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Mutation penicillin G acylase, recombinant expression plasmid and transformation engineering strains thereof

A technology of acylase and penicillin, applied in the field of genetic engineering, can solve the problems of reduction of antibiotics, low ratio, low conversion rate of mother nucleus, etc.

Active Publication Date: 2008-05-14
SHANXI WEIQIDA PHARMA IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the traditional chemical synthesis method, the main problem faced by the enzymatic synthesis of β-lactam antibiotics is that two side reactions occur while synthesizing the antibiotics, namely 1) hydrolysis activated acyl donor; 2) hydrolysis generated Antibiotics; resulting in a reduction in acyl donors and produced antibiotics, resulting in a low nuclear conversion rate, i.e. a low synthesis product / hydrolyzate (S / H) ratio

Method used

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  • Mutation penicillin G acylase, recombinant expression plasmid and transformation engineering strains thereof
  • Mutation penicillin G acylase, recombinant expression plasmid and transformation engineering strains thereof
  • Mutation penicillin G acylase, recombinant expression plasmid and transformation engineering strains thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 , Determine the sequence of the site-directed mutation gene

[0044] 1.1. Structural modeling of BmPGA-ligand complex

[0045] With the structure of penicillin G acylase derived from Escherichia coli (E.coli) and Presdenia (Providencia rettgeri) as template, utilize the Swiss-Model model (Kaplan and Littlejohn, Swiss-PDB Viewer, 2001, http: / / www.expasy.org / spdbv / ), to model PGA (BmPGA) derived from Bacillus megaterium; then using the modeled complex structure of E.coli PGA and penicillin G, by overlaying (PG) The complex structure of BmPGA-PG was obtained.

[0046] 1.2. Selection of mutation sites

[0047] BmPGA- The structure of the PG complex was energy-optimized to select suitable mutation sites and mutation directions.

[0048] The results of modeling and optimization are as follows figure 1 shown, according to figure 1 As a result, it was determined that the site-directed mutation sites were: α144, α145 and β24, and the mutation direction was: th...

Embodiment 2

[0049] Example 2 , Construction of mutant plasmids

[0050] 2.1. Primer design

[0051] According to the gene sequence (SEQ ID NO: 1) of PGA derived from Bacillus megaterium, and the selected mutation sites α144, α145, β24, the following 7 mutation primers were designed:

[0052] α144R: 5`-G AGA TTT ATG GAT AAT CAC CAG GAG TTA-3`;

[0053] α145Y: 5`-G TAT TAT ATG GAT AAT CAC CAG GAG TTA-3`;

[0054] α145A: 5`-G TAT GCT ATG GAT AAT CAC CAG GAG TTA-3`;

[0055] α145L:5`-G TAT CTT ATG GAT AAT CAC CAG GAG TTA-3`;

[0056] β24F2:5`-AA TTT GGT TTT GTT GCT CCT GGA TTT-3`;

[0057] #145U:5`-GT CAT CGA TAC CAT ATA AAC ACG GAC A-3`;

[0058] (Cla I)

[0059] β24F1:5`-G GGG CCC ACT GAA TAA TAA AGC ATT T-3`;

[0060] (Apa I)

[0061]Wherein, the underline in the primer indicates the introduced mutant sequence. Primers α145U and β24F1 are synonymous mutations, which are used to identify whether the mutation is successful.

[0062] 2.2. Construction of recombi...

Embodiment 3

[0087] Example 3 , the acquisition of engineered bacteria

[0088] According to the operation manual of TAKARA company, the mutated four kinds of plasmids (BmPGA α144R, BmPGAα145Y, BmPGAβ24F, BmPGAα144R+β24F) were digested with EcoR I, extracted with phenol chloroform, precipitated with ethanol, and then self-cyclized with T4 ligase e , and then transform Bacillus subtilis competent cells WB600 to obtain genetically engineered strains BmPGAα144R / WB600, BmPGAα145Y / WB600, BmPGAβ24F / WB600 and BmPGAα144R+β24F / WB600, as follows:

[0089] 3.1. Circularization of mutant plasmids

[0090] 1) Under the condition of 37°C, in 100 μl EcoR I enzyme digestion system (30 μl to obtain the mutant plasmid, 1 / 10 volume of H buffer from TaKaRa Company, EcoR I enzyme (final concentration is 0.05U / μl), supplemented with double Distilled water into 100 μl), digested the mutant plasmid, ran agarose gel electrophoresis after 2 hours, and recovered a 6.3 kb fragment with the gel recovery kit of Huas...

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Abstract

The invention relates to a gene, mutant plasmid and engineering bacteria which have improved synthesis performance to penicillin G acylase and are obtained by a gene site-directed mutagenesis method, and mutant enzyme can also be obtained with improved synthesis performance to penicillin G acylase by fermenting and purifying the engineering bacteria. Two enzymes Kpn I and Pst I are firstly used for cutting pUC18 by the invention, then T4 polymerase is adopted to make the ends blunt, and pZ01 is obtained through self-linkage; the enzyme of EcoR I is used for cutting pZ01, and then connected with pEES102 that is also cut by the enzyme of EcoR I, thereby obtaining the recombinant plasmid pY020; the pY020 is adopted as a template plasmid, and TaKaRa MuTanBEST Kit is utilized for conducting the site-directed mutagenesis to B.megaterium PGA, thereby obtaining the mutant plasmid with improved synthesis performance to the penicillin G acylase. The mutant plasmid is transformed to bacillus subtilis to obtain the required engineering bacteria. The engineering bacteria are amplified and fermented, and the mutant enzyme with improved maximum conversion rate of 7-ADCA and the ratio of synthetic product / hydrolysate can be obtained after the engineering bacteria are purified.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the penicillin G acylase gene, mutant plasmid, engineering bacteria and mutant enzyme obtained by gene site-directed mutation method with improved synthetic performance. Background technique [0002] Penicillin G Acylase (Penicillin G Acylase, E.C.3.5.1.11, referred to as PGA) is a heterodimer N-terminal nucleophilic serine hydrolase (Duggleby et al., Nature, 373 (6511), 264-268, 1995). Penicillin G acylase is an important enzyme used in the industry of semi-synthetic β-lactam antibiotics. The main purpose of this enzyme is to hydrolyze penicillin G and cephalosporin G respectively to generate the corresponding compound nucleus, 6-aminopenicillanic acid ( 6-AminoPenicillinic acid, 6-APA) and 7-Aminocephalosporanic acid (7-Amino-deacetoxy-cephalosporanic-acid, 7-ADCA) (Abian et al., Biotechnol Prog, 2003, 19(6), 1639-42 , 2003). The new use of penicillin...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/86C12N1/21
Inventor 黄鹤张磐王金刚袁中一杨晟姜卫红
Owner SHANXI WEIQIDA PHARMA IND
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