阅读说明：本技术 脱乙酰氧头孢菌素C合成酶突变体及其在β-内酰胺抗生素母核合成中的应用 (Deacetoxycephalosporin C synthetase mutant and application thereof in synthesis of beta-lactam antibiotic parent nucleus ) 是由 孙周通 杨大猛 曲戈 蒋迎迎 李超 刘保艳 于 2020-04-17 设计创作，主要内容包括：本发明公开了脱乙酰氧头孢菌素C合成酶突变体及其在β-内酰胺抗生素母核合成中的应用。本发明公开的脱乙酰氧头孢菌素C合成酶突变体为对scDAOCS7的第61、225、160、162、179、73、102和/或158位进行突变得到的蛋白质,scDAOCS7的氨基酸序列为序列表中SEQ ID No.2。实验证明,scDAOCS7及其突变体可以催化6-APA一步生成7-ADCA；还可以催化青霉素G钾盐合成G-7-ADCA,且催化能力相较于scDAOCS7有显著的提高,具有广泛的应用前景。(The invention discloses a deacetoxycephalosporin C synthetase mutant and application thereof in synthesis of a beta-lactam antibiotic parent nucleus. The deacetoxycephalosporin C synthetase mutant disclosed by the invention is a protein obtained by mutating the 61 th, 225 th, 160 th, 162 th, 179 th, 73 th, 102 th and/or 158 th positions of scDAOCS7, and the amino acid sequence of scDAOCS7 is SEQ ID No.2 in a sequence table. Experiments prove that the scDAOCS7 and the mutant thereof can catalyze 6-APA to generate 7-ADCA in one step; can also catalyze penicillin G potassium salt to synthesize G-7-ADCA, and the catalytic capability is obviously improved compared with that of scDAOCS7, thereby having wide application prospect.)

1. A method of preparing 7-ADCA comprising: carrying out catalytic reaction by using the mutant protein of the scDAOCS7 or scDAOCS7 by using 6-APA as a substrate to obtain 7-ADCA;

the amino acid sequence of the scDAOCS7 is SEQ ID No.2 in the sequence table; the mutant protein of the scDAOCS7 is obtained by mutating positions 61, 225, 160, 162, 179, 73, 102 and/or 158 of the scDAOCS 7.

2. The method of claim 1, wherein: the mutant protein of the scDAOCS7 is obtained by modifying all, any seven, any six, any five, any four, any three, any two or any one of the following eight types of scDAOCS 7:

x1, mutation of alanine residue at position 61 of scdaos 7 to aspartic acid residue or glutamic acid residue;

x2, mutation of phenylalanine residue at position 225 of scdaos 7 to leucine residue or isoleucine residue;

x3, mutation of arginine residue at position 160 of scdaos 7 to alanine residue, valine residue, threonine residue, isoleucine residue, cysteine residue, or glycine residue;

x4, mutation of arginine residue at position 162 of scdaos 7 to alanine residue, valine residue, threonine residue, isoleucine residue, cysteine residue, or glycine residue;

x5, mutation of arginine residue at position 179 of scdaos 7 to leucine, isoleucine, alanine, glycine, proline, valine or threonine residue;

x6, mutation of the threonine residue at position 73 of scdaos 7 to an alanine residue, a valine residue, an isoleucine residue, a leucine residue, a methionine residue, a cysteine residue, or a serine residue;

x7, mutation of serine residue at position 102 of scdaos 7 to alanine residue, valine residue, isoleucine residue, leucine residue, methionine residue, threonine residue or cysteine residue;

x8, mutation of leucine residue at position 158 of scdaos 7 to alanine residue, valine residue, isoleucine residue, methionine residue, threonine residue, cysteine residue or serine residue.

3. A method of preparing 7-ADCA comprising: carrying out a catalytic reaction using a recombinant cell expressing scdaos 7 or the scdaos 7 mutein of claim 1 or 2 or a lysate of said recombinant cell using 6-APA as a substrate to obtain 7-ADCA.

4. The method of claim 3, wherein: the recombinant cell is produced by introducing into a biological cell a recombinant vector capable of expressing scdaos 7 or the scdaos 7 mutein of claim 1 or 2.

5. A process for the preparation of G-7-ADCA by catalytic reaction of a penicillin G potassium salt substrate with scDAOCS7 or the scDAOCS7 mutein of claim 1 or 2 to obtain G-7-ADCA.

6. The scdaacs 7 mutein of claim 1 or 2.

7. The biomaterial related to the scdaos 7 mutant protein as claimed in claim 1 or 2, which is any one of the following B1) to B4):

B1) a nucleic acid molecule encoding the scdaos 7 mutein of claim 1 or 2;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector.

8. Any of the following products:

m1, a kit of parts consisting of 6-APA and the scdAACS 7 mutein of claim 1 or 2;

m2, a kit consisting of 6-APA and the biomaterial of claim 7;

m3, kit consisting of 6-APA and scDAOCS 7;

m4, a kit of parts consisting of penicillin G potassium salt, 6-APA and the scdAACS 7 mutein of claim 1 or 2;

m5, a kit consisting of penicillin G potassium salt, 6-APA and the biomaterial of claim 7;

m6, kit consisting of penicillin G potassium salt, 6-APA and scDAOCS 7.

9. Use of the scdaacs 7 mutein of claim 1 or 2 or of the biomaterial of claim 7 in any of the following applications:

z1, use in catalyzing 6-APA to produce 7-ADCA;

z2, in the preparation of 7-ADCA products generated by catalyzing 6-APA;

z3, use in the manufacture of 7-ADCA;

z4, application in preparing 7-ADCA products;

z5, application in catalyzing penicillin G potassium salt to generate G-7-ADCA;

z6, in preparing G-7-ADCA product of catalyzing penicillin G potassium salt;

z7, use in the manufacture of G-7-ADCA;

z8, and application in preparing G-7-ADCA products.

10. Use of the product of claim 8 in any of the following applications:

z3, use in the manufacture of 7-ADCA;

z4, and application in preparing 7-ADCA products.

Technical Field

The invention relates to a deacetoxycephalosporin C synthetase mutant and application thereof in synthesis of a parent nucleus of a beta-lactam antibiotic in the field of biotechnology.

Background

7-aminodesacetoxycephalosporanic acid (7-ADCA, molecular formula C)₈H₁₀N₂O₃S) is an important mother nucleus for synthesizing cephalosporin antibiotics, and the drugs synthesized by 7-ADCA at present comprise cefadriamine, cefradine, cefaclor and the like, which are antibiotic drugs with large market dosage at home and abroad. The 7-ADCA synthesis method mainly comprises two methods, namely a chemical method and an enzymatic method. Wherein, the chemical method firstly takes penicillin G sylvite as a raw material to oxidize the penicillin G sylvite into penicillin G sulfoxide, then performs ring expansion rearrangement to generate 7-phenylacetylamido-desacetoxycephalosporanic acid, and finally adopts a chemical hydrolysis method to prepare the 7-ADCA. Because the chemical method for synthesizing 7-ADCA has complex process, multiple working procedures and strict equipment requirements, a large amount of chemical raw materials and organic solvents are used in the reaction process, and the defects of labor protection, environmental pollution and the like exist, and the method is gradually replaced by the enzyme method.

The main process for producing 7-ADCA at present is a semi-synthetic enzyme method, which comprises the steps of using penicillin G potassium salt as a substrate, performing hydrogen peroxide catalytic ring expansion to generate 7-phenylacetylaminoacetoxycephalosporanic acid (G-7-ADCA), and hydrolyzing penicillin acylase to remove side chains to obtain the 7-ADCA. However, the process requires a large amount of hydrogen peroxide and has a large impact on the environment, and the bio-enzyme method is gradually favored by the industry due to the advantages of mild reaction conditions, environmental protection and the like, so that the research on the 7-ADCA synthesized by the bio-enzyme method has important scientific research and application values. The 7-ADCA synthesized by the biological enzyme method usually takes penicillin G potassium salt with low price as a raw material, and the route for synthesizing the 7-ADCA is as follows: the potassium salt of penicillin G is subjected to ring expansion reaction under the catalytic action of deacetoxycephalosporin C synthetase (DAOCS) to generate 7-phenylacetylaminoacetoxycephalosporanic acid (G-7-ADCA), the scheme of the reaction structural formula from penicillin G to G-7-ADCA is shown in figure 1, and G-7-ADCA is subjected to hydrolysis reaction under the catalytic action of penicillin acylase to generate 7-ADCA. Among them, penicillin acylases derived from escherichia coli discovered in the 60 th century have attracted attention due to the advantages of long-term operation stability and the like after being immobilized, and have been widely applied in the industrial field, so that penicillin acylases are not the rate-limiting step of enzymatic synthesis of 7-ADCA, and the key of enzymatic synthesis of 7-ADCA at present is to improve the catalytic activity of DAOCS on penicillin G, but the activity of DAOCS is low, and the industrial production requirements are difficult to meet.

DAOCS derived from cephalosporium acremonium (c.acremonium), streptomyces clavuligerus (s.clavuligerus) and nocardia (n.lactamdurans) have been purified for expression and proved to be Fe²⁺、O₂And alpha-ketoglutarate-dependent enzymes (Vallejo et al, 1987; Yeh and Dotzlaf., 1987). scdaos (s. clavuligerus-derived dacs, EC 1.14.20.1) is currently the only DAOCS enzyme that obtains single crystal resolution. The scdaos exists as a trimer and the carboxy terminus of each monomer (residues 308-311) is inserted into the active pocket of the adjacent monomer. When Fe is present in the system²⁺And alpha-ketoglutarate, induces the dissociation of scdaos into the monomeric form. Studies have shown that scdaos monomer is the active form that catalyzes the ring-expanding reaction, whereas its catalytic activity is related to the equilibrium between scdaos monomer and scdaos trimer. Although related mutation research is also carried out on deacetoxycephalosporin C synthetase of a substrate penicillin G potassium salt (Yang et al, 2012), the activity of the deacetoxycephalosporin C synthetase still cannot meet the industrial demand, and therefore, the improvement of catalytic activity to meet the industrial demand is important.

Disclosure of Invention

The invention aims to provide application of a deacetoxycephalosporin C synthetase mutant in preparation of 7-ADCA.

The present invention provides, first, a method of preparing 7-ADCA, the method comprising: carrying out catalytic reaction by using 6-aminopenicillanic acid (6-APA) as a substrate and using scDAOCS7 or scDAOCS7 mutant protein to obtain 7-ADCA;

the amino acid sequence of the scDAOCS7 is SEQ ID No.2 in the sequence table; the mutant protein of the scDAOCS7 is obtained by mutating positions 61, 225, 160, 162, 179, 73, 102 and/or 158 of the scDAOCS 7.

In the above method, the scdaacs 7 mutant protein may be obtained by modifying scdaacs 7 with all, any seven, any six, any five, any four, any three, any two or any one of the following eight proteins:

x1, mutation of alanine residue at position 61 of scdaos 7 to aspartic acid residue or glutamic acid residue;

x2, mutation of phenylalanine residue at position 225 of scdaos 7 to leucine residue or isoleucine residue;

x3, mutation of arginine residue at position 160 of scdaos 7 to alanine residue, valine residue, threonine residue, isoleucine residue, cysteine residue, or glycine residue;

x4, mutation of arginine residue at position 162 of scdaos 7 to alanine residue, valine residue, threonine residue, isoleucine residue, cysteine residue, or glycine residue;

x5, mutation of arginine residue at position 179 of scdaos 7 to leucine, isoleucine, alanine, glycine, proline, valine or threonine residue;

x6, mutation of the threonine residue at position 73 of scdaos 7 to an alanine residue, a valine residue, an isoleucine residue, a leucine residue, a methionine residue, a cysteine residue, or a serine residue;

x7, mutation of serine residue at position 102 of scdaos 7 to alanine residue, valine residue, isoleucine residue, leucine residue, methionine residue, threonine residue or cysteine residue;

x8, mutation of leucine residue at position 158 of scdaos 7 to alanine residue, valine residue, isoleucine residue, methionine residue, threonine residue, cysteine residue or serine residue.

In one embodiment of the present invention, the mutant protein of scdaacs 7 is scdaacs 7-a61D, scdaacs 7-a61E, scdaacs 7-F225I, scdaacs 7-F225L, scdaacs 7-a61D/F225I, scdaacs 7-a61D/F225L, scdaacs 7-a61E/F225I or scdaacs 7-a 61E/F225L. The scDAOCS7-A61D is a protein obtained by mutating alanine residue at position 61 of scDAOCS7 into aspartic acid residue. The scDAOCS7-A61E is a protein obtained by mutating alanine residue at position 61 of scDAOCS7 into glutamic acid residue. The scDAOCS7-F225I is a protein obtained by mutating a phenylalanine residue at position 225 of the scDAOCS7 into an isoleucine residue. The scDAOCS7-F225L is a protein obtained by mutating a phenylalanine residue at position 225 of the scDAOCS7 into a leucine residue. The scDAOCS7-A61D/F225I is a protein obtained by mutating alanine residue at position 61 of scDAOCS7 into aspartic acid residue and mutating phenylalanine residue at position 225 into isoleucine residue. The scDAOCS7-A61D/F225L is a protein obtained by mutating alanine residue at position 61 of scDAOCS7 into aspartic acid residue and mutating phenylalanine residue at position 225 into leucine residue. The scDAOCS7-A61E/F225I is a protein obtained by mutating alanine residue at position 61 of scDAOCS7 into glutamic acid residue and mutating phenylalanine residue at position 225 into isoleucine residue. The scDAOCS7-A61E/F225L is a protein obtained by mutating alanine residue at position 61 of scDAOCS7 to glutamic acid residue and mutating phenylalanine residue at position 225 to leucine residue.

In another embodiment of the invention, the mutant protein of the scDAOCS7 is scDAOCS7-R179L, scDAOCS7-R160A/R162A/R179I, scDAOCS7-R160I/R162C/R179A, scDAOCS7-R160G/R162A/R179I, scDAOCS7-T73S/S102V/L158T, scDAOCS7-T73L/S102C/L158I, scDAOCS7-T73M/S102T/L158I, scDAOCS 7-T73A/S102/L102L, scDAOCS L-T L/S102/L L, scDAOCS L/S L/L L, scDAOCS L/S L/L36158, scDAOCS L/L L/L L, scDAOCS L/S363672/L L/L, scDAOCS 363672/L/SCDAOCS 363672/L/36363672/L/SCDAOCS L/SCDAOCS L/36363672/SCDAOCS L/L, scDAOCS7-T73V/S102M/L158I, scDAOCS7-T73L/S102A/L158A, scDAOCS7-T73C/S102T, scDAOCS7-T73M/S102C/L158A, scDAOCS7-L158I, scDAOCS7-L158V, scDAOCS7-T73S/S102I, scDAOCS7-T73V/S102A/L158C or scDAOCS 7-T73C/S102I/L158S.

The scDAOCS7-R179L is a protein obtained by mutating the 179 th arginine residue of scDAOCS7 to a leucine residue.

The scDAOCS7-R160A/R162A/R179I is a protein obtained by mutating arginine residues at the 160 th, 162 th and 179 th positions of scDAOCS7 into alanine residues, alanine residues and isoleucine residues respectively.

The scDAOCS7-R160I/R162C/R179A is a protein obtained by mutating arginine residues at the 160 th, 162 th and 179 th positions of scDAOCS7 into isoleucine residue, cysteine residue and alanine residue respectively.

The scDAOCS7-R160G/R162A/R179I is a protein obtained by mutating arginine residues at the 160 th, 162 th and 179 th positions of scDAOCS7 into glycine residues, alanine residues and isoleucine residues respectively.

The scDAOCS7-T73S/S102V/L158T is a protein obtained by mutating threonine residue at position 73 to serine residue, serine residue at position 102 to valine residue and leucine residue at position 158 to threonine residue of scDAOCS 7.

The scDAOCS7-T73L/S102C/L158I is a protein obtained by mutating the 73 rd threonine residue of scDAOCS7 to leucine residue, the 102 th serine residue to cysteine residue and the 158 th leucine residue to isoleucine residue.

The scDAOCS7-T73M/S102T/L158I is a protein obtained by mutating the 73 rd threonine residue, the 102 th serine residue and the 158 th leucine residue of scDAOCS7 into methionine residues, threonine residues and isoleucine residues respectively.

The scDAOCS7-T73A/S102L/L158I is a protein obtained by mutating the 73 rd threonine residue, the 102 th serine residue and the 158 th leucine residue of scDAOCS7 into alanine residues, leucine residues and isoleucine residues respectively.

The scDAOCS7-T73L/S102M/L158T is a protein obtained by mutating threonine residue at position 73 to leucine residue, serine residue at position 102 to methionine residue and leucine residue at position 158 to threonine residue of scDAOCS 7.

The scDAOCS7-T73M/S102I/L158I is a protein obtained by mutating the 73 rd threonine residue, the 102 th serine residue and the 158 th leucine residue of scDAOCS7 into methionine residues, isoleucine residues and isoleucine residues respectively.

The scDAOCS7-T73V/S102T/L158S is a protein obtained by mutating threonine residue at position 73 to valine residue, serine residue at position 102 to threonine residue and leucine residue at position 158 to serine residue of scDAOCS 7.

The scDAOCS7-S102I/L158A is a protein obtained by mutating serine residue at position 102 of scDAOCS7 to isoleucine residue and leucine residue at position 158 to alanine residue.

The scDAOCS7-T73L/S102I/L158V is a protein obtained by mutating threonine residue at position 73 to leucine residue, serine residue at position 102 to isoleucine residue and leucine residue at position 158 to valine residue of scDAOCS 7.

The scDAOCS7-T73S/S102M/L158A is a protein obtained by mutating the 73 rd threonine residue, the 102 th serine residue and the 158 th leucine residue of scDAOCS7 into serine residues, and mutating the 102 th serine residue and the 158 th leucine residue into alanine residues.

The scDAOCS7-T73L/S102C/L158I is a protein obtained by mutating the 73 rd threonine residue of scDAOCS7 to leucine residue, the 102 th serine residue to cysteine residue and the 158 th leucine residue to isoleucine residue.

The scDAOCS7-T73M/S102C/L158I is a protein obtained by mutating the 73 rd threonine residue, the 102 th serine residue and the 158 th leucine residue of scDAOCS7 into methionine residues, cysteine residues and isoleucine residues respectively.

The scDAOCS7-T73S/S102L/L158I is a protein obtained by mutating the 73 rd threonine residue, the 102 th serine residue and the 158 th leucine residue of scDAOCS7 into serine residues and then mutating the 158 th serine residue and the 158 th leucine residue into isoleucine residues.

The scDAOCS7-T73L/S102C/L158A is a protein obtained by mutating threonine residue at position 73 to leucine residue, serine residue at position 102 to cysteine residue and leucine residue at position 158 to alanine residue of scDAOCS 7.

The scDAOCS7-T73V/S102M/L158I is a protein obtained by mutating the 73 rd threonine residue, the 102 th serine residue and the 158 th leucine residue of scDAOCS7 into valine residues, methionine residues and isoleucine residues respectively.

The scDAOCS7-T73L/S102A/L158A is a protein obtained by mutating threonine residue at position 73 to leucine residue, serine residue at position 102 to alanine residue and leucine residue at position 158 to alanine residue of scDAOCS 7.

The scDAOCS7-T73C/S102T is a protein obtained by mutating threonine residue at position 73 to cysteine residue and serine residue at position 102 to threonine residue of scDAOCS 7.

The scDAOCS7-T73M/S102C/L158A is a protein obtained by mutating the 73 rd threonine residue, the 102 th serine residue and the 158 th leucine residue of scDAOCS7 into methionine residues, cysteine residues and alanine residues respectively.

The scDAOCS7-L158I is a protein obtained by mutating the 158 th leucine residue of scDAOCS7 into an isoleucine residue.

The scDAOCS7-L158V is a protein obtained by mutating the 158 th leucine residue of scDAOCS7 into a valine residue.

The scDAOCS7-T73S/S102I is a protein obtained by mutating threonine residue at position 73 to serine residue and serine residue at position 102 to isoleucine residue of scDAOCS 7.

The scDAOCS7-T73V/S102A/L158C is a protein obtained by mutating the 73 rd threonine residue, the 102 th serine residue and the 158 th leucine residue of scDAOCS7 into valine residues, alanine residues and cysteine residues respectively.

The scDAOCS7-T73C/S102I/L158S is a protein obtained by mutating the 73 rd threonine residue, the 102 th serine residue and the 158 th leucine residue of scDAOCS7 into cysteine residues, isoleucine residues and serine residues.

In the above method, the reaction system for catalyzing the reaction may be adding the scDAOCS7 or the mutant protein scDAOCS7, FeSO to 100. mu.L of 50mM phosphate buffer (pH7.4)₄A system obtained from alpha-ketoglutaric acid, L-ascorbic acid and 6-APA, FeSO₄The final concentrations of alpha-ketoglutaric acid, L-ascorbic acid and 6-APA in the reaction system were 1.8mM, 4mM, 0.4mM and 2.5mM, respectively.

The catalytic reaction may be carried out at 25 ℃. The time for the catalytic reaction may be 5 hours.

The present invention also provides another method for preparing 7-ADCA, the method comprising: and (2) carrying out catalytic reaction by using the recombinant cell expressing the scDAOCS7 or the scDAOCS7 mutant protein or a lysate of the recombinant cell by using 6-APA as a substrate to obtain 7-ADCA.

In the above method, the recombinant cell can be obtained by introducing into a biological cell a recombinant cell capable of expressing scdaos 7 or the recombinant cell

Recombinant vector implementation of the scdaos 7 mutein.

The biological cell may be a microorganism. The microorganism can be Escherichia coli, or other fungi such as Bacillus subtilis, Corynebacterium glutamicum, yeast, Streptomyces, Penicillium or Cephalosporium acremonium. In one embodiment of the present invention, the microorganism is Escherichia coli BL21(DE3) for example.

The recombinant vector can be a recombinant plasmid obtained by replacing a small DNA fragment between NdeI and XhoI recognition sequences in a pET24a (+) vector by a gene scDAOCS7 or a coding gene of the mutant protein scDAOCS 7.

The gene of the scDAOCS7 can be a nucleic acid molecule shown as SEQ ID No.1 in a sequence table.

The gene encoding the mutant protein of scdaacs may be scdaacs-a 61 gene, scdaacs-F225 gene, scdaacs-a 61/F225 gene, scdaacs-R179 gene, scdaacs-R160/R162/R179 gene, scdaacs-T73/S102/L158 gene, scdaaocs-T102/L158 gene, scdaacs-T73/S102/L158 gene, scdaacs-T102/L158 gene, scdaaocs-T73/S102/L158 gene, scdaas 102/R158 gene, scdaas 102/L158 gene, scdaas 158 gene, An scDAOCS-S102/L158 gene, an scDAOCS-T73/S102/L158 gene, an scDAOCS-L158 gene, a DAOCS-L158 gene, an scDADADAOCS-T73/S102 gene, a DAOCS-T73/S102/L73 gene, or an scDAOCS-T73/S102/L158 gene.

The scDAOCS7-A61D gene is obtained by mutating the 181-183 th site of the scDAOCS7 gene from GCA to GAT or GAC.

The gene scDAOCS7-A61E is obtained by mutating the 181 rd and 183 th positions of the gene scDAOCS7 from GCA to GAA or GAG.

The gene scDAOCS7-F225I is obtained by mutating the 673 th 675 bit of the gene scDAOCS7 from TTC to ATT or ATC or ATA.

The gene of the scDAOCS7-F225L is obtained by mutating the 673 th 675 bit of the gene of the scDAOCS7 from TTC to TTA or TTG or CTT or CTC or CTA or CTG.

The gene scDAOCS7-A61D/F225I is obtained by mutating the 181 rd and 183 th positions of the gene scDAOCS7 from GCA to GAT or GAC and the 673 rd and 675 th positions from TTC to ATT or ATC or ATA.

The gene scDAOCS7-A61D/F225L is obtained by mutating the 181-183 site of the gene scDAOCS7 from GCA to GAT or GAC, and the 673-675 site of the gene scDAOCS from TTC to TTA or TTG or CTT or CTC or CTA or CTG.

The gene scDAOCS7-A61E/F225I is obtained by mutating the 181 rd and 183 th positions of the gene scDAOCS7 from GCA to GAA or GAG and the 673 rd and 675 th positions from TTC to ATT or ATC or ATA.

The gene scDAOCS7-A61E/F225L is obtained by mutating the 181 rd and 183 th positions of the gene scDAOCS7 from GCA to GAA or GAG, and the 673 rd and 675 th positions from TTC to TTA or TTG or CTT or CTA or CTG.

The gene scDAOCS7-R179L is obtained by mutating CGC at 535-537 of the gene scDAOCS7 into TTA, TTG, CTT, CTC, CTA or CTG.

The gene scDAOCS7-R160A/R162A/R179I is obtained by mutating the CGT at the 478-480 th site of the gene scDAOCS7 to GCT or GCC or GCA or GCG, and the CGT at the 484-486-th site to GCT or GCC or GCA or GCG, and the CGC at the 535-537 th site to ATT or ATC or ATA.

The gene scDAOCS7-R160I/R162C/R179A is obtained by mutating CGT at 478-480 position of the gene scDAOCS7 to ATT or ATC or ATA, CGT at 484-486 position to TGT or TGC, and CGC at 535-537 position to GCT or GCC or GCA or GCG.

The gene scDAOCS7-R160G/R162A/R179I is obtained by mutating the CGT at the 478-480 th site of the gene scDAOCS7 to GGT or GGC or GGA or GGG, the CGT at the 484-486 th site to GCT or GCC or GCA or GCG, and the CGC at the 535-537 th site to ATT or ATC or ATA.

The gene scDAOCS7-T73S/S102V/L158T is obtained by mutating ACA at position 217 and 219 of the gene scDAOCS7 into TCT, TCC, TCA, TCG, AGT, AGC at position 304 and 306 into GTT, GTC, GTA, GTG, CTG at position 472 and 474 into ACT, ACC, ACA or ACG.

The gene scDAOCS7-T73L/S102C/L158I is obtained by mutating ACA at position 217 and 219 of the gene scDAOCS7 to TTA or TTG or CTT or CTC or CTA or CTG, mutating AGC at position 304 and 306 to TGT or TGC, and mutating CTG at position 472 and 474 to ATT or ATC or ATA.

The gene scDAOCS7-T73M/S102T/L158I is obtained by mutating ACA at position 217 and 219 of the gene scDAOCS7 to ATG, AGC at position 304 and 306 to ACT or ACC or ACA or ACG, and CTG at position 472 and 474 to ATT or ATC or ATA.

The gene scDAOCS7-T73A/S102L/L158I is obtained by mutating ACA at position 217 and 219 of the gene scDAOCS7 to GCT or GCC or GCA or GCG, AGC at position 304 and 306 to TTA or TTG or CTT or CTC or CTA or CTG, and CTG at position 472 and 474 to ATT or ATC or ATA.

The gene scDAOCS7-T73L/S102M/L158T is obtained by mutating ACA at position 217 and 219 of the gene scDAOCS7 into TTA or TTG or CTT or CTC or CTA or CTG, mutating AGC at position 304 and 306 into ATG, and mutating CTG at position 472 and 474 into ACT or ACC or ACA or ACG.

The gene scDAOCS7-T73M/S102I/L158I is obtained by mutating ACA at position 217 and 219 of the gene scDAOCS7 to ATG, AGC at position 304 and 306 to ATT or ATC or ATA, and CTG at position 472 and 474 to ATT or ATC or ATA.

The gene scDAOCS7-T73V/S102T/L158S is obtained by mutating ACA at position 217 and 219 of the gene scDAOCS7 to GTT or GTC or GTA or GTG, AGC at position 304 and 306 to ACT or ACC or ACA or ACG, and CTG at position 472 and 474 to TCT or TCC or TCA or TCG or AGT or AGC.

The gene scDAOCS7-S102I/L158A is obtained by mutating AGC at the 304-th and 306-th positions of the gene scDAOCS7 to ATT or ATC or ATA, and CTG at the 472-th and 474-th positions to GCT or GCC or GCA or GCG.

The gene scDAOCS7-T73L/S102I/L158V is obtained by mutating ACA at position 217 and 219 of the gene scDAOCS7 to TTA or TTG or CTT or CTC or CTA or CTG, AGC at position 304 and 306 to ATT or ATC or ATA, and CTG at position 472 and 474 to GTT or GTC or GTA or GTG.

The gene scDAOCS7-T73S/S102M/L158A is obtained by mutating ACA at position 217 and 219 of the gene scDAOCS7 into TCT or TCC or TCA or TCG or AGT or AGC, mutating AGC at position 304 and 306 into ATG, and mutating CTG at position 472 and 474 into GCT or GCC or GCA or GCG.

The gene scDAOCS7-T73L/S102C/L158I is obtained by mutating ACA at position 217 and 219 of the gene scDAOCS7 to TTA or TTG or CTT or CTC or CTA or CTG, mutating AGC at position 304 and 306 to TGT or TGC, and mutating CTG at position 472 and 474 to ATT or ATC or ATA.

The gene scDAOCS7-T73M/S102C/L158I is obtained by mutating ACA at position 217 and 219 of the gene scDAOCS7 to ATG, AGC at position 304 and 306 to TGT or TGC, and CTG at position 472 and 474 to ATT or ATC or ATA.

The gene scDAOCS7-T73S/S102L/L158I is obtained by mutating ACA at position 217-219 of the gene scDAOCS7 to TCT or TCC or TCA or TCG or AGT or AGC, mutating AGC at position 304-306 to TTA or TTG or CTT or CTA or CTG, and mutating CTG at position 472-474 to ATT or ATC or ATA.

The gene scDAOCS7-T73L/S102C/L158A is obtained by mutating ACA at position 217 and 219 of the gene scDAOCS7 to TTA or TTG or CTT or CTC or CTA or CTG, mutating AGC at position 304 and 306 to TGT or TGC, and mutating CTG at position 472 and 474 to GCT or GCC or GCA or GCG.

The gene scDAOCS7-T73V/S102M/L158I is obtained by mutating ACA at position 217 and 219 of the gene scDAOCS7 to GTT or GTC or GTA or GTG, AGC at position 304 and 306 to ATG, and CTG at position 472 and 474 to ATT or ATC or ATA.

The gene scDAOCS7-T73L/S102A/L158A is obtained by mutating ACA at position 217 and 219 of the gene scDAOCS7 into TTA or TTG or CTT or CTC or CTA or CTG, mutating AGC at position 304 and 306 into GCT or GCC or GCA or GCG, and mutating CTG at position 472 and 474 into GCT or GCC or GCA or GCG.

The gene scDAOCS7-T73C/S102T is obtained by mutating ACA at positions 217 and 219 of the gene scDAOCS7 into TGT or TGC and AGC at positions 304 and 306 into ACT or ACC or ACA or ACG.

The gene scDAOCS7-T73M/S102C/L158A is obtained by mutating ACA at position 217 and 219 of the gene scDAOCS7 to ATG, AGC at position 304 and 306 to TGT or TGC, and CTG at position 472 and 474 to GCT or GCC or GCA or GCG.

The gene scDAOCS7-L158I is obtained by mutating CTG at 472-474 th site of the gene scDAOCS7 to ATT or ATC or ATA.

The gene scDAOCS7-L158V is obtained by mutating CTG at the 472-474 th site of the gene scDAOCS7 to GTT, GTC, GTA or GTG.

The gene scDAOCS7-T73S/S102I is obtained by mutating ACA at position 217 and 219 of the gene scDAOCS7 to TCT, TCC, TCA, TCG, AGT, AGC and AGC at position 304 and 306 to ATT, ATC or ATA.

The gene scDAOCS7-T73V/S102A/L158C is obtained by mutating ACA at position 217 and 219 of the gene scDAOCS7 to GTT or GTC or GTA or GTG, AGC at position 304 and 306 to GCT or GCC or GCA or GCG, and CTG at position 472 and 474 to TGT or TGC.

The gene scDAOCS7-T73C/S102I/L158S is obtained by mutating ACA at position 217 and 219 of the gene scDAOCS7 to TGT or TGC, mutating AGC at position 304 and 306 to ATT or ATC or ATA, and mutating CTG at position 472 and 474 to TCT or TCC or TCA or TCG or AGT or AGC.

The reaction system for catalyzing the reaction can be that the recombinant cell, glucose and FeSO are added into 10mL of 50mM phosphate buffer solution (pH7.4)₄And 6-APA, said recombinant cell, glucose, FeSO₄And the final concentrations of 6-APA in the reaction system are 0.1-0.3g/L, 2g/L, 1.8mM and 5mM respectively. The catalytic reaction may be carried out at 25 ℃. The catalytic reaction can be carried out in a shaker or in a reactor. The time of the catalytic reaction can be 0.5-5 h.

The reaction system for catalyzing the reaction may be FeSO or a lysate of the recombinant cell added to 100. mu.L of 50mM phosphate buffer (pH7.4)₄A system obtained from alpha-ketoglutaric acid, L-ascorbic acid and 6-APA, FeSO₄The final concentrations of alpha-ketoglutaric acid, L-ascorbic acid and 6-APA in the reaction system were 1.8mM, 4mM, 0.4mM and 2.5mM, respectively. The catalytic reaction may be carried out at 25 ℃. The time of the catalytic reaction can be 2-5 h.

The lysate of the recombinant cells may be obtained by lysing the recombinant cells.

The invention also provides a method for preparing G-7-ADCA, which takes penicillin G potassium salt as a substrate and utilizes the scDAOCS7 or the scDAOCS7 mutant protein to carry out catalytic reaction to obtain the G-7-ADCA.

In the above method, the reaction system for the catalytic reaction may be carried out using the recombinant cell.

In the above method, the reaction system for catalyzing the reaction may be a system obtained by adding the recombinant cell, glucose, ferrous sulfate, and penicillin G potassium salt to 50mM of phosphate buffer solution having a pH of 7.4, and the final concentrations of glucose, ferrous sulfate, and penicillin G potassium salt in the reaction system are 10G/L, 50 μ G/mL, and 5mM, respectively.

The catalytic reaction may be carried out at 25 ℃. The catalytic reaction can be carried out in a shaker or in a reactor.

The time for the catalytic reaction may be 2 hours.

The mutant protein of the scDAOCS7 also belongs to the protection scope of the invention.

The invention also provides a biomaterial related to the mutant protein of the scDAOCS7, wherein the biomaterial is any one of the following B1) to B4):

B1) a nucleic acid molecule encoding said scdaos 7 mutein;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector.

B1) The nucleic acid molecule may be the scDAOCS-A61 gene, the scDAOCS-F225 gene, the scDAOCS-A61/F225 gene, the scDAOCS-R179 gene, the scDAOCS-R160/R162/R179 gene, the scDAOCS-T73/S102/L158 gene, the scDAOCS 73/S102/L158 gene, the scDAOCS 158 gene, The scDAOCS-T73/S102/L158 gene, the scDAOCS-T73/S102/L158 gene, the DAscDAOCS-T73/S102 gene, the scDAOCS-T73/S102/L73/L158 gene, the scDAOCS-T73/S102/L158 gene, the scDAOCS-T158 gene, the scDAOCS 102/L158 gene, the scDAOCS 158 gene, the gene scDAOCS7-T73V/S102A/L158C or the gene scDAOCS 7-T73C/S102I/L158S.

The invention also provides any one of the following products:

m1, a kit consisting of 6-APA and the scdAACS 7 mutein;

m2, a kit consisting of 6-APA and the biomaterial;

m3, kit consisting of 6-APA and scDAOCS 7;

m4, kit consisting of potassium penicillin G salt, 6-APA and said scDAOCS7 mutein;

m5, a kit consisting of penicillin G potassium salt, 6-APA and the biological material;

m6, kit consisting of penicillin G potassium salt, 6-APA and scDAOCS 7.

Any of M1-M6 can be used to prepare 7-ADCA.

The invention also provides any one of the following uses of scdaacs 7, the scdaacs 7 mutein or the biological material:

z1, use in catalyzing 6-APA to produce 7-ADCA; z2, in the preparation of 7-ADCA products generated by catalyzing 6-APA; z3, use in the manufacture of 7-ADCA; z4, application in preparing 7-ADCA products; z5, application in catalyzing penicillin G potassium salt to generate G-7-ADCA; z6, in preparing G-7-ADCA product of catalyzing penicillin G potassium salt; z7, use in the manufacture of G-7-ADCA; z8, and application in preparing G-7-ADCA products.

Any of the following applications of the product also fall within the scope of the invention:

z3, use in the manufacture of 7-ADCA; z4, and application in preparing 7-ADCA products.

Experiments prove that the scDAOCS7 and the mutant protein thereof can catalyze 6-APA to generate 7-ADCA, in actual production, the 6-APA is used as a substrate, catalytic reaction is carried out by using scDAOCS7 or the mutant protein thereof, synthesis from 6-APA to 7-ADCA is realized, and the catalytic activity of the enzyme for synthesizing 7-ADCA is improved by further modifying deacetoxycephalosporin C synthetase through a directed evolution technology. Compared with the existing semi-synthetic enzyme method for producing 7-ADCA, the 7-ADCA production route has a plurality of advantages: first, the raw materials for this route are readily available, and 7-ADCA can be synthesized directly by adding cells expressing scdAOS 7 or its muteins to a solution containing high-purity 6-APA and then passing through whole cells or their lysates or crude enzyme powders, which is a novel 7-ADCA synthesis method. In addition, the mutant protein of the scDAOCS7 can also catalyze the synthesis of G-7-ADCA from penicillin G potassium salt, and the catalytic capability is obviously improved compared with that of scDAOCS 7. The 7-ADCA production route and the mutant protein of the scDAOCS7 have wide application prospect.

Drawings

FIG. 1 is a scheme showing the reaction scheme for penicillin G to G-7-ADCA.

FIG. 2 is a schematic representation of the reaction scheme for 6-APA to 7-ADCA.

FIG. 3 is a map of plasmid pET24a-scDAOCS 7.

FIG. 4 is a standard curve of 7-ADCA.

FIG. 5 is a schematic diagram of the design of primers for R160, R162 and R179 three-site combinatorial mutant libraries.

FIG. 6 is a graph of the standard curve for G-7-ADCA.

FIG. 7 is a schematic diagram of the design strategy of primers for T73, S102 and L158 three-site combinatorial mutation libraries.

Detailed Description

The invention provides a new 7-ADCA synthetic route, as shown in figure 2, 6-aminopenicillanic acid (6-APA) is taken as a substrate, a scdaOCS7 mutant protein with the function of catalyzing the synthesis of 6-APA to 7-ADCA is obtained by modifying scdaOCS7, the sequence of the scdaOCS7 gene is SEQ ID No.1 in a sequence table, the sequence of the coded scdaOCS7 protein is SEQ ID No.2, and the catalytic activity of the enzyme for synthesizing 7-ADCA is improved by further modifying deacetoxycephalosporin C synthetase through a directed evolution technology. Primers used to modify scdaos 7 are shown in table 1.

TABLE 1 primer sequence Listing

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

Penicillin G potassium salt: shanghai Michelle chemical technology, Inc., cat # F21739-500G.

The plasmid pET24a-scDAOCS7 in the following examples is a recombinant plasmid obtained by replacing a small DNA fragment between NdeI and XhoI recognition sequences in pET24a (+) vector (Novagen) with scDAOCS7 gene shown in SEQ ID No. 1. The map of pET24a-scDAOCS7 is shown in FIG. 3, and contains scDAOCS7 gene shown in SEQ ID No. 1.

Example 1 ScDAOCS7 mutant proteins catalyze the synthesis of 7-ADCA

In this example, the mutant protein scDAOCS7 was obtained by modifying scDAOCS7, specifically, single point saturation mutation was performed on the alanine residue at position 61 (A61) and the phenylalanine residue at position 225 (F225), respectively.

Synthesis of 7-ADCA by crude enzyme catalysis

Construction of a61 mutant library:

taking plasmid pET24a-scDAOCS7 as a template, and adding a primer A61-NDT-F, A61-VMA-F, A61-ATG-F, A61-TGG-F in the following molar ratio of 12: 6: 1: 1, 2. mu.L of the mixture of the above primers was used as an upstream primer, and 2. mu.L of A61-R was used as a downstream primer to perform two rounds of PCR to construct an A61 mutant library. The first round of PCR procedure was: pre-denaturation 98 ℃ for 2min, denaturation 98 ℃ for 15s, annealing 55 ℃ for 15s, elongation 72 ℃ for 30s, and finally denaturation-elongation at 72 ℃ for 10min, the procedure was set to 30 cycles. A second round of PCR was performed using 2. mu.L of the PCR product of the first round as primers and plasmid pET24a-scDAOCS7 as template, and the procedure of the second round of PCR was: pre-denaturation at 98 ℃ for 2min, denaturation at 98 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 4min, and final denaturation-extension program set to 30 cycles to obtain second round PCR products for constructing A61 mutant libraries.

Construction of F225 mutant pools: according to the construction steps of the A61 mutant library, the upstream primer is F225-NDT-F, F225-VMA-F, F225-ATG-F, F225-TGG-F according to the molar ratio of 12: 6: 1: 1, the downstream primer is F225-R, and other steps are not changed, so that a second round PCR product for constructing an F225 mutant library is obtained.

The two second round PCR products were subjected to the following operations:

to the product of the second round of PCR, 1. mu.L of Dpn I enzyme was added for digesting the plasmid template, and the mixture was treated at 37 ℃ for 3 hours. And 2 mu.L of the second round PCR product after enzyme digestion is electrically transformed into Escherichia coli BL21(DE3), the electrically transformed Escherichia coli BL21(DE3) bacterial liquid is uniformly coated on an LB plate with kanamycin resistance (the concentration is 50 mu g/mL), and a single colony, namely a mutant library, is grown after the culture is carried out for 14h at the constant temperature of 37 ℃. After all single colonies on one plate were scraped off and sent to sequencing to verify the diversity of the mutant library, negative control bacteria BL21(DE3)/pET24a (3 single colonies were picked), positive control bacteria BL21(DE3)/pET24a-scDAOCS7 (3 single colonies were picked) and single colonies grown on another mutant library LB plate (90 single colonies were picked) were individually picked with a sterile toothpick and transferred to a 96-well deep-well plate containing 300. mu.L of TB medium, kanamycin was added to a final concentration of 50. mu.g/mL, shaking culture was performed at 37 ℃ and 800rpm for 12 hours, 120. mu.L of bacterial solution was taken out of each well and subjected to conservation, then, 800. mu.L of TB medium was added to each well of the 96-well plate, kanamycin and isopropyl-. beta. -D-thiogalactoside (IPTG) were added to final concentrations of 50. mu.g/mL and 0.1mM, respectively, shaking and culturing at 20 deg.C and 800rpm for 12 h. Then, the cells were centrifuged at 4000rpm at 4 ℃ for 10min to collect the cells. The cells were washed once with 1000. mu.L of 50mM phosphate buffer (pH 7.4). Then 6U/mL DNase I and 1mg/mL lysozyme are added into the thalli, shaking culture is carried out for 2h at 25 ℃ to break the cells, and the cells are centrifuged at 4000rpm and 4 ℃ for 30min to collect supernatant of each single colony, and the supernatant is transferred to a new 96-well plate.

Among them, BL21(DE3)/pET24a is a recombinant bacterium obtained by introducing pET24a (+) into E.coli BL21(DE3), and BL21(DE3)/pET24a-scDAOCS7 is a recombinant bacterium obtained by introducing pET24a-scDAOCS7 into E.coli BL21(DE 3).

Preparing a catalytic reaction system: to 100. mu.L of 50mM phosphate buffer (pH7.4) was added 200. mu.L of the supernatant, FeSO₄Alpha-ketoglutaric acid, L-ascorbic acid, 6-APA, FeSO₄The final concentrations of alpha-ketoglutaric acid, L-ascorbic acid and 6-APA in the reaction system were 1.8mM, 4mM, 0.4mM and 2.5mM, respectively. The supernatant obtained from each individual colony was one system. 6-APA is provided by Shandong anti-medicine, Inc.

The resulting reaction was reacted at 25 ℃ for 5 hours, and then the amount of the reaction product 7-ADCA was measured by HPLC.

HPLC detection conditions for 6-APA and 7-ADCA: a chromatographic column: cosmosil 5C18-MS-II 4.6ID X150 mm; mobile phase: aqueous phase (50mM phosphate buffer, ph 7.4)/methanol 98/2 (vol); flow rate: 1 mL/min; detection wavelength: the amount of 7-ADCA was measured at 215nm and the amount of 6-APA was measured at 260 nm.

The standard substance is 7-ADCA (Shandong anti-medicine Co., Ltd.) with cas number of 22252-43-3. A standard curve for 7-ADCA was prepared using this standard, as shown in FIG. 4. The conversion was calculated as 100% x P/(2.5 x 10)^-3M), P is the yield (g/L) of 7-ADCA detected in the liquid phase, and M is the molar mass (g/mol) of 7-ADCA.

TABLE 2 conversion of 7-ADCA

The HPLC assay results are shown in table 2, and the conversion rates of the muteins containing the a61D, a61E, F225I, or F225L mutations were significantly higher in 90 single colonies of the a61 mutant pool and 90 single colonies of the F225 mutant pool than in the positive control. Extracting corresponding strain plasmids and sequencing the target DNA.

The mutein containing the A61D mutation was designated as scDAOCS7-A61D, and the strain and plasmid expressing the mutein were designated as BL21(DE3)/pET24a-scDAOCS7-A61D and pET24a-scDAOCS7-A61D, respectively. Wherein the scDAOCS7-A61D is a protein obtained by mutating alanine residue at position 61 of scDAOCS7 into aspartic acid residue, the position 181 and 183 of the scDAOCS7 gene in the strain and the plasmid are mutated into GAT by GCA, thus obtaining the scDAOCS7-A61D gene, and the strain and the plasmid only contain the scDAOCS7 mutant protein and the scDAOCS7 mutant gene which are mutated.

The mutein containing the A61E mutation was designated as scDAOCS7-A61E, and the strain and plasmid expressing the mutein were designated as BL21(DE3)/pET24a-scDAOCS7-A61E and pET24a-scDAOCS7-A61E, respectively. Wherein the scDAOCS7-A61E is a protein obtained by mutating alanine residue at position 61 of scDAOCS7 into glutamic acid residue, the position 181-183 of scDAOCS7 gene in the strain and plasmid is mutated from GCA into GAA, thus obtaining the scDAOCS7-A61E gene, and the strain and plasmid only contain the mutant protein scDAOCS7 and mutant gene scDAOCS7 which are mutated.

The mutein containing the F225I mutation was designated as scDAOCS7-F225I, and the strain and plasmid expressing the mutein were designated as BL21(DE3)/pET24a-scDAOCS7-F225I and pET24a-scDAOCS7-F225I, respectively. Wherein the scDAOCS7-F225I is a protein obtained by mutating the 225 th phenylalanine residue of the scDAOCS7 to isoleucine residue, the 673 rd and 675 th positions of the scDAOCS7 gene in the strain and the plasmid are mutated to ATT by TTC, so that the scDAOCS7-F225I gene is obtained, and the strain and the plasmid only contain the scDAOCS7 mutant protein and the scDAOCS7 mutant gene which are mutated.

The mutein containing the F225L mutation was designated as scDAOCS7-F225L, and the strain and plasmid expressing the mutein were designated as BL21(DE3)/pET24a-scDAOCS7-F225L and pET24a-scDAOCS7-F225L, respectively. Wherein the scDAOCS7-F225L is a protein obtained by mutating the 225 th phenylalanine residue of the scDAOCS7 to leucine residue, the 673 rd and 675 rd of the scDAOCS7 gene in the strain and the plasmid are changed into CTG by TTC, thus obtaining the scDAOCS7-F225L gene, and the strain and the plasmid only contain the scDAOCS7 mutant protein and the scDAOCS7 mutant gene which are mutated.

The activity of the mutants was then further verified using the screened crude enzyme powder of the mutants. The specific operation is as follows:

the monoclonals of negative control bacteria BL21(DE3)/pET24a, positive control bacteria BL21(DE3)/pET24a-scDAOCS7 and BL21(DE3)/pET24a-scDAOCS7-A61D, BL21(DE3)/pET24a-scDAOCS7-A61E, BL21(DE3)/pET24a-scDAOCS7-F225I, BL21(DE3)/pET24a-scDAOCS7-F225L were respectively picked up, and crude enzyme powders were prepared according to the following steps: the single clone was transferred to a tube containing 5mL of LB medium (kanamycin concentration 50. mu.g/mL), and cultured overnight at 37 ℃ with shaking at 220 rpm; then inoculating into 100mL TB medium (kanamycin concentration is 50 mug/mL) according to the inoculation amount of 1%, carrying out shake culture at 37 ℃ and 220rpm for about 3h, adding IPTG (isopropyl-beta-thiogalactoside) with the final concentration of 0.1mM in the system when the OD600 value of the bacterial liquid reaches 0.6, and continuing the shake culture at 25 ℃ and 220rpm for 18 h; then centrifuging at 4000rpm and 4 ℃ for 10min to collect thalli, carrying out ultrasonic cell disruption, centrifuging to collect supernatant, and drying the supernatant by using a freeze dryer to respectively obtain crude enzyme powder of six strains.

Preparation of a reaction system: the obtained crude enzyme powder and FeSO were added to 10mL of 50mM phosphate buffer solution (pH7.4)₄Alpha-ketoglutaric acid, L-ascorbic acid, 6-APA, crude enzyme powder, FeSO₄The final concentrations of alpha-ketoglutaric acid, L-ascorbic acid and 6-APA in the reaction system were 10g/L, 1.8mM, 4mM, 0.4mM and 2.5mM, respectively. One crude enzyme powder for each system.

The obtained reaction system was reacted at 25 ℃ for 2 hours at 800rpm, and then the amount of the reaction product 7-ADCA was measured by HPLC, and the conversion was calculated, as above.

TABLE 3 conversion of 7-ADCA

The results are shown in Table 3, and the catalytic activity of the scDAOCS7-A61D on 6-APA is slightly improved, while the catalytic activity of the scDAOCS7-A61E, the scDAOCS7-F225I and the scDAOCS7-F225L on 6-APA are all obviously improved.

Two, whole cell catalysis 7-ADCA synthesis

Single colonies of BL21(DE3)/pET24a, BL21(DE3)/pET24a-scDAOCS7, BL21(DE3)/pET24a-scDAOCS7-A61D, BL21(DE3)/pET24a-scDAOCS7-A61E, BL21(DE3)/pET24a-scDAOCS7-F225I, BL21(DE3)/pET24a-scDAOCS7-F225L were selected and prepared as follows:

single colonies were transferred to LB liquid medium containing 5mL of kanamycin resistance (kanamycin concentration 50. mu.g/mL), cultured at 37 ℃ for 14h at 220rpm, and cultured according to the following protocol 1: 100 to 50mL of TB liquid medium containing 50. mu.g/mL of kanamycin, and when the cells were cultured at 37 ℃ and 220rpm to an OD600 of 0.6, IPTG was added to the cells at a final concentration of 0.1mM to induce the cells at 25 ℃ and 220rpm for 18 hours, the cells were collected, washed once with a phosphate buffer (50mM) having a pH of 7.4, and centrifuged to collect the cells.

Carrying out whole-cell catalysis by using thalli, wherein a reaction system comprises the following steps: the obtained cells, glucose and FeSO were added to 10mL of 50mM phosphate buffer (pH7.4)₄6-APA, thallus, glucose, FeSO₄The final concentrations of 6-APA in the reaction system were 0.3g/L, 2g/L, 1.8mM, and 5mM, respectively. Each reaction system is a reaction system of a cell.

And (3) reacting the obtained reaction system at 25 ℃ and 220rpm, sampling after 5h of reaction, centrifuging at 1000rpm for 1min, taking supernate, performing liquid phase detection, and calculating the conversion rate, wherein the conversion rate calculation formula is the same as the above, and the result is shown in table 4. The liquid phase detection conditions were as above.

TABLE 4 conversion of 7-ADCA

As shown in Table 4, it is demonstrated that 7-ADCA can be synthesized by genetically modified whole cells, and the catalytic activity of 6-APA is significantly improved by scDAOCS7-A61D, scDAOCS7-A61E, scDAOCS7-F225I and scDAOCS 7-F225L.

Three, combinatorial mutant scDAOCS7 mutant proteins catalyze the synthesis of 7-ADCA

Based on scDAOCS7-A61D and scDAOCS7-A61E, mutation is carried out on the phenylalanine residue (F225) at the 225 th position to obtain mutant protein containing double mutation of A61D/F225I, A61D/F225L, A61E/F225I or A61E/F225L. The specific operation is as follows:

extracting the plasmid of BL21(DE3)/pET24a-scDAOCS7-A61D in the first step to obtain plasmid pET24a-scDAOCS7-A61D, and performing a first round of PCR by using the plasmid pET24a-scDAOCS7-A61D as a template and using upstream and downstream primers F225I-F and F225I-R, wherein the procedure of the first round of PCR is as follows: pre-denaturation 98 ℃ for 2min, denaturation 98 ℃ for 15s, annealing 55 ℃ for 15s, elongation 72 ℃ for 30s, and finally denaturation-elongation at 72 ℃ for 10min, the procedure was set to 30 cycles. Taking 2 μ L of the PCR product of the first round as a primer of the second round of PCR, carrying out PCR by taking the plasmid pET24a-scDAOCS7-A61D as a template, and carrying out the second round of PCR program: pre-denaturation at 98 ℃ for 2min, denaturation at 98 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 4min, and finally at 72 ℃ for 10min, the denaturation-extension program was set to 30 cycles. mu.L of Dpn I enzyme was added to the second round of PCR products and treated at 37 ℃ for 3h to remove the template plasmid. And 2 mu L of the PCR product after enzyme digestion is taken and electrically transformed into escherichia coli BL21(DE3), the bacterial liquid of the electrically transformed escherichia coli BL21(DE3) is evenly coated on an LB plate containing kanamycin resistance with the concentration of 50 mu g/mL, a single colony is grown after being cultured for 14h at the constant temperature of 37 ℃, the single colony is picked up to extract a plasmid for sequencing, and the plasmid pET24 a-scdaos 7-A61D/F225I containing double mutants is obtained after the sequencing is correct.

pET24a-scDAOCS7-A61D/F225L was constructed according to the construction procedure of pET24a-scDAOCS7-A61D/F225I, except that the primers F225L-F and F225L-R were used. pET24a-scDAOCS7-A61E/F225I was constructed according to the construction procedure of pET24a-scDAOCS7-A61D/F225I, except that pET24a-scDAOCS7-A61E was used as a template. pET24a-scDAOCS7-A61E/F225L was constructed according to the construction procedure of pET24a-scDAOCS7-A61D/F225I, except that pET24a-scDAOCS7-A61E was used as a template, and F225L-F and F225L-R were used as primers.

The strain containing pET24a-scDAOCS7-A61D/F225I is marked as BL21(DE3)/pET24a-scDAOCS7-A61D/F225I, the 181-183 bit of the scDAOCS7 gene in the plasmid and the strain is mutated by GCA to GAT, the 673-675 bit is mutated by TTC to ATT, the mutated gene is marked as scDAOCS7-A D/F225I gene, the scDAOCS7-A61D/F225I gene encodes scDAOCS7-A61D/F225I protein, the scDAOCS 7-A61D/F225D protein is obtained by mutating the alanine residue at the 61 bit of the scDAOCS D protein to aspartic acid residue, the phenylalanine residue at the 225 residue is mutated to isoleucine residue, and the mutant strain only contains the mutant gene of the scDAOCS D and the mutant strain contains the mutant gene of the scDAOCS D and the scDAOCS D.

The strain containing pET24a-scDAOCS7-A61D/F225L is marked as BL21(DE3)/pET24a-scDAOCS7-A61D/F225L, the 181-183 bit and the 183 bit of the scDAOCS7 gene in the plasmid and the strain are mutated by GCA to GAT, the 673-675 bit and the TTC to CTG, the mutated gene is marked as scDAOCS7-A D/F225L gene, the scDAOCS7-A61D/F225L gene encodes scDAOCS7-A61D/F225L protein, the scDAOCS 7-A61D/F225D protein is obtained by mutating the alanine residue at the 61 bit of the scDAOCS D protein to aspartic acid residue, the phenylalanine residue at the 225 bit is mutated to leucine residue, and the mutant strain contains only mutant protein of the scDAOCS D and the scDAOCS D gene.

The strain containing pET24a-scDAOCS7-A61E/F225I is marked as BL21(DE3)/pET24a-scDAOCS7-A61E/F225I, the 181-183 bit of the scDAOCS7 gene in the plasmid and the strain is mutated by GCA to GAA, the 673-675 bit is mutated by TTC to ATT, the mutated gene is marked as scDAOCS7-A E/F225I gene, the scDAOCS7-A61E/F225I gene encodes scDAOCS7-A61E/F225I protein, the scDAOCS 7-A61E/F225E protein is obtained by mutating the alanine residue at the 61 bit of the scDAOCS E protein to glutamic acid residue, the phenylalanine residue at the 225 to isoleucine residue, and the mutant strain only contains mutant strain of the mutant strain and the mutant strain contains the scDAOCS E gene with the mutant gene containing only the mutant strain and the mutant strain containing the scDAOCS E gene containing the mutant gene.

The strain containing pET24a-scDAOCS7-A61E/F225L is marked as BL21(DE3)/pET24a-scDAOCS7-A61E/F225L, the 181-183 bit of the scDAOCS7 gene in the plasmid and the strain is mutated by GCA to GAA, the 673-675 bit is mutated by TTC to CTG, the mutated gene is marked as scDAOCS7-A E/F225L gene, the scDAOCS7-A61E/F225L gene encodes scDAOCS7-A61E/F225L protein, the scDAOCS 7-A61E/F225E protein is obtained by mutating the alanine residue at the 61 bit of the scDAOCS E protein to glutamic acid residue, the phenylalanine residue at the 225 to leucine residue, and the mutant strain contains only mutant gene of the scDAOCS E and the mutant strain contains the mutant strain containing the mutant gene of the scDAOCS E and the mutant protein.

Preparing cells from BL (DE)/pET 24, BL (DE)/pET 24-scDAOCS and BL (DE)/pET 24-scDAOCS-A61, BL (DE)/pET 24-scDAOCS-F225 and the above-mentioned BL (DE)/pET 24-scDAOCS-A61/F225, BL (DE)/pET 24-scDAOCS-A61/F225 by the following methods:

the strain was inoculated into a tube containing 5mL of LB liquid medium for kanamycin resistance (kanamycin concentration 50. mu.g/mL), cultured at 37 ℃ at 220rpm for 14 hours in a volume ratio of 1: 100 to 50mL of TB liquid medium containing 50. mu.g/mL of kanamycin, culturing at 37 ℃ and 220rpm until the OD600 is 0.6, adding IPTG (0.1 mM) to the final concentration, inducing at 25 ℃ and 220rpm, culturing for 18 hours, collecting the cells, washing once with phosphate buffer (50mM) of pH7.4, and centrifuging to collect the cells.

Carrying out whole-cell catalysis by using thalli, wherein a reaction system comprises the following steps: the obtained cells, glucose and FeSO were added to 10mL of 50mM phosphate buffer (pH7.4)₄6-APA, thallus, glucose, FeSO₄6-APA in the reaction SystemThe final concentrations were 0.1g/L, 2g/L, 1.8mM, 5mM, respectively. Each reaction system is a reaction system of a cell.

The obtained reaction system was reacted at 25 ℃ and 220rpm, samples were taken at 5h of reaction, centrifuged at 1000rpm for 1min, and the supernatant was subjected to liquid phase detection, and the conversion was calculated, the results are shown in table 5. And the liquid phase detection conditions are the same as the second step.

TABLE 5 conversion of 7-ADCA

From the data in Table 5, it can be concluded that single mutants A61D, A61E, F225I, F225L and double mutants A61D/F225I, A61D/F225L, A61E/F225I and A61E/F225L have higher catalytic activity at the reaction time of 5h, which are all significantly higher than the control (BL21(DE3)/pET24a-scDAOCS 7).

The results of the mutant were further verified by increasing the amount of the bacterial cells, and the reaction system: the cells obtained above, glucose and FeSO were added to 10mL of 50mM phosphate buffer (pH7.4)₄6-APA, thallus, glucose, FeSO₄The final concentrations of 6-APA in the reaction system were 0.3g/L, 2g/L, 1.8mM, and 5mM, respectively. Each reaction system is a reaction system of a cell.

The obtained reaction system was reacted at 25 ℃ and 220rpm, samples were taken at 5h of reaction, centrifuged at 1000rpm for 1min, and the supernatant was subjected to liquid phase detection, and the conversion was calculated, the results are shown in table 6. And the liquid phase detection conditions are the same as the second step.

TABLE 6 conversion of 7-ADCA

As shown in Table 6, at the reaction time of 5h, compared with BL21(DE3)/pET24a-scDAOCS7, the catalytic efficiency of the single mutant and the double mutant is remarkably improved, wherein the catalytic efficiency of the double mutant scDAOCS7-A61E/F225L is the highest, and the double mutant can be used as a potential industrially applied enzyme.

In conclusion, the embodiment realizes the synthesis of 6-APA to 7-ADCA, and the crude enzyme method and the whole cell catalysis experiment are respectively carried out, so that the activity of the constructed mutant protein for catalyzing 6-APA to synthesize 7-ADCA is also obviously improved, and the constructed mutant protein and the encoding gene thereof can be used as potential industrial application enzymes.

Example 2 Synthesis of G-7-ADCA Using other mutant proteins of scDAOCS7

In this example, the mutant protein scDAOCS7 was obtained by modifying other sites of scDAOCS7, including arginine residues at positions 160, 162 and 179 (i.e., R160, R162 and R179), threonine residue at position 73, serine residue at position 102 and leucine residue at position 158 (T73, S102, L158).

One, R160, R162 and R179 muteins can catalyze the synthesis of G-7-ADCA

Mutating R179 into Arg, Gly, Pro, Ile, Leu, Val, Thr and Ala, wherein the degenerate codon is VBA; the two sites of R160 and R162 are respectively mutated into Arg, Ala, Val, Thr, Ile, Gly and Cys, and the degenerate codons are RBA. The mutant protein was prepared as follows:

a three-site combined mutant LIBRARY is constructed on the scDAOCS7, the strategy for designing the primers of the mutant LIBRARY is shown in FIG. 5, the sequences of the primers are shown in Table 1, plasmid pET24a-scDAOCS7 is used as a template, and two rounds of PCR are carried out on the upstream primer PG-LIBRARY-F, PG-LIBRARY-R to construct the combined mutant LIBRARY with the sites of R160, R162 and R179. The first round of PCR procedure was: pre-denaturation 98 ℃ for 2min, denaturation 98 ℃ for 15s, annealing 55 ℃ for 15s, elongation 72 ℃ for 30s, and finally denaturation-elongation at 72 ℃ for 10min, the procedure was set to 30 cycles. A second round of PCR was performed using 2. mu.L of the PCR product of the first round as primers and plasmid pET24a-scDAOCS7 as template, and the procedure of the second round of PCR was: pre-denaturation at 98 ℃ for 2min, denaturation at 98 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 4min, and finally at 72 ℃ for 10min, the denaturation-extension program was set to 30 cycles. To the product of the second round of PCR, 1. mu.L of Dpn I enzyme was added for digesting the plasmid template, and the mixture was treated at 37 ℃ for 3 hours. mu.L of the enzyme-digested second PCR product was transferred into E.coli BL21(DE3) by electroporation, and E.coli BL21(DE3) was spread on LB plate containing kanamycin resistance (kanamycin concentration 50. mu.g/mL) uniformly, and cultured at 37 ℃ for 14 hours to grow a single colony.

Single colonies growing on LB plates of a mutant library (864 single colonies picked) and negative control bacteria BL21(DE3)/pET24a (3 single colonies picked), positive control bacteria BL21(DE3)/pET24a-scDAOCS7 (3 single colonies picked) were picked respectively with sterile toothpicks, the picked single colonies were transferred to a 96-well deep-well plate containing 300. mu.L of LB medium, kanamycin antibiotic was added to a final concentration of 50. mu.g/mL, shaking culture was performed at 37 ℃ and 800rpm for 12h, 120. mu.L of bacterial liquid was taken from each well for preservation, 800. mu.L of TB medium was added to the 96-well plate, kanamycin and IPTG were added to a final concentration of 50. mu.g/mL and 0.1mM, shaking culture was performed at 20 ℃ and 800rpm for 12h, respectively. Then, the cells were centrifuged at 4000rpm at 4 ℃ for 10min to collect the cells. The cells were washed once with 1000. mu.L of 50mM phosphate buffer (pH 7.4). Then, a 50mM phosphate buffer solution (500. mu.L) having a pH of 7.4 and containing 10G/L of glucose, 50. mu.g/mL of ferrous sulfate and 5mM of penicillin G potassium salt was added to the cells, culturing at 25 deg.C and 800rpm for 2 hr, centrifuging at 4000rpm and 4 deg.C for 10min, collecting supernatant, transferring the supernatant into quartz 96-well plate, the enzyme-linked immunosorbent assay method is characterized in that a microplate reader is used for primary screening under the condition that the detection wavelength is 275nm, the mutant of the scDAOCS7 expressed in the strain with the absorption wavelength being larger than that of BL21(DE3)/pET24a-scDAOCS7 cultured in a 96-well plate has stronger catalytic activity, the result shows that the absorbance of 10 holes in 864 single colonies is higher than that of a positive control, and then the further determination is carried out through liquid phase detection of a supernatant, and sequencing the mutant sites of the strains with higher G-7-ADCA yield than the positive control.

Among them, BL21(DE3)/pET24a is a recombinant bacterium obtained by introducing pET24a (+) into E.coli BL21(DE3), and BL21(DE3)/pET24a-scDAOCS7 is a recombinant bacterium obtained by introducing pET24a-scDAOCS7 into E.coli BL21(DE 3).

The liquid phase detection conditions are as follows:

a chromatographic column: agilent ZORBAX SB-C18StableBond Analytical 4.6X 250mm, mobile phase: aqueous phase (20mM sodium phosphate buffer, ph 3.0)/methanol 55/45 (vol), flow rate: 1mL/min, detection wavelength: 215 nm.

The standard was G-7-ADCA (Shandong Lukang pharmaceutical Co., Ltd.), and a standard curve was prepared using G-7-ADCA, and the standard curve for G-7-ADCA is shown in FIG. 6. After the liquid phase detection, the conversion rate is calculated according to a standard curve, and the conversion rate is 100 percent multiplied by P/(5 multiplied by 10)^-3M), P is the yield (G/L) of G-7-ADCA detected in the liquid phase, and M is the molar mass (G/mol) of G-7-ADCA.

TABLE 7 conversion of G-7-ADCA

As shown in Table 7, the conversion rate of the four strains was higher than that of the positive control, that is, BL21(DE3)/pET24a-scDAOCS7-R179L, BL21(DE3)/pET24a-scDAOCS7-R160A/R162A/R179I, BL21(DE3)/pET24a-scDAOCS7-R160I/R162C/R179A and BL21(DE 21)/pET 24 21-scDAOCS 21-R160 21/R162/R179 21, wherein the positive control conversion rate of BL21(DE 21)/pET 24-scDAOCS 21-R160/R162/R21/BL 36179/pET 21/pET 24-scDAOCS 21/R162/21/R179 was higher than that of BL21(DE 21)/pET 24-scDAOCS 21/R36179.

BL21(DE3)/pET24a-scDAOCS7-R179L contains plasmid pET24a-scDAOCS7-R179L, the 179 th arginine residue of the scDAOCS7 protein in the strain and the plasmid is mutated into leucine residue, the 179 th arginine residue of the scDAOCS7 protein in the strain and the plasmid are denoted as scDAOCS7-R179L, the coding gene of the mutant is mutated into CTG from 535 and 537 th CGC of the scDAOCS7 gene, the mutated gene is denoted as scDAOCS7-R179L gene, and the strain and the plasmid only contain the mutant scDAOCS7 mutant protein and the mutant gene of the scDAOCS7 which are mutated.

BL21(DE3)/pET24a-scDAOCS7-R160A/R162A/R179I contains plasmid pET24a-scDAOCS7-R160A/R162A/R179I, the arginine residues at positions 160, 162 and 179 of the scDAOCS7 protein in the plasmid are mutated into alanine residue, alanine residue and isoleucine residue, respectively, the mutated protein is designated as scDAOCS7-R160A/R162A/R179I, the coding gene is mutated from CGT at position 478 and 480 of the scDAOCS7 gene into GCG, CGT at position 484 and 486 into GCG, CGC at position 535 and 537 into ATDAT, the mutated gene is designated as DAscOCS 7-R160A/R162/R179 gene, and the mutant strain contains only mutant of scOCS 160 and 828653 of the mutant OCS 8427.

BL21(DE3)/pET24a-scDAOCS7-R160I/R162C/R179A contains plasmid pET24a-scDAOCS7-R160I/R162C/R179A, the arginine residues at positions 160, 162 and 179 of the scDAOCS7 protein in the plasmid are mutated into isoleucine residue, cysteine residue and alanine residue, respectively, the mutated protein is designated as scDAOCS7-R160I/R162C/R179A, the coding gene is mutated from CGT at position 478 and 59480 of the scDAOCS 42 gene into ATT, from CGT at position 484 and 486 into TGT, from CGC at position 535 and 537 into GCG, the mutated gene is designated as scDAOCS7-R I/R6862/R179 gene, and the mutant strain contains only mutant of the scOCS 8653 gene and the scOCS 7 mutant gene.

BL21(DE3)/pET24a-scDAOCS7-R160G/R162A/R179I contains pET24a-scDAOCS7-R160G/R162A/R179I, the arginine residues at positions 160, 162 and 179 of the scDAOCS7 protein in the plasmid are mutated into glycine residue, alanine residue and isoleucine residue, the mutated protein is designated as scDAOCS7-R160G/R162A/R179I, the coding gene is mutated from CGT at position 478 480 to GGC, CGT at position 484-486 to GCG, CGC at position 535-537 to ATT, the mutated gene is designated as scDAOCS7-R160G/R162 2/R179 69556 gene, and the mutant strain contains only scDAOCS 160 and OCS7 mutations.

The four mutant proteins can be used as potential enzymes for industrial application to catalyze the synthesis of G-7-ADCA from penicillin G potassium salt.

Secondly, T73, S102 and L158 mutant proteins can catalyze and synthesize G-7-ADCA

T73, S102 and L158 are mutated into Ala, Val, Ile, Leu, Met, Thr, Cys and Ser, and the preparation method of the mutant protein is as follows:

a three-site combination mutant library is constructed on the scDAOCS7, the primer design strategy of the mutant library is shown in FIG. 7, the primer sequences are shown in Table 1, plasmid pET24a-scDAOCS7 is taken as a template, and an upstream primer T73-DYA-F, T73-ATG-F, T73-TGC-F is added according to the molar ratio of 6: 1: 1 as an upstream mixed primer T73-MIX-F, and mixing a downstream primer S102-DYA-F, S102-ATG-F, S102-TGC-F according to a molar ratio of 6: 1: 1 as downstream mixed primer S102-MIX-F, PCR products T73-S102 were obtained. Taking a plasmid pET24a-scDAOCS7 as a template, taking an upstream primer as L158-F, and taking a downstream primer L158-HRT-R, L158-TAC-R, L158-ACG-R according to a molar ratio of 6: 1: 1 as downstream mixed primer L158-MIX-F to carry out PCR amplification to obtain a PCR product L158. The PCR procedures were all as follows: pre-denaturation 98 ℃ for 2min, denaturation 98 ℃ for 15s, annealing 55 ℃ for 15s, elongation 72 ℃ for 30s, and finally denaturation-elongation at 72 ℃ for 10min, the procedure was set to 30 cycles. Performing electrophoresis on the two amplified PCR products, performing gel recovery to obtain purified gene fragments T73-S102 and L158, mixing the two gene fragments in an equimolar manner, adding primers T73-MIX-F and L158-MIX-F to perform PCR, and performing electrophoresis and gel recovery on the PCR products to obtain a gene fragment T73-S102-L158, wherein the PCR program is as follows: denaturation 98 ℃ for 15s, annealing 55 ℃ for 15s, elongation 72 ℃ for 30s, and finally 72 ℃ for 10min, the denaturation-to-elongation procedure was set to 30 cycles. Taking 2 mu L of T73-S102-L158 gene fragment as a primer, taking plasmid pET24a-scDAOCS7 as a template to carry out PCR, wherein the PCR program is as follows: pre-denaturation at 98 ℃ for 2min, denaturation at 98 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 4min, and final denaturation to extension at 72 ℃ for 10min, wherein the procedure was set to 30 cycles, adding 1. mu.L of Dpn I enzyme to the PCR product for digesting the plasmid template, and treating at 37 ℃ for 3 h. mu.L of the enzyme-digested PCR product was transferred into E.coli BL21(DE3) by electroporation, and E.coli BL21(DE3) was spread on an LB plate with kanamycin resistance (kanamycin concentration: 50. mu.g/mL) uniformly, and cultured at 37 ℃ for 14 hours to grow a single colony.

Negative control bacteria BL21(DE3)/pET24a (3 single colonies picked), positive control bacteria BL21(DE3)/pET24a-scDAOCS7 (3 single colonies picked) and single colonies (1536 single colonies picked) grown on LB plates of mutant libraries were picked with sterile toothpicks and transferred to a 96-well deep-well plate containing 300. mu.L of LB medium, kanamycin was added to a final concentration of 50. mu.g/mL, shaking culture was performed at 37 ℃ and 800rpm for 12 hours, 120. mu.L of bacterial liquid was taken from each well to preserve the bacteria, then 800. mu.L of TB medium was added to the 96-well plate, kanamycin and IPTG were added to final concentrations of 50. mu.g/mL and 0.1mM, and shaking culture was performed at 20 ℃ and 800rpm for 12 hours. Then, the cells were centrifuged at 4000rpm at 4 ℃ for 10min to collect the cells. The cells were washed once with 1000. mu.L of 50mM phosphate buffer (pH 7.4). Then adding 50mM phosphate buffer solution (500 mu L) containing 10G/L glucose, 50 mu G/mL ferrous sulfate and 5mM potassium penicillin G salt and having pH value of 7.4 into the thalli, culturing for 2h under the conditions of 25 ℃ and 800rpm, respectively, centrifuging for 10min under the conditions of 4000rpm and 4 ℃, collecting supernatant, transferring the supernatant into a quartz 96-well plate, performing primary screening by using a microplate reader under the condition that the detection wavelength is 275nm, and obtaining the scDAOCS7 mutant expressed in the strain with the absorption wavelength being more than that of BL21(DE3)/pET24a-scDAOCS7 cultured in the 96-well plate, wherein the scDAOCS7 mutant has stronger catalytic activity. The results showed that the conversion rate of the supernatant was higher than that of the positive control in 36 wells of 1536 single colonies, and then further determination was made by liquid phase detection of the supernatant, and the mutation sites of the strains with higher G-7-ADCA yield than that of the positive control were sequenced, and the liquid phase detection conditions were the same as above, and then the conversion rate was calculated.

TABLE 8 conversion of G-7-ADCA

The strains shown in Table 8 all had higher transformation rates than the positive control, wherein the transformation rates of BL (DE)/pET 24-scDAOCS-T73/S102/L158, BL (DE)/pET 24-scDAOCS-T73/S102/L158, BL (DE)/pET 24-scDAOCS-T73/S102/L158, BL (DE)/scDAOCS 24-scDAOCS-T73/S102/L158 were significantly higher than the positive control, all of the above expressed scdaos 7 muteins were found to be potentially industrially useful enzymes.

Wherein BL21(DE3)/pET24a-scDAOCS7-T73S/S102V/L158T comprises plasmid pET24a-scDAOCS7-T73S/S102V/L158T, the ACA mutation at the 217-position 219 of the scDAOCS7 gene of the strain and the plasmid is AGC, the AGC mutation at the 304-position 306 of the strain is GTG, the CTG mutation at the 472-position 474 of the strain is ACC, the mutated gene is recorded as scDAOCS7-T73S/S102V/L158T gene, the scDAOCS7-T73S/S102V/L158T gene codes scDAOCS7-T73S/S102V/L158T protein, the scDAOCS7-T73S/S102V/L158T protein is a mutant protein obtained by mutating the threonine residue at the 73 rd position of the scDAOCS7 protein into a serine residue, the serine residue at the 102 th position of the scDAOCS7 protein into a valine residue, and the leucine residue at the 158 th position of the scDAOCS7 protein into a threonine residue, the strain and plasmid only contain the mutant protein of the scDAOCS7 and the mutant gene of the scDAOCS7, which have the mutation.

BL21(DE3)/pET24a-scDAOCS7-T73L/S102C/L158I contains plasmid pET24a-scDAOCS7-T73L/S102C/L158I, the ACA at the 217-position 219 of the scDAOCS7 gene in the strain and the plasmid is mutated into CTG, the AGC at the 304-position 306 is mutated into TGC, the CTG at the 472-position 474 is mutated into ATT, the mutated gene is recorded as scDAOCS7-T73L/S102C/L158I gene, the scDAOCS7-T73L/S102C/L158I gene encodes scDAOCS7-T73L/S102C/L158I protein, the scDAOCS7-T73L/S102C/L158I protein is a mutant protein obtained by mutating the threonine residue at the 73-position of the scDAOCS7 protein into a leucine residue, mutating the serine residue at the 102-position into a cysteine residue and the leucine residue at the 158-position into isoleucine residue, the strain and plasmid only contain the mutant protein of the scDAOCS7 and the mutant gene of the scDAOCS7, which have the mutation.

BL21(DE3)/pET24a-scDAOCS7-T73M/S102T/L158I contains plasmid pET24a-scDAOCS7-T73M/S102T/L158I, the ACA at position 217 and 219 of the scDAOCS7 gene in the strain and the plasmid is mutated into ATG, the AGC at position 304 and 306 is mutated into ACC, the CTG at position 472 and 474 is mutated into ATT, the mutated gene is recorded as scDAOCS7-T73M/S102T/L158I gene, the scDAOCS7-T73M/S102T/L158I gene encodes scDAOCS7-T73M/S102T/L158I protein, the scDAOCS7-T73M/S102T/L158I protein is a mutant protein obtained by mutating the threonine residue at position 73 to a methionine residue, the serine residue at position 102 to a threonine residue and the leucine residue at position 158 to an isoleucine residue in the scDAOCS7 protein, the strain and plasmid only contain the mutant protein of the scDAOCS7 and the mutant gene of the scDAOCS7, which have the mutation.

BL21(DE3)/pET24a-scDAOCS7-T73A/S102L/L158I contains plasmid pET24a-scDAOCS7-T73A/S102L/L158I, the ACA at the 217-position 219 of the scDAOCS7 gene in the strain and the plasmid is mutated into GCG, the AGC at the 304-position 306 is mutated into CTG, the CTG at the 472-position 474 is mutated into ATT, the mutated gene is recorded as scDAOCS7-T73A/S102L/L158I gene, the scDAOCS7-T73A/S102L/L158I gene encodes scDAOCS7-T73A/S102L/L158I protein, the scDAOCS7-T73A/S102L/L158I protein is a mutant protein obtained by mutating the threonine residue at the 73-position of the scDAOCS7 protein into an alanine residue, mutating the serine residue at the 102-position into a leucine residue and mutating the leucine residue at the 158-position into isoleucine residue, the strain and plasmid only contain the mutant protein of the scDAOCS7 and the mutant gene of the scDAOCS7, which have the mutation.

BL21(DE3)/pET24a-scDAOCS7-T73L/S102M/L158T contains plasmid pET24a-scDAOCS7-T73L/S102M/L158T, the ACA at position 217 and 219 of the scDAOCS7 gene in the strain and the plasmid is mutated into CTG, the AGC at position 304 and 306 is mutated into ATG, the CTG at position 472 and 474 is mutated into ACC, the mutated gene is recorded as scDAOCS7-T73L/S102M/L158T gene, the scDAOCS7-T73L/S102M/L158T gene codes scDAOCS7-T73L/S102M/L158T protein, the scDAOCS7-T73L/S102M/L158T protein is a mutant protein obtained by mutating threonine residue at position 73 to leucine residue, serine residue at position 102 to methionine residue and leucine residue at position 158 to threonine residue of scDAOCS7 protein, the strain and plasmid only contain the mutant protein of the scDAOCS7 and the mutant gene of the scDAOCS7, which have the mutation.

BL21(DE3)/pET24a-scDAOCS7-T73M/S102I/L158I contains plasmid pET24a-scDAOCS7-T73M/S102I/L158I, the ACA at the 217-position 219 of the scDAOCS7 gene in the strain and the plasmid is mutated into ATG, the AGC at the 304-position 306 is mutated into ATT, the CTG at the 472-position 474 is mutated into ATT, the mutated gene is recorded as scDAOCS7-T73M/S102I/L158I gene, the scDAOCS7-T73M/S102I/L158I gene encodes scDAOCS7-T73M/S102I/L158I protein, the scDAOCS7-T73M/S102I/L158I protein is a mutant protein obtained by mutating the threonine residue at the 73-position of the scDAOCS7 protein into a methionine residue, mutating the serine residue at the 102-position into an isoleucine residue and mutating the leucine residue at the 158-position into isoleucine residue, the strain and plasmid only contain the mutant protein of the scDAOCS7 and the mutant gene of the scDAOCS7, which have the mutation.

BL21(DE3)/pET24a-scDAOCS7-T73V/S102T/L158S contains plasmid pET24a-scDAOCS7-T73V/S102T/L158S, the ACA at position 217 and 219 of the gene of the strain and the scDAOCS7 gene in the plasmid is mutated into GTG, the AGC at position 304 and 306 is mutated into ACC, the CTG at position 472 and 474 is mutated into AGC, the mutated gene is marked as scDAOCS7-T73V/S102T/L158S gene, the gene of scDAOCS7-T73V/S102T/L158S encodes scDAOCS7-T73V/S102T/L158S protein, the protein of scDAOCS7-T73V/S102T/L158S is a mutant protein obtained by mutating threonine residue at position 73 to valine residue, serine residue at position 102 to threonine residue and leucine residue at position 158 to serine residue, the strain and plasmid only contain the mutant protein of the scDAOCS7 and the mutant gene of the scDAOCS7, which have the mutation.

BL21(DE3)/pET24a-scDAOCS7-S102I/L158A contains plasmid pET24a-scDAOCS7-S102I/L158A, AGC mutation at position 304 and 306 of the scDAOCS7 gene in the plasmid is ATT, CTG mutation at position 472 and 474 is GCG, the mutated gene is scDAOCS7-S102I/L158A gene, scDAOCS7-S102I/L A gene encodes scDAOCS7-S102I/L158A protein, scDAOCS7-S102I/L158 OCS I protein is mutant protein obtained by mutating serine residue at position 102 to isoleucine residue at position 102 and leucine residue at position to alanine residue at position 158 of DAOCS I protein, and mutant protein of the mutant strain only contains mutant protein of scDAOCS I and scOCS I gene.

BL21(DE3)/pET24a-scDAOCS7-T73L/S102I/L158V contains plasmid pET24a-scDAOCS7-T73L/S102I/L158V, the ACA at position 217-219 of the scDAOCS7 gene in the strain and the plasmid is mutated into CTG, the AGC at position 304-306 is mutated into ATT, the CTG at position 472-474 is mutated into GTG, the mutated gene is recorded as scDAOCS7-T73L/S102I/L158V gene, the scDAOCS7-T73L/S102I/L158V gene encodes scDAOCS7-T73L/S102I/L158V protein, the scDAOCS7-T73L/S102I/L158V protein is a mutant protein obtained by mutating threonine residue at position 73 of the scDAOCS7 protein into leucine residue, serine residue at position 102 into isoleucine residue and leucine residue at position 158 into valine residue, the strain and plasmid only contain the mutant protein of the scDAOCS7 and the mutant gene of the scDAOCS7, which have the mutation.

BL21(DE3)/pET24a-scDAOCS7-T73S/S102M/L158A contains plasmid pET24a-scDAOCS7-T73S/S102M/L158A, the ACA at the 217-position 219 of the scDAOCS7 gene in the strain and the plasmid is mutated into AGC, the AGC at the 304-position 306 is mutated into ATG, the CTG at the 472-position 474 is mutated into GCG, the mutated gene is recorded as scDAOCS7-T73S/S102M/L158A gene, the scDAOCS7-T73S/S102M/L158A gene codes scDAOCS7-T73S/S102M/L158A protein, the scDAOCS7-T73S/S102M/L158A protein is a mutant protein obtained by mutating the threonine residue at the 73 th position of the scDAOCS7 protein into a serine residue, mutating the serine residue at the 102 th position into a methionine residue and mutating the leucine residue at the 158 position into an alanine residue, the strain and plasmid only contain the mutant protein of the scDAOCS7 and the mutant gene of the scDAOCS7, which have the mutation.

BL21(DE3)/pET24a-scDAOCS7-T73L/S102C/L158I contains plasmid pET24a-scDAOCS7-T73L/S102C/L158I, the ACA at the 217-position 219 of the scDAOCS7 gene in the strain and the plasmid is mutated into CTG, the AGC at the 304-position 306 is mutated into TGC, the CTG at the 472-position 474 is mutated into ATT, the mutated gene is recorded as scDAOCS7-T73L/S102C/L158I gene, the scDAOCS7-T73L/S102C/L158I gene encodes scDAOCS7-T73L/S102C/L158I protein, the scDAOCS7-T73L/S102C/L158I protein is a mutant protein obtained by mutating the threonine residue at the 73-position of the scDAOCS7 protein into a leucine residue, mutating the serine residue at the 102-position into a cysteine residue and the leucine residue at the 158-position into isoleucine residue, the strain and plasmid only contain the mutant protein of the scDAOCS7 and the mutant gene of the scDAOCS7, which have the mutation.

BL21(DE3)/pET24a-scDAOCS7-T73M/S102C/L158I contains plasmid pET24a-scDAOCS7-T73M/S102C/L158I, the ACA at the 217-position 219 of the scDAOCS7 gene in the strain and the plasmid is mutated into ATG, the AGC at the 304-position 306 is mutated into TGC, the CTG at the 472-position 474 is mutated into ATT, the mutated gene is recorded as scDAOCS7-T73M/S102C/L158I gene, the scDAOCS7-T73M/S102C/L158I gene encodes scDAOCS7-T73M/S102C/L158I protein, the scDAOCS7-T73M/S102C/L158I protein is a mutant protein obtained by mutating the threonine residue at the 73-position of the scDAOCS7 protein into a methionine residue, mutating the serine residue at the 102-position into a cysteine residue and the leucine residue at the 158-position into isoleucine residue, the strain and plasmid only contain the mutant protein of the scDAOCS7 and the mutant gene of the scDAOCS7, which have the mutation.

BL21(DE3)/pET24a-scDAOCS7-T73S/S102L/L158I contains plasmid pET24a-scDAOCS7-T73S/S102L/L158I, the ACA at the 217-position 219 of the scDAOCS7 gene in the strain and the plasmid is mutated into AGC, the AGC at the 304-position 306 is mutated into CTG, the CTG at the 472-position 474 is mutated into ATT, the mutated gene is recorded as scDAOCS7-T73S/S102L/L158I gene, the scDAOCS7-T73S/S102L/L158I gene codes scDAOCS7-T73S/S102L/L158I protein, the scDAOCS7-T73S/S102L/L158I protein is a mutant protein obtained by mutating the threonine residue at the 73-position of the scDAOCS7 protein into a serine residue, mutating the serine residue at the 102-position into a leucine residue and mutating the leucine residue at the 158-position into isoleucine residue, the strain and plasmid only contain the mutant protein of the scDAOCS7 and the mutant gene of the scDAOCS7, which have the mutation.

BL21(DE3)/pET24a-scDAOCS7-T73L/S102C/L158A contains plasmid pET24a-scDAOCS7-T73L/S102C/L158A, the ACA at position 217-219 of the scDAOCS7 gene in the strain and the plasmid is mutated into CTG, the AGC at position 304-306 is mutated into TGC, the CTG at position 472-474 is mutated into GCG, the mutated gene is recorded as scDAOCS7-T73L/S102C/L158A gene, the scDAOCS7-T73L/S102C/L158A gene encodes scDAOCS7-T73L/S102C/L158A protein, the scDAOCS7-T73L/S102C/L158A protein is a mutant protein obtained by mutating threonine residue at position 73 of the scDAOCS7 protein into leucine residue, serine residue at position 102 into cysteine residue and leucine residue at position 158 into alanine residue, the strain and plasmid only contain the mutant protein of the scDAOCS7 and the mutant gene of the scDAOCS7, which have the mutation.

BL21(DE3)/pET24a-scDAOCS7-T73V/S102M/L158I contains plasmid pET24a-scDAOCS7-T73V/S102M/L158I, the ACA at the 217-position 219 of the scDAOCS7 gene in the strain and the plasmid is mutated into GTG, the AGC at the 304-position 306 is mutated into ATG, the CTG at the 472-position 474 is mutated into ATT, the mutated gene is recorded as scDAOCS7-T73V/S102M/L158I gene, the scDAOCS7-T73V/S102M/L158I gene encodes scDAOCS7-T73V/S102M/L158I protein, the scDAOCS7-T73V/S102M/L158I protein is a mutant protein obtained by mutating the threonine residue at the 73-position of the scDAOCS7 protein into a valine residue, mutating the serine residue at the 102-position into a methionine residue and mutating the leucine residue at the 158-position into isoleucine residue, the strain and plasmid only contain the mutant protein of the scDAOCS7 and the mutant gene of the scDAOCS7, which have the mutation.

BL21(DE3)/pET24a-scDAOCS7-T73L/S102A/L158A contains plasmid pET24a-scDAOCS7-T73L/S102A/L158A, the ACA at the 217-position 219 of the scDAOCS7 gene in the strain and the plasmid is mutated into CTG, the AGC at the 304-position 306 is mutated into GCG, the CTG at the 472-position 474 is mutated into GCG, the mutated gene is recorded as scDAOCS7-T73L/S102A/L158A gene, the scDAOCS7-T73L/S102A/L158A gene codes scDAOCS7-T73L/S102A/L158A protein, the scDAOCS7-T73L/S102A/L158A protein is a mutant protein obtained by mutating the threonine residue at the 73 position of the scDAOCS7 protein into a leucine residue, mutating the serine residue at the 102 position into an alanine residue and mutating the leucine residue at the 158 position into an alanine residue, the strain and plasmid only contain the mutant protein of the scDAOCS7 and the mutant gene of the scDAOCS7, which have the mutation.

BL21(DE3)/pET24a-scDAOCS7-T73C/S102T contains plasmid pET24a-scDAOCS7-T73C/S102T, which is mutated from ACA at position 217 and 219 of scDAOCS7 gene in this plasmid to TGC, and AGC at position 304 and 306 to ACC, and the mutated gene is designated scDAOCS7-T73C/S102T, and scDAOCS7-T73C/S102T encodes scDAOCS7-T73C/S102T protein, and scDAOCS7-T73C/S102T is a mutant protein obtained by mutating threonine residue at position 73 to cysteine residue and serine residue at position 102 to threonine residue 7 of DAscDAOCS protein, and this mutant strain contains scOCS 7 and scOCS 7 mutant gene only.

BL21(DE3)/pET24a-scDAOCS7-T73M/S102C/L158A contains plasmid pET24a-scDAOCS7-T73M/S102C/L158A, the ACA at the 217-position 219 of the scDAOCS7 gene in the strain and the plasmid is mutated into ATG, the AGC at the 304-position 306 is mutated into TGC, the CTG at the 472-position 474 is mutated into GCG, the mutated gene is recorded as scDAOCS7-T73M/S102C/L158A gene, the scDAOCS7-T73M/S102C/L158A gene encodes scDAOCS7-T73M/S102C/L158A protein, the scDAOCS7-T73M/S102C/L158A protein is a mutant protein obtained by mutating the threonine residue at the 73-position of the scDAOCS7 protein into a methionine residue, mutating the serine residue at the 102-position into a cysteine residue and the leucine residue at the 158-position into an alanine residue, the strain and plasmid only contain the mutant protein of the scDAOCS7 and the mutant gene of the scDAOCS7, which have the mutation.

BL21(DE3)/pET24a-scDAOCS7-L158I contains plasmid pET24a-scDAOCS7-L158I, the CTG of 472-474 position of the strain and scDAOCS7 gene in the plasmid is mutated into ATT, the mutated gene is marked as scDAOCS7-L158I gene, the scDAOCS7-L158I gene codes scDAOCS7-L158I protein, the scDAOCS7-L158I protein is a mutant protein obtained by mutating leucine residue of 158-position of scDAOCS7 protein into isoleucine residue, and the strain and the plasmid only contain the mutant protein of DAscOCS 7 and the mutant gene of scDAOCS7 which are mutated.

BL21(DE3)/pET24a-scDAOCS7-L158V contains plasmid pET24a-scDAOCS7-L158V, the CTG of 472-474 position of the strain and scDAOCS7 gene in the plasmid is mutated into GTG, the mutated gene is marked as scDAOCS7-L158V gene, the scDAOCS7-L158V gene codes scDAOCS7-L158V protein, the scDAOCS7-L158V protein is a mutant protein obtained by mutating leucine residue of 158-position of scDAOCS7 protein into valine residue, and the strain and the plasmid only contain scDAOCS7 mutant protein and scDAS 7 mutant gene which are mutated.

BL21(DE3)/pET24a-scDAOCS7-T73S/S102I contains plasmid pET24a-scDAOCS7-T73S/S102I, the ACA mutation at position 217 and 219 of the scDAOCS7 gene in the plasmid is AGC, the AGC mutation at position 304 and 306 is ATT, the mutated gene is designated as scDAOCS7-T73S/S102I gene, the scDAOCS7-T73S/S102I gene encodes scDAOCS7-T73S/S102I protein, the scDAOCS 7-T73S/S102S gene is a mutant strain in which the threonine residue at position 73 of the DASCOCS S protein is mutated into serine residue, the serine residue at position 102 is mutated into isoleucine residue, and the mutant strain and the scDAOCS S gene only contain mutant gene of the scDAOCS S and the mutant strain.

BL21(DE3)/pET24a-scDAOCS7-T73V/S102A/L158C contains plasmid pET24a-scDAOCS7-T73V/S102A/L158C, the ACA at the 217-position 219 of the scDAOCS7 gene in the strain and the plasmid is mutated into GTG, the AGC at the 304-position 306 is mutated into GCG, the CTG at the 472-position 474 is mutated into TGC, the mutated gene is recorded as scDAOCS7-T73V/S102A/L158C gene, the scDAOCS7-T73V/S102A/L158C gene encodes scDAOCS7-T73V/S102A/L158C protein, the scDAOCS7-T73V/S102A/L158C protein is a mutant protein obtained by mutating the threonine residue at the 73-position of the scDAOCS7 protein into a valine residue, the serine residue at the 102-position into an alanine residue and the leucine residue at the 158-position into a cysteine residue, the strain and plasmid only contain the mutant protein of the scDAOCS7 and the mutant gene of the scDAOCS7, which have the mutation.

BL21(DE3)/pET24a-scDAOCS7-T73C/S102I/L158S contains plasmid pET24a-scDAOCS7-T73C/S102I/L158S, the ACA at the 217-position 219 of the scDAOCS7 gene in the strain and the plasmid is mutated into TGC, the AGC at the 304-position 306 is mutated into ATT, the CTG at the 472-position 474 is mutated into AGC, the mutated gene is recorded as scDAOCS7-T73C/S102I/L158S gene, the scDAOCS7-T73C/S102I/L158S gene codes scDAOCS7-T73C/S102I/L158S protein, the scDAOCS7-T73C/S102I/L158S protein is a mutant protein obtained by mutating the threonine residue at the 73 position of the scDAOCS7 protein into a cysteine residue, the serine residue at the 102 position into an isoleucine residue and the leucine residue at the 158 position into a serine residue, the strain and plasmid only contain the mutant protein of the scDAOCS7 and the mutant gene of the scDAOCS7, which have the mutation.

In conclusion, the R160, R162, R179, T73, S102 and L158 of the gene of the scDAOCS7 are transformed to realize the catalytic process from penicillin G potassium salt to G-7-ADCA, the conversion rate of the screened mutants is obviously higher than that of a positive control, and the mutants can be used as potential industrial application enzymes.

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

<120> mutant of deacetoxycephalosporin C synthetase and application thereof in synthesis of beta-lactam antibiotic parent nucleus

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 936

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

atggatacca ccgtgccgac ctttagtctg gcagaactgc agcagggtct gcatcaggat 60

gaatttcgcc gttgcctgcg cgataaaggt ctgttttatc tgaccgattg tggtctgacc 120

gataccgaac tgaaaagtgc caaagatttg gttattgatt tctttgaaca cggtagcgaa 180

gcagaaaaac gcgccgtgac cagtccggtg ccgaccacac gccgcggttt taccggtctg 240

gaaagcgaaa gtaccgcaca gattaccaat accggtagct atagtgatta tagtatgtgt 300

tatagcatgg gtacagccga taatctgttt ccgagcggcg attttgaacg catttggacc 360

cagtattttg atcgtcagta taccgcaagt cgcgcagttg cacgcgaagt gctgcgcgca 420

accggcaccg aaccggatgg tggtgtggaa gcatttctgg attatgaacc gctgctgcgt 480

tttcgttatt ttccgcaggt gccggaacat cgtagtgccg aagaacagcc gctgcgcatg 540

gcaccgcatc atgatctgag catggtgacc ctgattcagc agaccccgtg cgcaaatggc 600

tttgttagtc tgcaggccga agttggcggc gcctttgttg atctgccgta tcgtccggat 660

gcagtgctgg ttttctgtgg tgccattgcc accctggtga ccggcggcca ggttaaagca 720

ccgcgtcatc atgtggcagc accgcgtcgt gatcagattg ccggtagtag ccgtaccagc 780

agcgttttct ttctgcgccc gaatgcagat tttaccttta gcattccgct ggcacgtgaa 840

tatggttttg atgttagcct ggatggtgaa accgccacct ttcaggattg gattggcggc 900

aattatgtta atatgcgccg caccagcaaa gcctaa 936

<210> 2

<211> 311

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 2

Met Asp Thr Thr Val Pro Thr Phe Ser Leu Ala Glu Leu Gln Gln Gly

1 5 10 15

Leu His Gln Asp Glu Phe Arg Arg Cys Leu Arg Asp Lys Gly Leu Phe

20 25 30

Tyr Leu Thr Asp Cys Gly Leu Thr Asp Thr Glu Leu Lys Ser Ala Lys

35 40 45

Asp Leu Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg

50 55 60

Ala Val Thr Ser Pro Val Pro Thr Thr Arg Arg Gly Phe Thr Gly Leu

65 70 75 80

Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Ser Asp

85 90 95

Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser

100 105 110

Gly Asp Phe Glu Arg Ile Trp Thr Gln Tyr Phe Asp Arg Gln Tyr Thr

115 120 125

Ala Ser Arg Ala Val Ala Arg Glu Val Leu Arg Ala Thr Gly Thr Glu

130 135 140

Pro Asp Gly Gly Val Glu Ala Phe Leu Asp Tyr Glu Pro Leu Leu Arg

145 150 155 160

Phe Arg Tyr Phe Pro Gln Val Pro Glu His Arg Ser Ala Glu Glu Gln

165 170 175

Pro Leu Arg Met Ala Pro His His Asp Leu Ser Met Val Thr Leu Ile

180 185 190

Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val

195 200 205

Gly Gly Ala Phe Val Asp Leu Pro Tyr Arg Pro Asp Ala Val Leu Val

210 215 220

Phe Cys Gly Ala Ile Ala Thr Leu Val Thr Gly Gly Gln Val Lys Ala

225 230 235 240

Pro Arg His His Val Ala Ala Pro Arg Arg Asp Gln Ile Ala Gly Ser

245 250 255

Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr

260 265 270

Phe Ser Ile Pro Leu Ala Arg Glu Tyr Gly Phe Asp Val Ser Leu Asp

275 280 285

Gly Glu Thr Ala Thr Phe Gln Asp Trp Ile Gly Gly Asn Tyr Val Asn

290 295 300

Met Arg Arg Thr Ser Lys Ala

305 310