Penicillin g Acylase Mutant and Its Application in the Synthesis of Cephalosporin Antibiotics

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

Active Publication Date: 2015-10-21
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 Acylase Mutant and Its Application in the Synthesis of Cephalosporin Antibiotics
  • Penicillin g Acylase Mutant and Its Application in the Synthesis of Cephalosporin Antibiotics
  • Penicillin g Acylase Mutant and Its Application in the Synthesis of Cephalosporin Antibiotics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Site-directed mutagenesis

[0027] The Phe519 of penicillin G acylase was mutated to Ala, so the oligo7 software was used to design blunt-end primers, and then PCR site-directed mutagenesis was performed. This PCR mutation uses KOD-Plus-Neo DNA polymerase kit (purchased from Toyobo Technology Co., Ltd., Shanghai), and the base sequence of the primers 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

wxya 2 o

32μl

Total

50μl

[0033] The template is a plasmid containing the wild-type penicillin G acylase gene, w...

Embodiment 2

[0042] Embodiment 2 Expression and purification of penicillin G acylase mutant

[0043] 1. Expression of penicillin G acylase mutants

[0044] Transform the mutant library constructed in Example 1 into Escherichia coli JM109 competent cells, observe the growth status of the bacteria after 10 hours, pick the recombinant transformants for activation, and expand the culture for translation and expression.

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

[0046] (2) Inoculate the seed solution in the self-inducing medium at an inoculation amount of 1%, and incubate at 25°C for 48 hours.

[0047] The formula of self-induction medium 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 bacte...

Embodiment 3

[0056] Application of embodiment 3 penicillin G acylase mutants in the synthesis of cefaclor

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

[0058] Depend on figure 2 The reaction formula shows that the active enzyme (penicillin G acylase) can not only condense 7-amino-3-chloro-cephalosporanic acid (the core 7-ACCA) and phenylglycine methyl ester (side chain) to generate products, but also decompose the products The parent nucleus and side chains are generated, and the reversibility of the reaction depends on 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-cephemic acid (7-ACCA, CAS number: 53994-69-7), 40mM phenylglycine methyl ester (CAS number: 24461-61-8)) 0.525 ml, 0.1ml enzyme solution, react in a water bath...

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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, in particular to a penicillin G acylase mutant and its application in synthesizing cephalosporin antibiotics. Background technique [0002] At present, more and more drugs or their intermediates are synthesized by enzymatic catalysis instead of chemical synthesis. Obviously, enzymatic 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 the occurrence of side reactions. [0003] Acylase is a kind of hydrolase that catalyzes the hydrolysis of amides into corresponding carboxylic acids. It has a wide range of sources and a wide spectrum of substrates. It can be widely used to hydrolyze aliphatic, aromatic, and heterocyclic amino amides, but the enzyme activity varies greatly. When the substrate is an aromatic o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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