one of the following: SEQ ID NOs: 11-13.

[0011] In order to achieve the above object, according to the second aspect of the present invention, a method for preparing an immobilized enzyme is provided, the method comprising: fixing the enzyme on a carrier loaded with the enzyme to obtain the immobilized enzyme; wherein the enzyme is α-amino acid acyltransferase or polypeptide ligase; wherein the carrier is a polymethacrylate matrix carrier modified with a long-chain polymer; the polymethacrylate matrix carrier comprises an amino-type carrier or an epoxy-type carrier; wherein the long-chain polymer comprises polyethylene glycol M w=2~8KDa, polyvinyl alcohol M w =1.7~31 KDa, polyethyleneimine M w =1.8~70 KDa, polyacrylamide M w =50~300 KDa or poly-lysine M w =1.6~41 KDa.

[0012] Furthermore, the preparation method of the carrier includes: modifying the initial carrier with a long-chain polymer to obtain a long-chain polymer-modified polymethacrylate matrix carrier; wherein the initial carrier includes a polymethacrylate matrix amino type carrier or a polymethacrylate matrix epoxy type carrier; the mass volume ratio of the initial carrier to the long-chain polymer is 1g: (2~10)mL, wherein the volume concentration of the long-chain polymer is 5%~20%.

[0013] Furthermore, the carrier is an epoxy-type carrier of a polymethacrylate matrix modified by a long-chain polymer, and the preparation method includes: mixing the above-mentioned enzyme and the epoxy-type carrier of the polymethacrylate matrix modified by the long-chain polymer for ring opening and fixation; the ionic strength of the buffer of the above-mentioned enzyme is 0.2~1M; the above-mentioned buffer of the above-mentioned enzyme is selected from any one or more of the following: phosphate, borate, citrate or tris(hydroxymethyl)aminomethane.

[0014] Furthermore, the carrier is an epoxy-type carrier of a polymethacrylate matrix modified by a long-chain polymer or an amino-type carrier of a polymethacrylate matrix modified by a long-chain polymer, and the preparation method includes: activating the carrier to obtain an activated carrier; and fixing the enzyme on the activated carrier to obtain an immobilized enzyme.

[0015] Furthermore, the carrier is activated by a cross-linking agent to obtain an activated carrier, wherein the cross-linking agent includes glutaraldehyde, glyoxal, cyanuric chloride, genipin, epichlorohydrin or glycidyl ether or 1,4-butanediol glycidyl ether.

[0016] Furthermore, the mass volume ratio of the carrier and the cross-linking agent is 1 g: (2~10) mL, wherein the concentration of the cross-linking agent is 0.1%~5%.

[0017] Furthermore, the mass volume ratio of the activated carrier and the enzyme is 1 g: (2~10) mL, wherein the concentration of the enzyme is 10~50 mg / mL.

[0018] Furthermore, the α-amino acid acyltransferase is derived from Pedobacter or Sphingobacterium; and the polypeptide ligase is derived from Bacillus subtilis.

[0019] Furthermore, the amino acid sequence of the α-amino acid acyltransferase is selected from any one of the following: SEQ ID NOs: 1-10; the amino acid sequence of the polypeptide ligase is selected from any one of the following: SEQ ID NOs: 11-13.

[0020] To achieve the above object, according to the third aspect of the present invention, a method for synthesizing oligopeptides or polypeptides is provided, the method comprising: synthesizing oligopeptides or polypeptides using the above-mentioned immobilized enzyme or the immobilized enzyme prepared by the above-mentioned method for preparing the immobilized enzyme.

[0021] Furthermore, the method comprises: using an immobilized enzyme to catalyze a substrate to obtain an oligopeptide or a polypeptide; the substrate comprises a first substrate for synthesizing an oligopeptide or a second substrate for synthesizing a polypeptide.

[0022] Furthermore, the first substrate includes a first acyl donor and a first acyl acceptor; the first acyl donor is an ester hydrochloride of an amino acid, and the ester hydrochloride of an amino acid is selected from any one of the following: methyl hydrochloride of an amino acid, ethyl hydrochloride of an amino acid, or isopropyl hydrochloride of an amino acid; the first acyl acceptor includes an amino acid or a third peptide segment; and the third peptide segment is formed by condensation of 2 to 9 amino acids.

[0023] Furthermore, the second substrate includes a second acyl donor and a second acyl acceptor; the second acyl acceptor includes an amino acid or a first peptide segment; the second acyl donor includes a second peptide segment modified with carboxamide methyl ester; wherein the first peptide segment is condensed from 2 to 98 amino acids; and the second peptide segment is condensed from 2 to 98 amino acids.

[0024] Furthermore, the catalytic reaction temperature is 10-25° C. and the pH is 8.0-9.5.

[0025] In order to achieve the above object, according to the fourth aspect of the present invention, there is provided a use of the above immobilized enzyme or the above method for preparing the immobilized enzyme or the above method for synthesizing oligopeptides or polypeptides in synthesizing oligopeptides or polypeptides.

[0026] Furthermore, the application includes: placing the immobilized enzyme in a continuous flow device to achieve continuous synthesis of oligopeptides or polypeptides.

[0027] By applying the technical scheme of the present invention, α-amino acid acyltransferase or polypeptide ligase is fixed to an amino-type polymethacrylate matrix carrier modified by a long-chain polymer or an epoxy-type polymethacrylate matrix carrier modified by a long-chain polymer to obtain an immobilized enzyme for synthesizing oligopeptides or polypeptides. The immobilized enzyme of the present invention has good intermolecular dispersibility and relatively large spacing, can efficiently catalyze the substrate to synthesize oligopeptides or polypeptides, and has a high substrate conversion rate. In addition, the immobilized enzyme of the present invention can separate the enzyme from the reaction product (synthesized oligopeptide or polypeptide) immediately, prevent the hydrolysis of the reaction product caused by excessive reaction, and maximize the production capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0029] Figure 1 The device for continuous reaction involved in the embodiment of the present invention is shown. The substrate feeding mode of the device is single-channel feeding of premixed system. Figure 1 A in the equation is the reaction system. Figure 1 B in the figure is a peristaltic pump. Figure 1 C in the column is the reaction column, Figure 1 The D in it is the collection system. DETAILED DESCRIPTION

[0030] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0031] As mentioned in the background technology, enzymatic synthesis in the prior art methods for synthesizing oligopeptides or polypeptides has the advantages of simple steps, low production cost, no risk of product racemization, and more environmentally friendly. However, free enzymes cannot achieve immediate separation of the product (synthesized oligopeptide or polypeptide) from the enzyme, and the reaction can only be terminated by adjusting the acid or base. During the post-treatment process, due to the presence of free enzymes, there will also be a certain degree of emulsification, which makes post-treatment difficult. In the present invention, the inventors tried to screen an immobilized enzyme carrier with a high conversion rate, immobilize it with α-amino acid acyltransferase or polypeptide ligase, and achieve immediate separation of the reaction product and the enzyme while achieving a higher conversion rate, thereby proposing a series of protection schemes of the present invention.

[0032] In a first typical embodiment of the present invention, an immobilized enzyme is provided, which includes an enzyme and a carrier for loading the enzyme; wherein the enzyme is α-amino acid acyltransferase or polypeptide ligase; the carrier is a polymethacrylate matrix carrier modified with a long-chain polymer; the polymethacrylate matrix carrier includes an amino-type carrier or an epoxy-type carrier; wherein the long-chain polymer includes polyethylene glycol Mw=2~8 KDa, polyvinyl alcohol Mw=1.7~31 KDa, polyethyleneimine Mw=1.8~70KDa, polyacrylamide Mw=50~300 KDa or polylysine Mw=1.6~41 KDa.

[0033] The immobilized α-amino acid acyltransferase or immobilized polypeptide ligase of the present invention can not only realize the instant separation of the immobilized enzyme and the reaction product, thereby effectively reducing the occurrence of the hydrolysis reaction and simplifying the post-treatment process, but also can efficiently convert the substrate to generate oligopeptides or polypeptides, and has the beneficial effects of simple operation and high efficiency.

[0034] In a preferred embodiment of the present invention, the amino acid acyltransferase is derived from Pedobacter or Sphingobacterium; the polypeptide ligase is derived from Bacillus subtilis. In a more preferred embodiment of the present invention, the amino acid sequence of the α-amino acid acyltransferase is selected from any one of the following: SEQ ID NOs: 1 to 10; the amino acid sequence of the polypeptide ligase is selected from any one of the following: SEQ ID NOs: 11 to 13. The immobilized enzyme prepared by using the amino acid acyltransferase and the polypeptide ligase can efficiently synthesize oligopeptides and polypeptides.

[0035] In a second typical embodiment of the present invention, a method for preparing an immobilized enzyme is provided. The preparation method comprises: fixing the enzyme on a carrier loaded with the enzyme to obtain the immobilized enzyme; wherein the enzyme is α-amino acid acyltransferase or polypeptide ligase; wherein the carrier is a polymethacrylate matrix carrier modified with a long-chain polymer; the polymethacrylate matrix carrier comprises an amino-type carrier or an epoxy-type carrier; wherein the long-chain polymer comprises polyethylene glycol M w =2~8 KDa, polyvinyl alcohol M w =1.7~31 KDa, polyethyleneimine M w =1.8~70 KDa, polyacrylamide M w =50~300 KDa or poly-lysine M w =1.6~41 KDa.

[0036] The immobilized enzyme prepared by the preparation method of the present invention has good intermolecular dispersibility and relatively large spacing, and can efficiently catalyze the substrate to synthesize oligopeptides or polypeptides.

[0037] In a preferred embodiment of the present invention, the preparation method of the carrier comprises: modifying the initial carrier with a long-chain polymer to obtain a polymethacrylate matrix carrier modified with a long-chain polymer; wherein the initial carrier comprises a polymethacrylate matrix amino type carrier or a polymethacrylate matrix epoxy type carrier; the mass volume ratio of the initial carrier to the long-chain polymer is 1g: (2~10)mL, wherein the volume concentration of the long-chain polymer is 5%~20%. In a more preferred embodiment of the present invention, the mass volume ratio of the initial carrier to the long-chain polymer is 1g: 6mL, wherein the concentration of the long-chain polymer is 5%~10%. The use of the above parameter conditions is more conducive to the coating of the carrier by the long-chain polymer.

[0038] The above long-chain polymers are respectively combined with amino-type carriers or epoxy-type carriers and coated on the surface of the carriers by hydrogen bonding or adsorption. w =2~8 KDa or polyvinyl alcohol M w =1.7~31 KDa to modify the above initial carrier. Such long-chain polymers form a polymer coating on the carrier surface through hydrogen bonding or adsorption, which can provide more enzyme fixation binding sites, make the enzyme molecules evenly distributed, and avoid aggregation. At the same time, the long-chain polymer coating can increase the distance between the enzyme and the carrier, reduce steric hindrance, make the enzyme molecules easier to approach the substrate, and improve the activity of the immobilized enzyme.

[0039] In a preferred embodiment of the present invention, the carrier is an epoxy-type carrier of a polymethacrylate matrix modified by a long-chain polymer, and the preparation method comprises: placing the epoxy-type carrier of a polymethacrylate matrix modified by a long-chain polymer in an enzyme solution for epoxy ring opening and enzyme immobilization to obtain the immobilized enzyme; wherein the enzyme solution contains salt ions with an ionic strength of 0.2 to 1 M; wherein the salt ions are selected from any one or more of the following: phosphate, borate, citrate or tris(hydroxymethyl)aminomethane.

[0040] The modified epoxy-type carrier can directly immobilize the enzyme. The epoxy group can open the ring in one step in the enzyme solution containing 0.2~1 M salt ions and covalently bond with the amino group on the enzyme to achieve enzyme fixation.

[0041] In addition, the modified epoxy carrier can also be activated in the same manner as the amino-type carrier, and the enzyme can be immobilized under the action of an activator. For example, when the activator is glutaraldehyde, the amino or hydroxyl group coated on the modified epoxy carrier is connected to the aldehyde group at one end of glutaraldehyde, and the aldehyde group at the other end of glutaraldehyde is connected to the enzyme, thereby further realizing the combination of the carrier and the enzyme.

[0042] In another preferred embodiment of the present invention, the carrier is an epoxy-type carrier of a polymethacrylate matrix modified by a long-chain polymer or an amino-type carrier of a polymethacrylate matrix modified by a long-chain polymer, and the preparation method includes: activating the carrier to obtain an activated carrier; and fixing the enzyme on the activated carrier to obtain an immobilized enzyme.

[0043] In a preferred embodiment of the present invention, the carrier is activated by a cross-linking agent to obtain an activated carrier, wherein the cross-linking agent includes glutaraldehyde, glyoxal, cyanuric chloride, genipin, epichlorohydrin or glycidyl ether or 1,4-butanediol glycidyl ether.

[0044] The cross-linking agent is connected to the carrier and the enzyme respectively to fix the enzyme on the carrier. In a preferred embodiment of the present invention, the carrier is activated by glutaraldehyde. The exposed amino group or hydroxyl group (including the amino group or hydroxyl group on the long-chain polymer) on the modified amino-type carrier is connected to the aldehyde group at one end of the glutaraldehyde, and after removing the excess unconnected glutaraldehyde, it is mixed with the enzyme solution (protein), and the amino group on the enzyme solution is connected to the aldehyde group at the other end of the glutaraldehyde, and the unbound protein is removed, so that the enzyme can be fixed on the carrier through covalent action, thereby improving the binding force and stability of the enzyme.

[0045] The molar ratio of the carrier to the cross-linking agent plays an important role in obtaining an activated carrier. In a preferred embodiment of the present invention, the mass volume ratio of the carrier to the cross-linking agent is 1 g: (2-10) mL, wherein the concentration of the cross-linking agent is 0.1%-5%. Preferably, the volume ratio of the carrier to the cross-linking agent is 1:4, wherein the concentration of the cross-linking agent is 0.1%-5%. The use of the above mass volume ratio is more conducive to cross-linking between the cross-linking agent and the carrier.

[0046] The mass-volume ratio of the activated carrier and the enzyme may affect the preparation efficiency of the immobilized enzyme. In a preferred embodiment of the present invention, the mass-volume ratio of the activated carrier and the enzyme is 1g: (2-10) mL, wherein the concentration of the enzyme is 10-50 mg / mL. In a more preferred embodiment of the present invention, the volume ratio of the activated carrier and the enzyme is 1g: 4 mL; wherein the concentration of the enzyme is 5-10 mg / mL. The use of the above ratio helps to improve the preparation efficiency of the immobilized enzyme and enables the enzyme to be efficiently fixed to the carrier.

[0047] The purpose of stirring is to make the reaction proceed more thoroughly. In a preferred embodiment of the present invention, the initial carrier needs to be stirred for the first time after being mixed with the long-chain polymer; the carrier needs to be stirred for the second time after being mixed with the cross-linking agent; and the activated carrier needs to be stirred for the third time after being mixed with the enzyme.

[0048] In a preferred embodiment of the present invention, the first stirring time is 6-24 h, the temperature is 10-30° C., the second stirring time is 0.5-4 h, the temperature is 10-30° C., and the third stirring time is 16-24 h, the temperature is 10-30° C. The stirring helps to fully connect the initial carrier with the long-chain polymer, the carrier with the cross-linking agent, and the activated carrier with the enzyme solution, improve the preparation efficiency of the immobilized enzyme, and fully combine the enzyme with the carrier.

[0049] In a preferred embodiment of the present invention, before the initial carrier is modified with the long-chain polymer, the initial carrier is first washed to remove the impurities or organic solvents remaining on the initial carrier and reduce the influence of the impurities or organic solvents on the modification.

[0050] In a preferred embodiment of the present invention, before the carrier is activated by the cross-linking agent, the carrier is washed for a second time, and the purpose of the second washing is to remove excess unbound polymer.

[0051] In a preferred embodiment of the present invention, before the enzyme is fixed on the activated carrier, the activated carrier is washed for a third time. The purpose of the third washing is to remove excess unattached cross-linking agent.

[0052] In a preferred embodiment of the present invention, after the enzyme is fixed on the activated carrier and before the immobilized enzyme is obtained, the immobilized enzyme precursor is washed for the fourth time. The purpose of the fourth washing is to remove the unbound enzyme solution.

[0053] In a preferred embodiment of the present invention, purified water or 20 mM PB (pH 7.0) is used for the above-mentioned washing step, and generally 2-3 times of washing is sufficient.

[0054] In a preferred embodiment of the present invention, the α-amino acid acyltransferase is derived from Pedobacter or Sphingobacterium; the polypeptide ligase is derived from Bacillus subtilis. In a more preferred embodiment of the present invention, the amino acid sequence of the α-amino acid acyltransferase is selected from any one of the following: SEQ ID NOs: 1 to 10; the amino acid sequence of the polypeptide ligase is selected from any one of the following: SEQ ID NOs: 11 to 13.

[0055] In a third typical embodiment of the present invention, a method for synthesizing an oligopeptide or polypeptide is provided, the method comprising: synthesizing the oligopeptide or polypeptide using the above-mentioned immobilized enzyme or the immobilized enzyme prepared by the above-mentioned preparation method.

[0056] The use of this method to synthesize polypeptides or oligopeptides can immediately separate the enzyme solution and the reaction product (polypeptide or oligopeptide), preventing excessive hydrolysis of the reaction product, which leads to a low conversion rate of the reaction product. In addition, this method simplifies the post-processing and batch-to-batch reaction process because it does not require the addition of acid or base to terminate the reaction.

[0057] In a preferred embodiment of the present invention, the method comprises: using the immobilized enzyme to catalyze a substrate to obtain the oligopeptide or the polypeptide; the substrate comprises a first substrate for synthesizing the oligopeptide or a second substrate for synthesizing the polypeptide.

[0058] When the immobilized enzyme and the first substrate undergo a catalytic reaction, the oligopeptide is generated. The first substrate includes a first acyl donor and a first acyl acceptor. In a preferred embodiment of the present invention, the first acyl donor is an ester hydrochloride of an amino acid. The ester hydrochloride of the amino acid is selected from any one of the following: methyl hydrochloride of an amino acid, ethyl hydrochloride of an amino acid, or isopropyl hydrochloride of an amino acid. The first acyl acceptor includes an amino acid or a third peptide segment; the third peptide segment is formed by condensation of 2 to 9 amino acids; preferably, the third peptide segment is formed by condensation of 2 to 3 amino acids. The target oligopeptide can be efficiently synthesized using the first acyl donor and the first acyl acceptor.

[0059] It should be noted that different oligopeptides have different synthetic substrates (especially acyl acceptors). For example, when synthesizing a dipeptide, its acyl acceptor is an amino acid, when synthesizing a tripeptide, its acyl acceptor is a dipeptide, and when synthesizing a tetrapeptide, its acyl acceptor is a tripeptide.

[0060] When the immobilized enzyme and the second substrate undergo a catalytic reaction, the polypeptide is generated. The second substrate includes a second acyl donor and a second acyl acceptor. The second acyl acceptor includes an amino acid or a first peptide segment; the second acyl donor includes a second peptide segment modified with carboxamide methyl ester; the first peptide segment is formed by condensation of 2 to 98 amino acids; and the second peptide segment is formed by condensation of 2 to 98 amino acids.

[0061] In a more preferred embodiment of the present invention, the first peptide segment is formed by condensation of 2 to 48 amino acids; the second peptide segment is formed by condensation of 2 to 48 amino acids. The use of the second acyl donor and the second acyl acceptor has the beneficial effect of efficiently synthesizing polypeptides.

[0062] It should be noted that the polypeptide mentioned in this article refers to a peptide composed of 10 to 100 amino acid units. A polypeptide is a peptide synthesized by forming a peptide bond between two peptide segments under the action of a peptide ligase. The same polypeptide can be synthesized from multiple groups of different peptide segments. For example, when synthesizing a tridecapeptide, the acyl donor is an undecapeptide and the acyl acceptor is a dipeptide, or the acyl donor is a decapeptide and the acyl acceptor is a tripeptide, or the acyl donor is a nonapeptide and the acyl acceptor is a tetrapeptide, and so on.

[0063] In a preferred embodiment of the present invention, the above oligopeptide includes a dipeptide, a tripeptide or a tetrapeptide; the above polypeptide includes a decapeptide or a thirteenpeptide. In a more preferred embodiment of the present invention, the above dipeptide is selected from any one of the following: Gly-Thr, Gly-Gln, Thr-Phe, Ala-Gln or Gly-Ser; the above tripeptide is selected from any one of the following: Val-Gly-Gly; the above tetrapeptide is selected from any one of the following: Val-Glu-Cys-Gly (SEQ ID NO: 14) or Val-Gly-Gly-Gly (SEQ ID NO: 15); the above decapeptide is selected from any one of the following: Ac-DLSKQALKKA-NH2 (SEQ ID NO: 16) or Ac-DFSKLALKKA-NH2 (SEQ ID NO: 17); the above thirteenpeptide is selected from any one of the following: Ac-EGTFTSDLSKQAF-NH2 (SEQ ID NO: 18).

[0064] Different enzymes have different optimum catalytic temperatures and optimum catalytic pH. In a preferred embodiment of the present invention, the temperature for the above catalytic reaction is 10-25°C, and the pH of the reaction is 8.0-9.5. Carrying out the catalytic reaction under these conditions has the beneficial effect of high catalytic efficiency.

[0065] In a fourth typical embodiment of the present invention, there is provided an application of the above-mentioned immobilized enzyme or the above-mentioned method for preparing the immobilized enzyme or the above-mentioned method for synthesizing an oligopeptide or polypeptide in synthesizing the above-mentioned oligopeptide or polypeptide. In a preferred embodiment of the present invention, the above-mentioned application comprises: arranging the above-mentioned immobilized enzyme in a continuous flow device to realize continuous synthesis of the above-mentioned oligopeptide or polypeptide.

[0066] In a preferred embodiment of the present invention, the continuous flow device comprises a pump, a reaction system and a reaction column. In a more preferred embodiment of the present invention, the continuous flow device further comprises a pH automatic controller and a mixer.

[0067] In a preferred embodiment of the present invention, the reaction system comprises a substrate for synthesizing oligopeptides or polypeptides, and the pH of the reaction system is 8.0 to 9.5. In a more preferred embodiment, the reaction system comprises a substrate for synthesizing oligopeptides or polypeptides, and the pH of the reaction system is 5.5 to 7.0.

[0068] Since substrates such as methyl esters are prone to self-hydrolysis at a reaction pH of 8.0-9.5, the conversion rate will gradually decrease. However, the stability of substrates such as methyl esters will be greatly improved at a pH of 5.5-7.0. Therefore, the reaction system with a pH of 5.5-7.0 first enters a mixer connected to a pH automatic controller, and the pH is adjusted to 8.0-9.5 by the pH automatic controller, and then enters the reaction column. This method can maintain a high and stable conversion rate.

[0069] In a preferred embodiment of the present invention, the reaction column is filled with the immobilized enzyme for synthesizing oligopeptides or polypeptides. In a preferred embodiment of the present invention, the pump comprises one or more of a micro-peristaltic pump, a high-performance liquid phase pump or a syringe pump. In a preferred embodiment of the present invention, the mixer is selected from any one of the following: a mechanical stirring mixer, an air flow mixer, a high shear mixer or a static mixer. The use of the continuous flow device to catalyze the synthesis of oligopeptides or polypeptides has the beneficial effect of high efficiency.

[0070] The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

[0071] Example 1: Preparation method of immobilized α-amino acid acyltransferase

[0072] Amino carrier modification and immobilization method:

[0073] a. Carrier modification: The amino carrier used was first washed twice with 20 mM PB (pH 7.0), and then 6 volumes of 10% long-chain polymer solution relative to the carrier mass, including PEG 4K Da, PVA 2.4K Da, PEI 10K Da, PAM 2000KDa and PL 8.2K Da, were added, stirred at 20°C for 16 h, and then washed three times with 20 mM PB (pH 7.0), and the solid was collected by filtration to obtain the modified immobilized enzyme carrier;

[0074] b. Carrier activation: Add 0.5% glutaraldehyde solution to the modified immobilized enzyme carrier relative to 4 volumes of the carrier, stir at 20°C for 1 h, then wash three times with 20 mM PB (pH 7.0), collect the solid by filtration, and obtain the activated immobilized enzyme carrier;

[0075] c. Enzyme immobilization: Add 4 volumes of diluted enzyme solution with a protein concentration of about 30 mg / mL to the activated carrier bottle. The buffer used for diluting the enzyme solution is 20 mM PB (pH 7.0). Stir at 20°C for 24 h, and wash three times with 4 volumes of 20 mM PB (pH 7.0) to obtain immobilized α-amino acid acyltransferase.

[0076] Epoxy carrier modification and immobilization method:

[0077] a. Carrier modification: The epoxy carrier was first washed twice with 20 mM PB (pH 7.0), and then 6 volumes of 10% long-chain polymer solution relative to the carrier mass, including PEG 4K Da, PVA 2.4K Da, PEI 10K Da, PAM2000K Da and PL 8.2K Da, were added, stirred at 20°C for 16 h, and then washed three times with 20 mM PB (pH 7.0), and the solid was collected by filtration to obtain the modified immobilized enzyme carrier;

[0078] b. Carrier activation: Add 0.5% glutaraldehyde solution to the modified immobilized enzyme carrier relative to 4 volumes of the carrier, stir at 20°C for 1 h, then wash three times with 20 mM PB (pH 7.0), collect the solid by filtration, and obtain the activated immobilized enzyme carrier;

[0079] c. Enzyme immobilization: Add 4 volumes of diluted enzyme solution with a protein concentration of about 30 mg / mL to the activated carrier bottle. The buffer used for diluting the enzyme solution is 0.5 M PB (pH 7.0). Stir at 20°C for 24 h, and wash three times with 4 volumes of 20 mM PB (pH 7.0) to obtain immobilized α-amino acid acyltransferase.

[0080] Example 2: Immobilization of α-amino acid acyltransferase on different polymer-modified immobilized enzyme carriers to synthesize dipeptides

[0081] Taking α-amino acid acyltransferase as the target enzyme, amino resin, epoxy resin, amino resin modified with different polymers, and epoxy resin modified with different polymers were selected for immobilization. The method of using a polymer-modified carrier, the carrier activation method, and the enzyme immobilization method of this embodiment are the same as those of Example 1. In addition, the epoxy resin and epoxy resin modified with different polymers in this embodiment did not undergo a carrier activation step, and they were subjected to ring opening and enzyme covalent binding under 0.5 M PB (pH 7.0).

[0082] Taking the synthesis of dipeptides Gly-Thr, Gly-Gln, Thr-Phe, Ala-Gln and Gly-Ser as examples, the reaction conditions are shown in Table 1, and the reaction results are shown in Tables 2-1, 2-2, 2-3 and 2-4. The results show that the amino or epoxy carriers modified with polyethylene glycol Mw = 2~8 KDa, polyvinyl alcohol Mw = 1.7~31 KDa, polyethyleneimine Mw = 1.8~70 KDa, polyacrylamide Mw = 50~300 KDa or polylysine Mw = 1.6~41 KDa generally have higher catalytic activity of immobilized enzymes than unmodified carrier-immobilized enzymes.

[0083] α-amino acid acyltransferases with different amino acid sequences were fixed to the same carrier (4K Da PEG-modified LX1000HA) to prepare immobilized enzymes. Gly-Gln was synthesized using the immobilized enzymes. The reaction conditions are shown in Table 1, and the experimental results are shown in Tables 2-5.

[0084] It should be noted that, except for Table 2-5 of this example, the amino acid sequences of α-amino acid acyltransferases used in the remaining examples of the present invention and Tables 2-1, 2-2, 2-3 and 2-4 of this example are all SEQ ID NO: 1.

[0085] According to the HPLC test results, the conversion rate of all embodiments of the present invention is calculated as follows: conversion rate = peak area percentage of product % / (peak area percentage of product % + peak area percentage of acyl acceptors such as amino acids or dipeptides). In addition, due to the different structures or molecular weights of the product and the acyl acceptor, in the detection method, the peak areas of the product and the acyl acceptor are not completely consistent under the same amount of substance, that is, the absorption is inconsistent. Generally, the absorption ratio under the same amount of substance is detected, and then the conversion rate is calculated by the absorption ratio.

[0086] Table 1:

[0087]

[0088] Table 2-1:

[0089]

[0090] Table 2-2:

[0091]

[0092] Table 2-3:

[0093]

[0094] Table 2-4:

[0095]

[0096] It should be noted that LX1000HA and LX109S in the above table were purchased from Lanxiao Technology, and ECR8409 and ECR8285 were purchased from Purolite. The above carriers are all polymethacrylate matrix carriers.

[0097] Table 2-5:

[0098]

[0099] Example 3: Immobilization of α-amino acid acyltransferase on different polymer-modified immobilized enzyme carriers to synthesize tripeptides or tetrapeptides

[0100] Taking α-amino acid acyltransferase as the target enzyme, amino resin, epoxy resin, amino resin modified with different polymers, and epoxy resin modified with different polymers were selected for immobilization. The method of using a polymer-modified carrier, the carrier activation method, and the enzyme immobilization method of this embodiment are the same as those of Example 1. In addition, the epoxy resin and epoxy resin modified with different polymers in this embodiment did not undergo a carrier activation step, and they were subjected to ring opening and enzyme covalent binding under 0.5 M sodium citrate (pH 7.0).

[0101] Taking the synthesis of tripeptide Val-Gly-Gly, tetrapeptide Val-Glu-Cys-Gly and tetrapeptide Val-Gly-Gly-Gly as examples, the reaction conditions are shown in Table 3, and the reaction results are shown in Table 4-1, Table 4-2, Table 4-3 and Table 4-4. The results show that the amino or epoxy carriers modified with polyethylene glycol Mw = 2~8 KDa, polyvinyl alcohol Mw = 1.7~31 KDa, polyethyleneimine Mw = 1.8~70 KDa, polyacrylamide Mw = 50~300 KDa or polylysine Mw = 1.6~41 KDa generally have higher immobilized enzyme activity than the unmodified carrier-immobilized enzyme.

[0102] Table 3:

[0103]

[0104] Table 4-1:

[0105]

[0106] Table 4-2:

[0107]

[0108] Table 4-3:

[0109]

[0110] Table 4-4:

[0111]

[0112] It should be noted that LX1000HA and LX109S in the above table were purchased from Lanxiao Technology, and ECR8409 and ECR8285 were purchased from Purolite. The above carriers are all polymethacrylate matrix carriers.

[0113] Example 4: Immobilization of polypeptides on immobilized enzyme carriers modified with different polymers to connect enzymes to synthesize polypeptides

[0114] With the polypeptide ligase as the target enzyme, amino resin, epoxy resin, amino resin modified with different polymers, and epoxy resin modified with different polymers were selected for immobilization. The method of using a polymer-modified carrier, the carrier activation method, and the enzyme immobilization method of this embodiment are the same as those of Example 1. In addition, the epoxy resin and epoxy resin modified with different polymers in this embodiment did not undergo a carrier activation step, and they were subjected to ring opening and enzyme covalent binding under 0.5 M Tris-HCl (pH 8.0).

[0115] Taking the synthetic peptides Ac-DLSKQALKKA-NH2, Ac-EGTFTSDLSKQAF-NH2 and Ac-DFSKLALKKA-NH2 as examples, the reaction conditions are shown in Table 5, and the reaction results are shown in Table 6-1, Table 6-2, Table 6-3 and Table 6-4. The results show that the amino or epoxy carriers modified with polyethylene glycol Mw = 2~8 KDa, polyvinyl alcohol Mw = 1.7~31 KDa, polyethyleneimine Mw = 1.8~70 KDa, polyacrylamide Mw = 50~300 KDa or polylysine Mw = 1.6~41 KDa generally have higher immobilized enzyme activity than the unmodified carrier-immobilized enzyme. -OCam in the substrate of the present invention indicates that the C-terminus is protected by carboxamide methyl ester; Ac- indicates that the N-terminus is protected by acetyl; -NH2 indicates that the C-terminus is amino-modified; H- indicates that the N-terminal amino group is not protected.

[0116] The peptide ligases with different amino acid sequences were fixed to the same carrier (4K Da PEG-modified LX1000HA) to prepare immobilized enzymes. Ac-DLSKQALKKA-NH2 was synthesized using this immobilized enzyme. The reaction conditions are shown in Table 5, and the experimental results are shown in Table 6-5.

[0117] It should be noted that, except for Table 6-5 of this example, the amino acid sequences of the polypeptide ligases used in the remaining examples of the present invention and Tables 6-1, 6-2, 6-3 and 6-4 of this example are all SEQ ID NO: 13.

[0118] Table 5:

[0119]

[0120] Table 6-1:

[0121]

[0122] Table 6-2:

[0123]

[0124] Table 6-3:

[0125]

[0126] Table 6-4:

[0127]

[0128] It should be noted that LX1000HA and LX109S in the above table were purchased from Lanxiao Technology, and ECR8409 and ECR8285 were purchased from Purolite. The above carriers are all polymethacrylate matrix carriers.

[0129] Table 6-5:

[0130]

[0131] Example 5: Continuous synthesis of dipeptide Thr-Phe by immobilized α-amino acid acyltransferase

[0132] According to the results in Example 2, an immobilized α-amino acid acyltransferase with LX1000HA as the enzyme immobilization carrier and PEG 8K Da as the modification group was selected to run a continuous reaction. The reaction conditions are shown in Table 1. The prepared immobilized enzyme was added to a 1 mL reaction column ( Figure 1 C), approximately 0.75 g of immobilized enzyme was filled and pumped through a peristaltic pump ( Figure 1 B) at 20°C, the reaction system ( Figure 1 A) in the reaction column continuously, and the reaction products are collected in the collection system ( Figure 1 D in the figure). Set the retention time to 0.5~5 min, and take samples for testing every 10 min to determine the optimal retention time.

[0133] The samples were tested by HPLC, and the results showed that the product conversion rate reached more than 80%, and the conversion rate could be continuously operated for about 24 h without significant decrease. However, since L-threonine methyl ester hydrochloride will undergo self-hydrolysis at the reaction pH, the system needs to be reconfigured every 1 to 2 h. Compared with free enzymes, information such as production capacity is shown in Table 7. The experimental results show that compared with free enzymes, immobilized enzymes have higher production capacity and more economical use of enzymes.

[0134] The steps of the free enzyme batch reaction were as follows: L-threonine methyl ester hydrochloride: phenylalanine = 75:50 mM, 0.1 mM Tris-HCl, pH = 9.5, adding 0.03 wt of free enzyme (relative to the amino acid phenylalanine), and then tracking the progress of the reaction, taking samples for detection every 0.5 h.

[0135] Table 7:

[0136]

[0137] Example 6: Continuous synthesis of tripeptide Val-Gly-Gly by immobilized α-amino acid acyltransferase

[0138] According to the results in Example 3, an immobilized α-amino acid acyltransferase with LX1000HA as an enzyme immobilization carrier and PL 41KDa as a modification group was selected to run a continuous reaction. The reaction conditions are shown in Table 3. The prepared immobilized enzyme was added to a 1 mL reaction column, about 0.75 g of the immobilized enzyme was filled, and the reaction system was continuously passed through the reaction column (reaction device as shown in FIG. 1 ) at 20°C by a peristaltic pump. Figure 1 As shown in the figure, the retention time is set to 1-10 min, and samples are tested every 20 min to determine the optimal retention time.

[0139] The sample was tested by HPLC, and the results showed that the product conversion rate reached more than 70%, and it could be operated continuously for about 34 hours without a significant decrease in conversion rate. However, since L-valine methyl ester hydrochloride will undergo self-hydrolysis at the reaction pH, the system needs to be reconfigured every 1 to 2 hours. Compared with free enzyme, the production capacity and other information are shown in Table 8.

[0140] The free enzyme batch reaction system is shown in Example 3, and 0.2 wt of free enzyme (relative to the acyl acceptor) is added, and then the reaction progress is tracked, and samples are taken every 0.5 h for detection.

[0141] Table 8:

[0142]

[0143] Example 7: Continuous synthesis of the tetrapeptide Val-Glu-Cys-Gly by immobilized α-amino acid acyltransferase

[0144] According to the results in Example 3, an immobilized α-amino acid acyltransferase with LX1000HA as an enzyme immobilization carrier and PEI 70KDa as a modification group was selected to run a continuous reaction. The reaction conditions are shown in Table 3. The prepared immobilized enzyme was added to a 1 mL reaction column, about 0.75 g of the immobilized enzyme was filled, and the reaction system was continuously passed through the reaction column (reaction device as shown in Figure 2) at 10°C by a peristaltic pump. Figure 1 As shown in the figure, the retention time is set to 1-10 min, and samples are tested every 20 min to determine the optimal retention time.

[0145] The sample was tested by HPLC, and the results showed that the product conversion rate reached more than 90%, and it could be operated continuously for about 30 h without a significant decrease in conversion rate. However, since L-valine methyl ester hydrochloride will undergo self-hydrolysis at the reaction pH, the system needs to be reconfigured every 1 to 2 h. Compared with the free enzyme, the production capacity and other information are shown in Table 9.

[0146] The free enzyme batch reaction system is shown in Example 3, 0.05 wt % of free enzyme (relative to the acyl acceptor) is added, and then the reaction progress is tracked, and samples are taken every 0.5 h for detection.

[0147] Table 9:

[0148]

[0149] Example 8: Continuous synthesis of Ac-DLSKQALKKA-NH2 using immobilized polypeptide ligase

[0150] According to the results in Example 4, LX1000HA was selected as the enzyme immobilization carrier and PEG 4KDa was used as the modified group of the immobilized polypeptide ligase to run the continuous reaction. The reaction conditions are shown in Table 5. The prepared immobilized enzyme was added to a 0.2 mL reaction column, about 0.15 g of the immobilized enzyme was filled, and the reaction system was continuously passed through the reaction column (reaction device as shown in Figure 5) at 25 °C by the action of a peristaltic pump. Figure 1 As shown in the figure), the retention time was set to 0.5-5 min, and samples were taken every 10 min for testing to determine the optimal retention time. The samples were tested by HPLC, and the results showed that the product conversion rate reached more than 79%. Compared with the free enzyme, the production capacity and other information are shown in Table 10.

[0151] The free enzyme batch reaction system is shown in Example 5, and 0.05 wt % of free enzyme (relative to the acyl acceptor) is added, and then the reaction progress is tracked, and samples are taken every 0.5 h for detection.

[0152] Table 10:

[0153]

[0154] Example 9: Continuous synthesis of Ac-EGTFTSDLSKQAF-NH2 by immobilized polypeptide ligase

[0155] According to the results in Example 4, LX1000HA was selected as the enzyme immobilization carrier and PL 1.6K Da was selected as the immobilized polypeptide ligase for continuous reaction. The reaction conditions are shown in Table 5. The prepared immobilized enzyme was added to a 0.2 mL reaction column, about 0.15 g of the immobilized enzyme was filled, and the reaction system was continuously passed through the reaction column (reaction device as shown in Figure 5) at 25 °C by the action of a peristaltic pump. Figure 1 As shown in the figure, the retention time was set to 0.5-5 min, and samples were taken for testing every 10 min to determine the optimal retention time. The samples were tested by HPLC, and the results showed that the product conversion rate reached more than 50%. Compared with the free enzyme, the production capacity and other information are shown in Table 11.

[0156] The free enzyme batch reaction system is shown in Example 5, and 0.05 wt % of free enzyme (relative to the acyl acceptor) is added, and then the reaction progress is tracked, and samples are taken every 0.5 h for detection.

[0157] Table 11:

[0158]

[0159] Example 10: Continuous synthesis of dipeptide Ala-Gln by immobilized α-amino acid acyltransferase

[0160] Referring to the method of continuous synthesis of Ala-Gln in the prior art, α-amino acid acyltransferase is immobilized in the form of agarose embedding, and the preparation method is as follows: 1 g of enzyme cells are taken, resuspended in 10 mL of phosphate buffer, then mixed with 10 mL of 4% (w / v) agar solution instantly, and placed in a 55°C water bath for insulation. Then the mixture is slowly dripped into an ice water-oil immiscible solvent to form uniform spherical beads (about 3-4 mm in diameter), and the agar beads are washed to obtain agarose-embedded immobilized enzyme.

[0161] Compared with the above existing methods, the present invention selects LX1000HA selected in Example 2 as the enzyme immobilization carrier, uses PVA 31K Da as the immobilized α-amino acid acyltransferase with a modification group, and verifies the activity of the two prepared immobilized enzymes respectively. The reaction conditions refer to Table 1 in Example 2, and the detection is performed every 30 minutes for reaction tracking. The comparison results are shown in Table 12. The results show that the carrier-immobilized enzyme prepared in the present invention has a higher conversion rate than the immobilized cells obtained by agarose embedding.

[0162] Table 12:

[0163]

[0164] Example 11: Synthesis of Ac-DFSKLALKKA-NH2 by Immobilized Peptide Ligase

[0165] Usually, no activation step is required before preparing immobilized enzymes on epoxy carriers. However, the epoxy carriers in the present application are first modified with a polymer, and amino or hydroxyl groups are attached to the surface of the carrier. Therefore, in this example, the modified epoxy carriers are activated (the activation method is the same as in Example 1) or not activated before immobilizing the enzyme to compare the activity of the immobilized enzyme.

[0166] LX109S and ECR8285 were used as enzyme immobilization carriers, modified with different polymers (modification method was the same as in Example 1), and peptide ligase was used as the target enzyme for immobilization, the immobilization method was the same as in Example 1, the reaction system was shown in Table 5, and the comparison results were shown in Table 13. The results showed that the activity of the polymer-modified epoxy carrier was further improved if it was activated by glutaraldehyde before enzyme immobilization.

[0167] Table 13:

[0168]

[0169] Example 14: Synthesis of the tetrapeptide Val-Gly-Gly-Gly using different ester hydrochlorides as substrates

[0170] According to the results in Example 3, an immobilized α-amino acid acyltransferase using LX1000HA as an enzyme immobilization carrier and PEG 4K Da as a modification group was selected. Reaction conditions: L-valine methyl ester hydrochloride or L-valine ethyl ester hydrochloride or L-valine isopropyl ester hydrochloride: triglycine = 100: 50 mM, 0.1 M Tris-HCl, pH = 8.5, reaction temperature 20 ° C, sampling at different times, the results are shown in Table 14, the results show that after the substrate is changed from methyl ester hydrochloride to ethyl ester hydrochloride and isopropyl ester hydrochloride, the corresponding activity is also detected, proving that methyl ester hydrochloride can be replaced by other derivative esters.

[0171] Table 14:

[0172]

[0173] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects:

[0174] The α-amino acid acyltransferase or polypeptide ligase is fixed on an immobilized enzyme carrier modified with a long-chain polymer to obtain an immobilized enzyme for synthesizing oligopeptides or polypeptides. The immobilized enzyme synthesized by the present invention has the enzymes on the carrier surface dispersed evenly and at a certain distance, so that the synthesis reaction can be carried out stably and the decomposition rate is greatly reduced.

[0175] By combining the immobilized enzyme with a continuous reactor to synthesize oligopeptides or polypeptides, the product can be separated from the enzyme in time, so that the production capacity of the product can be maximized, and the method has the advantages of simple preparation, low production cost, low environmental pollution, and easy post-treatment. The preparation method of the immobilized enzyme of the present invention makes the enzyme uniformly dispersed and fixed on the surface of the carrier in small quantities, overcoming the disadvantage that the synthesis rate and decomposition rate of the product are too fast and it is difficult to control the product yield due to excessive contact between the substrate and the enzyme active site due to large-scale aggregation of the enzyme.

[0176] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An immobilized enzyme, characterized in that: The immobilized enzyme comprises an enzyme and a carrier for loading the enzyme; Wherein, the enzyme is α-amino acid acyltransferase or polypeptide ligase; The carrier is a polymethacrylate matrix carrier modified by a long-chain polymer; The polymethacrylate matrix carrier includes an amino type carrier or an epoxy type carrier; Wherein, the long-chain polymer includes polyethylene glycol M w =2~8 KDa, polyvinyl alcohol M w =1.7~31 KDa, polyethyleneimine M w =1.8~70 KDa, polyacrylamide M w =50~300 KDa or poly-lysine M w =1.6~41 KDa.

2. The immobilized enzyme according to claim 1, characterized in that The α-amino acid acyltransferase is derived from Pedobacter or Sphingobacterium; and the polypeptide ligase is derived from Bacillus subtilis.

3. The immobilized enzyme according to claim 2, characterized in that The amino acid sequence of the α-amino acid acyltransferase is selected from any one of the following: SEQ ID NOs: 1 to 10; The amino acid sequence of the polypeptide ligase is selected from any one of the following: SEQ ID NOs: 11-13.

4. A method for preparing an immobilized enzyme, characterized in that: The preparation method comprises: fixing the enzyme on a carrier loaded with the enzyme to obtain the immobilized enzyme; Wherein, the enzyme is α-amino acid acyltransferase or polypeptide ligase; Wherein, the carrier is a polymethacrylate matrix carrier modified by a long-chain polymer; The polymethacrylate matrix carrier includes an amino type carrier or an epoxy type carrier; Wherein, the long-chain polymer includes polyethylene glycol M w =2~8 KDa, polyvinyl alcohol M w =1.7~31 KDa, polyethyleneimine M w =1.8~70 KDa, polyacrylamide M w =50~300 KDa or poly-lysine M w =1.6~41 KDa.

5. The preparation method according to claim 4, characterized in that: The preparation method of the carrier comprises: The initial carrier is modified by the long-chain polymer to obtain a polymethacrylate matrix carrier modified by the long-chain polymer; Wherein, the initial carrier comprises a polymethacrylate matrix amino type carrier or a polymethacrylate matrix epoxy type carrier; The mass volume ratio of the initial carrier to the long-chain polymer is 1 g: (2-10) mL, wherein the volume concentration of the long-chain polymer is 5%-20%.

6. The preparation method according to claim 5, characterized in that: The carrier is an epoxy-type carrier of a polymethacrylate matrix modified by a long-chain polymer, and the preparation method comprises: The enzyme is mixed with the epoxy type carrier of the polymethacrylate matrix modified by the long-chain polymer to perform ring opening and fixation; The ionic strength of the enzyme buffer is 0.2~1M; The buffer of the enzyme is selected from any one or more of the following: phosphate, borate, citrate or tris(hydroxymethylaminomethane).

7. The preparation method according to claim 5, characterized in that: The carrier is an epoxy-type carrier of a polymethacrylate matrix modified by a long-chain polymer or an amino-type carrier of a polymethacrylate matrix modified by the long-chain polymer, and the preparation method comprises: activating the carrier to obtain an activated carrier; The enzyme is fixed on the activation carrier to obtain the immobilized enzyme.

8. The preparation method according to claim 7, characterized in that: The carrier is activated by a cross-linking agent to obtain the activated carrier, wherein the cross-linking agent includes glutaraldehyde, glyoxal, cyanuric chloride, genipin, epichlorohydrin or glycidyl ether or 1,4-butanediol glycidyl ether.

9. The preparation method according to claim 8, characterized in that: The mass volume ratio of the carrier and the cross-linking agent is 1 g: (2-10) mL, wherein the concentration of the cross-linking agent is 0.1%-5%.

10. The preparation method according to claim 7, characterized in that: The mass volume ratio of the activated carrier and the enzyme is 1 g: (2-10) mL, wherein the concentration of the enzyme is 10-50 mg / mL.

11. The preparation method according to any one of claims 4 to 10, characterized in that: The α-amino acid acyltransferase is derived from Pedobacter or Sphingobacterium; and the polypeptide ligase is derived from Bacillus subtilis.

12. The preparation method according to claim 11, characterized in that: The amino acid sequence of the α-amino acid acyltransferase is selected from any one of the following: SEQ ID NOs: 1 to 10; The amino acid sequence of the polypeptide ligase is selected from any one of the following: SEQ ID NOs: 11-13.

13. A method for synthesizing an oligopeptide or a polypeptide, characterized in that: The method comprises: synthesizing the oligopeptide or the polypeptide using the immobilized enzyme described in any one of claims 1 to 3 or the immobilized enzyme prepared by the preparation method of the immobilized enzyme described in any one of claims 4 to 12.

14. The method according to claim 13, characterized in that The method comprises: using the immobilized enzyme to catalyze a substrate to obtain the oligopeptide or the polypeptide; The substrate includes a first substrate for synthesizing the oligopeptide or a second substrate for synthesizing the polypeptide.

15. The method according to claim 14, characterized in that The first substrate includes a first acyl donor and a first acyl acceptor; The first acyl donor is an amino acid ester hydrochloride, and the amino acid ester hydrochloride is selected from any one of the following: amino acid methyl ester hydrochloride, amino acid ethyl ester hydrochloride or amino acid isopropyl ester hydrochloride; The first acyl acceptor includes amino acids or a third peptide segment; the third peptide segment is formed by condensation of 2 to 9 amino acids.

16. The method according to claim 14, characterized in that The second substrate includes a second acyl donor and a second acyl acceptor; The second acyl acceptor comprises an amino acid or a first peptide segment; the second acyl donor comprises a second peptide segment modified with carboxamide methyl ester; Wherein, the first peptide segment is formed by condensation of 2 to 98 amino acids; The second peptide segment is formed by condensation of 2 to 98 amino acids.

17. The method according to claim 14, characterized in that The catalytic reaction temperature is 10-25° C. and the pH is 8.0-9.

5.

18. Use of the immobilized enzyme according to any one of claims 1 to 3, the method for preparing the immobilized enzyme according to any one of claims 4 to 12, or the method for synthesizing an oligopeptide or polypeptide according to any one of claims 13 to 17 in synthesizing the oligopeptide or polypeptide.

19. The use according to claim 18, characterized in that The application comprises: arranging the immobilized enzyme in a continuous flow device to realize continuous synthesis of the oligopeptide or the polypeptide.

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