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 polysaccharide matrix amino carrier; the polysaccharide matrix amino carrier comprises an agarose matrix amino carrier, a glucan matrix amino carrier or a cellulose matrix amino carrier. The immobilized enzyme provided by the invention can efficiently synthesize oligopeptide or polypeptide, and has the advantages of saving labor, improving productivity and simplifying production process.

Description

Technical Field

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

[0002] Oligopeptides and polypeptides are collectively referred to as peptides. Peptides composed of 2 to 10 amino acids are usually called oligopeptides or small molecule peptides, and peptides composed of 10 to 100 amino acids are called polypeptides. The molecular weight is generally less than 10,000Da. Oligopeptides and polypeptide drugs have a variety of physiological functions in the human body, including but not limited to regulating physiological functions, promoting wound healing, anti-oxidation and anti-aging, regulating endocrine, regulating the nervous system and immune system. In recent years, the research and development of peptide drugs has remained high. Not only have there been a variety of approved drugs, but hundreds of candidate drugs are in different stages of clinical development.

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

[0004] Enzymatic synthesis of oligopeptides mainly includes the use of α-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, and enzymatic synthesis of polypeptides mainly includes the use of polypeptide ligase to combine the solid-phase synthesized acyl donor with the acyl acceptor substrate. Most of the current research uses free α-amino acid ester acyltransferase and free polypeptide ligase to synthesize oligopeptides and polypeptides, which has the disadvantages of severe hydrolysis side reactions, difficult post-treatment, and the inability to recover the enzyme, resulting in increased costs.

[0005] However, the synthesis of oligopeptides with α-amino acid ester acyltransferase and the synthesis of polypeptides with polypeptide ligase 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 a timely separation of the product and the enzyme, and the reaction can only be terminated by adjusting the acid and alkali. During the post-processing process, due to the presence of free enzymes, there will also be a certain degree of emulsification, making post-processing difficult.

[0006] Immobilized enzymes, however, can significantly ease the pressure of post-processing and product separation because they introduce very little protein residue into the reaction system; and immobilized enzymes can be reused, thereby saving enzyme usage and reducing costs. Further development of the process of continuous production of peptide drugs using immobilized enzymes has incomparable advantages over intermittent reactions: easy to achieve automated control, stable product quality and output; can shorten reaction time and improve production efficiency; can produce products uninterruptedly, saving labor, increasing production capacity and simplifying production processes.

[0007] In the prior art, there are few reports on the synthesis of oligopeptides using immobilized enzymes. Among them, the α-amino acid ester acyltransferase prepared in the form of calcium alginate or agarose embedding synthesized alanine-glutamyl dipeptide, with a molar conversion rate of only ~50%, a continuous production capacity of about 3 mg / (min*mL), and no long-term operation data reported. There are currently no reports on the immobilization process of peptide ligase. Therefore, it is of great significance to develop an immobilized enzyme with a high conversion rate for synthesizing oligopeptides or polypeptides. Summary of the invention

[0008] The main purpose of the present invention is to provide an immobilized enzyme, a preparation method and application thereof, so as to solve the problem of lack of immobilized enzymes for synthesizing polypeptides and low conversion rate of substrates catalyzed by immobilized enzymes for synthesizing oligopeptides in the prior art.

[0009] In order to achieve the above object, according to a first aspect of the present invention, an immobilized enzyme is provided, the immobilized enzyme comprising an enzyme and a carrier loaded with the enzyme;

[0010] Wherein, the enzyme is α-amino acid acyltransferase or polypeptide ligase;

[0011] The above-mentioned carrier is a polysaccharide matrix amino carrier;

[0012] Wherein, the above-mentioned polysaccharide matrix amino carrier includes agarose matrix amino carrier, dextran matrix amino carrier or cellulose matrix amino carrier.

[0013] Furthermore, the agarose matrix amino carrier is selected from any one or more of the following: AminoLink, EAHSepharose 4B, ToyoPearl AF-Amino-650M, Fractogel EMD Amino or Purolite ACR; the cellulose matrix amino carrier is selected from IB-ANI-13.

[0014] Furthermore, the above-mentioned polysaccharide matrix amino carrier is a polysaccharide matrix carrier with amino modification;

[0015] The above-mentioned polysaccharide matrix carrier includes an agarose matrix carrier, a dextran matrix carrier or a cellulose matrix carrier;

[0016] The agarose matrix carrier is selected from Sepharose 6 FF;

[0017] The above-mentioned dextran matrix carrier is selected from any one or more of the following: Sephacryl S-100 HR or TandexG250C;

[0018] The cellulose matrix carrier is selected from DEAE-Cellulose.

[0019] Furthermore, the polysaccharide matrix amino carrier is a glutaraldehyde-activated polysaccharide matrix amino carrier.

[0020] Furthermore, the α-amino acid acyltransferase is derived from Pedobacter or Sphingobacterium; and the polypeptide ligase is derived from Bacillus subtilis.

[0021] Furthermore, the amino acid sequence of the above-mentioned α-amino acid acyltransferase is any one of the following: SEQ ID NOs: 1 to 10;

[0022] The amino acid sequence of the above polypeptide ligase is any one of the following: SEQ ID NOs: 11-13.

[0023] In order to achieve the above object, according to a second aspect of the present invention, a method for preparing an immobilized enzyme is provided, the method comprising:

[0024] activating the polysaccharide matrix amino carrier to obtain an activated carrier;

[0025] Immobilizing the enzyme on the above-mentioned activated carrier to obtain an immobilized enzyme;

[0026] Wherein, the above-mentioned polysaccharide matrix amino carrier includes an agarose matrix amino carrier, a dextran matrix amino carrier or a cellulose matrix amino carrier;

[0027] The enzyme is α-amino acid acyltransferase or polypeptide ligase.

[0028] Furthermore, the polysaccharide matrix amino carrier is obtained by amino-modifying the polysaccharide matrix carrier, and the preparation method comprises:

[0029] The polysaccharide matrix carrier is mixed with an amine compound and stirred for the first time to obtain the polysaccharide matrix amino carrier;

[0030] The polysaccharide matrix amino carrier is mixed with glutaraldehyde and stirred for a second time to obtain the activated carrier;

[0031] The activated carrier and the enzyme are mixed and stirred for a third time to obtain the immobilized enzyme.

[0032] Furthermore, the amine compound is selected from any one of the following: ammonia, ethylenediamine or polyethyleneimine.

[0033] Furthermore, the mass volume ratio of the polysaccharide matrix carrier and the amine compound is 1 g: (1-4) mL; and the volume concentration of the amine compound is 10%-40%.

[0034] Furthermore, the mass volume ratio of the polysaccharide matrix amino carrier and the glutaraldehyde is 1 g: (2-8) mL; the volume concentration of the glutaraldehyde is 0.5%-5%.

[0035] Furthermore, the mass volume ratio of the above-mentioned activated carrier and the above-mentioned enzyme is 1 g: (2-8) mL, and the concentration of the above-mentioned enzyme is 10-50 mg / mL.

[0036] Furthermore, the first stirring time is 2 to 16 h, and the temperature is 30°C to 50°C; the second stirring time is 0.5 to 4 h, and the temperature is 10°C to 30°C; the third stirring time is 8 to 48 h, and the temperature is 10°C to 25°C.

[0037] Furthermore, the α-amino acid acyltransferase is derived from Pedobacter or Sphingobacterium; and the polypeptide ligase is derived from Bacillus subtilis.

[0038] Furthermore, the amino acid sequence of the above-mentioned α-amino acid acyltransferase is any one of the following: SEQ ID NOs: 1 to 10;

[0039] The amino acid sequence of the above polypeptide ligase is any one of the following: SEQ ID NOs: 11-13.

[0040] In order 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 the above oligopeptides or polypeptides using the above immobilized enzyme or the immobilized enzyme prepared by the above immobilized enzyme preparation method.

[0041] Furthermore, the method comprises: using an immobilized enzyme to catalyze a substrate to obtain the oligopeptide or polypeptide;

[0042] The above-mentioned substrate includes a first substrate for synthesizing the above-mentioned oligopeptide or a second substrate for synthesizing the above-mentioned polypeptide.

[0043] Further, the first substrate comprises a first acyl donor and a first acyl acceptor;

[0044] 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;

[0045] The first acyl acceptor comprises amino acids or a third peptide segment; the third peptide segment is formed by condensation of 2 to 9 amino acids.

[0046] Furthermore, the second substrate comprises a second acyl donor and a second acyl acceptor;

[0047] 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;

[0048] The first peptide segment is composed of 2 to 98 amino acids condensed together;

[0049] The second peptide segment is formed by condensation of 2 to 98 amino acids.

[0050] In order to achieve the above object, according to the fourth aspect 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 oligopeptides or polypeptides in synthesizing oligopeptides or polypeptides.

[0051] Furthermore, the above application includes: placing the above immobilized enzyme in a continuous flow device to achieve continuous synthesis of the above oligopeptide or the above polypeptide.

[0052] By applying the technical scheme of the present invention, the immobilized enzyme formed by covalently bonding the polysaccharide matrix amino carrier with α-amino acid ester acyltransferase and polypeptide ligase respectively has a high conversion rate and can efficiently synthesize oligopeptides and polypeptides. In addition, compared with free enzymes, the immobilized enzyme of the present invention can be separated from oligopeptides and polypeptides immediately, avoiding product hydrolysis caused by excessive reaction, maximizing the production capacity of oligopeptides and polypeptides, and at the same time greatly improving the S / H ratio (synthesis to hydrolysis ratio), accelerating the rate of synthesizing oligopeptides and polypeptides, and not only having the reusability of the enzyme, but also having the advantages of simple preparation, low production cost, low environmental pollution and easy post-processing. DETAILED DESCRIPTION

[0053] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0054] As mentioned in the background art, the yield of immobilized enzymes used to synthesize oligopeptides in the prior art is low, and there is no report on the synthesis of polypeptides by immobilized enzymes. In the present invention, the inventors tried to use a polysaccharide matrix amino carrier as an immobilized enzyme carrier. The immobilized enzyme prepared by using this immobilized enzyme carrier has the advantage of high conversion rate when synthesizing oligopeptides or polypeptides, and thus proposed a series of protection schemes of the present invention.

[0055] In a first typical embodiment of the present invention, an immobilized enzyme is provided, which includes an enzyme and a carrier loaded with the enzyme; wherein the enzyme is α-amino acid acyltransferase or polypeptide ligase; the carrier is a polysaccharide matrix amino carrier; wherein the polysaccharide matrix amino carrier includes an agarose matrix amino carrier, a dextran matrix amino carrier or a cellulose matrix amino carrier.

[0056] Compared with free enzymes and immobilized enzymes prepared by other immobilized enzyme carriers, the use of this immobilized enzyme to prepare oligopeptides or polypeptides has the beneficial effect of high conversion rate.

[0057] The polysaccharide matrix amino carrier has the characteristics of multiple hydroxyl groups, strong biocompatibility and relatively green and environmentally friendly. In some embodiments of the present invention, the polysaccharide matrix amino carrier includes an agarose matrix amino carrier, a dextran matrix amino carrier or a cellulose matrix amino carrier.

[0058] In a more preferred embodiment of the present invention, the above-mentioned polysaccharide matrix amino carrier is an agarose matrix amino carrier. Agarose can form a stable gel state at an appropriate temperature and has good elasticity and structural stability. More activatable hydroxyl groups on agarose can be accessed under certain conditions to form different ligands as the medium of affinity chromatography, hydrophobic, reverse phase, ion exchange chromatography and immobilized enzyme carrier, so adopting agarose as the immobilized enzyme carrier matrix generally has the advantage of high activity recovery.

[0059] It should be noted that there are commercial polysaccharide matrix carriers and polysaccharide matrix amino carriers in the prior art. When using polysaccharide matrix amino carriers, there is no need to further perform the above amino modification. When using polysaccharide matrix carriers, since they have no amino groups, the above amino modification is required. The above polysaccharide matrix carriers are also called microspheres.

[0060] In a preferred embodiment of the present invention, the agarose matrix amino carrier is selected from any one or more of the following: AminoLink, EAH Sepharose 4B, ToyoPearl AF-Amino-650M, Fractogel EMD Amino or Purolite ACR; the cellulose matrix amino carrier is selected from IB-ANI-13.

[0061] In a preferred embodiment of the present invention, the polysaccharide matrix amino carrier is a polysaccharide matrix carrier with amino modification; the polysaccharide matrix carrier includes an agarose matrix carrier, a dextran matrix carrier or a cellulose matrix carrier; the agarose matrix carrier is selected from Sepharose 6 FF; the dextran matrix carrier is selected from any one or more of the following: Sephacryl S-100 HR or Tandex G250C; the cellulose matrix carrier is selected from DEAE-Cellulose. The polysaccharide matrix carrier or the polysaccharide matrix amino carrier can be used to prepare immobilized enzymes for synthesizing oligopeptides or polypeptides.

[0062] In a preferred embodiment of the present invention, the polysaccharide matrix amino carrier is a glutaraldehyde-activated polysaccharide matrix amino carrier. The amino group of the glutaraldehyde-activated polysaccharide matrix amino carrier is connected to an aldehyde group of glutaraldehyde, and the other aldehyde group of glutaraldehyde is connected to the amino group of the enzyme, so that the enzyme is finally fixed on the polysaccharide matrix amino carrier.

[0063] 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 coding gene of the α-amino acid acyltransferase has any one of the following nucleotide sequences: SEQ ID NOs: 1 to 10; the polypeptide ligase has any one of the following nucleotide sequences: SEQ ID NOs: 11 to 13. The immobilized enzyme prepared by using the above enzyme has the beneficial effect of high catalytic efficiency.

[0064] In a second typical embodiment of the present invention, a method for preparing an immobilized enzyme is provided, which comprises: activating the above-mentioned polysaccharide matrix amino carrier to obtain an activated carrier; immobilizing the enzyme on the above-mentioned activated carrier to obtain an immobilized enzyme; wherein the above-mentioned polysaccharide matrix amino carrier comprises an agarose matrix amino carrier, a dextran matrix amino carrier or a cellulose matrix amino carrier; and the above-mentioned enzyme is an α-amino acid acyltransferase or a polypeptide ligase.

[0065] When preparing immobilized enzymes for synthesizing oligopeptides or polypeptides, the amino groups on the polysaccharide matrix amino carrier are first connected to the aldehyde groups on one side of glutaraldehyde, and the excess unconnected glutaraldehyde is removed. The enzyme is then mixed with the enzyme solution (protein), and the aldehyde groups on the other side of the glutaraldehyde are combined with the amino groups on the protein. The unbound protein is removed, and the enzyme can be fixed to the carrier through covalent action, thereby improving the binding force and stability of the enzyme.

[0066] In a preferred embodiment of the present invention, the above-mentioned polysaccharide matrix amino carrier is obtained by amino-modification of a polysaccharide matrix carrier, and the above-mentioned preparation method comprises: mixing the above-mentioned polysaccharide matrix carrier with an amine compound and stirring it for the first time to obtain the above-mentioned polysaccharide matrix amino carrier; mixing the above-mentioned polysaccharide matrix amino carrier with glutaraldehyde and stirring it for the second time to obtain the above-mentioned activated carrier; mixing the above-mentioned activated carrier and the above-mentioned enzyme and stirring it for the third time to obtain the above-mentioned immobilized enzyme.

[0067] In a preferred embodiment of the present invention, the amine compound is selected from any one of the following: ammonia, ethylenediamine or polyethyleneimine. In a preferred embodiment of the present invention, the mass volume ratio of the polysaccharide matrix carrier and the amine compound is 1g: (1-4)mL; the volume concentration of the amine compound is 10%-40%.

[0068] In a more preferred embodiment of the present invention, the mass volume ratio of the polysaccharide matrix carrier and the amine compound is 1 g: 1 mL; the volume concentration of the amine compound is 20%. The amino group modification of the polysaccharide matrix carrier using the above ratio can form a relatively saturated amino group on the carrier. If the volume of the added amino compound is small, sufficient amino modification cannot be achieved.

[0069] It should be noted that before activating the polysaccharide matrix amino carrier, it is also necessary to wash the polysaccharide matrix amino carrier, which is called the first wash. The purpose of the first wash is to remove organic solvents or impurities that may be contained on the carrier, and to remove the effects of other substances on the immobilized enzyme. After the second stirring is completed, before obtaining the above-mentioned activated carrier, the polysaccharide matrix amino carrier needs to be washed for the second time to remove excess unattached glutaraldehyde. After the third stirring is completed, before obtaining the immobilized enzyme, the fixed activated carrier needs to be washed for the third time to remove unbound proteins. Use purified water or 20 mM PB (pH7.0) for the above washing steps, and generally wash 2-3 times.

[0070] The molar ratio of the polysaccharide matrix amino carrier and glutaraldehyde plays an important role in obtaining the activated carrier. In a preferred embodiment of the present invention, the mass volume ratio of the polysaccharide matrix amino carrier and the glutaraldehyde is 1g: (2~8)mL; the volume concentration of the glutaraldehyde is 0.5%~5%. In a more preferred embodiment of the present invention, the mass volume ratio of the polysaccharide matrix amino carrier and the glutaraldehyde is 1 g: 4 mL; the concentration of the glutaraldehyde is 2%. The use of the above concentration of glutaraldehyde to activate the polysaccharide matrix amino carrier can ensure the full utilization of the amino groups on the carrier and increase the enzyme immobilization capacity in the next step.

[0071] The 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 above-mentioned activated carrier and the above-mentioned enzyme is 1g: (2~8) mL, and the protein concentration of the above-mentioned enzyme is 10~50mg / mL. In a more preferred embodiment of the present invention, the mass volume ratio of the above-mentioned activated carrier and the above-mentioned enzyme is 1 g: 4mL, and the protein concentration of the above-mentioned enzyme is 20~30 mg / mL. The above-mentioned added amount of enzyme is used to save the cost of enzyme protein and carrier. If the enzyme is too little, the carrier will have an empty load phenomenon and cannot fully exert the catalytic effect, which will increase the cost of the carrier. If the enzyme is too much, there will be a large amount of enzyme protein loss, which increases the cost of the enzyme.

[0072] Wherein, the purpose of stirring is to make the reaction more thorough. In a preferred embodiment of the present invention, the time of the first stirring is 2h~16h, the temperature is 30℃~50℃; the speed is 100~300rpm. In a more preferred embodiment of the present invention, the time of the first stirring is 4h, the temperature is 45℃; the speed is 150~250rpm. In a preferred embodiment of the present invention, the time of the second stirring is 0.5h~4h, the temperature is 10℃~30℃; the speed is 100~300rpm. In a more preferred embodiment of the present invention, the time of the second stirring is 1h, the temperature is 20℃; the speed is 150~250rpm. In a preferred embodiment of the present invention, the time of the third stirring is 8~48h, the temperature is 10℃~25℃; the speed is 100~300rpm. In a more preferred embodiment of the present invention, the time of the third stirring is 16h, the temperature is 20℃; the speed is 100~200rpm.

[0073] The above stirring allows the amino modification to proceed fully, quickly and stably, so that the two aldehyde groups of glutaraldehyde are fully connected with the amino groups of the polysaccharide matrix amino carrier and the amino groups of the enzyme solution (protein), so that more enzyme solution is stably fixed on the polysaccharide matrix amino carrier, thereby improving the preparation efficiency and stability of the immobilized enzyme.

[0074] In a preferred embodiment of the present invention, after the amino modification reaction and before obtaining the polysaccharide matrix amino carrier, the preparation method further comprises: rinsing the polysaccharide matrix amino carrier with pure water until the pH of the effluent is 6.5-8.0. The purpose of rinsing is to reduce the pH of the amino-modified polysaccharide matrix amino carrier to neutral under alkaline reaction conditions, so as to provide milder conditions for the preparation of the immobilized enzyme in the next step.

[0075] 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 any one of the following: SEQ ID NOs: 1-10; the amino acid sequence of the polypeptide ligase is any one of the following: SEQ ID NOs: 11-13.

[0076] In a third typical embodiment of the present invention, a method for synthesizing a synthetic polypeptide or oligopeptide is provided. The synthesis method comprises: synthesizing the above-mentioned oligopeptide or the above-mentioned polypeptide using the above-mentioned immobilized enzyme or the immobilized enzyme prepared by the above-mentioned method for preparing the immobilized enzyme. The method for synthesizing polypeptides or oligopeptides can not only separate the enzyme solution and the reaction product (polypeptide or oligopeptide) immediately, and prevent the excessive hydrolysis of the reaction product, resulting in a low conversion rate of the reaction product, but also can efficiently promote the synthesis of oligopeptides or polypeptides from substrates, and the conversion rate of the substrates is high. In addition, since this method does not require the addition of acid or base to terminate the reaction, the process of post-treatment and batch-to-batch reaction is simplified.

[0077] In a preferred embodiment of the present invention, an immobilized enzyme is used to catalyze a substrate to obtain the above-mentioned oligopeptide or the above-mentioned polypeptide; the above-mentioned substrate includes a first substrate for synthesizing the above-mentioned oligopeptide or a second substrate for synthesizing the above-mentioned polypeptide.

[0078] In a preferred embodiment of the present invention, 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 an amino acid or a third peptide segment; the third peptide segment is formed by condensation of 2 to 9 amino acids. The acyl donor can provide an amino acid residue to form a new peptide bond with the acyl acceptor through an acyl transfer reaction, thereby achieving the purpose of extending the peptide chain.

[0079] In another preferred embodiment of the present invention, 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; the second acyl donor comprises a second peptide modified by carboxamide methyl ester; wherein the first peptide is condensed from 2 to 98 amino acids; the second peptide is condensed from 2 to 98 amino acids. Using the second peptide modified by carboxamide methyl ester as the acyl donor has the beneficial effects of improving the reaction activity, enhancing the substrate specificity, avoiding side reactions and simplifying the reaction conditions.

[0080] 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 includes: arranging the above-mentioned immobilized enzyme in a continuous flow device to realize continuous synthesis of the above-mentioned oligopeptide or polypeptide. The use of the above-mentioned continuous flow device to catalyze the synthesis of oligopeptides or polypeptides has the beneficial effect of high efficiency.

[0081] The present invention is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed by the present invention.

[0082] Example 1: Amino modification of polysaccharide matrix carrier

[0083] The amino modification methods of commercial agarose-based microspheres Sepharose 6 FF without amino groups, dextran-based microspheres Sephacryl S-100 HR and Tandex G250C, and cellulose-based microspheres DEAE-Cellulose are the same. Agarose-based microspheres Sepharose 6 FF were selected as the target carrier, and the amino modification method was as follows: 100g agarose microspheres, 240mL purified water, and 60mL 50% ammonia water were added to a 500mL four-necked bottle, mechanical stirring was turned on at 200rpm, and the reaction was carried out at 45℃ for 4h. The modified agarose microspheres were rinsed with purified water until the effluent was 7.0 to obtain the amino-modified agarose carrier Sepharose6FF.

[0084] Example 2: Preparation of polysaccharide matrix amino carrier immobilized enzyme

[0085] The immobilization method of α-amino acid ester acyltransferase is the same as that of polypeptide ligase. α-amino acid ester acyltransferase is selected as the target enzyme for immobilization, and the immobilization method is as follows:

[0086] Carrier activation: The amino carrier used was first washed twice with 20 mM PB (pH 7.0), then 4 volumes of 2% glutaraldehyde solution was added relative to the carrier, stirred at 20 °C for 1 h, and then washed three times with 20 mM PB (pH 7.0);

[0087] Enzyme immobilization: Add 4 volumes of diluted enzyme solution with a protein concentration of ~20 mg / mL to the activated carrier bottle. The buffer used for diluting the enzyme solution is 20 mM PB (pH 7.0), and stir at 20 °C for 16-24 h.

[0088] Washing: Wash three times with 4 volumes of 20 mM PB (pH 7.0) to obtain the immobilized enzyme.

[0089] Example 3: Synthesis of dipeptide Gly-Thr by immobilized α-amino acid ester acyltransferase

[0090] The structure of dipeptide Gly-Thr is shown in Formula I:

[0091] .

[0092] Reaction conditions: glycine methyl ester hydrochloride: L-threonine = 200:100 mM, 0.1 M Tris-HCl, pH = 8.5, reaction temperature 20°C.

[0093] Different immobilized enzymes were prepared using different α-amino acid ester acyltransferases and the same carrier AminoLink. The immobilization method was the same as in Example 2. The experimental results are shown in Table 1-1.

[0094] The α-amino acid acyltransferase was immobilized using amino-type carriers of different matrices. The amino modification method of the polysaccharide matrix carrier without amino groups was the same as in Example 1, and the amino carrier after amino modification and other carriers containing amino groups were immobilized in the same way as in Example 2. The highest conversion rate of the reaction is shown in Table 1-2, and the results show that the conversion rate of the immobilized enzyme prepared by the amino carrier of the polysaccharide matrix is ​​significantly improved compared with other matrices.

[0095] 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.

[0096] Table 1-1

[0097]

[0098] Table 1-2:

[0099]

[0100] Example 4: Synthesis of tripeptide Val-Gly-Gln by immobilized α-amino acid ester acyltransferase

[0101] The structure of the tripeptide Val-Gly-Gln is shown in Formula II:

[0102] .

[0103] The amino-type carriers of different matrices were used to immobilize α-amino acid acyltransferase. The amino modification method of the polysaccharide matrix carrier without amino groups was the same as in Example 1. The amino-modified amino carriers and other carriers containing amino groups themselves were immobilized in the same manner as in Example 2. Reaction conditions: L-valine methyl ester hydrochloride: glycine dipeptide = 75:50 mM, 0.1 mM Tris-HCl, pH = 8.5, 20°C. The highest conversion rate of the reaction is shown in Table 2. The results show that the conversion rate of the immobilized enzyme prepared by the amino carrier of the polysaccharide matrix is ​​significantly improved compared with other matrices.

[0104] Table 2:

[0105]

[0106] Example 5: Synthesis of tetrapeptide Ile-Glu-Cys-Gly by immobilized α-amino acid ester acyltransferase

[0107] The structure of the tetrapeptide Ile-Glu-Cys-Gly (SEQ ID NO: 14) is shown in Formula III:

[0108] .

[0109] The amino-type carriers of different matrices were used to immobilize α-amino acid acyltransferase. The amino modification method of the polysaccharide matrix carrier without amino groups was the same as in Example 1. The amino carriers after amino modification and other carriers containing amino groups themselves were immobilized in the same way as in Example 2. Reaction conditions: L-leucine methyl ester hydrochloride: glutathione = 75: 50 mM, 0.1 mM Tris-HCl, pH = 8.0. The highest conversion rate of the reaction is shown in Table 3. The results show that the conversion rate of the immobilized enzyme prepared by the amino carrier of the polysaccharide matrix is ​​significantly improved compared with other matrices.

[0110] Table 3:

[0111]

[0112] Example 6: Synthesis of Ac-FIEWLAF-NH2 (SEQ ID NO: 15) by Immobilized Peptide Ligase

[0113] Reaction conditions: Ac-Phe-Ile-Glu-Trp-Leu-OCam (SEQ ID NO: 16): H-Ala-Phe-NH2 = 15: 10 mM, 0.1 M Tricine buffer, pH = 8.5, reaction temperature 25 °C.

[0114] Different immobilized enzymes were prepared using different α-amino acid ester acyltransferases and the same carrier AminoLink. The immobilization method was the same as in Example 2, and the experimental results are shown in Table 4-1.

[0115] The peptide ligase was immobilized using amino-type carriers of different matrices. The amino modification method of the polysaccharide matrix carrier without amino groups was the same as in Example 1. The amino-modified amino carriers and other carriers containing amino groups were immobilized in the same manner as in Example 2. The highest conversion rate of the reaction is shown in Table 4-2. The results show that the conversion rate of the immobilized enzyme prepared by the amino carrier of the polysaccharide matrix is ​​significantly improved compared with other matrices.

[0116] It should be noted that in the present invention, -OCam 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; and H- indicates that the N-terminal amino group is not protected.

[0117] Table 4-1:

[0118]

[0119] Table 4-2:

[0120]

[0121] Example 7: Synthesis of Ac-DFSKLSLR-NH2 (SEQ ID NO: 17) by Immobilized Peptide Ligase

[0122] The peptide ligase was immobilized using amino-type carriers of different matrices. The amino modification method of the polysaccharide matrix carrier without amino groups was the same as in Example 1. The amino-modified amino carriers and other carriers containing amino groups themselves were immobilized in the same manner as in Example 2. Reaction conditions: 10 mM Ac-DFSKL-OCam (SEQ ID NO: 18), 5 mM H-SLR-NH2, 0.1 M PB8.0, reaction temperature 25°C. The highest conversion rate of the reaction is shown in Table 5. The results show that the conversion rate of the immobilized enzyme prepared by the amino carrier of the polysaccharide matrix is ​​significantly improved compared with other matrices.

[0123] Table 5:

[0124]

[0125] Example 8: Continuous synthesis of dipeptide Gly-Arg by immobilized α-amino acid ester acyltransferase

[0126] The structure of the dipeptide Gly-Arg is shown in Formula IV:

[0127] .

[0128] The amino-type carriers of different matrices were used to immobilize α-amino acid ester acyltransferase. The amino modification method of the polysaccharide matrix carrier without amino groups was the same as in Example 1. The amino carriers after amino modification and other carriers containing amino groups themselves were immobilized in the same manner as in Example 2. Reaction conditions: glycine methyl ester hydrochloride: L-arginine = 200: 100mM, 0.1 mM Tris-HCl, reaction pH = 8.0-8.5. The highest conversion rate of the reaction is shown in Table 6. The results show that the conversion rate of the immobilized enzyme prepared by the amino carrier of the polysaccharide matrix is ​​significantly improved compared with other matrices.

[0129] Table 6:

[0130]

[0131] Select the amino carrier AminoLink immobilized α-amino acid ester acyltransferase on the agarose matrix in the above table, and run the continuous reaction. The reaction system is the same as above. Add the prepared immobilized enzyme to a 1 mL reaction column, fill with about 0.75 g of the immobilized enzyme, and use the peristaltic pump to continuously pass the reaction system through the reaction column at 20 °C. Set the retention time to 0.5~5min, and take samples for detection every 20 minutes to determine the optimal retention time.

[0132] The sample was subjected to HPLC detection, and the results showed that the product conversion rate reached more than 85%, and it could be continuously operated for about 24 hours without a significant decrease in conversion rate. Compared with the free enzyme, the production capacity and other information are shown in Table 7. The reaction system of the free enzyme batch reaction in this implementation is consistent with the reaction system of the immobilized enzyme continuous reaction. After the system configuration is completed, 0.03wt (relative to the acyl acceptor) α-amino acid acyltransferase is added to the reaction system, and then the reaction progress is tracked, and samples are taken every 0.5 hours for detection.

[0133] Table 7:

[0134]

[0135] Example 9: Continuous synthesis of the tripeptide Val-Gly-Tyr by immobilized α-amino acid ester acyltransferase

[0136] The structure of the tripeptide Val-Gly-Tyr is shown in Formula V:

[0137] .

[0138] The amino-type carriers of different matrices were used to immobilize α-amino acid ester acyltransferase. The amino modification method of the polysaccharide matrix carrier without amino groups was the same as in Example 1. The amino carriers after amino modification and other carriers containing amino groups themselves were immobilized in the same way as in Example 2. Reaction conditions: L-valine methyl ester hydrochloride: tyrosine dipeptide = 75:50 mM, 0.1 mM Tris-HCl, pH = 9.0. The highest conversion rate of the reaction is shown in Table 8. The results show that the conversion rate of the immobilized enzyme prepared by the amino carrier of the polysaccharide matrix is ​​significantly improved compared with other matrices.

[0139] Table 8:

[0140]

[0141] The amino carrier AminoLink immobilized α-amino acid ester acyltransferase of the agarose matrix in the above table was selected to run the continuous reaction, and the reaction system was the same as above. The prepared immobilized enzyme was added to a 1 mL reaction column, and about 0.75 g of the immobilized enzyme was filled. The reaction system was continuously passed through the reaction column at 20 ° C by the action of the peristaltic pump, and the retention time was set to 0.5~5min. At the same time, the sample was taken every 20 min for detection to determine the optimal retention time. The sample was subjected to HPLC detection, and the results showed that the product conversion rate was about 50%, and the conversion rate did not decrease significantly after continuous operation for about 24 h. Compared with the free enzyme, the production capacity and other information are shown in Table 9. The reaction system of the free enzyme batch reaction in this implementation is consistent with the reaction system of the immobilized enzyme continuous reaction. After the system configuration is completed, 0.1wt (relative to the acyl acceptor) α-amino acid acyltransferase is added to the reaction system, and then the reaction progress is tracked, and samples are taken every 0.5 h for detection.

[0142] Table 9:

[0143]

[0144] Example 10: Continuous synthesis of tetrapeptide Ser-Glu-Cys-Gly by immobilized α-amino acid acyltransferase

[0145] The structure of the tetrapeptide Ser-Glu-Cys-Gly (SEQ ID NO: 19) is shown in Formula VI:

[0146] .

[0147] The amino-type carriers of different matrices were used to immobilize α-amino acid ester acyltransferase. The amino modification method of the polysaccharide matrix carrier without amino groups was the same as in Example 1. The amino carriers after amino modification and other carriers containing amino groups themselves were immobilized in the same way as in Example 2. Reaction conditions: L-serine methyl ester hydrochloride: glutathione = 75: 50mM, 0.1 mM Tris-HCl, pH = 8.0. The highest conversion rate of the reaction is shown in Table 10. The results show that the conversion rate of the immobilized enzyme prepared by the amino carrier of the polysaccharide matrix is ​​significantly improved compared with other matrices.

[0148] Table 10:

[0149]

[0150] The amino carrier AminoLink immobilized α-amino acid ester acyltransferase on the agarose matrix in the above table was selected to run the continuous reaction, and the reaction system was the same as above. The prepared immobilized enzyme was added to a 1 mL reaction column, and about 0.75 g of the immobilized enzyme was filled. The reaction system was continuously passed through the reaction column at 20 °C by the action of a peristaltic pump, and the retention time was set to 0.5~5min. At the same time, the sample was taken every 20 min for detection to determine the optimal retention time. The sample was subjected to HPLC detection, and the results showed that the product conversion rate reached more than 75%, and it could be continuously operated for about 24 h without a significant decrease in the conversion rate. Compared with the free enzyme, the production capacity and other information are shown in Table 11. The reaction system of the free enzyme batch reaction in this implementation is consistent with the reaction system of the immobilized enzyme continuous reaction. After the system configuration is completed, 0.03 wt (relative to the acyl acceptor) α-amino acid acyltransferase is added to the reaction system, and then the reaction progress is tracked, and samples are taken every 0.5h for detection.

[0151] Table 11:

[0152]

[0153] Example 11: Continuous Synthesis of Exenatide by Immobilized Peptide Ligase

[0154] The peptide ligase was immobilized using amino-type carriers of different matrices. The amino modification method of the polysaccharide matrix carrier without amino groups was the same as in Example 1. The amino carrier after amino modification and other carriers containing amino groups themselves were immobilized in the same way as in Example 2. Reaction conditions: 10 mM HGEGTFTSDLSKQMEEEAVRL-OCam-L-OH (SEQ ID NO: 20), 10 mM FIEWLKNGGPSSGAPPPS-NH2 (SEQ ID NO: 21), 1 M PB8.0. The highest conversion rate of the reaction is shown in Table 12. The results show that the conversion rate of the immobilized enzyme prepared by the amino carrier of the polysaccharide matrix is ​​significantly improved compared with other matrices.

[0155] Table 12:

[0156]

[0157] Select the carrier-immobilized peptide ligase in the above table and run the continuous reaction. The reaction system is the same as above. Add the prepared immobilized enzyme to the 0.2 mL reaction column, fill with about 0.15 g of the immobilized enzyme, and use the peristaltic pump to continuously pass the reaction system through the reaction column at 25 °C. Set the retention time to 0.5-5 min, and take samples for detection every 10 min to determine the optimal retention time.

[0158] The samples were subjected to HPLC testing, and the results showed that the product conversion rate reached more than 50%, and the conversion rate could be continuously operated for about 24 hours without a significant decrease. Compared with the free enzyme, the production capacity and other information are shown in Table 13. The reaction system of the free enzyme batch reaction in this implementation is consistent with the reaction system of the immobilized enzyme continuous reaction. After the system configuration is completed, 0.1wt (relative to the acyl receptor) of polypeptide ligase is added to the reaction system, and then the reaction progress is tracked, and samples are taken every 0.5h for testing.

[0159] Table 13:

[0160]

[0161] Example 12: Comparison of modification effects of different amine compounds

[0162] The agarose-based Sepharose 6 FF, dextran-based Sephacryl S-100 HR and Tandex G250C, and cellulose-based DEAE-Cellulose selected in this patent application are commercial microspheres without amino groups, and need to be modified by amino groups for carrier activation and enzyme immobilization. The amino groups were modified with different amine compounds, the amino modification method was the same as in Example 1, the immobilization method was the same as in Example 2, the activation substrate and system were the same as in Example 8, and the amino acid sequence of α-amino acid acyltransferase was SEQ ID NO: 1. The specific results are shown in Tables 14, 15 and 16. The results show that amino groups can be successfully modified with ammonia water, ethylenediamine and polyethyleneimine (1.8KDa, 10KDa), and the concentration range of amine compounds is 10%~40%.

[0163] Table 14:

[0164]

[0165] Table 15:

[0166]

[0167] Table 16:

[0168]

[0169] Example 13: Amine compound modification time and temperature

[0170] Referring to Example 12, this example uses Sepharose 6 FF microspheres with agarose matrix for amino modification, optimizes amino modification conditions, and uses the same amino modification method as Example 1 (using 3V 10% ammonia water), the same immobilization method as Example 2, the same test substrate and system as Example 8, and the amino acid sequence of α-amino acid acyltransferase is SEQ ID NO: 1. The specific results are shown in Table 17. The results show that the modification effect is better when the modification time is 2 to 16 hours and the temperature range is 30°C to 50°C.

[0171] Table 17:

[0172]

[0173] Example 14: Concentration and volume of glutaraldehyde activation and enzyme immobilization

[0174] In this example, the amino carrier AminoLink of agarose matrix was selected for optimization of activation conditions. The immobilization method was the same as in Example 2, wherein the volume and concentration of glutaraldehyde during activation were different, or the concentration and volume of the enzyme during enzyme immobilization were different. The activation substrate and system were the same as in Example 8, and the amino acid sequence of α-amino acid acyltransferase was SEQ ID NO: 1. The specific results are shown in Tables 18 and 19. The results showed that the preferred volume ratio of glutaraldehyde was 2~8V, i.e. 1g: (2~8)mL, and the concentration of glutaraldehyde was 0.5%~5%; the volume ratio of the enzyme solution during immobilization was 2~8V, i.e. 1 g: (2~8)mL, and the protein concentration of the enzyme solution was 10~50 mg / mL.

[0175] Table 18:

[0176]

[0177] Table 19:

[0178]

[0179] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the amino-type carrier prepared by the present invention with a water-insoluble polysaccharide as a matrix has a faster reaction speed, better stability, and high continuous reaction capacity compared to other amino-type carriers or other types of carriers with water-insoluble polysaccharide matrices, and can synthesize peptide products more efficiently.

[0180] 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 polysaccharide matrix amino carrier; Wherein, the polysaccharide matrix amino carrier includes an agarose matrix amino carrier, a dextran matrix amino carrier or a cellulose matrix amino carrier.

2. The immobilized enzyme according to claim 1, characterized in that The agarose matrix amino carrier is selected from any one or more of the following: AminoLink, EAH Sepharose 4B, ToyoPearl AF-Amino-650M, Fractogel EMDAmino or Purolite ACR; the cellulose matrix amino carrier is selected from IB-ANI-13.

3. The immobilized enzyme according to claim 2, characterized in that The polysaccharide matrix amino carrier is a polysaccharide matrix carrier with amino modification; The polysaccharide matrix carrier includes an agarose matrix carrier, a dextran matrix carrier or a cellulose matrix carrier; The agarose matrix carrier is selected from Sepharose 6 FF; The dextran matrix carrier is selected from any one or more of the following: Sephacryl S-100 HR or TandexG250C; The cellulose matrix carrier is selected from DEAE-Cellulose.

4. The immobilized enzyme according to claim 3, characterized in that The polysaccharide matrix amino carrier is a polysaccharide matrix amino carrier activated by glutaraldehyde.

5. The immobilized enzyme according to claim 4, characterized in that The α-amino acid acyltransferase is derived from Pedobacter or Sphingobacterium; and the polypeptide ligase is derived from Bacillus subtilis.

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

7. A method for preparing an immobilized enzyme, characterized in that: The preparation method comprises: activating the polysaccharide matrix amino carrier to obtain an activated carrier; Immobilizing the enzyme on the activated carrier to obtain an immobilized enzyme; Wherein, the polysaccharide matrix amino carrier includes an agarose matrix amino carrier, a dextran matrix amino carrier or a cellulose matrix amino carrier; The enzyme is α-amino acid acyltransferase or polypeptide ligase.

8. The preparation method according to claim 7, characterized in that: The polysaccharide matrix amino carrier is obtained by modifying the polysaccharide matrix carrier with amino groups, and the preparation method comprises: The polysaccharide matrix carrier is mixed with an amine compound and stirred for the first time to obtain the polysaccharide matrix amino carrier; The polysaccharide matrix amino carrier is mixed with glutaraldehyde and stirred for a second time to obtain the activated carrier; The activated carrier and the enzyme are mixed and stirred for a third time to obtain the immobilized enzyme.

9. The preparation method according to claim 8, characterized in that: The amine compound is selected from any one of the following: ammonia, ethylenediamine or polyethyleneimine.

10. The preparation method according to claim 9, characterized in that: The mass volume ratio of the polysaccharide matrix carrier and the amine compound is 1 g: (1-4) mL; the volume concentration of the amine compound is 10%-40%.

11. The preparation method according to claim 9, characterized in that: The mass volume ratio of the polysaccharide matrix amino carrier and the glutaraldehyde is 1 g: (2-8) mL; the volume concentration of the glutaraldehyde is 0.5%-5%.

12. The preparation method according to claim 9, characterized in that: The mass volume ratio of the activated carrier and the enzyme is 1 g: (2-8) mL, and the concentration of the enzyme is 10-50 mg / mL.

13. The preparation method according to claim 9, characterized in that: The first stirring time is 2 to 16 h, and the temperature is 30°C to 50°C; the second stirring time is 0.5 to 4 h, and the temperature is 10°C to 30°C; the third stirring time is 8 to 48 h, and the temperature is 10°C to 25°C.

14. The preparation method according to claim 7, characterized in that: The α-amino acid acyltransferase is derived from Pedobacter or Sphingobacterium; and the polypeptide ligase is derived from Bacillus subtilis.

15. The preparation method according to claim 14, characterized in that: The amino acid sequence of the α-amino acid acyltransferase is any one of the following: SEQ ID NOs: 1 to 10; The amino acid sequence of the polypeptide ligase is any one of the following: SEQ ID NOs: 11-13.

16. 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 6 or the immobilized enzyme prepared by the method for preparing the immobilized enzyme described in any one of claims 7 to 15.

17. The method according to claim 16, 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.

18. The method according to claim 17, 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.

19. The method according to claim 18, 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.

20. Use of the immobilized enzyme according to any one of claims 1 to 6, the method for preparing the immobilized enzyme according to any one of claims 7 to 15, or the method for synthesizing an oligopeptide or polypeptide according to any one of claims 16 to 19 in synthesizing an oligopeptide or polypeptide.

21. The use according to claim 20, 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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