Gamma-glutamyltranspeptidase mutant with improved transpeptide vitality as well as construction method thereof

A technology of glutamyl transpeptidase and mutant, applied in the field of genetic engineering, can solve the existing problems and achieve the effect of improving the potential of industrial application

Active Publication Date: 2018-10-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The outstanding problem in the synthesis of L-theanine catalyzed by γ-glutamyl transpeptidase is that there is a hydrolysis reaction, resulting in the synthesis of by-product L-glutamic acid
In general, the reactio

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0021] Example 1 Construction of recombinant vector containing γ-glutamyl transpeptidase mutant

[0022] (1) Obtaining the T463D mutant: Using the nucleotide sequence shown in SEQ ID NO.4 as a template, Fprimer (sequence shown in SEQ ID NO.5) and Rprimer (sequence shown in SEQ ID NO.6) are Primer, PCR is performed to obtain the recombinant gene shown in SEQ ID NO.3.

[0023] (2) The recombinant gene and pMA5 were digested with BamHI and MluI respectively, and then purified and ligated with T4 DNA ligase overnight at 16°C. The ligation product chemically transforms JM109 competent cells. The transformation solution was coated on an LB plate containing kanamycin (50 mg / L), the plasmid was extracted, and the recombinant plasmid constructed by double enzyme digestion was verified and named pMA5-T463D. The sequencing work was completed by Shanghai Shenggong.

Example Embodiment

[0024] Example 2 Construction of Bacillus subtilis engineering bacteria producing γ-glutamyl transpeptidase

[0025] The recombinant γ-glutamyl transpeptidase plasmid pMA5-T463D obtained in Example 1 was chemically transformed into B.subtilis 168 competent cells, the specific method is as follows:

[0026] (1) The solution required for the transformation experiment is as follows (g / L):

[0027] Sp-A: (NH 4 ) 2 SO 4 4. K 2 HPO 4 28, Sodium Citrate 12 Sp-B: MgSO 4 ·7H 2 O 0.4

[0028] 100×CAYE: Casamino acid 20, yeast powder 100Sp I medium: Sp-A49%, Sp-B 49%, 50% glucose 2%, 100×CAYE 2% Sp II medium: Sp I medium 98%, 50mmol / LCaCl 2 1%, 250mmol / LMgCl 2 1%. Sterilized by moist heat at 115℃.

[0029] (2) Inoculate a single colony of B. Subtilis 168 into 2 mL Sp I medium (50 mL centrifuge tube), and cultivate overnight at 37°C and 200 r / min;

[0030] (3) Take 100μL of culture solution into 5mL Sp I medium, culture it to logarithmic phase (OD600 value is about 1) at 37℃, 200r / min, about ...

Example Embodiment

[0032] Example 3 High-efficiency expression of recombinant strain pMA5-T463D / B.subtilis 168γ-glutamyl transpeptidase and enzyme activity determination.

[0033] (1) The recombinant strain pMA5-T463D / B.subtilis 168 constructed in Example 2 and the control strain pMA5-ggt / B.subtilis 168 expressing the unmutated enzyme were respectively inoculated in 10 mL of LB medium containing kanamycin Cultivate overnight at 37°C with shaking, transfer to Bacillus subtilis fermentation medium at 4% of the inoculum the next day, culture at 37°C for 24h, take the fermentation broth and centrifuge at 4°C, 10000r / min for 10 minutes, and supernatant is extracellular Crude enzyme solution and cell disruption supernatant are crude intracellular enzyme solution, which is used for the determination of enzyme activity.

[0034] (2) Bacillus subtilis fermentation medium: sucrose 25g / L, tryptone 5g / L, corn steep liquor 15g / L, MgSO40.3g and K 2 HPO 4 ·3H 2 O 1g / L, add kanamycin to a final concentration of 50μg...

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PUM

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Abstract

The invention discloses a gamma-glutamyltranspeptidase mutant with improved transpeptide vitality as well as a construction method thereof and belongs to the field of gene engineering. The mutant is that the 463rd amino acid is mutated from threonine to aspartic acid on the basis of amino acid as shown in SEQ ID NO.3. The mutant is expressed in bacillus subtilis, the transpeptide reaction vitalityof the gamma-glutamyltranspeptidase mutant enzyme is increased by 22.6 percent by compared with that before mutation, and the hydrolytic reaction vitality is reduced by 90 percent. The invention indicates that the 463rd amino acid residue has big influence on the transpeptide reaction vitality of the gamma-glutamyltranspeptidase, certain basis is provided for research on the catalytic mechanism of the enzyme, and the industrial application potential of the enzyme is improved. The gamma-glutamyltranspeptidase mutant provided by the invention can be applied to preparation of L-theanine.

Description

technical field [0001] The invention relates to a gamma-glutamyl transpeptidase mutant with improved transpeptide activity and a construction method thereof, belonging to the technical field of genetic engineering. Background technique [0002] L-theanine is a natural amino acid that exists in tea plants. It determines the quality and flavor of tea and has many health effects on the human body. Its demand as a food component and beverage additive is increasing day by day. . At present, the research on the production of theanine mainly focuses on the enzymatic conversion method, and among them, γ-glutamyl transpeptidase stands out with its unique advantages. [0003] γ-glutamyltranspeptidase (γ-glutamyltranspeptidase, GGT, EC2.3.2.2) is responsible for catalyzing the transfer of γ-glutamyl molecules of γ-glutamyl compounds to other receptor molecules such as amino acids and short peptides ( Transpeptide reaction) or on water molecules (hydrolysis reaction), widely exists in...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N1/21C12P13/04C12R1/125
CPCC12N9/104C12P13/04C12Y203/02002
Inventor 杨套伟饶志明杨晨张显徐美娟刘会灵
Owner JIANGNAN UNIV
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