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A kind of γ-glutamyl transpeptidase mutant with improved enzyme activity and its construction method

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: 2020-08-04
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 reaction is optimized by controlling the dosage ratio between the donor and the acceptor, adjusting pH and other conditions to reduce the synthesis of by-products, but the accumulation of by-product L-glutamic acid still exists

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1 Construction of recombinant vector containing γ-glutamyl transpeptidase mutant

[0016] (1) Obtainment of T413C 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 primers and PCR to obtain the recombinant gene shown in SEQ ID NO.3.

[0017] (2) The recombinant gene and pMA5 were digested with BamHI and MluI respectively, and then ligated with T4 DNA ligase at 16°C overnight after purification. The ligation products were chemically transformed into JM109 competent cells. The transformation solution was coated on LB plate containing kanamycin (50 mg / L), the plasmid was extracted, and the constructed recombinant plasmid was verified by double enzyme digestion and named pMA5-T413C. The sequencing work was done by Shanghai Shenggong.

Embodiment 2

[0018] Example 2 Construction of Bacillus subtilis engineering bacteria producing γ-glutamyl transpeptidase

[0019] The recombinant γ-glutamyl transpeptidase particle pMA5-T413C obtained in Example 1 was chemically transformed into B.subtilis 168 competent cells, and the specific method was as follows:

[0020] (1) The solution required for the transformation experiment is as follows (g / L):

[0021] Sp-A: (NH 4 ) 2 SO 4 4. K 2 HPO 4 28. Sodium Citrate 12Sp-B:MgSO 4 ·7H 2 O 0.4

[0022] 100×CAYE: Casamino acid 20, yeast powder 100Sp I medium: Sp-A 49%, Sp-B 49%, 50% glucose 2%, 100×CAYE 2% Sp II medium: Sp I medium 98%, 50mmol / LCaCl 2 1%, 250mmol / LMgCl 2 1%. 115°C moist heat sterilization.

[0023] (2) Inoculate a single colony of B.Subtilis 168 into 2mL Sp I medium (50mL centrifuge tube), and cultivate overnight at 37°C and 200r / min;

[0024] (3) Take 100 μL of culture solution into 5 mL of Sp I medium, and culture at 37°C at 200 r / min to logarithmic phase (OD60...

Embodiment 3

[0026] Example 3 High expression of recombinant bacterium pMA5-T413C / B.subtilis 168γ-glutamyl transpeptidase and determination of enzyme activity.

[0027] (1) The recombinant strain pMA5-T413C / 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 , 37 °C shaking culture overnight, transferred to Bacillus subtilis fermentation medium at 4% of the inoculum the next day, cultured at 37 °C for 24 h, the fermentation broth was centrifuged at 4 °C, 10000 r / min for 10 min, the supernatant was extracellular Crude enzyme solution, cell disruption supernatant is intracellular crude enzyme solution, which is used for the determination of enzyme activity.

[0028] (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 1 g / L, kanamycin was added to a final concentration of 50 μ...

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Abstract

The invention discloses an enzyme activity increased gamma-glutamyltranspeptidase mutant, and a construction method thereof, and belongs to the field of gene engineering. According to the constructionmethod, an amino acid represented by SEQ ID NO.2 is taken as a base, at the 413th site, mutation of threonine into cysteine is realized. The expression of the obtained enzyme activity increased gamma-glutamyltranspeptidase mutant is carried out in bacillus subtilis, the transpeptidation reaction activity of the gamma-glutamyltranspeptidase mutant enzyme is increased by 12% of that before mutation, and hydrolysis reaction activity is reduced by 50%. It is shown that the amino acid residue on the 413th site possesses relatively high influences on the transpeptidation reaction activity of gamma-glutamyltranspeptidase, certain foundation is provided for study on the catalytic mechanisms of gamma-glutamyltranspeptidase, the industrial application potential value of gamma-glutamyltranspeptidaseis increased; and the enzyme activity increased gamma-glutamyltranspeptidase mutant can be used for preparing L-theanine.

Description

technical field [0001] The invention relates to a γ-glutamyl transpeptidase mutant with improved enzyme activity and a construction method thereof, belonging to the technical field of genetic engineering. Background technique [0002] L-theanine, a natural amino acid existing in tea plants, determines the quality and flavor of tea leaves, and has various 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 theanine production mainly focuses on the enzymatic conversion method, among which γ-glutamyl transpeptidase stands out for its unique superiority. [0003] γ-glutamyl transpeptidase (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 ( Transpeptidation reaction) or water molecule (hydrolysis reaction), widely present in mammals and bact...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/75C12N1/21C12P13/04C12R1/125
CPCC12N9/104C12N15/75C12P13/04C12Y203/02002
Inventor 饶志明杨套伟张显徐美娟刘会灵
Owner JIANGNAN UNIV
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