Glutamate decarboxylase mutant establishment improving enzyme activity and application thereof

A glutamic acid decarboxylase and mutant technology, applied in the field of genetic engineering, can solve the problems of low glutamic acid decarboxylase activity and low protein expression, and achieve the effect of improving the potential of industrial application

Active Publication Date: 2016-01-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The outstanding problems of heterologous expression of glutamic acid decarboxylase are low protein expression and low activity of glutamic acid decarboxylase

Method used

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  • Glutamate decarboxylase mutant establishment improving enzyme activity and application thereof
  • Glutamate decarboxylase mutant establishment improving enzyme activity and application thereof
  • Glutamate decarboxylase mutant establishment improving enzyme activity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0009] Embodiment 1 Contains the construction of the recombinant vector of glutamic acid decarboxylase mutant

[0010] (1) Obtaining the Y172C mutant: using the nucleotide sequence shown in SEQIDNO.2 as a template, Fprimer (sequence shown in SEQIDNO.3) and Rprimer (sequence shown in SEQIDNO.4) as primers, PCR is performed to obtain The recombinant gene shown in SEQ ID NO.2.

[0011] (2) Digest the recombinant gene and pET-28a with BamHI and EcoRI, respectively, and ligate with T4 DNA ligase overnight at 16°C after purification. The ligation product was chemically transformed into JM109 competent cells. The transformation solution was applied to an LB plate containing kanamycin (50mg / L), the plasmid was extracted, and the recombinant plasmid constructed was verified by double enzyme digestion, which was named pET-28a-Y172C. The sequencing work was completed by Shanghai Sangong.

[0012] (3) The recombinant gene and pXMJ19 were digested with BamHI and EcoRI respectively, and ...

Embodiment 2

[0013] Embodiment 2 produces glutamic acid decarboxylase escherichia coli engineering bacterium construction

[0014] The recombinant plasmid pET-28a-Y172C obtained in Example 1 was chemically transformed into E.coliBL21 competent cells, the specific method is as follows:

[0015] (1) The medium required for the transformation experiment is as follows (g / L):

[0016] LB medium: peptone 10, yeast powder 5, NaCl10.

[0017] (2) Conversion method:

[0018] Add 10 μl of recombinant plasmid pET-28a-Y172C to 120 μl of competent E.coilBL21, place on ice for 45 minutes, heat shock at 42°C for 90 seconds, place on ice for 2 minutes, add 800 μl of LB liquid medium, incubate at 37°C for 1 hour, centrifuge , Pour off most of the supernatant, leave 150μL to mix with the precipitate, spread it on a Kanamycin plate (Km+LB), incubate in a 37°C incubator for about 9 hours, pick the positive colonies on the plate to 10ml of liquid In LB medium, culture overnight at 37°C on a shaker. After e...

Embodiment 3

[0019] Embodiment 3 produces glutamic acid decarboxylase Corynebacterium glutamicum engineering bacterium construction

[0020] The recombinant plasmid pXMJ19-Y172C obtained in Example 1 was transformed into C. glutamicum13032 competent cells by click method, the specific method is as follows:

[0021] (1) The medium required for the transformation experiment is as follows (g / L):

[0022] LBG medium: peptone 10, yeast powder 5, NaCl 10, glucose 5.

[0023] (2) Conversion method:

[0024] Add 10 μl of recombinant plasmid pXMJ19-Y172C to 120 μl of competent C.glutamicum13032, place on ice for 5 minutes, 1800V electric shock for 5 ms, place on ice for 2 minutes, add 800 μl of LBG liquid medium, culture on a shaker at 37°C for 2 hours, centrifuge, and discard the large Part of the supernatant, keep 150 μL and mix it with the precipitate, spread it on a kanamycin plate (Km+LBG), culture it in a 37°C incubator for about 20 hours, pick the positive colonies on the plate and put it ...

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Abstract

The invention discloses a glutamate decarboxylase mutant improving enzyme activity and an establishment method thereof, and belongs to the field of gene engineering. On the basis of an amino acid shown as SEQ ID NO.1, a 172 tyrosine is mutated to form cysteine. The obtained mutant is expressed in colibacillus, after being fermented for 24h in a shake flask, the enzyme activity is 28.6U / mL, the mutant enzyme activity is improved by 81 percent, compared with the original enzyme, the substrate affinity is reduced by 53 percent, the enzyme activity is improved by 83 percent, and the half-time period of the enzyme at 35 DEG C is increased from 16h to 24h. The recombinase is expressed in the colibacillus, and the glutamic acid is converted in a total cell manner for 18h to obtain 283.8g / L gamma-aminobutyric acid; the recombinase is expressed in glutamic acid coryneform bacteria, the glutamic acid is converted for 18h in a total cell manner to obtain 126.7g / L gamma-aminobutyric acid. The result shows that the 172 amino acid residue can severely influence the catalytic effect and stability of the enzyme, a foundation is set for researching the catalytic mechanism of the enzyme, and the industrial application potential of the enzyme is improved.

Description

technical field [0001] The invention relates to the construction and application of a glutamic acid decarboxylase mutant with improved enzyme activity, which belongs to the technical field of genetic engineering. Background technique [0002] Glutamate decarboxylase (glutamate decarboxylase, GAD, EC4.1.1.15) is the rate-limiting enzyme in the biosynthesis of γ-aminobutyric acid (GABA), which catalyzes the decarboxylation of glutamate to form CO 2 and GABA. Glutamate decarboxylase widely exists in microorganisms, animals, human body, and plants. The prominent problems of heterologous expression of glutamic acid decarboxylase are low protein expression and low activity of glutamic acid decarboxylase. Therefore, site-directed mutagenesis to modify glutamic acid decarboxylase and increase extracellular enzyme activity is of great significance for improving the industrial application prospect of glutamic acid decarboxylase. Contents of the invention [0003] The present inve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N15/77C12N1/21C12P13/00
CPCC12N9/88C12P13/005C12Y401/01015
Inventor 饶志明张显杨套伟徐美娟
Owner JIANGNAN UNIV
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