Penicillin G acylase mutant, and coding gene and application thereof

Abstract

The invention discloses a penicillin G acylase mutant, and coding gene and application thereof; the amino acid sequence of the penicillin G acylase mutant is displayed by SE Q ID NO.1; the nucleotide sequence of the coding gene is presented by the SEQ ID NO.2; the invention also provides the application of the penicillin G acylase mutant in synthesis of cephalo-type antibiotic. Compared with wild type, the novel penicillin G acylase mutant has higher activity in synthesis of the cephalo-type antibiotic like cephalosporin propylene, cofactor or cephalosporin amoxicillin, and has highly improved enzyme vitality when catalyzing 7-APRA and side chain 2-hydroxy ethyl to react with hydroxy benzenes glycine ester so as to synthesis the cephalosporin propylene; specific vitality is improved from 1.5U / mg to 35U / mg, and synthesis hydrolysis vitality ratio is not reduced, and the synthesis hydrolysis vitality ratio can reach 1.8.

Description

Technical field

[0001] The invention belongs to the field of biocatalysis, and particularly relates to a penicillin G acylase mutant and its coding gene and application. Background technique

[0002] At present, more and more drugs or their intermediates are synthesized through a biological enzyme catalysis instead of chemical synthesis. Obviously, enzyme catalysis has great advantages over traditional chemical synthesis: (1) Toxic reagents or solvents can be better avoided in biological reactions; (2) Enzymes have the characteristics of high efficiency and substrate specificity. ; (3) Better avoid side reactions.

[0003] Acylase is a type of hydrolase that catalyzes the hydrolysis of amides into corresponding carboxylic acids. It has a wide range of sources and a broad spectrum of substrates. It can be widely used to hydrolyze fatty, aromatic, and heterocyclic amino amides, but the enzyme activity varies greatly. When the substrate is aromatic and heterocyclic amides, especiall...

Examples

Embodiment 1

[0026] Example 1 Site-directed mutation

[0027] The Ser621 of penicillin G acylase was mutated to Gly, so oligo7 software was used to design blunt-end primers, and then PCR site-directed mutation was performed. This PCR mutation uses the KOD-Plus-Neo DNA polymerase kit (purchased from Toyobo Technology Co., Ltd., Shanghai). The base sequence of the primer is:

[0028] Ser-gly-F: 5’-GGGGCTCTGCCGCGTCTGGCTGGTGACG

[0029] Ser-gly-anti: 5’-CGCGGCAGAGCAGAGTCGATAGCA

[0030] The PCR reaction system is shown in Table 1.

[0031] Table 1 Site-directed mutagenesis reaction system

[0032] 10×reaction buffering

5μl

dNTPs (each 2mM)

5μl

Mg 2+ (25mM)

3μl

Template

1μl

Ser-gly-F (10mM)

1.5μl

Ser-gly-anti (10mM)

1.5μl

KOD enzyme (1U / μl)

1μl

ddH2O

32μl

Total

50μl

[0033] The template is a plasmid containing wild-type penicillin G acylase gene, which was commissioned to synthesize by the company. Among them, the plasmid vector is PET-28a, the wild-type penicillin G acylas...

Embodiment 2

[0042] Example 2 Expression and Purification of Penicillin G Acylase Mutant

[0043] 1. Expression of Penicillin G Acylase Mutant

[0044] The recombinant plasmid obtained in Example 1 was transformed into E. coli JM109 competent cells. After 10 hours, the growth of the bacteria was observed, the recombinant transformants were selected for activation, and the culture was expanded for translation and expression.

[0045] (1) Inoculate the recombinant transformants in 5ml of LB medium containing kanamycin (50μg / ml), and place them in a shaker at 37°C and 200rpm for shaking culture until the OD600 reaches about 0.4-0.5 to obtain seeds liquid.

[0046] (2) The seed solution was inoculated into the auto-induction medium at 1% of the inoculum, and cultivated at 25°C for 48 hours.

[0047] The auto-induction medium formula is: arabinose 3g / L, glucose 0.5g / L, glycerol 5g / L, peptone 10g / L, phosphate 6.8g / L, sulfate 1.2g / L, NH 4 Cl2.65g / L, MgSO 4 0.98g / L, CaCl 2 0.1g / L.

[0048] (3) The bacterial...

Embodiment 3

[0056] Example 3 Application of Penicillin G Acylase Mutant in the Synthesis of Cefprozil

[0057] The technical solution adopted in this embodiment is as follows figure 2 Shown.

[0058] by figure 2 The reaction formula shows that penicillin G acylase can not only condense the nucleus and side chains to produce products, but also decompose the products to produce nuclei and side chains. The reversibility of the reaction depends on the equilibrium constant of the reaction. However, 2-hydroxyethyl p-hydroxyphenylglycinate itself can be decomposed into p-hydroxyphenylglycine, so it is of great significance to improve the synthetic activity of penicillin G acylase.

[0059] Reaction system: substrate solution (40mM 7-amino-3-[(Z)-allyl-1-yl]-3-cephalosporin-4-carboxylic acid (7-APRA, CAS number: 120709-09-3) , 40mM 2-hydroxyethyl p-hydroxyphenylglycinate (CAS number: 2030007-73-2)) 0.525ml, 0.1ml enzyme solution, 28℃ water bath reaction, after 20min, take 10μl reaction solution and d...

Technical field

[0001] The invention belongs to the field of biocatalysis, and particularly relates to a penicillin G acylase mutant and its coding gene and application. Background technique

[0002] At present, more and more drugs or their intermediates are synthesized through a biological enzyme catalysis instead of chemical synthesis. Obviously, enzyme catalysis has great advantages over traditional chemical synthesis: (1) Toxic reagents or solvents can be better avoided in biological reactions; (2) Enzymes have the characteristics of high efficiency and substrate specificity. ; (3) Better avoid side reactions.

[0003] Acylase is a type of hydrolase that catalyzes the hydrolysis of amides into corresponding carboxylic acids. It has a wide range of sources and a broad spectrum of substrates. It can be widely used to hydrolyze fatty, aromatic, and heterocyclic amino amides, but the enzyme activity varies greatly. When the substrate is aromatic and heterocyclic amides, especiall...

Examples

Embodiment 1

[0026] Example 1 Site-directed mutation

[0027] The Ser621 of penicillin G acylase was mutated to Gly, so oligo7 software was used to design blunt-end primers, and then PCR site-directed mutation was performed. This PCR mutation uses the KOD-Plus-Neo DNA polymerase kit (purchased from Toyobo Technology Co., Ltd., Shanghai). The base sequence of the primer is:

[0028] Ser-gly-F: 5’-GGGGCTCTGCCGCGTCTGGCTGGTGACG

[0029] Ser-gly-anti: 5’-CGCGGCAGAGCAGAGTCGATAGCA

[0030] The PCR reaction system is shown in Table 1.

[0031] Table 1 Site-directed mutagenesis reaction system

[0032] 10×reaction buffering

5μl

dNTPs (each 2mM)

5μl

Mg 2+ (25mM)

3μl

Template

1μl

Ser-gly-F (10mM)

1.5μl

Ser-gly-anti (10mM)

1.5μl

KOD enzyme (1U / μl)

1μl

ddH2O

32μl

Total

50μl

[0033] The template is a plasmid containing wild-type penicillin G acylase gene, which was commissioned to synthesize by the company. Among them, the plasmid vector is PET-28a, the wild-type penicillin G acylas...

Embodiment 2

[0042] Example 2 Expression and Purification of Penicillin G Acylase Mutant

[0043] 1. Expression of Penicillin G Acylase Mutant

[0044] The recombinant plasmid obtained in Example 1 was transformed into E. coli JM109 competent cells. After 10 hours, the growth of the bacteria was observed, the recombinant transformants were selected for activation, and the culture was expanded for translation and expression.

[0045] (1) Inoculate the recombinant transformants in 5ml of LB medium containing kanamycin (50μg / ml), and place them in a shaker at 37°C and 200rpm for shaking culture until the OD600 reaches about 0.4-0.5 to obtain seeds liquid.

[0046] (2) The seed solution was inoculated into the auto-induction medium at 1% of the inoculum, and cultivated at 25°C for 48 hours.

[0047] The auto-induction medium formula is: arabinose 3g / L, glucose 0.5g / L, glycerol 5g / L, peptone 10g / L, phosphate 6.8g / L, sulfate 1.2g / L, NH 4 Cl2.65g / L, MgSO 4 0.98g / L, CaCl 2 0.1g / L.

[0048] (3) The bacterial...

Embodiment 3

[0056] Example 3 Application of Penicillin G Acylase Mutant in the Synthesis of Cefprozil

[0057] The technical solution adopted in this embodiment is as follows figure 2 Shown.

[0058] by figure 2 The reaction formula shows that penicillin G acylase can not only condense the nucleus and side chains to produce products, but also decompose the products to produce nuclei and side chains. The reversibility of the reaction depends on the equilibrium constant of the reaction. However, 2-hydroxyethyl p-hydroxyphenylglycinate itself can be decomposed into p-hydroxyphenylglycine, so it is of great significance to improve the synthetic activity of penicillin G acylase.

[0059] Reaction system: substrate solution (40mM 7-amino-3-[(Z)-allyl-1-yl]-3-cephalosporin-4-carboxylic acid (7-APRA, CAS number: 120709-09-3) , 40mM 2-hydroxyethyl p-hydroxyphenylglycinate (CAS number: 2030007-73-2)) 0.525ml, 0.1ml enzyme solution, 28℃ water bath reaction, after 20min, take 10μl reaction solution and d...

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