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Penicillin G acylation enzyme mutant, and application thereof in synthesis of cephalosporin antibiotics

A technology of acylase mutants and penicillin, applied in applications, enzymes, enzymes, etc., can solve problems such as heavy computational workload, long time, and inability to achieve high activity and improved enzyme activity

Active Publication Date: 2014-06-18
ZHEJIANG APELOA TOSPO PHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the calculation workload in the early stage is huge, and it takes a long time to accumulate more knowledge in the early stage. At this stage, it is still impossible to achieve a program that calculates first and then performs fixed-point directional mutations.

Method used

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  • Penicillin G acylation enzyme mutant, and application thereof in synthesis of cephalosporin antibiotics
  • Penicillin G acylation enzyme mutant, and application thereof in synthesis of cephalosporin antibiotics
  • Penicillin G acylation enzyme mutant, and application thereof in synthesis of cephalosporin antibiotics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Site-directed mutation

[0027] The Phe519 of penicillin G acylase was mutated to Ala, 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] PHE TO ALA-F: 5’-GCATAACTTCTCTCACGTTGACCGTGTTAA

[0029] PHE TO ALA-ANTI: 5’-GAGTCAGAACGCAGACCAGCGTAA

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

[0031] Table 1 Site-directed mutagenesis reaction system

[0032] 10×reaction buffering

5μl

dNTPs (2mM each)

5μl

Mg 2+ (25mM)

3μl

Template

1μl

PHE TO ALA-F (10mM)

1.5μl

PHE TO ALA-ANTI (10mM)

1.5μl

KOD enzyme (1U / μl)

1μl

ddH 2 O

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, and the wild-type...

Embodiment 2

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

[0043] 1. Expression of Penicillin G Acylase Mutant

[0044] The mutant library constructed 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 transformant in 5ml of LB medium containing kanamycin (50μg / ml), and place it 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 liqu...

Embodiment 3

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

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

[0058] by figure 2 The reaction formula shows that the active enzyme (penicillin G acylase) can not only condense 7-amino-3-chloro-cephalosporanic acid (nucleus 7-ACCA) and phenylglycine methyl ester (side chain) to produce products, but also decompose products The nucleus and side chains are generated, and the reversibility of the reaction is determined by the equilibrium constant of the reaction. However, phenylglycine methyl ester itself can be decomposed into phenylglycine, so it is of great significance to improve the activity of penicillin acylase.

[0059] Reaction system: substrate solution (40mM 7-amino-3-chloro-cephalosporin (7-ACCA, CAS number: 53994-69-7), 40mM phenylglycine methyl ester (CAS number: 24461-61-8)) 0.525 ml, 0.1ml enzyme solution, 28℃ water bath reaction, after 20 minutes, take ...

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Abstract

The invention discloses a penicillin G acylation enzyme mutant, and application thereof in synthesis of cephalosporin antibiotics. An amino acid sequence of the penicillin G acylation enzyme mutant is shown in SEQ ID NO.1; a nucleotide sequence of an encoding gene is shown in SEQ ID NO.2. The invention also provides application of the penicillin G acylation enzyme mutant in synthesis of cefaclor. The invention provides a novel penicillin acylation enzyme mutant. Compared with the wild penicillin acylation enzyme mutant, the penicillin G acylation enzyme mutant has higher activity in synthesis of cephalosporin antibiotics, such as cefprozil, cefaclor or cefadroxil; especially when the penicillin G acylation enzyme mutant is used for catalyzing 7-ACCA and phenylglycine methyl ester to synthesize the cefaclor, the vitality is improved to 29.8U / mg from 1.2U / mg in the past, the synthesis and hydrolysis activity is almost consistent with that of the wild penicillin acylation enzyme mutant, the synthesis and hydrolysis ratio can be up to 1.7, and the yield of the cefaclor can be up to 71.5%.

Description

Technical field [0001] The invention belongs to the field of biocatalysis, and particularly relates to a penicillin G acylase mutant and its application in the synthesis of cephalosporin antibiotics. 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 compared with 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 arom...

Claims

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Application Information

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IPC IPC(8): C12N9/84C12N15/55C12N15/70C12N1/21C12P35/04
CPCC12N9/84C12P35/04C12Y305/01011
Inventor 任红阳李兰杰陈振明周硕陈治黄艳芳赖敦岳陈亮厉昆
Owner ZHEJIANG APELOA TOSPO PHARMA
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