D,D-carboxypeptidase DacA mutant with improved catalysis efficiency and preparation method thereof

A carboxypeptidase and mutant technology, which is applied in the field of D,D-carboxypeptidase DacA mutant and its preparation, can solve the problem of discontinuous protein production, shorten the time of enzymatic property modification and improve the secretion of extracellular protein The effect of improving expression level and catalytic efficiency

Active Publication Date: 2018-08-17
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in E. coli, most proteins are transported to the periplasmic space of the cell and cannot directly reach the outside of the cell, which is prone to cause many problems (such as discontinuous protein production, etc.)

Method used

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  • D,D-carboxypeptidase DacA mutant with improved catalysis efficiency and preparation method thereof
  • D,D-carboxypeptidase DacA mutant with improved catalysis efficiency and preparation method thereof
  • D,D-carboxypeptidase DacA mutant with improved catalysis efficiency and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0015] Example 1: D, D-carboxypeptidase DacA catalytic efficiency site-directed mutation analysis and method

[0016] D,D-carboxypeptidase DacA (SEQ ID NO.1) derived from Escherichia coli was simulated by Swiss-model software to obtain a 3D structure model of D,D-carboxypeptidase DacA. Select the key amino acid residue located in the "active pocket" - lysine, and replace it with arginine, glycine or histidine.

[0017] According to the E.coli D,D-carboxypeptidase DacA sequence, the E.coli BL21 genome was used as a template and cloned between the restriction sites XhoI and EcoRI of the plasmid pET-28a(+) by PCR method to construct the recombinant plasmid pET28a- dacA.

[0018] For site-directed mutagenesis, design corresponding site-directed mutagenesis primers (Table 1). D,D-carboxypeptidase DacA was subjected to site-directed mutation using site-directed mutagenesis primers and recombinant plasmid pET28a-dacA. PCR enzyme was used to amplify the recombinant plasmid pET28a-d...

Embodiment 2

[0021] Embodiment 2: D, D-carboxypeptidase DacA enzyme activity determination and analysis method

[0022] (1) Preparation of D-alanine standard curve: D-alanine solutions with different concentrations of 0-60 mM were prepared. Add 5 μL o-dianisidine solution, 70 μL enzyme and coenzyme (39:19:10:2) mixture, and react at 37°C for 5 minutes. Immediately add 400 μL of methanol / water (1:1) mixture, react at 37°C for 2 min, and measure the absorbance value at 460 nm. Take the concentration of D-alanine as the abscissa and the absorbance as the ordinate to make a standard curve.

[0023] (2) The reaction system includes: 15 μL substrate Park nucleotide analog (Na,Ne-Diacetyl-Lys-D-Ala-D-Ala), 3 μL Tris-HCl buffer (pH 7.5) and 12 μL enzyme solution. Mix well and react at 37°C for 10min. Add 5 μL o-dianisidine, 70 μL enzyme and coenzyme mixture, and react at 37°C for 5 minutes. Immediately add 400 μL of methanol / water mixture, react at 37°C for 2 min, and measure the absorbance va...

Embodiment 3

[0027] Example 3: Determination and analysis of D, D-carboxypeptidase DacA and its mutant catalytic efficiency

[0028] By measuring the catalytic efficiency of D,D-carboxypeptidase DacA and its mutants, it was found that the catalytic efficiency of the mutants K113R and K113G (Table 2) was increased, but the catalytic efficiency of the mutant K113H was decreased. Catalytic efficiency constant k of mutants K113R and K113G cat values ​​were 6543.42s from wild enzyme -1 Increased to 8541.56s -1 、9428.13s -1 . Among them, the effect of the mutant K113G is the most significant, and the catalytic efficiency constant k cat The value is increased to 1.4 times of the original value. Meanwhile, the K of mutant K113G m The value decreased from 0.85mmol / L of the wild enzyme to 0.66mmol / L, indicating that the binding ability of the enzyme to the substrate was enhanced after the mutation.

[0029] Table 2 Kinetic parameters of D,D-carboxypeptidase DacA mutants

[0030]

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Abstract

The invention discloses a D,D-carboxypeptidase DacA mutant with improved catalysis efficiency and a preparation method thereof, belonging to the fields of genetic engineering and enzyme engineering. D,D-carboxypeptidase DacA from Escherichia coli BL21 is used as a female parent, key sites and a mutation manner are determined based on enzyme structure analysis, and the DacA mutant with improved catalysis efficiency is obtained through fixed point mutation by using a molecular biological technology. Under the modification condition, the catalysis efficiency constant k<cat> of E.coli D,D-carboxypeptidase DacA mutant K113G is improved at most, from 6543.4s<-1> to 9428.1s<-1>. Due to adoption of the method, the catalysis efficiency of the D,D-carboxypeptidase DacA is improved remarkably, layinga foundation for the application of metabolizing and modifying E.coli to improve exocytosis level and the like. The method has an important guiding significance in property modification of other enzymes.

Description

technical field [0001] The invention relates to a D,D-carboxypeptidase DacA mutant with improved catalytic efficiency and a preparation method thereof, belonging to the field of enzyme engineering. Background technique [0002] Escherichia coli is one of the most widely used bacterial species for recombinant protein production, which has many advantages (such as post-translational modification - disulfide bond formation, etc.). But in Escherichia coli, most proteins are transported to the periplasmic space of the cell and cannot directly reach the outside of the cell, which is prone to cause many problems (such as discontinuous protein production, etc.). As a major component of the cell wall, peptidoglycan helps maintain the robustness of the cell structure. Extracellular production levels of macromolecules are enhanced by disrupting the peptidoglycan structure surrounding cells. D,D-carboxypeptidase DacA can excise the terminal D-alanine residue from the peptidoglycan sid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/48C12N15/57C12N15/70C12N1/21C12P13/06C12R1/19
CPCC12N9/485C12N15/70C12P13/06C12Y304/16004
Inventor 许菲杨海泉陈媛卢潇王浩坤王拂祥代瑶
Owner JIANGNAN UNIV
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