CN117286192B

CN117286192B - Use of amide synthetases in the preparation of litaxetil intermediates and/or litaxetil

Info

Publication number: CN117286192B
Application number: CN202311587909.7A
Authority: CN
Inventors: 苏影; 刘璨; 谢磊; 李斌
Original assignee: Xinya Lihua Biotechnology Shanghai Co ltd
Current assignee: Xinya Lihua Biotechnology Shanghai Co ltd
Priority date: 2023-11-27
Filing date: 2023-11-27
Publication date: 2024-02-23
Anticipated expiration: 2043-11-27
Also published as: CN117286192A

Abstract

The application relates to application of an amide synthase in preparing a litaxetil intermediate and/or litaxetil, wherein the amide synthase comprises an amino acid sequence shown as SEQ ID NO. 1. The application prepares the ritodynamic enzyme through the amide synthase and/or the amino acid ligase, and has the advantages of simple operation, environmental friendliness and high yield.

Description

Use of amide synthetases in the preparation of litaxetil intermediates and/or litaxetil

Technical Field

The application relates to the technical field of genetic engineering, in particular to application of amide synthetase in preparation of a litaxetil intermediate and/or litaxetil.

Background

Lifiteglast chemical name @S) -2- [2- (benzofuran-6-carbonyl) -5,7-dichloro-1, 2,3, 4-tetrahydroisoquinoline-6-carboxamide]-3- (3-methylsulfonylphenyl) propionic acid, the English name of which is [ ]S) -2- (2- (benzofuranyl-6-carboyl) -5,7-dichloro-1-2-3-4-tetrahydroisoquinoline-6-carboxamido-3- (3- (methylstyryl) phenyl) propanoic acid of formula C ₂₉ H ₂₄ C _l2 N ₂ O ₇ S, the chemical structural formula is shown as follows:

。

litaset, under the trade name Xidra, is a prescribed drug for the treatment of dry eye, which acts as a small molecule integrin inhibitor, inhibiting the binding of lymphocyte function-associated antigen 1 (LFA-1) to intercellular adhesion molecule 1 (ICAM-1), and reducing the inflammatory response mediated by T lymphocytes. It was developed by Shire, usa, filed a new drug application to the us Food and Drug Administration (FDA) in 2015, and obtained FDA approval for marketing in 2016, becoming the first new drug to treat dry eye symptoms and signs.

The currently reported synthetic routes of ritalst mainly include the following:

first reaction scheme:

according to the route, methyl isoquinoline carboxylate 1 protected by a Boc group is used as a starting material, the Boc group is removed under an acidic condition to prepare 2, a compound 2 is condensed with benzofuran carboxylic acid 3 to form 4, an intermediate 5 is prepared through hydrolysis reaction, the compound 5 is condensed with phenylpropionate 6 to obtain an intermediate 7, and finally the intermediate 7 is subjected to hydrolysis or hydrogenation degreasing to prepare the final product ritgaset.

The second reaction scheme:

the compound 8 is used as an initial substrate, boc groups are used for protection and then are condensed with phenylpropionate 11 to prepare an intermediate 12, the compound 2 is deprotected under acidic conditions to form an intermediate 13, and finally the intermediate is condensed with benzofuran carboxylic acid 3 to form the target product Litaset.

Third reaction scheme:

the route uses 6-isoquinolinecarboxylic acid-5, 7-dichloro-1, 2,3, 4-tetrahydro-14 as an initial substrate, and is condensed with benzofuran-6-carboxylic acid 3 to form intermediate 15, compound 15 is activated to generate active intermediate 16, and then condensed with 3- (methylsulfonyl) -L-phenylalanine 17 to prepare the final product Litaset.

At present, the production route of the ritalst is mainly a chemical total synthesis method, or the synthesis of the final product of the ritalst is realized through a series of upper protection and deprotection modes, but the reaction steps are more, and the yield is lower; or a one-pot synthesis method, the feeding sequence is controlled, the method is unprotected, the operation is simple, but the product has more impurities, and the method can reach the pharmaceutical grade standard by column separation and purification.

Therefore, how to provide a synthetic route which is simple to operate and easy to separate and purify the product is a difficulty in industrial production of ritalst.

Disclosure of Invention

In order to provide a synthesis route of the litaxetil, which is simple to operate and easy to separate and purify the product, the first object of the application is to provide an amide synthetase, comprising an amino acid sequence shown as SEQ ID NO. 1.

A second object of the present application is to provide an isolated nucleic acid encoding the above amide synthase;

alternatively, the nucleic acid comprises the nucleotide sequence shown as SEQ ID NO. 3.

A third object of the present application is to provide a recombinant amide synthase expression vector that satisfies at least one of the following features (1) - (2):

(1) For expressing the above amide synthase;

(2) Contains nucleic acid encoding the above amide synthase.

A fourth object of the present application is to provide a host cell which satisfies at least one of the following characteristics (1) to (3):

(1) For expressing the above amide synthase;

(2) Nucleic acids encoding the above amide synthetases;

(3) Contains the recombinant amide synthetase expression vector.

A fifth object of the present application is to provide the method for producing an amide synthase as described above, wherein the step of producing the amide synthase includes:

connecting an amide synthase gene with a protein expression vector to construct a recombinant amide synthase expression vector;

after transforming the recombinant amide synthase expression vector into a target strain to obtain a recombinant strain, inducing and culturing the recombinant strain to express the amide synthase;

alternatively, the amide synthase gene comprises a nucleotide sequence shown as SEQ ID NO. 3;

alternatively, the protein expression vector comprises pET30a (+);

alternatively, the strain of interest comprises an E.coli strain.

A sixth object of the present application is to provide the use of an amide synthase comprising the amino acid sequence shown in SEQ ID No.1 as a catalyst in the preparation of a rituximab intermediate and/or rituximab.

In one embodiment, the amide synthase catalyzed reaction conditions satisfy at least one of the following features (1) - (5):

(1) The catalytic reaction is carried out in an aqueous medium;

(2) The pH of the catalytic reaction is 7.0-8.5;

(3) The temperature of the catalytic reaction is 20-50 ℃;

(4) The catalytic reaction is carried out under stirring, and optionally, the stirring rotation speed is 200-500 r/min;

(5) The reaction time of the catalytic reaction is 20-72 h.

In one embodiment, the reaction system of the catalytic reaction satisfies at least one of the following characteristics (1) - (3):

(1) The structure of the litaxetil intermediate is shown below:

；

(2) The reaction system further comprises a first cofactor, optionally the first cofactor comprises ATP;

(3) The reaction system further comprises a buffer reagent, optionally, the buffer reagent comprises at least one of Tris-HCl buffer reagent, phosphate buffer reagent, and HEPES buffer reagent.

In one embodiment, optionally, the mass ratio of 6-isoquinolinecarboxylic acid-5, 7-dichloro-1, 2,3, 4-tetrahydrohydrochloride, benzofuran carboxylic acid, amide synthase and ATP in the reaction system is (1.0-2.0): (1.0 to 2.0): (1.0 to 2.0): (0.1 to 1.0).

In one embodiment, the amide synthase is prepared by the steps of:

alternatively, the amide synthase gene comprises the nucleotide sequence shown in SEQ ID NO. 3;

alternatively, the protein expression vector comprises pET30a (+);

alternatively, the strain of interest comprises a strain of E.coli.

In one embodiment, the rituximab is prepared from an intermediate of rituximab.

In one embodiment, the amide synthase catalyzes the formation of the intermediate of ritodynamic acid-5, 7-dichloro-1, 2,3, 4-tetrahydrohydrochloride and benzofurancarboxylic acid by forming intermolecular amide bonds.

A seventh object of the present application is to provide an amino acid ligase comprising the amino acid sequence shown in SEQ ID No. 2.

An eighth object of the present application is to provide an isolated nucleic acid encoding the above amino acid ligase;

alternatively, the nucleic acid comprises the nucleotide sequence shown as SEQ ID NO. 4.

A ninth object of the present application is to provide a recombinant amino acid ligase expression vector which satisfies at least one of the following characteristics (1) to (2):

(1) For expressing the above amino acid ligase;

(2) Contains a nucleic acid encoding the above amino acid ligase.

A tenth object of the present application is to provide a host cell which satisfies at least one of the following characteristics (1) to (3):

(1) Expressing the amino acid ligase;

(2) Nucleic acids encoding the above amino acid ligases;

(3) Contains the recombinant amino acid ligase expression vector.

An eleventh object of the present application is to provide a method for preparing the amino acid ligase, wherein the amino acid ligase is prepared by a genetic engineering method;

the genetic engineering method comprises the following steps:

connecting an amino acid ligase gene and a protein expression vector to construct a recombinant amino acid ligase expression vector;

after transforming the recombinant amino acid ligase expression vector into a target strain to obtain a recombinant strain, inducing and culturing the recombinant strain to express the amino acid ligase;

optionally, the amino acid ligase gene comprises a nucleotide sequence shown as SEQ ID NO. 4;

alternatively, the protein expression vector comprises pET30a (+);

alternatively, the strain of interest comprises an E.coli strain.

A twelfth object of the present application is to provide the use of an amino acid ligase comprising the amino acid sequence shown in SEQ ID No.2 as a catalyst in the preparation of ritodlast.

In one embodiment, the preparation of ritalst satisfies at least one of the following characteristics (1) - (7):

(1) The reaction system for preparing the ritalst comprises the intermediate of the ritalst:

；

(2) The reaction system for preparing the ritodynamic t also comprises 3- (methylsulfonyl) -L-phenylalanine;

(3) The reaction system for preparing the ritodynamic also includes a second cofactor, optionally the second cofactor also includes ATP;

(4) The reaction system for preparing the rituximab further comprises a buffer reagent, optionally, the buffer reagent comprises at least one of a Tris-HCl buffer reagent, a phosphate buffer reagent and a HEPES buffer reagent;

(5) The pH value of a reaction system for preparing the rituximab is 8.5-9.5;

(6) The reaction temperature for preparing the rituximab is 20-50 ℃;

(7) The reaction time for preparing the rituximab is 30-60 hours;

optionally, the mass ratio of the intermediate of the rituximab, the 3- (methylsulfonyl) -L-phenylalanine, the amino acid ligase and the ATP in the reaction system for preparing the rituximab is (1.0-6.0): (1.0 to 4.0): (1.0 to 4.0): (0.1 to 1.0).

A thirteenth object of the present application is to provide the use of the above amide synthase in combination with the above amino acid ligase in the preparation of ritodynamic.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the detection results of crude enzyme liquid prepared in example 1 of the present application, wherein "1" in FIG. 1 represents a protein Marker; "2" represents the result of the detection of the crude enzyme solution of the recombinant amide synthase McbA; "3" represents the result of the detection of the crude enzyme solution of the recombinant amide synthase McbA;

FIG. 2 is a HPLC conversion chart of the first step reaction provided in example 3 of the present application;

FIG. 3 is a HPLC conversion profile of the second step reaction provided in example 3 of the present application.

Detailed Description

Reference now will be made in detail to the embodiments of the application, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the present application. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the scope or spirit of the present application. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Accordingly, it is intended that the present application cover such modifications and variations as fall within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present application are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present application.

To at least partially solve at least one of the above technical problems, a first aspect of the present application provides an amide synthase comprising an amino acid sequence as shown in SEQ ID NO. 1.

The method adopts the amide synthetase as the catalyst to prepare the intermediate of the rituximab, has high catalytic activity on a substrate, good substrate selectivity and high reaction conversion rate, and can be used as the first-step reaction of the prepared rituximab to prepare the rituximab based on the intermediate of the rituximab generated in the first-step reaction.

In a second aspect the present application provides an isolated nucleic acid encoding an amide synthase as described above;

In a third aspect the present application provides a recombinant amide synthase expression vector for expressing an amide synthase as described above.

In this application, the term "expression vector" refers to a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide operably linked to control sequences for expression thereof.

In some embodiments, the recombinant amide synthase expression vector comprises the nucleotide sequence set forth in SEQ ID NO. 3.

The recombinant expression vector may be constructed in any suitable manner. Recombinant expression vectors may include, for example, vectors comprising i) a collection of genetic elements, such as promoters and enhancers, that have a regulatory effect on gene expression; ii) a structural or coding sequence transcribed into mRNA and translated into protein; and iii) transcriptional subunits of appropriate transcription and translation initiation and termination sequences. The nature of the vector is not critical and any vector may be used, including plasmids, viruses, phages and transposons. Possible vectors for use in the present application include, but are not limited to, chromosomal, nonchromosomal and synthetic DNA sequences, such as bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from combinations of plasmids and phage DNA, DNA from viruses such as vaccinia, adenovirus, chicken pox, baculovirus, SV40, and pseudorabies.

In some embodiments, the recombinant amide synthase expression vector is derived from pET30a (+).

In a fourth aspect, the present application provides a host cell for expressing an amide synthase as described above.

In the present application, the term "host cell" means any cell type that is readily transformed, transfected, transduced, or the like with a polynucleotide or recombinant expression vector comprising a mutant polypeptide, encoding a mutant polypeptide of the present application. The host cell may be a prokaryotic cell or a eukaryotic cell, and may be any cell into which a polynucleotide encoding a polypeptide or a recombinant polypeptide having an amide synthase activity of the present application can be introduced.

In some embodiments, the host cell contains a nucleic acid encoding the above-described amide synthase or contains the above-described recombinant amide synthase expression vector.

In some embodiments, the host cell comprises an E.coli strain.

In a fifth aspect, the present application provides a method for preparing the above amide synthase, specifically, the amide synthase is prepared by using a genetic engineering method. Genetic engineering (genetic engineering) is also known as gene splicing technology and DNA recombination technology. The genetic engineering is a complex technique for manipulating a gene at a molecular level, and is a manipulation of introducing a foreign gene into a recipient cell after in vitro recombination, so that the gene can be replicated, transcribed, and translated in the recipient cell.

In some embodiments, the amide synthase is prepared by steps comprising:

and after the recombinant amide synthase expression vector is transformed into a target strain to obtain a recombinant strain, the recombinant strain is induced to express the amide synthase.

In some embodiments, the amide synthase gene comprises the nucleotide sequence set forth in SEQ ID NO. 3.

In some embodiments, the protein expression vector comprises pET30a (+).

In some embodiments, the strain of interest comprises a strain of E.coli.

In a sixth aspect the present application provides the use of an amide synthase comprising the amino acid sequence shown in SEQ ID No.1 as a catalyst in the preparation of a rituximab intermediate and/or rituximab.

In some embodiments, the structure of the rituximab intermediate is as follows:

。

further, the intermediate of the rituximab can be used for preparing the rituximab.

In some embodiments, for the catalytic synthesis of the aforementioned litaxetil intermediates, the reaction system for preparing the litaxetil intermediates comprises 6-isoquinolinecarboxylic acid-5, 7-dichloro-1, 2,3, 4-tetrahydrohydrochloride and benzofurancarboxylic acid.

Specifically, the amide synthase catalyzes 6-isoquinolinecarboxylic acid-5, 7-dichloro-1, 2,3, 4-tetrahydrohydrochloride and benzofuran carboxylic acid to generate a litaxetil intermediate through intermolecular amide bond formation, the stereoselectivity of the amide synthase is high, the conversion rate of the catalytic reaction can reach more than 94%, and the amide synthase can be used as the first step reaction of the prepared litaxetil.

In some embodiments, the reaction system further comprises a first cofactor. Specifically, cofactor (Cofactor) refers to a non-protein compound that binds to an enzyme and is necessary in a catalytic reaction, for example, a coenzyme, prosthetic group, or metal ion of the enzyme, which, when bound to an enzyme protein, enables the enzyme to be catalytically active or otherwise catalytically inactive.

In some embodiments, the first cofactor comprises ATP.

In some embodiments, the reaction system further comprises a buffer reagent for providing a stable pH environment for the catalyzed reaction of the enzyme.

In some embodiments, the buffer reagent comprises at least one of Tris-HCl buffer reagent, phosphate buffer reagent, and HEPES buffer reagent.

In some embodiments, the reaction system optionally comprises 6-isoquinolinecarboxylic acid-5, 7-dichloro-1, 2,3, 4-tetrahydrohydrochloride, benzofuran carboxylic acid, amide synthase and ATP in a mass ratio of (1.0-2.0): (1.0 to 2.0): (1.0 to 2.0): (0.1 to 1.0).

In some embodiments, the catalysis is performed in an aqueous medium.

In some embodiments, the catalyzed pH is 7.0 to 8.5, more preferably 7.0 to 8.0, and still more preferably 7.0 to 7.5.

In some embodiments, the temperature of the catalysis is 20 ℃ to 50 ℃, further may be 25 ℃ to 45 ℃, and further may be 25 ℃ to 37 ℃.

In some embodiments, the catalysis is performed under stirring, optionally, the stirring speed is 200-500 r/min, further may be 200-400 r/min, and further may be 200-400 r/min.

In some embodiments, the catalytic reaction time is 20h to 72h, more preferably 20h to 50h, still more preferably 20h to 30h.

In a seventh aspect, the present application provides an amino acid ligase comprising the amino acid sequence shown in SEQ ID NO. 2.

An eighth aspect of the present application provides an isolated nucleic acid encoding an amino acid ligase as described above.

In some embodiments, the nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO. 4.

In a ninth aspect, the present application provides a recombinant amino acid ligase expression vector for expressing the above amino acid ligase. As above, the explanation of the recombinant amino acid ligase expression vector with respect to "expression vector" and "recombinant expression vector" is not described in detail herein.

In some embodiments, the recombinant amino acid ligase expression vector contains nucleic acid encoding an amino acid ligase as described above.

In a tenth aspect, the present application provides a host cell for expressing the amino acid ligase described above. As described above, in this application, the term "host cell" means any cell type that is readily transformed, transfected, transduced, or the like with a polynucleotide or recombinant expression vector comprising a mutant polypeptide, encoding a mutant polypeptide of the present disclosure. The host cell may be a prokaryotic cell or a eukaryotic cell, and may be any cell into which a polynucleotide encoding a polypeptide or a recombinant polypeptide having an amino acid ligase activity of the present application can be introduced.

In some embodiments, the host cell contains a nucleic acid encoding an amino acid ligase as described above.

In some embodiments, the host cell contains a recombinant amino acid ligase expression vector as described above.

In some embodiments, the host cell comprises an E.coli strain.

An eleventh aspect of the present application provides a method for preparing the amino acid ligase, wherein the amino acid ligase is prepared by adopting a genetic engineering method;

the genetic engineering method comprises the following steps:

and after the recombinant amino acid ligase expression vector is transformed into the target strain to obtain the recombinant strain, the recombinant strain is induced to be cultured to express the amino acid ligase.

In some embodiments, an amino acid ligase gene is used to encode an amino acid ligase as described above.

In some embodiments, the amino acid ligase gene comprises the nucleotide sequence of SEQ ID No. 4.

In some embodiments, the protein expression vector comprises pET30a (+).

In some embodiments, the strain of interest comprises a strain of escherichia coli.

In a twelfth aspect the present application provides the use of an amino acid ligase comprising the amino acid sequence shown in SEQ ID NO.2 as a catalyst in the preparation of ritodlast.

In some embodiments, the reaction system for preparing ritalst comprises a ritalst intermediate:

。

specifically, the amino acid ligase can shrink the carboxyl of the intermediate of the rituximab and the carboxyl of the 3- (methylsulfonyl) -L-phenylalanine to generate the rituximab, and the reaction is used as a second step for preparing the rituximab, does not need to activate the intermediate of the rituximab, and has simple operation and higher conversion rate which can reach more than 65 percent.

Thus, in some embodiments, the reaction system for preparing ritatistat further comprises 3- (methylsulfonyl) -L-phenylalanine.

In some embodiments, the mass ratio of the intermediate of the rituximab, the 3- (methylsulfonyl) -L-phenylalanine, the amino acid ligase and the ATP in the reaction system for preparing the rituximab is (1.0-6.0): (1.0 to 4.0): (1.0 to 4.0): (0.1 to 1.0).

In some embodiments, the reaction system for preparing ritodynamic properties further comprises a second cofactor, optionally the second cofactor further comprises ATP.

In some embodiments, the reaction system for preparing ritodynamic properties further comprises a buffer reagent, optionally comprising at least one of Tris-HCl buffer reagent, phosphate buffer reagent, and HEPES buffer reagent, for providing a stable pH environment for the catalytic reaction of the amino acid ligase.

In some embodiments, the pH of the reaction system for preparing ritodynamic agents is 8.5 to 9.5, and may further be 9.0 to 9.5.

In some embodiments, the reaction temperature for preparing ritodynamic properties is 20 ℃ to 50 ℃, further may be 30 ℃ to 50 ℃, still further 30 ℃ to 37 ℃. For providing a suitable temperature for the catalytic reaction of the amino acid ligase.

A thirteenth aspect of the present application provides the use of an amide synthase in combination with an amino acid ligase in the preparation of ritalast. Specifically, the amide synthase is used as a catalyst to catalyze 6-isoquinolinecarboxylic acid-5, 7-dichloro-1, 2,3, 4-tetrahydrohydrochloride and benzofurancarboxylic acid to generate a litaxel intermediate through intermolecular amide bond formation, and the amino acid ligase is used as a catalyst to catalyze the litaxel intermediate and 3- (methylsulfonyl) -L-phenylalanine to generate the litaxel through intermolecular carboxyl condensation, so that the method is simple to operate, environment-friendly, high in yield, good in enzymatic catalytic reaction specificity, good in conversion rate, few in byproducts and easy to separate and purify products.

Embodiments of the present application will be described in detail below with reference to examples.

The sources of materials in the embodiments of the present application are:

the recombinant plasmid pET30a (+) -McbA and the recombinant plasmid pET30a (+) -YwfE are constructed by adopting the conventional technical means in the field;

the empty plasmid vector pET-30a (+) was purchased from Novagen;

2 x PrimeSTAR HS was purchased from Beijing Bao Ri doctor materials technology Co., ltd;

E. the competent cells of the coll BL21 (DE 3) and the agarose gel DNA recovery kit are all purchased from Beijing Tiangen Biochemical technology Co., ltd;

the restriction endonucleases Nde I, hind III and T4 DNA ligase are commercially available from NewEngland Biolabs (NEB) company.

Unless otherwise indicated, the specific experiments in the following examples were performed according to methods and conditions conventional in the art, or following the commercial specifications of the kit.

EXAMPLE 1 selection of an amide synthetase

13 sequences with different sequence consistencies are selected from NCBI database by using sequence alignment method, namely ligase1 to ligase13 in table 1, through complete gene synthesis by An Sheng company, pET30a (+) is used as expression vector, and Nde I and Hind III restriction enzyme sites are respectively added at two ends of coding region. Transformation of the resulting plasmid intoE. coliBL21 (DE 3) competent cells, 13 recombinant expression strains containing recombinant plasmid pET30a (+) -ligase were obtained, and glycerol was added and stored at-80℃for use.

6 mu L of bacteria liquid is taken from glycerol bacteria (13 recombinant bacteria) stored at minus 80 ℃ in a laboratory, placed into a first-stage 96-well plate (containing 200 mu L of LB culture medium), placed into a shaking table at 37 ℃ and 400 rpm for culturing about 12 h, then 40 mu L of seed liquid is transferred into a second-stage plate (containing 320 mu L of LB culture medium) for culturing for 3-4 hours at 37 ℃ and 450 rpm, then inducer IPTG (final concentration is 0.2 mM) is added, and placed into the shaking table at 20 ℃ and 800rpm for induced expression of target protein.

And (5) after induction for 20-24 hours, centrifugally collecting thalli. The cells were resuspended using 0.5. 0.5 mg/mL lysozyme and disrupted in a shaker at 30℃and 800rpm for 3 h. After the completion of the crushing, the mixture was centrifuged for 30 minutes by using a horizontal centrifuge at 4℃and 4000 rpm, and the supernatant was collected for reaction. The reaction system is as follows: 300. mu.L of a reaction system (KPB buffer,100 mM, pH 7.0) containing 0.5 mg of 6-isoquinolinecarboxylic acid-5, 7-dichloro-1, 2,3, 4-tetrahydrohydrochloride and 0.5. 0.5 mg benzofuran carboxylic acid, 0.2 mg of ATP, 240. Mu.L of enzyme lysate were reacted at 30℃and 1000 rpm for 48 h. The peak position of the product is determined by comparing with a standard substance of the intermediate of the litaxet, the conversion rate is calculated, and the recombinase with the highest conversion rate is selected for the subsequent cascade reaction. The screening results are shown in Table 1 below, and it is clear from Table 1 that the conversion efficiency of the recombinant enzyme Ligase1 (i.e., mcbA) is highest.

TABLE 1

^a And n.d. means that no reaction product was detected.

The following sequences of ligase1 to ligase13 were obtained:

Ligase1(McbA):

MVGYARRVMDGIGEVAVTGAGGSVTGARLRHQVRLLAHALTEAGIPPGRGVACLHANTWRAIALRLAVQAIGCHYVGLRPTAAVTEQARAIAAADSAALVFEPSVEARAADLLERVSVPVVLSLGPTSRGRDILAASVPEGTPLRYREHPEGIAVVAFTSGTTGTPKGVAHSSTAMSACVDAAVSMYGRGPWRFLIPIPLSDLGGELAQCTLATGGTVVLLEEFQPDAVLEAIERERATHVFLAPNWLYQLAEHPALPRSDLSSLRRVVYGGAPAVPSRVAAARERMGAVLMQNYGTQEAAFIAALTPDDHARRELLTAVGRPLPHVEVEIRDDSGGTLPRGAVGEVWVRSPMTMSGYWRDPERTAQVLSGGWLRTGDVGTFDEDGHLHLTDRLQDIIIVEAYNVYSRRVEHVLTEHPDVRAAAVVGVPDPDSGEAVCAAVVVADGADPDPEHLRALVRDHLGDLHVPRRVEFVRSIPVTPAGKPDKVKVRTWFTD（SEQ ID NO.1）；

Ligase2:

MALRIPISPLASIPWFHRVSVTEISSIAHCASVRLSESSLWRNPIPPEPTSEELVDYVRRVLAGIAPQDD

VLVSENRSLNGAETHRLVAGMAAVLARFGVGPGVRVACLHGNTPEAVLLRLAVQWAGGCYVGLRPMFSPTVHAACLAHAAPEVLVHDQQREERATELLRRASVPRVLSLGPSALGEDLAALAAAAVADAPTLPAALDRPASLAYTSGTTGSPKGVVHGTTAMAACLDVARAMYGPPPWRFLVAIPLSDLGGELAQWILACGGVVVLREDLDPLATLALLERERITHLFTAPSSLYQLAEHPDLDHFDLTALRLAVYGGAPAVPARTSAALRRLGPRLMQNYGTQETGFVCALLPEDHGHPELLDHAGRLLPGVEVEIRDTSGAVVPPGVVGEIWVRSPMTMSGYHADPARTGEVLVDGWVRTGDLGHLSEADLLRILDRAKDLIIVEAYNVYSRRVEDVLCHHPKVLQAGVVGLPDVDRGERVCAAVVLASGTADADELREHVEAQLGEPHVPRLIRFVDTIPLTDGGKPDKSALRALFAPDTPSH；

Ligase3：

MRRVLAGIAPQDDVLVSENRSLNGAETHRLVAGMAAVLARFGVGPGVRVACLHGNTPEAVLLRLAVQWAGGCYVGLRPMFSPTVHAACLAHAAPEVLVHDQQREERATELLRRASVPRVLSLGPSALGEDLVALAAAAVADAPALPAALDRPASLAYTSGTTGSPKGVVHGTTAMAACLDVARAMYGPPPWRFLVAIPLSDLGGELAQWILACGGVVVLREDLDPLATLALLERERITHLFTAPSSLYQLAEHPDLDHFDLTALRLAVYGGAPAVPARTSAALRRLGPRLMQNYGTQETGFVCALLPEDHGHPELLDHAGRLLPGVEVEIRDTSGAVVPPGVVGEIWVRSPMTMSGYHADPARTGEVLVDGWVRTGDLGHLSEADLLRILDRAKDLIIVEAYNVYSRRVEDVLCHHPKVLQAGVVGLPDVDRGERVCAAVVLASGTADADELREHVEAQLGEPHVPRLIRFVDTIPLTDGGKPDKSALRALFAPDTPSR；

Ligase4：

MSPVGYVSRIVRGLESIGTKEAVVLDGHRVSGEQVLRRCSALAGALADQGLGRGSSVAYLHGNSIESVAVRLAVQALGACYVGLRPFFATGEKLKFLVEGRPTLFLFDPDQSDEAALLCSRSGISRAFSVGASDFGENLLALADGRPDAPLDCADRGDDVSMVTFTSGSTGEPKGITHTFASSSGFFDNALHMYGPGPWRFLVAIPLSDLGGEIVQWTLAGGGTAVLMDDFRADEAVRILGRERITHFFGSPSMVHALVHEPGLRNTRLPDLRLVAYGGAVSSPSRTADAVAGLGPVLMHNYGMQEAGFISFLSRSDHIRAGRYLLNSVGRPLPGVEIAVRDAEGGELPAGEIGEVCVRSATIMAGYRHRPDLTAQVLREGWFRTGDLGRIDDEGYLYLVGRIKDIIIVDAYNVYPQQVEQVLSAHPGVAQAAVVGVPDPDTGESVYAAVVPSSEIPAGRTAEFTAGLAAAVRDTLGSVHEPARIDLLASLPLTPRGKPDREALRGRAAETAASPRT；

Ligase5：

MAEVGPTYTGLVLDGLARDPGRVAIRSGNDTLTGQDCLDAVHRMARALERAGLGRGDGVTVLAGNRPEALLVRSAANLLGCRVAMPYPDGPVAEQVALAGFAGTSALVFDPLRCSAAAAGIAQAVPGAALLSLGPAPRGLGTDLLESAADCSPAAFEPRYRPEDVMAVRFTSGTTGRPKGVLRRFARPPRPALPSGSTFLLCTPLCHGGGTTADLALAAGGTVVLQDGFDAGAVLAAVERYRVSRTYLPPHLLYRLLDHPLLSATDTGSLRRVGYTGCPPSPRRLAEATRRLGRVLHQTYSLTECGPVARLSPDEHLDPRLLTTAGRPYPDTAVRVLDEDGTELPPGRTGEVWVRTPTTMAGYWRDPELTARVLRDGWLDTGDLGSLDAAGYLTVAGRRDPMAIVEGHNVFPREVEEPLRTHPDVREAVMFTTADPDRLERVHAAVALASGSRTTAEQLRRWSREHGYARCAPDTVLLLPAIPLNGLGKPDLAVLRALVADGGTGRDGGRSGRVDGGTERADGRSGLTAATAGACEGV；

Ligase6：

MSGISAQSDELYRGAFLPDLLIAALRRDPGKPAIYLGDGVLTAADLSAQISRYAQVYAAKGVSPGSGVATLSKNRPEVLYSMGAYMVTGSRNTALHPLGSLDDHAYVLADAQIETLVFDESFAERAALLQERVSTLRRLLSFGPCDVGEDLVALAAGFEPKPLVGPDVQAEDVAAVAYTGGTTGEPKGVMTAYRGSAAMTQILLTEWQWPTDLRHLVCTPLSHAGASFIVPVLHQGGSVVVLPRFEAGAVLEAIEEHRVTSVFLVPSMIYAILDHPRFAETDLSSLETVFYGASAMSPSRLREAIERIGPVFFQFYGQTEAPQSVFVLRKEEHDPDDLARLASCGRPVPWVKVALLDDEGQRVPRGEPGEICVRGPLVMKGYWNKPQQTAEAFAGGWLHTGDIAREDADGFYTIVDRKKDMIVSGGFNVFPREIEDVLSTHPAIAAAAVIGVPDEKWGEAVKAVVVLRPGTTVEAAELMALVKERKGAHHAPKTVDVVDALPLTAVGKPDKKALRARYWSLADRQVG；

Ligase7：

MQIHGAYSSNYTVTVLDRLQAKGQHDAVVAGDRRISGTDATNMVLRFSAALRRVGLKQGDGVALLSGNSPEALLLCLAVHFSGCRLVFVPPEPGAGELAAYIRQADVATLVFDPVFEPLVDRVTRLVSIPLYLSIGPSQLASDFLAEVPDTTEASARDAADTRHVTTLLYTGGTTGAPKPVTHNRNYYAAFVRASTGFSSRSPEPRTLICTLTTHSSGHNGAVNGFLTGQTIVLMKSFQPASALSLMKSESVTSVVLVTPMLYELLDTPEWANTRFPALEKLYYTGAPAAASRLRQAAVRFGPVLHQVYGASESGVVTVLGPEEHDVTRPELFMSCGRPAAGVEVQLRDEAGAPVPVGDVGELWVRSPMVMTGYWKDPERSAEVLGRGGWFRSGDVARQDKNGYLYLIDRVRDIIVTGVTADNVYSRLLDDFLLSLPNIKDAATIGVPDNDGMERVHVALVPRNQAEQPDFTKLTRLIVDELGALYAPASYSVEASLPRTPVGKTDKKALRAALLASGDATAEAAEGVCRRHGTHLG；

Ligase8：

MTARAEQKEDQMTDEFADLHRPVYGPDLLITALERNHDKPALYLGDVILTGGQMRDQISCFAQALASLGITQGTSTAMLSKNRPEVLISMGATMITGCRASALNPMGSLDDHKYIVDDAEIETLIFDPNAFEERAAELKASSPSLTNVLSMGPSEVGIDILALAATFEPAPLKAAHVDAEDASSMVYTGGTTGKPKGVVGSFRSGAALNQIQMSEWQWPEDNRFLICTPLSHAGAAFFIPTLLRGGALIVLPAFEPGAVLEAIEKHKITATMLVPTMIYMLMDHPDLPKRDVSSLQTLFYGASAMSPARLQEGIKKFGQIFFQFYGQTECGMTISVLRKEEHLADDPARLATCGRPVPWLDVRLLDDDLNEVPQGELGEICVRGPLIMKGYWKKPAETAEAFRGGWLHTGDIARKDKDGFMSIVDRKKDMIVTGGFNVFPREIEDVISAHPSVASVAVVGVPDDKWGEAVKACVVLRAGLTVDTEELVEKVKAAKGSVHAPKSVDFVDSLPLTPLGKLDKKALRARYWETSARAV；

Ligase9：

MTARAEQKEDQMTDEFADLHRPVYGPDLLITALERNHDKPALYLGDVILTGGQMRDQISCFAQALASLGISQGTSTAMLSKNRPEVLISMGATMITGCRASALNPMGSLDDHKYIVDDAEIETLIFDPNAFEERAAELKASSPSLTNVLSMGPSEVGIDILALAATFEPAPLKAAHVDAEDASSMVYTGGTTGKPKGVVGSFRSGAALNQIQMSEWQWPEDNRFLICTPLSHAGAAFFIPTLLRGGALIVLPAFEPGAVLEAIEKHKITATMLVPTMIYMLMDHPDLPKRDVSSLQTLFYGASAMSPARLQEGIKKFGQIFFQFYGQTECGMTISVLRKEEHLADDPARLATCGRPVPWLDVRLLDDDLNEVPQGELGEICVRGPLIMKGYWKKPAETAEAFRGGWLHTGDIARKDKDGFMSIVDRKKDMIVTGGFNVFPREIEDVISAHPSVASVAVVGVPDDKWGEAVKACVVLRAGLTVDTEELVDKVKAAKGSVHAPKSIDFVDSLPLTPLGKLDKKALRARYWETSARAV；

Ligase10：

MRLVDYLDKGAQLGADAPCLTMGDADLSYADVQRISHRVARGLQRSGIAAGDKVAVLSSNHALAFACVFGISRAGAVWCPINPRNEASENRYVLDAFDCACVIFHSNYGPMVEQMRAGLPKLRLLVCLDQPAPFAAGFDDWLDGLGDEPLHVEPPDDLAMIAGTGGTTGSPKGVMLTGRNLETMSALTLMGYPFDGRPSYLALAPLTHAAGVLCLPVMALGGRVVIMPKPDLGEFLALIERHRITHTFLPPTLIYMLLQHERLATTRLDSLQCFWYGAAPISAARLEEALTKIGPVMAQLFGQTEAPMMISMMSPREHFQADGAVARARLSSAGRPGPLVQVATMDGEGRLLPTGETGEIVVRGSLVMAGYYKDPKATAEAGRYGWHHTGDIGRLDADGFLFIVDRAKDMIITGGFNVYSVEVEQALMQHPDVQDSAVIGLPDDKWGERVVAVLQLHAGRSVKPEDIQAFVKARIGSVKSPKQVEIWSDLPRSKVGKVLKKDIRASLLKPAESS；

Ligase11：

MSTQSMRQPIHSGHLTVGALKRNRDRPVLFLGDTTLTGGQLAERISQYIQAFDALGADATGLLSLNRPEVLMIIGAGQIRGYRRTALHPLGSLDDHAYVLADAEVATLIIDPTPAFVERALGLLEKVPTLRQVLTIGPVPAALEGKAVDLAAEAAKYEPQPLVPADLPADHIGGLTYTGGTTGKPKGVMGTVSSITAMTTTQLAEWEWPEHPKFLMCTPLSHAGAAFFVPTIVKGGELVVLTKFDPAEVLRVIEEQKITATMLVPSMIYALLDHPDSHTRDLSSLETVYYGASAMNPVRLQEAIDRFGPIFAQYYGQSEAPMVITYLAKGDHDQKRLTSCGRPTLFARVALLGDDGKPVKQGEVGEICVSGPLLSGGYWKLPEATAETFKDGWMHTGDLAREDEDGFYFIVDRTKDMIVTGGFNVFPREVEDVIAEHPSVAQVCVIGTPDEKWGEAVTAVVVLRPGADSDAEAIATMTAEIQSAVKERKGSVQAPKQVIVADSVPVTALGKPDKKAVRAQFWSGAARSVG；

Ligase12：

MAIIDFFDRGWRIAPNGIAYIQGERSYSFQEIGELSCRIANGLLAAGFAKETKAAVWADNDVTAWACALGLWRAGLAYIPVNGRSTPAENQYVLDAFDCEVLFFQQAFATAIDTLRASLPKVKLWVCIDADLPWAPSLATWSAGRPSTMPFVDYDMDDVVTLSATGGTTGSPKGVMNTHRSFQTYFAQFMMACPYGAERPVNLAAAPMTHTAGMMSLPCTACGGTVVVLPKPDPALLLGAIAKHRVTEFFLPPTVIYRLLDIPGIEKLDYSSLKYFLYGAAPMSVEKLKRAIEVFGPVMAGGYGQTEAPASIANMTPAEHFVDGKLASDERLSSVGRPNPLVRVEILNDRGEVLPQGETGEICVRGDLVMKGYYNAPDKTADTIVDGWLHTGDIGHLDADGYLHITDRKKDMIISGGFNVYPSEIEQVIWAHPAVQDCAVIGVPDDKWGEAVKAVVELNAGQSVSAEELVALCKEKLGSVKAPKSVDFVAALPRSTVGKVLKKDLREQYWQGQQRRI；

Ligase13：

MRLVDYLDKGAQLGAHSPCLTMGGSSLSYAQVQRISWRVARGLQRAGIRPGDKVAVLSSNDATAFATVFGISRAGCVWCPINPRNEAGENAYVLDAFDCACLVFHGNYAAMVEQMRPQLPGLRALVCLDQRQAFAPSLEDWLEGLDDSPFDIAPPDDLAMIAGTGGTTGQPKGVMLSGRNLEAMSALTLMGYPFEGRPVYLALAPLTHAAGVLCLPVMALGGQVVIMPRPDLGEFLGLIEAHGVTHTFLPPTLIYMLLQHPQLAHTKLDSLQCFWYGAAPMSAARLEEALQKIGPVMAQLFGQTEAPMMISMMSPREHFNADGTIARHRLSSAGRPGPLVQVGVMNADGVLLPTSESGEIVVRGSLVMLGYYKNPGATEEASRHGWHHTGDIGYLDADGFLYIVDRAKDMIISGGFNVYSAEVEQALLQHPDVQDSAVVGLPDEKWGERVVAVLQLHEGRQVDVEDVKAFVKARIGSVKAPKQIEVWLDLPRSRVGKVLKKEVRATLLQGADQPSRS。

EXAMPLE 2 construction of recombinant amide synthase McbA and amino acid ligase YwfE expression System

The coding gene fragments of the recombinant amide synthetase McbA and the amino acid ligase YwfE are synthesized by An Sheng company through total genes, and NdeI and HindIII restriction enzyme sites are respectively added at two ends of the coding region. The target gene fragment and empty vector pET30a (+) are subjected to double digestion for 4 hours at 37 ℃ through restriction enzymes Nde I and Hind III, and then the digested target gene fragment and vector are subjected to gel cutting recovery by using an agarose gel DNA recovery kit. Subsequently, the target gene and the vector were ligated overnight at 16℃using T4 DNA ligase, the ligation solution was transformed into E.coli DH 5. Alpha. And spread on LB medium plates containing 50. Mu.g/mL kanamycin, cultured at 37℃for 12 hours, single colonies were picked up and subjected to sequencing verification (Biotechnology Co., ltd.), after the sequencing verification was successful, plasmids were extracted, and the extracted plasmids were transformed into E.coli BL21 (DE 3) competent cells to obtain recombinant expression strains containing the recombinant plasmid pET30a (+) -McbA/YwfE.

EXAMPLE 3 preparation of recombinant amide synthase McbA and amino acid ligase YwfE catalysts

The expression strain as described in example 1 was inoculated into TB medium containing 50. Mu.g/mL kanamycin, shake-cultured overnight at 37℃and inoculated into 2L triangular flasks containing 500 mL of TB medium at 1% (v/v), 10 shake flasks were placed in each recombinant expression strain, shake-cultured at 200 rpm at 37℃until the OD600 of the culture solution reached 1.2, IPTG was added as an inducer at a final concentration of 0.2 mmol/L, after induction at 20℃for 24 hours, the culture solution was centrifuged to collect the cells, and washed twice with physiological saline and suspended in 400 mL phosphate buffer (10 mM, pH 7.0), homogenized at 4℃for 45 minutes, and the supernatant was collected to obtain a crude enzyme solution, SDS-PAGE detection results of the crude enzyme solution were as shown in FIG. 1, "1" represents protein marker, "2" represents crude enzyme solution detection results of recombinant amide synthase McbA, "3" represents crude enzyme solution fwE detection results of amino acid ligase YwE. The successful preparation of the recombinant amide synthase McbA and the amino acid ligase YwfE can be judged by translating the gene sequence into an amino acid sequence and calculating the theoretical molecular weight of the amino acid sequence and comparing the amino acid sequence with an SDS-PAGE result.

The crude enzyme solution was frozen overnight at-80℃and subsequently lyophilized to give 5 g lyophilized recombinant amide synthetase McbA (sequence shown as SEQ ID No. 1) and 5 g lyophilized amino acid ligase YwfE (sequence shown as SEQ ID No. 2). The lyophilized enzyme powder was stored in a refrigerator at 4 ℃ for later use.

In this example, SEQ ID No.2:

MGPLGSKTVLVIADLGGCPPHMFYKSAAEKYNLVSFIPRPFAITASHAALIEKYSVAVIKDKDYFKSLADFEHPDSIYWAHEDHNKPEEEVVEQIVKVAEMFGADAITTNNELFIAPMAKACERLGLRGAGVQAAENARDKNKMRDAFNKAGVKSIKNKRVTTLEDFRAALEEIGTPLILKPTYLASSIGVTLITDTETAEDEFNRVNDYLKSINVPKAVTFEAPFIAEEFLQGEYGDWYQTEGYSDYISIEGIMADGEYFPIAIHDKTPQIGFTETSHITPSILDEEAKKKIVEAAKKANEGLGLQNCATHTEIKLMKNREPGLIESAARFAGANMIPNIKKVFGLDMAQLLLDVLCFGKDADLPDGLLDQEPYYVADCHLYPQHFKQNGQIPETAEDLVIEAIDIPDGLLKGDTEIVSFSAAAPGTSVDLTLFEAFNSIAAFELKGSNSQDVAESIRQIQQHAKLTAKY。

in this example, SEQ ID No.3:

ATGGTGGGCTATGCGCGCCGCGTGATGGATGGCATTGGCGAAGTGGCGGTGACCGGCGCGGGCGGCAGCGTGACCGGCGCGCGCCTGCGCCATCAGGTGCGCCTGCTGGCGCATGCGCTGACCGAAGCGGGCATTCCGCCGGGCCGCGGCGTGGCGTGCCTGCATGCGAACACCTGGCGCGCGATTGCGCTGCGCCTGGCGGTGCAGGCGATTGGCTGCCATTATGTGGGCCTGCGCCCGACCGCGGCGGTGACCGAACAGGCGCGCGCGATTGCGGCGGCGGATAGCGCGGCGCTGGTGTTTGAACCGAGCGTGGAAGCGCGCGCGGCGGATCTGCTGGAACGCGTGAGCGTGCCGGTGGTGCTGAGCCTGGGCCCGACCAGCCGCGGCCGCGATATTCTGGCGGCGAGCGTGCCGGAAGGCACCCCGCTGCGCTATCGCGAACATCCGGAAGGCATTGCGGTGGTGGCGTTTACCAGCGGCACCACCGGCACCCCGAAAGGCGTGGCGCATAGCAGCACCGCGATGAGCGCGTGCGTGGATGCGGCGGTGAGCATGTATGGCCGCGGCCCGTGGCGCTTTCTGATTCCGATTCCGCTGAGCGATCTGGGCGGCGAACTGGCGCAGTGCACCCTGGCGACCGGCGGCACCGTGGTGCTGCTGGAAGAATTTCAGCCGGATGCGGTGCTGGAAGCGATTGAACGCGAACGCGCGACCCATGTGTTTCTGGCGCCGAACTGGCTGTATCAGCTGGCGGAACATCCGGCGCTGCCGCGCAGCGATCTGAGCAGCCTGCGCCGCGTGGTGTATGGCGGCGCGCCGGCGGTGCCGAGCCGCGTGGCGGCGGCGCGCGAACGCATGGGCGCGGTGCTGATGCAGAACTATGGCACCCAGGAAGCGGCGTTTATTGCGGCGCTGACCCCGGATGATCATGCGCGCCGCGAACTGCTGACCGCGGTGGGCCGCCCGCTGCCGCATGTGGAAGTGGAAATTCGCGATGATAGCGGCGGCACCCTGCCGCGCGGCGCGGTGGGCGAAGTGTGGGTGCGCAGCCCGATGACCATGAGCGGCTATTGGCGCGATCCGGAACGCACCGCGCAGGTGCTGAGCGGCGGCTGGCTGCGCACCGGCGATGTGGGCACCTTTGATGAAGATGGCCATCTGCATCTGACCGATCGCCTGCAGGATATTATTATTGTGGAAGCGTATAACGTGTATAGCCGCCGCGTGGAACATGTGCTGACCGAACATCCGGATGTGCGCGCGGCGGCGGTGGTGGGCGTGCCGGATCCGGATAGCGGCGAAGCGGTGTGCGCGGCGGTGGTGGTGGCGGATGGCGCGGATCCGGATCCGGAACATCTGCGCGCGCTGGTGCGCGATCATCTGGGCGATCTGCATGTGCCGCGCCGCGTGGAATTTGTGCGCAGCATTCCGGTGACCCCGGCGGGCAAACCGGATAAAGTGAAAGTGCGCACCTGGTTTACCGAT。

in this example, SEQ ID No.4:

ATGGGCCCGCTGGGCAGCAAAACCGTGCTGGTGATTGCGGATCTGGGCGGCTGCCCGCCGCATATGTTTTATAAAAGCGCGGCGGAAAAATATAACCTGGTGAGCTTTATTCCGCGCCCGTTTGCGATTACCGCGAGCCATGCGGCGCTGATTGAAAAATATAGCGTGGCGGTGATTAAAGATAAAGATTATTTTAAAAGCCTGGCGGATTTTGAACATCCGGATAGCATTTATTGGGCGCATGAAGATCATAACAAACCGGAAGAAGAAGTGGTGGAACAGATTGTGAAAGTGGCGGAAATGTTTGGCGCGGATGCGATTACCACCAACAACGAACTGTTTATTGCGCCGATGGCGAAAGCGTGCGAACGCCTGGGCCTGCGCGGCGCGGGCGTGCAGGCGGCGGAAAACGCGCGCGATAAAAACAAAATGCGCGATGCGTTTAACAAAGCGGGCGTGAAAAGCATTAAAAACAAACGCGTGACCACCCTGGAAGATTTTCGCGCGGCGCTGGAAGAAATTGGCACCCCGCTGATTCTGAAACCGACCTATCTGGCGAGCAGCATTGGCGTGACCCTGATTACCGATACCGAAACCGCGGAAGATGAATTTAACCGCGTGAACGATTATCTGAAAAGCATTAACGTGCCGAAAGCGGTGACCTTTGAAGCGCCGTTTATTGCGGAAGAATTTCTGCAGGGCGAATATGGCGATTGGTATCAGACCGAAGGCTATAGCGATTATATTAGCATTGAAGGCATTATGGCGGATGGCGAATATTTTCCGATTGCGATTCATGATAAAACCCCGCAGATTGGCTTTACCGAAACCAGCCATATTACCCCGAGCATTCTGGATGAAGAAGCGAAAAAAAAAATTGTGGAAGCGGCGAAAAAAGCGAACGAAGGCCTGGGCCTGCAGAACTGCGCGACCCATACCGAAATTAAACTGATGAAAAACCGCGAACCGGGCCTGATTGAAAGCGCGGCGCGCTTTGCGGGCGCGAACATGATTCCGAACATTAAAAAAGTGTTTGGCCTGGATATGGCGCAGCTGCTGCTGGATGTGCTGTGCTTTGGCAAAGATGCGGATCTGCCGGATGGCCTGCTGGATCAGGAACCGTATTATGTGGCGGATTGCCATCTGTATCCGCAGCATTTTAAACAGAACGGCCAGATTCCGGAAACCGCGGAAGATCTGGTGATTGAAGCGATTGATATTCCGGATGGCCTGCTGAAAGGCGATACCGAAATTGTGAGCTTTAGCGCGGCGGCGCCGGGCACCAGCGTGGATCTGACCCTGTTTGAAGCGTTTAACAGCATTGCGGCGTTTGAACTGAAAGGCAGCAACAGCCAGGATGTGGCGGAAAGCATTCGCCAGATTCAGCAGCATGCGAAACTGACCGCGAAATAT。

EXAMPLE 4 recombinant amide synthase McbA and amino acid ligase YwfE Cascade catalytic Synthesis of Tast

The reaction was carried out in a reaction flask of 100 mL, followed by adding 20 mL potassium phosphate buffer (100 mM, pH 7.0), 3g lyophilized enzyme powder catalyst prepared in example 2 using recombinant amide synthase McbA expression strain, 1g ATP,3g 6-isoquinolinecarboxylic acid-5, 7-dichloro-1, 2,3, 4-tetrahydrohydrochloride, and 3g benzofurancarboxylic acid, reacting at 30℃and 200 rpm, detecting the conversion rate of the reaction by batch sampling liquid chromatography, determining the peak position of the product by comparing with a litaxetil intermediate standard after 24 hours of the reaction, and calculating the conversion rate to 94%, and the relevant detection results are shown in FIG. 2. The liquid chromatography conditions were: using a C18 column (Φ4.6 mm ×250 mm), the mobile phase was acetonitrile: water (1% formic acid), gradient elution, column temperature 35 ℃, flow rate 0.8 mL/min, detection wavelength 254 nm.

The pH of the reaction solution was adjusted to 9.0 using NaOH, and 4g lyophilized enzyme powder catalyst prepared using recombinant amino acid ligase YwfE expression strain in example 2, 4g of 3- (methylsulfonyl) -L-phenylalanine and 1g cofactor ATP were sequentially added to react at 37℃and 200 rpm, the conversion rate of the reaction was detected by batch sampling liquid chromatography, after 48 hours of reaction, the peak position of the product was determined by comparison with a standard of rituximab, and the conversion rate was calculated to be 65%, and the results of the related detection are shown in FIG. 3. The liquid chromatography conditions were: using a C18 column (Φ4.6 mm ×250 mm), the mobile phase was acetonitrile: water (1% formic acid) =50:50, column temperature 35 ℃, flow rate 0.8 mL/min, detection wavelength 254 nm.

Therefore, the recombinant amide synthase McbA of example 2 has higher catalytic activity on the substrate 6-isoquinolinecarboxylic acid-5, 7-dichloro-1, 2,3, 4-tetrahydrohydrochloride and benzofuran carboxylic acid, and can produce high-yield litaxetil intermediate through the first-step reaction catalysis. The recombinant amino acid ligase of example 2 directly catalyzes the reaction of the first step reaction solution and 3- (methylsulfonyl) -L-phenylalanine to produce betahistine. The preparation method of the rituximab intermediate or the rituximab in the embodiment has the advantages of simple operation, environmental friendliness, high yield, good specificity of enzymatic catalytic reaction, good conversion rate, few byproducts and easy separation and purification of the rituximab product.

EXAMPLE 5 catalytic combination of recombinant amide synthetase McbA and amino acid ligase YwfE Cascade to Tast

The reaction was carried out in a reaction flask of 100 mL, followed by the addition of 20 mL potassium phosphate buffer (100 mM, pH 7.0), 3g lyophilized enzyme powder catalyst prepared in example 2 using recombinant amide synthase McbA expression strain, 1g ATP,6g 6-isoquinolinecarboxylic acid-5, 7-dichloro-1, 2,3, 4-tetrahydrohydrochloride, and 3g benzofurancarboxylic acid, and the reaction was carried out at 30℃and 200 rpm, the conversion rate of the reaction was examined by batch sampling liquid chromatography, and after 24 hours of the reaction, the conversion rate reached 84%.

The pH of the reaction solution was adjusted to 9.0 using NaOH, and 3g lyophilized enzyme powder catalyst prepared by using recombinant amino acid ligase YwfE expression strain in example 2, 3g of 3- (methylsulfonyl) -L-phenylalanine and 1g cofactor ATP were sequentially added to react at 37℃and 200 rpm, and the conversion rate of the reaction was detected by batch sampling liquid chromatography and reached 49% after 48 hours of reaction.

EXAMPLE 6 recombinant amide synthase McbA and amino acid ligase YwfE Cascade catalytic Synthesis of Tast

The reaction was carried out in a reaction flask of 100 mL, followed by the addition of 20 mL potassium phosphate buffer (100 mM, pH 7.0), 6g lyophilized enzyme powder catalyst prepared in example 2 using recombinant amide synthase McbA expression strain, 1g ATP,3g 6-isoquinolinecarboxylic acid-5, 7-dichloro-1, 2,3, 4-tetrahydrohydrochloride, and 6g benzofurancarboxylic acid, and the reaction was carried out at 30℃and 200 rpm, and the conversion rate of the reaction was examined by batch sampling liquid chromatography and after 24 hours of reaction, the conversion rate reached 74%.

The pH of the reaction solution was adjusted to 9.0 using NaOH, and 3g lyophilized enzyme powder catalyst prepared by using recombinant amino acid ligase YwfE expression strain in example 2, 3g of 3- (methylsulfonyl) -L-phenylalanine and 1g cofactor ATP were sequentially added to react at 37℃and 200 rpm, and the conversion rate of the reaction was detected by batch sampling liquid chromatography and reached 41% after 48 hours of reaction.

In conclusion, the method for synthesizing the tasselt by the cascade catalytic combination of the recombinant amide synthetase McbA and the amino acid ligase YwfE is simple to operate, environment-friendly, high in yield and easy to separate and purify the product.

The technical features of the above embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The foregoing examples represent only a few embodiments of the present application, which are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. Accordingly, the scope of protection of the present application is to be determined by the claims appended hereto.

Claims

1. The application of the amide synthase as a catalyst in preparing a litaxetil intermediate and/or litaxetil is characterized in that the amino acid sequence of the amide synthase is shown as SEQ ID NO.1, and the structure of the litaxetil intermediate is shown as follows:；

the rituximab is prepared from an intermediate of the rituximab prepared by catalyzing the amide synthase.

2. The use according to claim 1, characterized in that the reaction conditions for the preparation of the litaxest intermediate as catalyst by the amide synthase meet at least one of the following features (1) - (5):

(1) The reaction for preparing the litaxetil intermediate is carried out in an aqueous medium;

(2) The reaction pH of the preparation of the rituximab intermediate is 7.0-8.5;

(3) The reaction temperature of the preparation of the rituximab intermediate is 20-50 ℃;

(4) The reaction for preparing the litaxetil intermediate is carried out under stirring;

(5) The reaction time for preparing the rituximab intermediate is 20-72 h.

3. The use according to claim 2, wherein the reaction system for preparing the litaxetil intermediate satisfies at least one of the following characteristics (1) - (3):

(1) The reaction system for preparing the litaxetil intermediate comprises 6-isoquinolinecarboxylic acid-5, 7-dichloro-1, 2,3, 4-tetrahydrohydrochloride and benzofurancarboxylic acid;

(2) The reaction system for preparing the rituximab intermediate further comprises a first cofactor, wherein the first cofactor comprises ATP;

(3) The reaction system for preparing the litaxetil intermediate also comprises a buffer reagent.

4. The use according to claim 3, wherein the reaction system for preparing the litaxetil intermediate satisfies at least one of the following characteristics (1) - (2):

(1) In the reaction system for preparing the litaxetil intermediate, the mass ratio of 6-isoquinolinecarboxylic acid-5, 7-dichloro-1, 2,3, 4-tetrahydrohydrochloride, benzofuran carboxylic acid, amide synthase and the first cofactor is (1.0-2.0): (1.0 to 2.0): (1.0 to 2.0): (0.1 to 1.0);

(2) The buffer reagent includes at least one of Tris-HCl buffer reagent, phosphate buffer reagent and HEPES buffer reagent.

5. The use according to any one of claims 1 to 4, wherein the amide synthase preparation step comprises:

6. The use according to claim 5, wherein the nucleotide sequence of the amide synthase gene is shown in SEQ ID NO. 3.

7. The use according to claim 5, wherein the protein expression vector comprises pET30a (+).

8. The use according to claim 5, wherein the strain of interest comprises a strain of escherichia coli.

9. The use according to any one of claims 1 to 4, wherein the amide synthase and amino acid ligase are used in combination to prepare ritodynamic.

10. Use according to claim 9, characterized in that the amino acid ligase is used for catalyzing the preparation of the ritalst intermediate.

11. The use according to claim 10, wherein the amino acid sequence of the amino acid ligase is as shown in SEQ ID No. 2.

12. The use according to claim 9, characterized in that the reaction for preparing the rituximab satisfies at least one of the following characteristics (1) to (6):

(1) The reaction system for preparing the ritatist also comprises 3- (methylsulfonyl) -L-phenylalanine;

(2) The reaction system for preparing the rituximab further comprises a second cofactor comprising ATP;

(3) The reaction system for preparing the rituximab also comprises a buffer reagent;

(4) The pH value of the reaction system for preparing the rituximab is 8.5-9.5;

(5) The reaction temperature for preparing the rituximab is 20-50 ℃;

(6) The reaction time for preparing the rituximab is 30-60 hours.

13. The use according to claim 12, wherein the reaction system for preparing ritalst satisfies at least one of the following characteristics (1) - (2):

(1) The mass ratio of the intermediate of the rituximab, the 3- (methylsulfonyl) -L-phenylalanine, the amino acid ligase and the second cofactor in the reaction system for preparing the rituximab is (1.0-6.0): (1.0 to 4.0): (1.0 to 4.0): (0.1 to 1.0);

14. The use according to claim 9, wherein the amino acid ligase is prepared by genetic engineering;

the genetic engineering method comprises the following steps:

and after the recombinant amino acid ligase expression vector is transformed into a target strain to obtain a recombinant strain, the recombinant strain is induced to be cultured to express the amino acid ligase.

15. The use according to claim 14, wherein the nucleotide sequence of the amino acid ligase gene is shown in SEQ ID No. 4.

16. The use according to claim 14, wherein the protein expression vector comprises pET30a (+).

17. The use according to claim 14, wherein the strain of interest comprises a strain of escherichia coli.