Immobilized enzyme, method for its preparation and use

Abstract

The application provides an immobilized enzyme, a preparation method and application thereof. The immobilized enzyme comprises an enzyme and a carrier for loading the enzyme; the enzyme is alpha-amino acid acylase or polypeptide ligase; the carrier is a polymethacrylate matrix carrier; the polymethacrylate matrix carrier comprises an amino type carrier or an epoxy type carrier; the amino type carrier is a long-chain polymer modified amino type carrier; the epoxy type carrier is a long-chain polymer modified epoxy type carrier; the long-chain polymer comprises polyethylene glycol M w =2~8 KDa, polyvinyl alcohol M w =1.7~31 KDa, polyethylene imine M w =1.8~70 KDa, polyacrylamide M w =50~300 KDa or polylysine M w =1.6~41 KDa. The immobilized enzyme of the application has good intermolecular dispersity and relatively large spacing, can efficiently catalyze the synthesis of oligopeptide or polypeptide from a substrate, and has high substrate conversion rate.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of oligopeptide and polypeptide synthesis, in particular, to an immobilized enzyme, its preparation method and application. BACKGROUND

[0002] Peptides composed of 2-10 amino acids are generally referred to as oligopeptides (small molecule peptides) in the literature, and peptides composed of 10-100 amino acids are referred to as 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 have the effects of promoting physical development, digestive system development, neurobehavioral development, skeletal development, immune system development, etc. In recent years, people's interest in polypeptides has grown, and there are currently about 60 approved polypeptide drugs on the market, in addition to about 100 more candidate drugs in different stages of clinical development. The importance of polypeptides has been recognized in the nutrition and cosmetics industries, etc.

[0003] Currently, 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 racemization risk of products, and more green and environmentally friendly. In the process of solid-phase synthesis, the amino acid substrate needs to go through the steps of protection and deprotection, resulting in a complex procedure, high production cost, and a large amount of organic waste.

[0004] The main routes for enzymatic synthesis of oligopeptides include using amino acid ester acyltransferase to catalyze the reaction of amino acid methyl ester with another amino acid or dipeptide / tripeptide to synthesize oligopeptides, or using amino acid ligase to catalyze the reaction of two amino acids or dipeptide / tripeptide to synthesize oligopeptides. However, the use of amino acid ester acyltransferase to synthesize oligopeptides cannot avoid hydrolysis, which reduces the yield of the product, and the use of amino acid ligase to synthesize oligopeptides has relatively low conversion rate and poor selectivity, with a large amount of self-ligation product.

[0005] The main routes for enzymatic synthesis of polypeptides include chemical-enzymatic coupling method, which further synthesizes polypeptides by catalysis of proteases. Since the synthesis of peptide bonds is thermodynamically unfavorable in the presence of water, the use of pure organic solvents with very low water activity can be beneficial for peptide coupling. However, proteases exhibit very low activity in pure organic solvents, and peptide substrates are often difficult to dissolve when the side chain (part) is protected. Another method is to perform coupling reactions in water using some effective ligases known in nature, but they require specific recognition sequences. Another method is to use substrate mimics and proteases, however, the synthesis of high-activity peptide esters required is challenging, and some amino acids may undergo side reactions such as Cys, Met, Trp, His and Tyr.

[0006] At present, the oligopeptide and polypeptide are mainly synthesized by connecting the alpha-amino acid ester acyltransferase and polypeptide ligase, which not only has the synthesis function, but also has the hydrolysis function. If the product is not separated from the enzyme in time, the product will be hydrolyzed, resulting in the reduction of yield. The free enzyme reaction cannot separate the product from the enzyme in time, and can only terminate the reaction by adjusting the acid and alkali, and the emulsification phenomenon will occur to a certain extent in the post-processing process, resulting in the difficulty in post-processing. The continuous reaction of the immobilized enzyme can realize the separation of the enzyme and the reaction substrate in time, and has the advantages of saving labor, improving productivity and simplifying the production process. Therefore, how to develop an immobilized alpha-amino acid ester acyltransferase and polypeptide ligase for efficiently synthesizing oligopeptide or polypeptide is crucial. SUMMARY

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

[0008] In order to achieve the above-mentioned purpose, according to the first aspect of the present application, an immobilized enzyme is provided, which comprises an enzyme and a carrier for loading the enzyme; wherein the enzyme is alpha-amino acid ester acyltransferase or polypeptide ligase; the carrier is a long-chain polymer modified polymethacrylate matrix carrier; 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, polyethylene imine Mw=1.8~70 KDa, polyacrylamide Mw=50~300 KDa or polylysine Mw=1.6~41 KDa.

[0009] Further, the alpha-amino acid ester acyltransferase is derived from Pedobacter or Sphingobacterium; the polypeptide ligase is derived from Bacillus subtilis.

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

[0011] In order to achieve the above-mentioned purpose, according to the second aspect of the present application, a preparation method of the immobilized enzyme is provided, which comprises: fixing the enzyme on the carrier for loading the enzyme to obtain the immobilized enzyme; wherein the enzyme is alpha-amino acid ester acyltransferase or polypeptide ligase; wherein the carrier is a long-chain polymer modified polymethacrylate matrix carrier; 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, polyethylene imine Mw=1.8~70 KDa, polyacrylamide Mw=50~300 KDa or polylysine Mw=1.6~41 KDa. w= 2~8 KDa, polyvinyl alcohol M w = 1.7~31 KDa, polyethylene imine M w = 1.8~70 KDa, polyacrylamide M w = 50~300 KDa or polylysine M w = 1.6~41 KDa.

[0012] Further, the preparation method of the carrier comprises: modifying an initial carrier by using a long-chain polymer to obtain a long-chain polymer modified polymethacrylate matrix carrier; 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%.

[0013] Further, the carrier is a long-chain polymer modified polymethacrylate matrix epoxy-type carrier, and the preparation method comprises: mixing the enzyme and the long-chain polymer modified polymethacrylate matrix epoxy-type carrier to perform ring opening and fixation; the ionic strength of the buffer solution of the enzyme is 0.2-1 M; the buffer solution of the enzyme is selected from any one or more of the following: phosphate, borate, citrate or tris-hydroxymethyl aminomethane.

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

[0015] Further, the carrier is a long-chain polymer modified polymethacrylate matrix epoxy-type carrier or a long-chain polymer modified polymethacrylate matrix amino-type carrier, and the preparation method comprises: activating the carrier to obtain an activated carrier; and fixing the enzyme on the activated carrier to obtain an immobilized enzyme.

[0016] Further, 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%.

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

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

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

[0020] To achieve the above object, according to a third aspect of the present application, there is provided a method for synthesizing an oligopeptide or a polypeptide, comprising: synthesizing the oligopeptide or the polypeptide by using the immobilized enzyme or the immobilized enzyme prepared by the preparation method of the immobilized enzyme.

[0021] Further, the method comprises: catalyzing the substrate by the immobilized enzyme to obtain the oligopeptide or the polypeptide; and the substrate comprises a first substrate for synthesizing the oligopeptide or a second substrate for synthesizing the polypeptide.

[0022] Further, the first substrate comprises a first acyl donor and a first acyl acceptor; the first acyl donor is an ester hydrochloride of an amino acid, which is selected from any one of a methyl ester hydrochloride of an amino acid, an ethyl ester hydrochloride of an amino acid or an isopropyl ester hydrochloride of an amino acid; and the first acyl acceptor comprises an amino acid or a third peptide segment; the third peptide segment is condensed by 2-9 amino acids.

[0023] Further, the second substrate comprises a second acyl donor and a second acyl acceptor; the second acyl acceptor comprises an amino acid or a first peptide segment; and the second acyl donor comprises a second peptide segment modified by carboxamide methyl ester; wherein the first peptide segment is condensed by 2-98 amino acids; and the second peptide segment is condensed by 2-98 amino acids.

[0024] Further, the reaction temperature for catalysis is 10-25°C, and the pH is 8.0-9.5.

[0025] To achieve the above object, according to a fourth aspect of the present application, there is provided an application of the immobilized enzyme or the preparation method of the immobilized enzyme or the method for synthesizing the oligopeptide or the polypeptide in synthesizing the oligopeptide or the polypeptide.

[0026] Further, the application comprises: arranging the immobilized enzyme in a continuous flow device to realize continuous synthesis of the oligopeptide or the polypeptide.

[0027] By applying the technical solution of this invention, α-amino acid acyltransferases or polypeptide ligases are immobilized on long-chain polymer-modified amino-type polymethacrylate matrix supports or long-chain polymer-modified epoxy-type polymethacrylate matrix supports to obtain immobilized enzymes for synthesizing oligopeptides or polypeptides. The immobilized enzymes of this invention exhibit good intermolecular dispersion and relatively large spacing, enabling efficient catalysis of substrate synthesis into oligopeptides or polypeptides with high substrate conversion rates. Furthermore, the immobilized enzymes of this invention allow for immediate separation of the enzyme from the reaction products (synthesized oligopeptides or polypeptides), preventing over-reaction and subsequent hydrolysis of the reaction products, thereby maximizing production capacity. Attached Figure Description

[0028] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0029] Figure 1 The apparatus shown in the embodiments of the present invention is a continuous reaction apparatus in which the substrate is fed into the column via a single-channel premixed system. Figure 1 In this context, A represents the reaction system. Figure 1 B in the text stands for peristaltic pump. Figure 1 C in the text represents the reaction column. Figure 1 In this context, D represents the collection system. Detailed Implementation

[0030] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0031] As mentioned in the background section, enzymatic synthesis of oligopeptides or polypeptides in existing technologies has advantages such as simple steps, low production cost, no risk of product racemization, and being more environmentally friendly. However, free enzymes cannot achieve immediate separation of the product (synthesized oligopeptide or polypeptide) from the enzyme; the reaction can only be terminated by adjusting the acid or base. Furthermore, the presence of free enzymes during post-processing can lead to emulsification, making post-processing difficult. In this invention, the inventors attempted to screen an immobilized enzyme carrier with high conversion rate, immobilizing it with α-amino acid acyltransferases or polypeptide ligases to achieve immediate separation of the reaction product and enzyme while maintaining a high conversion rate. Therefore, a series of protection schemes for this invention have been proposed.

[0032] In a first typical embodiment of the present invention, an immobilized enzyme is provided, comprising an enzyme and a carrier for loading the enzyme; wherein the enzyme is an α-amino acid acyltransferase or a 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=1.8~70 kDa, 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 achieve immediate separation of immobilized enzyme and reaction product, thereby effectively reducing the occurrence of hydrolysis reaction and simplifying the post-processing, but also efficiently convert substrates into oligopeptides or polypeptides, with the beneficial effects of simple operation and high efficiency.

[0034] In a preferred embodiment of the present invention, the above-mentioned amino acid acyltransferase is derived from Pedobacter or Sphingobacterium; the above-mentioned polypeptide ligase is derived from Bacillus subtilis. In a more preferred embodiment of the present invention, the amino acid sequence of the above-mentioned α-amino acid acyltransferase is selected from any of the following: SEQ ID NOs: 1-10; the amino acid sequence of the above-mentioned polypeptide ligase is selected from any of the following: SEQ ID NOs: 11-13. The immobilized enzyme prepared using the above-mentioned amino acid acyltransferase and the above-mentioned 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 method includes: immobilizing an enzyme on a carrier to obtain an immobilized enzyme; wherein the enzyme is an α-amino acid acyltransferase or a polypeptide ligase; wherein 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 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 polylysine M w =1.6~41 KDa.

[0036] The immobilized enzyme prepared by the method of the present invention has good intermolecular dispersion and relatively large spacing, and can efficiently catalyze the synthesis of oligopeptides or polypeptides from substrates.

[0037] In a preferred embodiment of the present application, the preparation method of the carrier comprises: modifying the initial carrier with long-chain polymers to obtain a long-chain polymer modified polymethacrylate matrix carrier; 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, and the volume concentration of the long-chain polymer is 5%-20%. In a more preferred embodiment of the present application, the mass-volume ratio of the initial carrier to the long-chain polymer is 1 g:6 mL, and the concentration of the long-chain polymer is 5%-10%. The use of the above parameters is more conducive to the coating of the long-chain polymer on the carrier.

[0038] The long-chain polymer is combined with the amino-type carrier or the epoxy-type carrier respectively to form a polymer coating on the surface of the carrier by hydrogen bonding or adsorption. In a preferred embodiment of the present application, polyethylene glycol M w =2-8 KDa or polyvinyl alcohol M w =1.7-31 KDa is used to modify the initial carrier. Such long-chain polymers form a polymer coating on the surface of the carrier by hydrogen bonding or adsorption, which can provide more enzyme immobilization sites, make the enzyme molecules uniformly 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, and make the enzyme molecules more easily accessible to the substrate, thereby improving the activity of the immobilized enzyme.

[0039] In a preferred embodiment of the present application, the carrier is a long-chain polymer modified polymethacrylate matrix epoxy-type carrier, and the preparation method comprises: placing the long-chain polymer modified polymethacrylate matrix epoxy-type carrier in an enzyme solution to perform epoxy ring opening and enzyme immobilization, thereby obtaining the immobilized enzyme; wherein the enzyme solution contains salt ions with an ionic strength of 0.2-1 M; and 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 be opened in one step and covalently combined with the amino group on the enzyme to immobilize the enzyme in the enzyme solution containing 0.2-1 M salt ions.

[0041] In addition, the modified epoxy-type carrier can also be activated in the same way as the amino-type carrier to immobilize the enzyme under the action of an activating agent. For example, when the activating agent is glutaraldehyde, the amino group or the hydroxyl group coated on the modified epoxy-type carrier is connected to the aldehyde group at one end of the glutaraldehyde, and the aldehyde group at the other end of the 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 application, the carrier is an epoxy-type carrier of long-chain polymer-modified polymethacrylate matrix or an amino-type carrier of long-chain polymer-modified polymethacrylate matrix, and the preparation method comprises: activating the carrier to obtain an activated carrier; and immobilizing the enzyme on the activated carrier to obtain the immobilized enzyme.

[0043] In a preferred embodiment of the present application, the carrier is activated by using a cross-linking agent to obtain an activated carrier, wherein the cross-linking agent comprises glutaraldehyde, glyoxal, trichloro cyanogen, genipin, epichlorohydrin or glycidyl ether or 1,4-butanediol glycidyl ether.

[0044] The cross-linking agent is connected with the carrier and the enzyme respectively to immobilize the enzyme on the carrier. In a preferred embodiment of the present application, the carrier is activated by using 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 with the aldehyde group at one end of the glutaraldehyde, and after removing the excess unconnected glutaraldehyde, the enzyme solution (protein) is mixed, the amino group on the enzyme solution is connected with the aldehyde group at the other end of the glutaraldehyde, and after removing the unbound protein, the enzyme can be immobilized on the carrier through covalent action to improve 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 the activated carrier. In a preferred embodiment of the present application, 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%. Using the above mass-volume ratio is more conducive to cross-linking of the cross-linking agent and the carrier.

[0046] The mass-volume ratio of the activated carrier to the enzyme can affect the preparation efficiency of the immobilized enzyme. In a preferred embodiment of the present application, the mass-volume ratio of the activated carrier to the enzyme is 1 g: (2-10) mL, wherein the concentration of the enzyme is 10-50 mg / mL. In a more preferred embodiment of the present application, the volume ratio of the activated carrier to the enzyme is 1 g: 4 mL, wherein the concentration of the enzyme is 5-10 mg / mL. Using the above ratio is conducive to improving the preparation efficiency of the immobilized enzyme and efficiently immobilizing the enzyme on the carrier.

[0047] The purpose of stirring is to make the reaction more thorough. In a preferred embodiment of the present application, 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 application, the first stirring is performed for 6-24 h at 10-30 °C, the second stirring is performed for 0.5-4 h at 10-30 °C, and the third stirring is performed for 16-24 h at 10-30 °C. The stirring helps the initial carrier to be fully connected with the long-chain polymer, the carrier to be fully connected with the cross-linking agent, and the activated carrier to be fully connected with the enzyme solution, thereby improving the preparation efficiency of the immobilized enzyme and allowing the enzyme to be fully combined with the carrier.

[0049] In a preferred embodiment of the present application, the initial carrier is washed for the first time before being modified by the long-chain polymer. The first washing is performed to remove the impurities or organic solvents remaining on the initial carrier, thereby reducing the influence of the impurities or organic solvents on the modification.

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

[0051] In a preferred embodiment of the present application, the activated carrier is washed for the third time before the enzyme is immobilized on the activated carrier. The third washing is performed to remove the excess unbound cross-linking agent.

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

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

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

[0055] In a third typical embodiment of the present application, a method for synthesizing an oligopeptide or a polypeptide is provided, which comprises: synthesizing the oligopeptide or the polypeptide by using the immobilized enzyme or the immobilized enzyme prepared by the preparation method.

[0056] The use of this method to synthesize polypeptides or oligopeptides can separate the enzyme solution and the reaction product (polypeptides or oligopeptides) in real time, prevent excessive hydrolysis of the reaction product, and result in low conversion of the reaction product. In addition, this method does not require the addition of acid or base to terminate the reaction, thereby simplifying the post-processing and reaction process between batches.

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

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

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

[0060] When the above immobilized enzyme and the above second substrate undergo a catalytic reaction, the above polypeptide is generated. The above second substrate comprises a second acyl donor and a second acyl acceptor. The above second acyl acceptor comprises an amino acid or a first peptide segment; the above second acyl donor comprises a carboxamide methyl ester modified second peptide segment; the above first peptide segment is condensed from 2 to 98 amino acids; and the above second peptide segment is condensed from 2 to 98 amino acids.

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

[0062] It should be noted that the polypeptide mentioned herein refers to a peptide composed of 10-100 amino acid units, and the polypeptide is a peptide segment synthesized after a peptide bond is formed between two peptide segments under the action of a polypeptide ligase. The same polypeptide can be synthesized by different groups of peptide segments, for example, when synthesizing a tridecapeptide, the acyl donor is a undecapeptide, the acyl acceptor is a dipeptide, or the acyl donor is a decapeptide, the acyl acceptor is a tripeptide, or the acyl donor is a nonapeptide, the acyl acceptor is a tetrapeptide, and so on.

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

[0064] The optimal catalytic temperature and the optimal catalytic pH of different enzymes are different. In a preferred embodiment of the present application, the temperature for performing the catalytic reaction described above is 10-25°C, and the pH of the reaction is 8.0-9.5. Performing the catalytic reaction under this condition has the beneficial effect of high catalytic efficiency.

[0065] In a fourth typical embodiment of the present application, the application of the immobilized enzyme described above or the preparation method of the immobilized enzyme described above or the method for synthesizing the oligopeptide or the polypeptide described above in the synthesis of the oligopeptide or the polypeptide described above is provided. In a preferred embodiment of the present application, the application comprises arranging the immobilized enzyme described above in a continuous flow device to realize the continuous synthesis of the oligopeptide or the polypeptide described above.

[0066] In a preferred embodiment of the present application, the continuous flow device comprises a pump, a reaction system and a reaction column. In a more preferred embodiment of the present application, the continuous flow device further comprises a pH self-control instrument and a mixer.

[0067] In a preferred embodiment of the present application, the reaction system comprises a substrate for synthesizing an oligopeptide or a polypeptide; and the pH of the reaction system is 8.0-9.5. In a more preferred embodiment, the reaction system comprises a substrate for synthesizing an oligopeptide or a polypeptide; and the pH of the reaction system is 5.5-7.0.

[0068] Since the substrate such as methyl ester is prone to self-hydrolysis at a reaction pH of 8.0-9.5, the conversion rate gradually decreases. The stability of the substrate such as methyl ester is greatly improved at a pH of 5.5-7.0. Therefore, the reaction system with a pH of 5.5-7.0 is first introduced into a mixer connected with a pH self-controller, and the pH is adjusted to 8.0-9.5 by the pH self-controller, and then introduced into the reaction column. This method can maintain a high and stable conversion rate.

[0069] In a preferred embodiment of the present application, the reaction column is filled with the immobilized enzyme for synthesizing an oligopeptide or a polypeptide. In a preferred embodiment of the present application, the pump comprises one or more of a micro-ceramic pump, a high-performance liquid pump, or a syringe pump. In a preferred embodiment of the present application, the mixer is selected from any one of a mechanical stirring mixer, an air flow mixer, a high-shear mixer, or a static mixer. The continuous flow device has the beneficial effect of high efficiency in catalytic synthesis of an oligopeptide or a polypeptide.

[0070] The present application will be further described in detail below with reference to specific examples, which should not be construed as limiting the scope of 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 is first washed with 20 mM PB (pH 7.0) for 2 times, then 6 volumes of 10% long-chain polymer solution relative to the mass of the carrier is added, including PEG 4K Da, PVA 2.4K Da, PEI 10K Da, PAM 2000KDa, and PL 8.2K Da, stirred at 20°C for 16 h, then washed with 20 mM PB (pH 7.0) for 3 times, and the solid is collected by filtration to obtain the modified immobilized enzyme carrier;

[0074] b. Carrier activation: The modified immobilized enzyme carrier is added with 4 volumes of 0.5% glutaraldehyde solution relative to the mass of the carrier, stirred at 20°C for 1 h, then washed with 20 mM PB (pH 7.0) for 3 times, and the solid is collected by filtration to obtain the activated immobilized enzyme carrier;

[0075] c. Enzyme immobilization: 4 volumes of diluted enzyme solution with a protein concentration of about 30 mg / mL were added to the activated carrier bottle, and the diluted enzyme solution used Buffer was 20 mM PB (pH7.0), which was stirred at 20°C for 24 h. Then, the immobilized α-amino acid acylase was obtained by washing the immobilized enzyme carrier with 4 volumes of 20 mM PB (pH7.0) for 3 times.

[0076] Epoxy carrier modification and immobilization method:

[0077] a. Carrier modification: The epoxy carrier was first washed with 20 mM PB (pH7.0) for 2 times, then 6 volumes of 10% long-chain polymer solution was added, including PEG 4K Da, PVA 2.4K Da, PEI 10K Da, PAM2000K Da and PL 8.2K Da, which was stirred at 20°C for 16 h. Then, the modified immobilized enzyme carrier was obtained by washing with 20 mM PB (pH7.0) for 3 times, filtering and collecting the solid.

[0078] b. Carrier activation: The modified immobilized enzyme carrier was added with 4 volumes of 0.5% glutaraldehyde solution, which was stirred at 20°C for 1 h. Then, the activated immobilized enzyme carrier was obtained by washing with 20 mM PB (pH7.0) for 3 times, filtering and collecting the solid.

[0079] c. Enzyme immobilization: 4 volumes of diluted enzyme solution with a protein concentration of about 30 mg / mL were added to the activated carrier bottle, and the diluted enzyme solution used Buffer was 0.5 M PB (pH7.0), which was stirred at 20°C for 24 h. Then, the immobilized α-amino acid acylase was obtained by washing the immobilized enzyme carrier with 4 volumes of 20 mM PB (pH7.0) for 3 times.

[0080] Example 2: Immobilized enzyme carrier with different polymer modifications for synthesizing dipeptide by immobilizing α-amino acid acylase

[0081] The amino resin, epoxy resin, amino resin with different polymer modifications and epoxy resin with different polymer modifications were selected for immobilization of α-amino acid acylase as the target enzyme. The polymer modification carrier method, carrier activation method and enzyme immobilization method of this embodiment were the same as those of Example 1. In addition, the epoxy resin and the epoxy resin with different polymer modifications in this embodiment did not undergo the carrier activation step, and the ring opening and covalent binding of the enzyme were carried out in 0.5 M PB (pH7.0).

[0082] The reaction conditions are shown in Table 1, and the reaction results are shown in Table 2-1, Table 2-2, Table 2-3 and Table 2-4. The results show that the catalytic activity of the immobilized enzyme on the amino or epoxy carrier modified by polyethylene glycol Mw=2-8 KDa, polyvinyl alcohol Mw=1.7-31 KDa, polyethylene imine Mw=1.8-70 KDa, polyacrylamide Mw=50-300 KDa or polylysine Mw=1.6-41 KDa is generally higher than that of the immobilized enzyme on the unmodified carrier.

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

[0084] It should be noted that in addition to Table 2-5 of the present embodiment, the amino acid sequences of the α-amino acid acyltransferases used in the remaining embodiments of the present application and Tables 2-1, 2-2, 2-3 and 2-4 of the present embodiment are all SEQ ID NO: 1.

[0085] According to the HPLC detection results, the calculation formula of the conversion rate of all embodiments in the present application is: conversion rate=product peak area ratio% / (product peak area ratio%+amino acid or dipeptide acyl acceptor peak area ratio%). In addition, due to the difference in structure or molecular weight between the product and the acyl acceptor, the peak area of the same amount of substance of the product and the acyl acceptor is not completely consistent, that is, the absorption is not consistent. Generally, the absorption ratio of the same amount of substance is detected, and then the absorption ratio is calculated to calculate the conversion rate.

[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 are purchased from Blue Sky Bio, ECR8409 and ECR8285 are purchased from Proligo, and the above carriers are polymethacrylate matrix carriers.

[0097] Table 2-5:

[0098]

[0099] Example 3: Immobilization of α-amino acid acylase for synthesis of tripeptide or tetrapeptide by different polymer-modified enzyme carrier

[0100] Taking α-amino acid acylase as the target enzyme, amino resin, epoxy resin, different polymer-modified amino resin, and different polymer-modified epoxy resin were selected for immobilization. The method of using polymer-modified carrier, carrier activation method, and enzyme immobilization method in this example are the same as those in Example 1. In addition, the epoxy resin and different polymer-modified epoxy resin in this example do not undergo the carrier activation step, and the ring opening and covalent binding of the enzyme are carried out in 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 Tables 4-1, 4-2, 4-3, and 4-4. The results show that the immobilized enzyme activity of the amino or epoxy carrier modified by 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 is generally higher than that of the carrier immobilized enzyme without modification.

[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 are purchased from Blue Sky Bio, ECR8409 and ECR8285 are purchased from Proligo, and the above carriers are polymethacrylate matrix carriers.

[0113] Example 4: Immobilization of polypeptide ligase on different polymer-modified immobilized enzyme carriers to synthesize polypeptides

[0114] Taking polypeptide ligase as the target enzyme, amino resin, epoxy resin, different polymer-modified amino resin, and different polymer-modified epoxy resin were selected for immobilization. The method of using polymer-modified carriers, the carrier activation method, and the enzyme immobilization method in this embodiment are the same as those in Example 1. In addition, the epoxy resin and the different polymer-modified epoxy resin in this embodiment do not undergo the carrier activation step, and the ring opening and covalent binding of the enzyme are carried out in 0.5 M Tris-HCl (pH 8.0).

[0115] Taking the synthesis of polypeptides 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 Tables 6-1, 6-2, 6-3, and 6-4. The results show that the immobilized enzyme activity of the amino or epoxy carrier 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 is generally higher than that of the immobilized enzyme of the unmodified carrier. In the substrate of the present application, -OCam represents C-terminal carboxamide methyl ester protection; Ac- represents N-terminal acetyl protection; -NH2 represents C-terminal amino modification; and H- represents N-terminal amino group without protection.

[0116] Different polypeptide ligases with different amino acid sequences were immobilized with the same carrier (4 KDa PEG-modified LX1000HA) to prepare immobilized enzymes. The immobilized enzyme was used to synthesize Ac-DLSKQALKKA-NH2, and the reaction conditions are shown in Table 5, and the experimental results are shown in Table 6-5.

[0117] It should be noted that in addition to Table 6-5 of this embodiment, the amino acid sequences of the polypeptide ligases used in the remaining embodiments of the present application and Tables 6-1, 6-2, 6-3, and 6-4 of this embodiment 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 are purchased from Blue Sky Bio, ECR8409 and ECR8285 are purchased from Proligo, and the above carriers are polymethacrylate matrix carriers.

[0129] Table 6-5:

[0130]

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

[0132] According to the results in Example 2, the immobilized α-amino acid lipoacyltransferase with LX1000HA as the enzyme immobilization carrier and PEG 8K Da as the modification group was selected to run a continuous reaction, and the reaction conditions are shown in Table 1. The prepared immobilized enzyme was added to a 1 mL reaction column (C in Figure 1 ), about 0.75 g of immobilized enzyme was filled, and the reaction system (A in Figure 1 ) was continuously passed through the reaction column at 20°C by the action of a peristaltic pump (B in Figure 1 ), and the reaction product was collected in a collection system (D in Figure 1 ). The retention time was set to 0.5-5 min, and samples were taken every 10 min for detection to determine the preferred retention time.

[0133] The sample was detected by HPLC, and the results showed that the product conversion rate reached more than 80%, and the continuous operation could be carried out for about 24 h without significant decrease in conversion rate, but since L-threonine methyl ester hydrochloride would self-hydrolyze at the reaction pH, the system needed to be reconfigured every 1-2 h. Compared with free enzyme, the production capacity and other information are shown in Table 7, and the experimental results showed that the production capacity of the immobilized enzyme was higher than that of the free enzyme, and the use of the enzyme was more economical.

[0134] Procedure of batch reaction of free enzyme: L-threonine methyl ester hydrochloride: phenylalanine = 75: 50 mM, 0.1 mM Tris-HCl, pH = 9.5, 0.03 wt of free enzyme was added (relative to amino acid phenylalanine), then the reaction progress was tracked, and sample was taken every 0.5 h for detection.

[0135] Table 7:

[0136]

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

[0138] According to the results in Example 3, the immobilized α-amino acid lipoyl transferase with LX1000HA as the enzyme immobilization carrier and PL 41KDa as the modification group was selected to run a continuous reaction, and 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 immobilized enzyme was filled, and the reaction system was continuously passed through the reaction column (reaction device as shown in Figure 1 The preferred retention time was determined by setting the retention time to 1-10 min, and taking samples every 20 min for detection.

[0139] The sample was detected by HPLC, and the results showed that the product conversion rate reached more than 70%, and the continuous operation could be performed for about 34 h without significant reduction in conversion rate. However, since L-valine methyl ester hydrochloride would self-hydrolyze at the reaction pH, the system needed to be reconfigured every 1-2 h. 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 acyl acceptor) was added, and then the reaction progress was tracked, and sample was taken every 0.5 h for detection.

[0141] Table 8:

[0142]

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

[0144] According to the results in Example 3, the immobilized α-amino acid acylase with LX1000HA as the enzyme immobilization carrier and PEI 70KDa as the modification group was selected to run a continuous reaction, and 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 immobilized enzyme was filled, and the reaction system was continuously passed through the reaction column (the reaction device is shown in Figure 1 Table 1) at 10°C by the action of a peristaltic pump, the retention time was set to 1-10 min, and samples were taken every 20 min for detection to determine the preferred retention time.

[0145] HPLC detection of the sample found that the product conversion rate reached more than 90%, and the continuous operation could be performed for about 30 h without significant reduction in conversion rate, but since L-valine methyl ester hydrochloride would self-hydrolyze at the reaction pH, the system needed to be reconfigured every 1-2 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, and 0.05wt 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 by immobilized polypeptide ligase

[0150] According to the results in Example 4, the immobilized polypeptide ligase with LX1000HA as the enzyme immobilization carrier and PEG 4KDa as the modification group was selected to run a continuous reaction, and 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 immobilized enzyme was filled, and the reaction system was continuously passed through the reaction column (the reaction device is shown in Figure 1 Table 2) at 25°C by the action of a peristaltic pump, the retention time was set to 0.5-5 min, and samples were taken every 10 min for detection to determine the preferred retention time. HPLC detection of the sample found that the product conversion rate reached more than 79%, and 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 0.05wt 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, the immobilized polypeptide ligase with LX1000HA as the enzyme immobilization carrier and PL 1.6K Da as the modification group was selected to run the continuous reaction, and 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 immobilized enzyme was filled, and the reaction system was continuously passed through the reaction column (the reaction device is shown in Figure 1 The sample was detected by HPLC, and it was found that the product conversion rate reached more than 50%, and 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 0.05wt of free enzyme (relative to the acyl acceptor) is added, and then the reaction process is tracked every 0.5 h to take a sample for detection.

[0157] Table 11:

[0158]

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

[0160] Referring to the method for continuous synthesis of Ala-Gln in the prior art, the α-amino acid lipoyl transferase is immobilized in the form of agarose embedding, and the preparation method is as follows: 1 g of enzyme cells is resuspended in 10 mL of phosphate buffer, then mixed with 10 mL of 4% (w / v) agar solution, and placed in a 55°C water bath for incubation. Then the mixture is slowly dropped into 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 prior art method, LX1000HA selected in Example 2 is used as the enzyme immobilization carrier, and PVA 31K Da is used as the modification group of the immobilized α-amino acid lipoyl transferase, and the prepared two kinds of immobilized enzymes are respectively verified for activity, and the reaction conditions are referred to Table 1 in Example 2, and detection is performed every 30 min for reaction tracking. The comparison results are shown in Table 12. The results show that the carrier immobilized enzyme prepared in the present application has higher conversion rate compared with the immobilized cells obtained in the form of agarose embedding.

[0162] Table 12:

[0163]

[0164] Example 11: Synthesis of Ac-DFSKLALKKA-NH2 by immobilized polypeptide ligase

[0165] Generally, no activation step is needed before preparing immobilized enzyme with epoxy carrier, but the epoxy carrier in this application is first modified with polymer, and the carrier surface will be attached with groups containing amino or hydroxyl groups, so this example uses the modified epoxy carrier, activated (activation method is the same as example 1) or not activated, and then the enzyme is immobilized, and the activity of the immobilized enzyme is compared.

[0166] LX109S and ECR8285 are used as enzyme immobilization carriers, modified with different polymers (modification method is the same as example 1), and polypeptide ligase is used as the target enzyme for immobilization, the immobilization method is the same as example 1, and the reaction system is shown in table 5, and the comparison results are shown in table 13. The results show that the activity of the enzyme immobilized on the epoxy carrier modified with polymer will be further improved after activation by glutaraldehyde.

[0167] Table 13:

[0168]

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

[0170] According to the results in example 3, LX1000HA is selected as the enzyme immobilization carrier, and PEG 4K Da is selected as the modification group of immobilized α-amino acid acylase. 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℃, sample at different times, and the results are shown in table 14. The results show that after changing the substrate from methyl ester hydrochloride to ethyl ester hydrochloride and isopropyl ester hydrochloride, the corresponding activity is detected, which proves 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-mentioned examples of the present application achieve the following technical effects:

[0174] The α-amino acid acyltransferase or polypeptide ligase is fixed on the long-chain polymer modified immobilized enzyme carrier to obtain the immobilized enzyme for synthesizing oligopeptide or polypeptide. The synthesized immobilized enzyme of the present application has the enzyme evenly dispersed on the surface of the carrier with a certain interval, which can make the synthesis reaction stably proceed, and the decomposition rate is greatly reduced.

[0175] The immobilized enzyme is combined with the continuous reactor to synthesize oligopeptide or polypeptide, the product can be separated from the enzyme in time, the product yield reaches the maximum, and the preparation is simple, the production cost is low, the environmental pollution is small, and the post-processing is easy. The preparation method of the immobilized enzyme of the present application makes the enzyme evenly dispersed and fixed on the surface of the carrier in a small amount, which overcomes the defects that the product synthesis rate and decomposition rate are too fast to control the product yield due to the too much contact between the substrate and the enzyme active site caused by the large amount of enzyme aggregation.

[0176] The above only describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for the preparation of an immobilized enzyme, characterized by, The preparation method is selected from: 1) activating the carrier with glutaraldehyde to obtain an activated carrier; fixing the enzyme on the activated carrier to obtain the immobilized enzyme; or 2) mixing the enzyme and the carrier to perform ring opening and fixation; wherein the ionic strength of the buffer solution of the enzyme is 0.2-1 M; wherein the buffer solution of the enzyme is selected from any one or more of the following: phosphate, borate, citrate or tris; wherein the enzyme is an α-amino acid acyltransferase or a polypeptide ligase; wherein the preparation method of the carrier comprises: modifying an initial carrier with a long-chain polymer to obtain the carrier; wherein the long chain polymer is polyethylene glycol M w = 2-8 KDa, polyvinyl alcohol M w = 1.7-31 KDa, polyethylene imine M w = 1.8-70 KDa, polyacrylamide M w = 50-300 KDa or polylysine M w = 1.6-41 Kda; 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%; wherein the initial carrier is a polymethacrylate matrix amino-type carrier or a polymethacrylate matrix epoxy-type carrier; wherein the polymethacrylate matrix amino-type carrier is selected from any one of the following: LX1000HA or ECR8409; and the polymethacrylate matrix epoxy-type carrier is selected from any one of the following: LX109S or ECR8285; when the initial carrier is LX1000HA or ECR8409, the preparation method is selected from 1); when the initial carrier is LX109S or ECR8285, the preparation method is selected from 1) or 2).

2. The production method according to claim 1, characterized by, The mass-volume ratio of the carrier to the glutaraldehyde is 1 g:(2-10) mL, wherein the concentration of the glutaraldehyde is 0.1%-5%.

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

4. The production method according to any one of claims 1 to 3, characterized by, The alpha-amino acid acyltransferase is derived from Pedobacter or Sphingobacterium ; the polypeptide ligase is derived from Bacillus subtilis .

5. The preparation method according to claim 4, characterized in that, The amino acid sequence of the α-amino acid acyltransferase is selected from any one of the following: SEQ ID NOs: 1-10; The amino acid sequence of the polypeptide ligase is selected from any one of the following: SEQ ID NOs: 11-13.

6. An immobilized enzyme prepared by the preparation method of any one of claims 1-5.

7. A method of synthesizing an oligopeptide or polypeptide, characterized by, The method comprises: synthesizing the oligopeptide or the polypeptide by using the immobilized enzyme prepared by the preparation method of any one of claims 1-5 or the immobilized enzyme of claim 6.

8. The method of claim 7, wherein, The method comprises: catalyzing a substrate by using the immobilized enzyme 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.

9. The method of claim 8, wherein, The first substrate comprises a first acyl donor and a first acyl acceptor; The first acyl donor is an ester hydrochloride of an amino acid, which is selected from any one of the following: a methyl ester hydrochloride of an amino acid, an ethyl ester hydrochloride of an amino acid or an isopropyl ester hydrochloride of an amino acid; The first acyl acceptor comprises an amino acid or a third peptide segment; and the third peptide segment is condensed from 2-9 amino acids.

10. The method of claim 8, wherein, The second substrate comprises a second acyl donor and a second acyl acceptor; The second acyl acceptor comprises an amino acid or a first peptide segment; and the second acyl donor comprises a carboxyamidomethyl-modified second peptide segment. The first peptide segment is condensed from 2 to 98 amino acids. The second peptide segment is condensed from 2 to 98 amino acids.

11. The method of claim 8, wherein, The catalyzed reaction is carried out at a temperature of 10 to 25°C and a pH of 8.0 to 9.

5.

12. Use of the method for preparing an immobilized enzyme according to any one of claims 1-5 or the immobilized enzyme according to claim 6 or the method for synthesizing an oligopeptide or polypeptide according to any one of claims 7-11 in synthesizing the oligopeptide or the polypeptide.

13. Use according to claim 12, characterized in that, The use comprises: disposing the immobilized enzyme in a continuous flow device to realize continuous synthesis of the oligopeptide or the polypeptide.

Images