CN113025671B

CN113025671B - Application of nitrile hydratase derived from sinorhizobium meliloti in preparation of amide pyrazine compounds

Info

Publication number: CN113025671B
Application number: CN202110424332.2A
Authority: CN
Inventors: 姚红; 倪泉明; 王伟文; 黄晓飞; 陈文斌
Original assignee: Zhejiang Hengkang Pharmaceutical Co ltd
Current assignee: Zhejiang Hengkang Pharmaceutical Co ltd
Priority date: 2020-06-23
Filing date: 2020-06-23
Publication date: 2023-01-31
Anticipated expiration: 2040-06-23
Also published as: CN113025671A; CN111662938A; CN113106130B; CN113106130A; CN111662938B

Abstract

The invention provides application of nitrile hydratase derived from sinorhizobium meliloti in preparation of amide pyrazine compounds; belongs to the technical field of biochemical engineering; the nitrile hydratase catalyzes the hydration reaction of the cyanopyrazine compound to generate the amide pyrazine compound; the nitrile hydratase is derived from Sinorhizobium meliloti (Sinorhizobium meliloti). The invention takes the cyanopyrazine compound as a substrate, and takes isolated nitrile hydratase or cells expressing the nitrile hydratase as a catalyst to carry out hydration reaction, thereby obtaining the amide pyrazine compound. The invention has high conversion rate of the applied raw materials, no side reaction, easy separation and purification of the product and higher purity; compared with the traditional chemical catalysis process, the process disclosed by the invention is green and environment-friendly, is simple and convenient to operate, and has a yield of over 99%.

Description

Application of nitrile hydratase derived from sinorhizobium meliloti in preparation of amide pyrazine compounds

The application is a divisional application with the application date of 2020, 06, 23 and the application number of 202010580723.9, and the invention is named as application of nitrile hydratase in catalyzing hydration reaction of cyano pyrazine compounds to generate amide pyrazine compounds.

Technical Field

The invention relates to the technical field of biochemical engineering, in particular to application of nitrile hydratase derived from sinorhizobium meliloti in preparation of amide pyrazine compounds.

Background

The amide pyrazine (or pyrazinamide) is an important antituberculosis drug and has good curative effect on tuberculosis. In recent years, amide pyrazine has a good bactericidal effect on stubborn bacteria, so that the amide pyrazine is widely applied clinically; in addition, the amide pyrazine can be used as an intermediate for preparing various antibacterial and antiviral medicaments. For example, derivatives of amide pyrazines: 6-fluoro-3-hydroxy-2-amidopyrazine, also known as Favipiravir (English: favipiravir, also known as Avigan or Favilavivir), is an antiviral drug developed by professor Baumi Gongkang, department of medicine, fushan university, japan, together with Fushan chemical industry, under Fuji Gum Fushan chemical, which is capable of fighting against a variety of RNA viruses. Favipiravir has the effect of reducing the activity of influenza virus, west Nile virus, yellow fever, foot and mouth disease virus and other various viruses; it has also been shown to inhibit activities such as enterovirus and rift valley heat; the drug also exhibits efficacy against rabies and has been used experimentally to treat patients infected with rabies virus. In 2020 and 2 months, favipiravir is used for experimental treatment of 2019 coronavirus disease (COVID-19) in China, and in 3 and 17 days, the drug is found to be effective in treating infected patients in experiments performed by Wuhan and Shenzhen, and early reports indicate that the drug can be effective in treating the disease.

The preparation method of Favipiravir mainly comprises a chemical synthesis method and a biological catalysis method. In terms of chemical synthesis, patent document WO0010569 discloses a preparation method of 6-bromo-3-amino-2-pyrazinecarboxylic acid methyl ester by multi-step reaction; patent document WO2010087117A1 discloses a preparation method of 3-hydroxy-2-pyrazinamide through multi-step reaction after bromination. Patent document CN102603658A uses 6-bromo-3-amino-2-pyrazinamide as a raw material, and is prepared by four steps of amino protection, halogen substitution, deprotection and azidation. However, these known chemical synthetic preparation methods have the following disadvantages: the synthesis is complex, the process is complicated, and the conversion rate and the total yield are low.

The application of biological catalysis in organic synthesis process has great potential and has the advantages that chemical methods cannot replace the prior methods, such as: mild reaction conditions, environmental protection, low cost, no side reaction and the like. In the preparation of amide compounds, nitrile hydratase (NHase, e.c. 4.2.1.84) is preferably used to catalyze the hydration reaction of nitrile compounds to produce amides. Among them, some nitrile hydratase-producing microorganisms have been used in the production of acrylamide, nicotinamide, 2-amino-2, 3-dimethylbutanamide, and the like: for example, U.S. Pat. No.5,692,5064,652 discloses Comamonas Testosteroni 5-MGAM-4D, and U.S. Pat. No. ZL88106735 discloses Rhodococcus rhodochrous J-1, and U.S. Pat. No. ZL103834600 discloses Pseudomonas putida, and these wild strains are used in the production of acrylamide. However, nitrile hydratases used for pyrazinamide production are currently reported to be few. World patent document WO2006049618A1 reports that E.coli SW132 is used as an expression host, and nitrile hydratase derived from Comamonas testosteoni 5-MGAM-4D is cloned and expressed to produce 2-amide pyrazine, and recombinant E.coli SW132 cells catalytically convert 2-cyano pyrazine with the concentration of 0.5. M for 15min in a phosphate buffer system (pH 7.0) at 23 ℃, and the conversion rate is 100%. Chinese patent CN101481713A discloses a method for producing 2-amide pyrazine by using 2-cyano pyrazine as a substrate and Serratia marcescens ZJB09104 expressing nitrile hydratase as a biocatalyst.

Although the gene resources of nitrile hydratase in various databases are abundant at present, the nitrile hydratase capable of catalyzing substrate cyanopyrazine to prepare amide pyrazine is few. More importantly, no document reports nitrile hydratase capable of catalyzing cyanopyrazine compounds to directly prepare amide pyrazine compounds (such as Favipiravir) at present.

Disclosure of Invention

The invention aims to provide application of nitrile hydratase in catalyzing hydration reaction of a cyanopyrazine compound to generate an amide pyrazine compound.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides application of nitrile hydratase in catalyzing hydration reaction of cyanopyrazine compounds to generate amide pyrazine compounds; the nitrile hydratase is derived from Agrobacterium tumefaciens (Agrobacterium tumefaciens), staphylococcus aggregatus (Stappia aggregatata IAM 12614) or Sinorhizobium meliloti (Sinorhizobium meliloti).

Preferably, the amino acid sequence of the alpha subunit of the nitrile hydratase derived from Agrobacterium tumefaciens (Agrobacterium tumefaciens) is shown as SEQ ID NO.1, and the amino acid sequence of the beta subunit is shown as SEQ ID NO. 2; the amino acid sequence of the alpha subunit of the nitrile hydratase derived from Staphylococcus aggregatus (Stappia aggregata IAM 12614) is shown as SEQ ID NO.3, and the amino acid sequence of the beta subunit is shown as SEQ ID NO.4; the amino acid sequence of the alpha subunit of the nitrile hydratase derived from Sinorhizobium meliloti (Sinorhizobium meliloti) is shown as SEQ ID NO.5, and the amino acid sequence of the beta subunit is shown as SEQ ID NO. 6.

Preferably, the chemical formula of the cyanopyrazine compound is shown as the formula I; the chemical formula of the amide pyrazine compound is shown as a formula II;

in formula I and formula II:

R ₁ selected from H, CH ₃ F, cl, br, or I;

R ₂ selected from H, NH ₂ Or OH.

Preferably, the cyanopyrazine compound includes 2-cyanopyrazine, 3-methyl-2-cyanopyrazine, 6-methyl-3-hydroxy-2-cyanopyrazine, 6-bromo-3-amino-2-cyanopyrazine, 6-fluoro-3-hydroxy-2-cyanopyrazine, 6-chloro-3-hydroxy-2-cyanopyrazine, 6-bromo-3-hydroxy-2-cyanopyrazine or 6-iodo-3-hydroxy-2-cyanopyrazine.

Preferably, the application comprises the steps of: taking a cyanopyrazine compound as a substrate, and carrying out hydration reaction in an aqueous solution by using isolated nitrile hydratase or cells for expressing the nitrile hydratase in vitro as a catalyst to obtain the amide pyrazine compound.

Preferably, the concentration of the substrate in water is 2 to 100g/L; when the cells for expressing nitrile hydratase in vitro are used as a catalyst, the adding amount of the catalyst is calculated by the wet weight of the cells after centrifugation at 12000rpm for 10min, and the adding amount of the cells is 1-20% of the weight of the reaction solution; when isolated nitrile hydratase is used as a catalyst, the amount of the isolated nitrile hydratase is 10 to 10000U per liter of the reaction solution.

Preferably, the temperature of the hydration reaction is 10-50 ℃, and the pH value of the hydration reaction is 5-10.

Preferably, the temperature of the hydration reaction is 20-30 ℃; the pH value of the hydration reaction is 6-8.

The invention has the beneficial effects that: the invention provides application of nitrile hydratase in catalyzing hydration reaction of cyanopyrazine compounds to generate amide pyrazine compounds; the nitrile hydratase is derived from Agrobacterium tumefaciens (Agrobacterium tumefaciens), staphylococcus aggregatus (Stappia aggregatata IAM 12614) or Sinorhizobium meliloti (Sinorhizobium meliloti). The method takes the cyanopyrazine compound as a substrate and nitrile hydratase as a catalyst to carry out water and reaction, so as to obtain the amide pyrazine compound. The method has the advantages of high conversion rate of the application raw materials, no side reaction, easy separation and purification of the product and high purity; compared with the traditional chemical catalysis process, the process is environment-friendly and simple and convenient to operate, and the yield exceeds 99%.

Drawings

FIG. 1 is a reaction formula of nitrile hydratase of the invention catalyzing hydration reaction of cyanopyrazine compounds to form amidopyrazine compounds;

FIG. 2 is a HPLC analysis chart of 6-fluoro-3-hydroxy-2-cyanopyrazine converted by nitrile hydratase.

Detailed Description

The invention provides application of nitrile hydratase in catalyzing cyano pyrazine compounds to carry out hydration reaction to generate amide pyrazine compounds, wherein the reaction formula is shown in figure 1; the nitrile waterThe synthase is derived from Agrobacterium tumefaciens (Agrobacterium tumefaciens), staphylococcus aureus (Stappia aggregatata IAM 12614) or Rhizobium meliloti (Sinorhizobium meliloti), preferably Staphylococcus agglomerans (Stappia aggregatata IAM 12614). The nitrile hydratase of the invention is a cobalt-type nitrile hydratase consisting of an alpha subunit and a beta subunit, and is a dimer (. Alpha. Beta.) ₂ Exist in the form of (1). The nitrile hydratase has the activity of catalyzing the hydration reaction of the cyanopyrazine compound to generate the amide pyrazine compound.

In the present invention, the amino acid sequence of the α subunit of the nitrile hydratase derived from Agrobacterium tumefaciens (Agrobacterium tumefaciens) is shown in SEQ ID No.1 (GenBank accession No. CAD 54074), specifically:

MSHDHDHTEPPTEIALRVKALESLLTEKGLVDPAALDELVDTYENRIGPRNGALVVAKAWTDPAYKQRLLTNATEAIAELGFSGVQGEDMLVVENSPTVHNMTVCTLCSCYPWPTLGLPPAWYKSAPYRSRVVIDPRGVLAEFGVSVPADKEVRVWDSSAELRYLVLPERPAGTEGWSEEQLVELVTRDSMIGTGFPKNPADLH；

the amino acid sequence of the beta subunit is shown as SEQ ID NO.2 (GenBank accession number CAD 54075), and specifically comprises the following components:

MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA。

in the present invention, the amino acid sequence of the α subunit of the nitrile hydratase derived from Staphylococcus aggregatus (Stappia aggregata IAM 12614) is shown in SEQ ID NO.3 (GenBank accession EAV 46030), specifically:

MSAHDHDHPHDHDHSELSEIELRVRALETLLTEKGYIDPPALDELIETYETKIGPRNGALVVAKAWTDPDYRERLMKDATAAIAELGFTGRQGEHMVAVENTDDIHNMVVCTLCSCYPWTVLGLPPVWYKSAPYRSRAVRDPRGVLAEFKVELPDDTEIRVWDSTAEIRYLVIPKRPAGTDGWSEERLADLVTRNSMIGTGLALDPSSLEDAA；

the amino acid sequence of the beta subunit is shown as SEQ ID NO.4 (GenBank accession number EAV 46029), and specifically comprises the following components:

MNGAQDLGGQMGFGPIELEENEPNFHAKWEERAFAVTLAMGATGSWTLDASRFARESLPPVTYLSSSYYEIWTRGLEKLLLSNGLVTEEELKEGRKLKEAKPIKRVLSADAVAGVLAKGSSVDREEHQPARFRTGDRVKTRRMNPEHHTRLPRYARDAVGTIEAIHGVHVFPDTNAHGEGEQPAWLYGVLFKGTDIWGPDSDPKLTLRIDLWEPYLDLAR。

in the present invention, the amino acid sequence of the α subunit of nitrile hydratase derived from Sinorhizobium meliloti (Sinorhizobium meliloti) is shown in SEQ ID No.5 (NCBI accession No. WP _ 015007805.1), specifically:

MSEHHHGHGDDHGHHHDNHLTDMEARVKALETVLTEKGLIDPAAIDAIVDAYETKVGPRNGARVVAKAWSDPGFADWLKRDATAAIASLGFTGRQGEHMRAVFNTSETHNLIVCTLCSCYPWAVLGLPPVWYKAPPYRSRAVIDPRGVLAEFGLELSAEKKGTVRNSVRWAMMMKRRESSHGYRERTSGPAPGWT；

the amino acid sequence of the beta subunit is shown in SEQ ID NO.6 (NCBI accession number WP _ 010969705.1), and specifically:

MNGPHDLGGQHGMGPIAPERNEPIFHAEWEKRALGITLSCGAFGAWTLDESRHARESLAPATYLSASYYEIWTRALETLLKRHGFVTQAELDAGHMLDKGREPKRVLTADMVAGVLAKGGPCDRPVEAPPRFAAGDSVRTKNFNPESHTRLPRYARARTGMVEAVQGSFVFPDDNAHGKGENPQWLYMVVFDAGEIWGEGADPTLTVSIDAWESYLEHA。

in the invention, the chemical formula of the cyanopyrazine compound is preferably as shown in formula I; the chemical formula of the amide pyrazine compound is preferably as shown in formula II;

in formula I and formula II:

R ₁ preferably selected from H, CH ₃ F, cl, br, or I;

R ₂ preferably selected from H, NH ₂ Or OH.

In the present invention, the cyanopyrazine-based compound preferably includes 2-cyanopyrazine, 3-methyl-2-cyanopyrazine, 6-methyl-3-hydroxy-2-cyanopyrazine, 6-bromo-3-amino-2-cyanopyrazine, 6-fluoro-3-hydroxy-2-cyanopyrazine, 6-chloro-3-hydroxy-2-cyanopyrazine, 6-bromo-3-hydroxy-2-cyanopyrazine or 6-iodo-3-hydroxy-2-cyanopyrazine, more preferably 6-fluoro-3-hydroxy-2-cyanopyrazine. The nitrile hydratase p-cyanopyrazine derivative, especially 6-fluoro-3-hydroxy-2-cyanopyrazine, has high catalytic activity.

In the invention, nitrile hydratase catalyzes hydration reaction of 2-cyanopyrazine to generate 2-amidopyrazine, catalyzes 3-methyl-2-amidopyrazine to generate 3-methyl-2-amidopyrazine, catalyzes 6-methyl-2-amidopyrazine to generate 6-methyl-2 amidopyrazine, catalyzes 6-bromo-3-amino-2-amidopyrazine to generate 6-bromo-3-amino-2-amidopyrazine, catalyzes 6-fluoro-3-hydroxy-2-amidopyrazine to generate 6-fluoro-3-hydroxy-2-amidopyrazine, catalyzes 6-chloro-3-hydroxy-2-amidopyrazine to generate 6-chloro-3-hydroxy-2-amidopyrazine, catalyzes 6-bromo-3-hydroxy-2-amidopyrazine to generate 6-bromo-3-hydroxy-2-amidopyrazine, and catalyzes 6-iodo-3-hydroxy-2-amidopyrazine to generate 6-iodo-3-hydroxy-2-amidopyrazine.

In the present invention, the application preferably comprises the steps of: taking a cyanopyrazine compound as a substrate, and carrying out hydration reaction in an aqueous solution by using isolated nitrile hydratase or cells for expressing the nitrile hydratase in vitro as a catalyst to obtain the amide pyrazine compound.

In the present invention, the concentration of the substrate in water is preferably 2 to 4g/L, more preferably 3g/L; when the cells expressing nitrile hydratase in vitro are used as the catalyst, the amount of the catalyst is preferably 1-20%, more preferably 5-15%, and most preferably 10% of the weight of the reaction solution, based on the wet weight of the cells after centrifugation at 12000rpm for 10 min; when in vitro nitrile hydratase is used as a catalyst, the addition amount of in vitro nitrile hydratase is preferably 10-10000U per liter of reaction solution, more preferably 100-5000U, and most preferably 500-1000U; the reaction solution consists of a substrate and water.

In the present invention, the temperature of the hydration reaction is preferably 10 to 50 ℃, more preferably 20 to 30 ℃, and the pH of the hydration reaction is preferably 5 to 10, more preferably 6 to 8, and most preferably 7.

In the present invention, the method for producing the nitrile hydratase in vitro-expressing cell (genetically engineered bacterium) comprises: constructing a gene for expressing nitrile hydratase into a target plasmid vector, and introducing the gene into an expression host bacterium to obtain a cell for expressing nitrile hydratase in vitro; the target plasmid vector is preferably pET-30a (+), pET-21a (+), pET-22b (+), pET-28a (+), pETDuet-1 or pACYCDuet-1, but is not limited to the vectors, and is preferably pET-28a (+), and the restriction sites are preferably EcoRI/BamHI and HindIII; the expression host bacterium is preferably E.coli, more preferably E.coliBL21 (DE 3). The present invention is not particularly limited with respect to the specific method for constructing the gene expressing nitrile hydratase into a target plasmid vector and introducing the gene into an expression host bacterium, and may be carried out by a conventional method in the art.

In the present invention, the nucleotide (gene) sequence (GenBank accession No. AJ 511276.1) of the nitrile hydratase derived from said Agrobacterium tumefaciens (Agrobacterium tumefaciens) is shown in SEQ ID No.7, specifically:

atgtcacatgatcatgatcataccgaaccgccgactgagatcgcgctgcgtgtcaaggccctggagtccctgcttacggaaaagggacttgtcgatcccgctgcactcgatgagctcgttgacacgtacgagaacaggatcggaccccgcaacggggcgcttgttgtcgcaaaagcctggacggatcctgcctacaaacaacgcttgctgaccaacgctacggaggcaatcgccgagctcggcttttccggtgttcagggcgaggacatgctggtggtggaaaattcgcccaccgtgcataacatgaccgtctgcacgctttgctcctgctacccctggccgaccctcgggctgccgccggcgtggtacaaatccgcgccctatcgttcccgcgtcgtcatcgatccgcgcggcgtccttgccgaattcggcgtcagcgttcccgccgacaaggaagtccgtgtttgggacagcagcgccgagcttcgctacctggtcctgccagaacgcccggcgggaacggagggctggagcgaagagcagcttgtcgaactcgtgacacgcgattcgatgatcgggaccggctttcccaaaaacccggccgatcttcactaactccaatccaagaaggggaatgagcatgaatggagtacatgatctcggcggaatgcacgggcttggcccgatcgcgccgcctgccgacgagccggtgttcgcccaccagtgggagcggcgcatttttgcgctgttcgttccgttgttcgggggcgggcatttcaatgtcgaccagttccgccacgctatcgagcggatggatccggcgcattatcttcaagggacctactacgagcactggctgcatgcgttcgaaacccttctgatagagggtggtgcaatctcgcgagcggaactcgacgcccggatcaaacagatcggaggggcacagataatggccgtcgtcaccagagatatgatcgagccgattgtcaggaccggcgcctccgctcgcgtcgctgcagacgtcgccgcgagattcaaagtgggcgacacggtccgcgccaaaaatatcaatccgacgacccatacgcggttgccgcgctacgtccgcggcagggtcggaacaatcgagatcgatcacggcgtcttcgtgacgcctgataccgttgcccatggcaagggcgagcatccgcagcatgtctattgcgtgcggttcgccgcagtcgagctttggggttcggatgtttccggcaccgataatgtccgcatcgacttgtgggatgactatctggagaaggcataa。

in the present invention, the nucleotide (gene) sequence (GenBank accession No. AAUW 01000001.1) of the nitrile hydratase derived from the Staphylococcus aggregatum (Stappia aggregata IAM 12614) is shown in SEQ ID NO.8, specifically:

atgtccgctcacgaccacgatcatccccacgatcacgaccattccgaattgagcgagatcgagctgcgtgtgcgcgccctggaaacgctgctgacggaaaaggggtacatcgatcctcctgccctggatgaactcatcgagacctacgagacaaaaatcggcccccggaacggcgctctggtggtggcaaaggcctggacggatccggactatcgggaacggctgatgaaagacgcaacggcggccattgccgaacttggtttcaccggccggcagggcgaacacatggtagcggtcgagaataccgacgacattcacaacatggtcgtctgcacgctgtgctcctgttatccatggacagtgctgggcctgccgccggtctggtacaagtctgcgccctaccgctccagagccgtacgcgacccgcgcggcgttctggctgagttcaaggtagaactgcccgatgacaccgagatccgcgtttgggattcgacggcagaaatccggtatctggtgatcccgaaacgtcctgccggaaccgatggctggagcgaagagcgcctcgccgatctggtcacccgcaacagcatgatcggaacgggccttgccctcgatccttcctccctggaggacgcagcatgaacggtgcacaggatctgggcggacagatgggctttggccccattgaactggaagaaaacgagccgaactttcacgccaaatgggaagaacgcgcctttgccgtgacattggccatgggcgcgaccggatcctggacgctggatgcctcgcgctttgcccgcgaatccttgccgccggtcacctacctctcttccagctattacgagatctggacccgcgggcttgaaaaactcctgctctccaacggtctggtcacggaagaagagctgaaggaaggacgcaaacttaaagaggccaagccgatcaagcgtgtgctctcggcggacgctgtcgccggggtcctggcgaagggatcgtcggtcgaccgtgaagagcatcagcctgccaggttccgcaccggtgacagggttaagacacggcgcatgaaccccgaacatcacacccgcctgccccgttacgcccgcgatgccgtcggcacgatcgaggccattcacggcgttcacgtgtttccggacaccaatgcccacggcgaaggcgaacagcctgcctggctctatggcgtcctcttcaagggcactgacatctgggggccggacagcgatcccaagcttaccctgcgcatcgatctctgggagccatatcttgaccttgcccgctga。

in the present invention, the nucleotide (gene) sequence (NCBI accession No. NC — 018700.1) of the nitrile hydratase derived from Sinorhizobium meliloti (Sinorhizobium meliloti) is shown in SEQ ID No.9, which specifically is:

atgtccgaacatcatcatgggcatggcgacgatcacggccatcaccacgacaaccacctgaccgacatggaagcccgggtaaaggcgttggagacggtgctgacggaaaagggtttgatcgatcctgcggcgatcgacgcgatcgtcgacgcgtacgagacgaaggtgggaccgcgaaacggcgcacgcgtcgtcgccaaggcctggagcgatccgggttttgccgattggctgaaacgcgacgcgaccgcggcgatcgccagcctcggctttaccgggcgccagggcgagcacatgcgtgcggtgttcaacacgtccgagacgcacaacctcatcgtctgtacgctgtgctcctgctatccctgggcagtgctggggctgccgccggtctggtacaaggcgccgccctatcgttcgcgcgccgtcatcgatccccgcggcgtgcttgccgaattcgggctcgagctgtcggctgaaaagaaggggactgtcaggaattctgtgcggtgggccatgatgatgaaaaggagagaatcatcacatggctatcgagaaagaacttctggaccagctcctggctggacgtgaatgaacggtccgcatgatctcggcggccagcatggcatgggcccgatcgctccggaacgcaatgagcccattttccatgcagagtgggagaagcgggcgctcggcatcacgctttcctgtggcgctttcggtgcctggacgctggacgaaagccggcacgcgcgcgaaagcctggccccggcgacctatctctccgcgagctattatgaaatctggactcgagccctggaaacgcttctcaagcgccatggcttcgtgacgcaggccgagctcgatgccggccatatgctcgacaaggggcgggagccgaaacgggtgctcacagcggacatggtggccggcgtgctcgccaagggcgggccatgcgaccggccggtcgaagcgccgccgcgctttgccgccggcgacagtgtgcgtacgaagaacttcaatccggagagccacacgcgcctgccgcgctacgcccgcgcccggacaggcatggtggaggccgtgcagggcagcttcgtcttcccggacgacaacgcccacggcaagggcgagaacccgcaatggctctacatggtggtcttcgatgccggcgagatctggggtgagggcgcggacccgacgcttaccgtctccatcgatgcctgggagagctatcttgaacacgcttag。

in the specific implementation process of the invention, a nitrile hydratase gene sequence is synthesized by the whole gene of Shanghai biological engineering technology GmbH, and is constructed on a plasmid vector pET-28a (+), and the restriction enzyme sites are EcoRI/BamHI and HindIII; then the constructed plasmid is introduced into an expression host E.coliBL21 (DE 3) strain to obtain a genetic engineering strain E.coliBL21 (DE 3)/pET-28 a (+) -NHx.

After obtaining the genetically engineered bacterium E.coliBL21 (DE 3)/pET-28 a (+) -NHx, the invention inoculates the E.coliBL21 (DE 3)/pET-28 a (+) -NHx in a liquid culture medium, and cultures until the thallus density OD600 value reaches 0.8-1.0, obtains the culture solution of the genetically engineered bacterium; the liquid culture medium takes water as a solvent and comprises the following components: 10g/L of peptone, 5g/L of yeast extract and 10g/L of NaCl; the pH value of the liquid culture medium is preferably 7.0; the temperature of the culture is preferably 35 to 40 ℃, and the culture rotation speed is preferably 180 to 220rpm.

The genetically engineered bacterium E.coliBL21 (DE 3)/pET-28 a (+) -NHx can efficiently express nitrile hydratase protein under the conventional IPTG induction condition; the final concentration of IPTG is preferably 0.5mM.

In the catalytic reaction system of the invention, the catalyst is used in the form of crude enzyme liquid after the cells of the genetically engineered bacteria are crushed, engineering bacteria resting cells expressing recombinase, purified pure enzyme or immobilized enzyme.

In the present invention, the medium for the hydration reaction is preferably pure water or an aqueous solution to which a buffer salt is added; the buffer salt is preferably Tris-HCl; the concentration of Tris-HCl is preferably 50mM.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It should be apparent that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

The materials and methods in the examples were:

the experimental methods in the present invention are conventional methods unless otherwise specified, and can be specifically referred to the molecular cloning laboratory Manual, edited by J. Sambuque et al.

The restriction enzyme DNA ligase and other tool enzymes used in the embodiment of the invention are all purchased from TaKaRa, takara Bio engineering (Dalian) Co., ltd; the genome extraction kit, the plasmid extraction kit and the DNA recovery and purification kit are purchased from Axygen Hangzhou limited company; coli DH5 α, E.coli BL21 (DE 3), plasmids pET-30a (+), pET-21a (+), pET-22b (+), and the like, from Novagen; DNAmarker, fastPFuDNA polymerase, low molecular weight standard protein and agarose electrophoresis reagent are purchased from Beijing all-style gold biotechnology limited; the gene synthesis and sequence sequencing work is completed by Shanghai Bioengineering technology, inc. The method of using the above reagent refers to the commercial specification. The raw materials of the cyanopyrazines are all purchased from Zhejiang Hengkang pharmaceutical industry, inc. (China).

Example 1

1. Acquisition of nitrile hydratase Gene

The reported amino acid sequences of cobalt-type nitrile hydratase are collected, and the nitrile hydratase is subjected to multiple sequence alignment by the method of ClustalW2 (http:// www.ebi.ac.uk/Tools/msa/ClustalW 2) to determine the conserved regions in the sequences. And taking the amino acid sequence of the conserved region as a molecular probe for subsequent search of nitrile hydratase genes in a gene information database. A conserved amino acid sequence-CTLCSC-in the alpha subunit of the reported nitrile hydratase is selected as a molecular probe by aligning the amino acid sequences, and the GenBank bioinformatic data (https:// www.ncbi.nlm.nih.gov) is searched for homologous sequences using BasiclocalalignmentSearchTool (http:// blast.ncbi.nlm.n.gov). Genes reported and annotated as nitrile hydratase after whole genome sequencing were selected. The searched amino acid sequences of the nitrile hydratases are respectively gathered, multi-sequence comparison is carried out by using a ClustalW2 method, the amino acid sequences with the similarity of more than 95 percent are removed, and the specific information of the obtained nitrile hydratases is shown in a table 1.

TABLE 1 nitrile hydratase enzyme library information

2. Construction of a Strain expressing nitrile hydratase Gene

The nitrile hydratase gene sequence is submitted to a gene synthesis company for complete gene synthesis, and is constructed on a plasmid vector pET-28a (+), and the restriction enzyme cutting sites are EcoRI/BamHI and HindIII; and then introducing the constructed plasmid into an expression host E.coliBL21 (DE 3) strain, namely the genetic engineering strain E.coliBL21 (DE 3)/pET-28 a (+) -NHx.

If the nitrile hydratase gene is constructed on other expression plasmids, a plasmid pET-28a (+) can be extracted, the target gene fragment and the target empty plasmid which are purified and recovered after double enzyme digestion are respectively subjected to double enzyme digestion by using corresponding restriction enzymes, and the enzyme digestion conditions and the system are shown in Table 2; then, the target gene fragment was ligated to the target plasmid using T4DNA ligase, and the ligation system is shown in Table 3.

TABLE 2 double digestion System and conditions

TABLE 3 construction of ligation System of recombinant expression plasmids

3. Recombinant expression of nitrile hydratase

The engineering bacteria successfully constructed are inoculated into 5mL of liquid culture medium, shaken at 37 ℃ for 10h with 200 rpm. Inoculating the cultured culture solution into a fresh liquid culture medium according to the inoculation amount of 10%. Shaking and culturing in a shaker at 37 ℃ for 2-3 h at 200rpm, cooling to 18 ℃ when the density OD600 value of the thalli reaches 0.8, and adding IPTG until the final concentration is 0.5mM. The flask was then transferred to a shaker at 18 ℃ and incubated for a further 16h at 200 rpm.

4. Enzyme activity assay for recombinant nitrile hydratase

The enzyme activity of the recombinant nitrile hydratase is detected by taking 6-fluoro-3-hydroxy-2-cyanopyrazine as a substrate, and specific operation steps and a detection method are as follows in consideration of different optimal pH values and temperatures of the recombinant nitrile hydratase.

The activity determination system of the recombinant nitrile hydratase is as follows: the total reaction system is 5.0mL, phosphate buffer (100mM, pH7.0), 5mM substrate, adding appropriate amount of engineering bacteria cell expressing nitrile hydratase. The reaction was shaken at 25 ℃ for 60min, and 500. Mu.L of a sample was taken and 500. Mu.L of pure acetonitrile was immediately added to terminate the reaction. The reaction mixture was centrifuged at 12,000 Xg for 5min to remove cells and enzyme proteins. The 6-fluoro-3-hydroxy-2-amidopyrazine produced in the reaction system was determined by high performance liquid chromatography with C18 column (5 μm, 4.6X 250mm, agilent) as the mobile phase at 10mMKH ₂ PO ₄ Buffer solution: acetonitrile =8 (v/v) and flow rate 0.5mL/min. The UV detector detects the light with a wavelength of 225nm. The analytical map is shown in FIG. 2, and the peak appearance time is 6-fluoro-3-hydroxy-2-acylThe retention time of the aminopyrazine and 6-fluoro-3-hydroxy-2-cyanopyrazine is 11.2min and 16.5min respectively. Definition of enzyme activity: at 25 ℃, the enzyme amount which can convert the substrate (6-fluoro-3-hydroxy-2-cyanopyrazine) into 1 mu mol of the product (6-fluoro-3-hydroxy-2-amidopyrazine) is 1U within 1 min. The activity of all the recombinant nitrile hydratases was measured and the results are shown in Table 4. As can be seen from Table 4, only three nitrile hydratases (NH 16, NH25, NH 27) derived from Agrobacterium tumefaciens (Agrobacterium tumefaciens), staphylococcus aggregatus (Stappia aggregatata IAM 12614) and Sinorhizobium meliloti (Sinorhizobium meliloti) have corresponding catalytic activities.

TABLE 4 determination of nitrile hydratase Activity

Example 2 Gene engineering bacteria catalyze 2-cyanopyrazine to produce 2-amide pyrazine

25ml of the fermentation broth of the engineered bacterium E.coli BL21 (DE 3)/pET-28 a (+) -NH16 constructed in example 1 was centrifuged at 12000rpm for 10min to collect the cells, and then 250ml of a 50mM Tris-HCl (pH 8.0) buffer was used to resuspend the collected cells, and the enzyme activity of the resuspended enzyme solution was 10U/ml. 1.0g of 2-cyanopyrazine was added to the resuspension solution, and the reaction was carried out at 20 ℃ for 24 hours. And detecting the contents of the 2-cyanopyrazine, the 2-amidopyrazine and the 2-carboxypyrazine in the reaction system by using a liquid chromatography. The conversion rate of the substrate is more than 99 percent, the yield of the 2-amide pyrazine is more than 92 percent, and the generation of the 2-carboxyl pyrazine is not found in the reaction system.

Example 3 genetically engineered bacteria catalyze the formation of 3-methyl-2-amidopyrazine from 3-methyl-2-cyanopyrazine

25ml of the fermentation broth of the engineered bacterium E.coli BL21 (DE 3)/pET-28 a (+) -NH27 constructed in example 1 was centrifuged at 12000rpm for 10min to collect the cells, and then the collected cells were resuspended in 250ml of 50mM Tris-HCl (pH 6.0) buffer. 0.5g of 3-methyl-2-cyanopyrazine was added to the resuspended solution, and hydration reaction was carried out at 30 ℃ for 120 hours. And then detecting the content of the 3-methyl-2-cyano pyrazine and the 3-methyl-2-amide pyrazine in the reaction system by using a high performance liquid chromatography. The substrate conversion rate is more than 99 percent, and the yield of the 3-methyl-2-amide pyrazine is more than 89 percent.

Example 4 genetically engineered bacteria catalyze the formation of 6-methyl-2-aminopyrazine from 6-methyl-2-cyanopyrazine

25ml of the fermentation broth of the engineered bacterium E.coli BL21 (DE 3)/pET-28 a (+) -NH25 constructed in example 1 was centrifuged at 12000rpm for 10min to collect the cells, and then the collected cells were resuspended in 10ml of 50mM Tris-HCl (pH 7.5) buffer. 1.0g of 6-methyl-2-cyanopyrazine was added to the resuspended solution, and hydration reaction was carried out at 25 ℃ for 12 hours. And then detecting the contents of the 6-methyl-2-cyanopyrazine and the 6-methyl-2-amide pyrazine in the reaction system by using a high performance liquid chromatography. The substrate conversion rate is more than 99%, and the yield of 6-methyl-2 amide pyrazine is more than 99%.

Example 5 genetically engineered bacteria catalyze the formation of 6-methyl-3-hydroxy-2-pyrazinamide from 6-methyl-3-hydroxy-2-pyrazinamide

25ml of the fermentation broth of the engineered bacterium E.coli BL21 (DE 3)/pET-28 a (+) -NH25 constructed in example 1 was centrifuged at 12000rpm for 10min to collect the cells, and then 250ml of 50mM Tris-HCl (pH 8.0) buffer was used to resuspend the collected cells. 0.6g of 6-methyl-3-hydroxy-2-cyanopyrazine was added to the resuspended solution, and hydration reaction was carried out at 30 ℃ for 26 hours. Then detecting the content of 6-methyl-3-hydroxy-2-cyanopyrazine and 6-methyl-3-hydroxy-2-amide pyrazine in the reaction system by using high performance liquid chromatography. The substrate conversion rate is more than 99%, and the yield of 6-methyl-3-hydroxy-2-amide pyrazine is more than 99%.

Example 6 genetically engineered bacteria catalyze the formation of 6-bromo-3-amino-2-amidopyrazine from 6-bromo-3-amino-2-amidopyrazine

25ml of the fermentation broth of the engineered bacterium E.coliBL21 (DE 3)/pET-30 a (+) -NH25 constructed in example 1 was centrifuged at 12000rpm for 10min to collect the cells, and then the collected cells were resuspended in 25ml of 50mM Tris-HCl buffer (pH 7.0). 0.5g of 6-bromo-3-amino-2-cyanopyrazine was added to the resuspended solution, and hydration reaction was carried out at 35 ℃ for 48 hours. And then detecting the contents of the 6-bromo-3-amino-2-cyanopyrazine and the 6-bromo-3-amino-2-amidopyrazine in the reaction system by using a high performance liquid chromatography. The substrate conversion rate is more than 99%, and the yield of the 6-bromo-3-amino-2-amide pyrazine is more than 99%.

Example 7 genetically engineered bacteria catalyze 6-fluoro-3-hydroxy-2-cyanopyrazine to form 6-fluoro-3-hydroxy-2-amidopyrazinyl

25ml of the fermentation broth of the engineered bacterium E.coli BL21 (DE 3)/pET-21 a (+) -NH25 constructed in example 1 was centrifuged at 12000rpm for 10min to collect the cells, and then the collected cells were resuspended in 250ml of 50mM Tris-HCl (pH 7.0) buffer. 0.75g of 6-fluoro-3-hydroxy-2-cyanopyrazine was added to the resuspension solution, and hydration reaction was carried out at 25 ℃ for 18 hours. And then detecting the contents of the 6-fluoro-3-hydroxy-2-cyanopyrazine and the 6-fluoro-3-hydroxy-2-amidopyrazine in the reaction system by using a high performance liquid chromatography. The substrate conversion rate is more than 99%, and the yield of 6-fluoro-3-hydroxy-2-amide pyrazine is more than 99%.

Example 8 catalysis of 6-chloro-3-hydroxy-2-cyanopyrazine by genetically engineered bacteria to 6-chloro-3-hydroxy-2-amidopyrazinyl

25ml of the fermentation broth of the engineered bacterium E.coli BL21 (DE 3)/pET-22 b (+) -NH25 constructed in example 1 was centrifuged at 12000rpm for 10min to collect the cells, and then 250ml of a 50mM Tris-HCl (pH 7.0) buffer solution was used to resuspend and sonicate the collected cells to obtain an in vitro crude enzyme solution of in vitro nitrile hydratase NH25, and the enzyme activity of the nitrile hydratase was determined to be 1035.16U/L. 0.75g of 6-chloro-3-hydroxy-2-cyanopyrazine was added to the crude enzyme solution, and hydration reaction was carried out at 40 ℃ for 23 hours. And then detecting the contents of the 6-chloro-3-hydroxy-2-cyanopyrazine and the 6-chloro-3-hydroxy-2-amidopyrazine in the reaction system by using a high performance liquid chromatography. The substrate conversion rate is more than 99%, and the yield of the 6-chloro-3-hydroxy-2-amide pyrazine is more than 99%.

Example 9 Gene engineering bacteria catalyzed 6-bromo-3-hydroxy-2-cyanopyrazine to 6-bromo-3-hydroxy-2-amidopyrazine

25ml of the fermentation broth of the engineered bacterium E.coli BL21 (DE 3)/pETDuet-NH 25 constructed in example 1 was centrifuged at 12000rpm for 10min to collect the cells, and then the collected cells were resuspended in 10ml of 50mM Tris-HCl (pH 7.0) buffer. 0.75g of 6-bromo-3-hydroxy-2-cyanopyrazine was added to the resuspension solution, and hydration reaction was carried out at 20 ℃ for 54 hours. And then detecting the contents of the 6-bromo-3-hydroxy-2-cyanopyrazine and the 6-bromo-3-hydroxy-2-amidopyrazine in the reaction system by using a high performance liquid chromatography. The conversion rate of the substrate is more than 99 percent, and the yield of the 6-bromo-3-hydroxy-2-amide pyrazine is more than 99 percent.

Example 10 catalysis of 6-iodo-3-hydroxy-2-cyanopyrazine by genetically engineered bacteria to 6-iodo-3-hydroxy-2-amidopyrazinyl

25ml of the fermentation broth of the engineered bacterium E.coliBL21 (DE 3)/pACYCDuet-NH 25 constructed in example 1 was centrifuged at 12000rpm for 10min to collect the cells, and then the collected cells were resuspended in 250ml of 50mM Tris-HCl (pH 7.0) buffer. 0.75g of 6-iodo-3-hydroxy-2-cyanopyrazine was added to the resuspension solution, and hydration reaction was carried out at 20 ℃ for 103 hours. And detecting the content of the 6-iodo-3-hydroxy-2-cyanopyrazine and the 6-iodo-3-hydroxy-2-amidopyrazine in the reaction system by using a high performance liquid chromatography. The substrate conversion rate is more than 99%, and the yield of 6-iodo-3-hydroxy-2-amide pyrazine is more than 99%.

Comparative example 1

25ml of the fermentation broth of the engineered bacterium E.coli BL21 (DE 3)/pET-28 a (+) -NH4 constructed in example 1 was centrifuged at 12000rpm for 10min to collect the cells, and then the collected cells were resuspended in 250ml of 50mM Tris-HCl (pH 7.0) buffer. 0.75g of 6-fluoro-3-hydroxy-2-cyanopyrazine was added to the resuspension solution, and hydration reaction was carried out at 25 ℃ for 120 hours. And then detecting the contents of the 6-fluoro-3-hydroxy-2-cyanopyrazine and the 6-fluoro-3-hydroxy-2-amidopyrazine in the reaction system by using a high performance liquid chromatography, wherein the generation of the product is not monitored.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Zhejiang Hengkang pharmaceutical industry Co., ltd

Application of <120> nitrile hydratase derived from sinorhizobium meliloti in preparation of amide pyrazine compounds

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<213> Agrobacterium tumefaciens (Agrobacterium tumefaciens)

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<213> Agrobacterium tumefaciens (Agrobacterium tumefaciens)

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<212> PRT

<213> Sinorhizobium meliloti (Sinorhizobium meliloti)

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<213> Sinorhizobium meliloti (Sinorhizobium meliloti)

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<213> Agrobacterium tumefaciens (Agrobacterium tumefaciens)

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<213> Staphylococcus aggregatus (Stappia aggregata IAM 12614)

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ggccttgccc tcgatccttc ctccctggag gacgcagcat gaacggtgca caggatctgg 660

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cggcggacgc tgtcgccggg gtcctggcga agggatcgtc ggtcgaccgt gaagagcatc 1020

agcctgccag gttccgcacc ggtgacaggg ttaagacacg gcgcatgaac cccgaacatc 1080

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gcgtcctctt caagggcact gacatctggg ggccggacag cgatcccaag cttaccctgc 1260

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<210> 9

<211> 1248

<212> DNA

<213> Sinorhizobium meliloti (Sinorhizobium meliloti)

<400> 9

atgtccgaac atcatcatgg gcatggcgac gatcacggcc atcaccacga caaccacctg 60

accgacatgg aagcccgggt aaaggcgttg gagacggtgc tgacggaaaa gggtttgatc 120

gatcctgcgg cgatcgacgc gatcgtcgac gcgtacgaga cgaaggtggg accgcgaaac 180

ggcgcacgcg tcgtcgccaa ggcctggagc gatccgggtt ttgccgattg gctgaaacgc 240

gacgcgaccg cggcgatcgc cagcctcggc tttaccgggc gccagggcga gcacatgcgt 300

gcggtgttca acacgtccga gacgcacaac ctcatcgtct gtacgctgtg ctcctgctat 360

ccctgggcag tgctggggct gccgccggtc tggtacaagg cgccgcccta tcgttcgcgc 420

gccgtcatcg atccccgcgg cgtgcttgcc gaattcgggc tcgagctgtc ggctgaaaag 480

aaggggactg tcaggaattc tgtgcggtgg gccatgatga tgaaaaggag agaatcatca 540

catggctatc gagaaagaac ttctggacca gctcctggct ggacgtgaat gaacggtccg 600

catgatctcg gcggccagca tggcatgggc ccgatcgctc cggaacgcaa tgagcccatt 660

ttccatgcag agtgggagaa gcgggcgctc ggcatcacgc tttcctgtgg cgctttcggt 720

gcctggacgc tggacgaaag ccggcacgcg cgcgaaagcc tggccccggc gacctatctc 780

tccgcgagct attatgaaat ctggactcga gccctggaaa cgcttctcaa gcgccatggc 840

ttcgtgacgc aggccgagct cgatgccggc catatgctcg acaaggggcg ggagccgaaa 900

cgggtgctca cagcggacat ggtggccggc gtgctcgcca agggcgggcc atgcgaccgg 960

ccggtcgaag cgccgccgcg ctttgccgcc ggcgacagtg tgcgtacgaa gaacttcaat 1020

ccggagagcc acacgcgcct gccgcgctac gcccgcgccc ggacaggcat ggtggaggcc 1080

gtgcagggca gcttcgtctt cccggacgac aacgcccacg gcaagggcga gaacccgcaa 1140

tggctctaca tggtggtctt cgatgccggc gagatctggg gtgagggcgc ggacccgacg 1200

cttaccgtct ccatcgatgc ctgggagagc tatcttgaac acgcttag 1248

Claims

1. The application of nitrile hydratase derived from sinorhizobium meliloti in preparing amide pyrazine compounds; the nitrile hydratase catalyzes the cyano pyrazine compound to carry out hydration reaction to generate the amide pyrazine compound; the nitrile hydratase is derived from Sinorhizobium meliloti (Sinorhizobium meliloti);

the amino acid sequence of the alpha subunit of the nitrile hydratase of Sinorhizobium meliloti (Sinorhizobium meliloti) is shown as SEQ ID NO.5, and the amino acid sequence of the beta subunit is shown as SEQ ID NO. 6;

the cyano pyrazine compound is 3-methyl-2-cyano pyrazine or 6-fluoro-3-hydroxy-2-cyano pyrazine.

2. The application according to claim 1, characterized in that it comprises the following steps: taking a cyanopyrazine compound as a substrate, and carrying out hydration reaction in an aqueous solution by using isolated nitrile hydratase or cells for expressing the nitrile hydratase in vitro as a catalyst to obtain the amide pyrazine compound.

3. Use according to claim 2, wherein the concentration of the substrate in water is between 2 and 100g/L; when the cells expressing nitrile hydratase in vitro are used as a catalyst, the adding amount of the catalyst is calculated by the wet weight of the cells after centrifugation at 12000rpm for 10min, and the adding amount of the cells is 1-20% of the weight of the reaction solution; when the isolated nitrile hydratase is used as a catalyst, the amount of the isolated nitrile hydratase added is 10 to 10000U per liter of the reaction solution.

4. Use according to claim 2, wherein the temperature of the hydration reaction is between 10 and 50 ℃ and the pH of the hydration reaction is between 5 and 10.

5. Use according to claim 2, wherein the temperature of the hydration reaction is between 20 and 30 ℃; the pH value of the hydration reaction is 6-8.