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L-lactate dehydrogenase mutant with improved catalysis efficiency and building method thereof

A technology for improving the catalytic activity of lactate dehydrogenase, which is applied in the field of genetic engineering and can solve the problems of limiting the wide application of L/D-LDH and low activity

Active Publication Date: 2018-05-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the wild-type L / D-LDHs found to have long aliphatic or aromatic side chain substrates (benzoylformic acid, PPA, oxaloacetate, etc.) show lower activity. To some extent, it limits the wide application of L / D-LDH

Method used

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  • L-lactate dehydrogenase mutant with improved catalysis efficiency and building method thereof
  • L-lactate dehydrogenase mutant with improved catalysis efficiency and building method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Embodiment 1: Construction of mutant enzyme gene and its expression plasmid

[0025] The amplified nucleotide sequence is the gene Lcldh shown in SEQ ID NO.4, the amplified product is ligated with pUCm-T, transformed into E.coli JM109, and subjected to blue-white screening, PCR verification of bacteria solution and DNA sequencing. The recombinant plasmid with correct sequencing was named pUCm-T-Lcldh. Digested pUCm-T-Lcldh with NdeI and XhoI, recovered Lcldh, ligated with pET-28a(+) which had been digested with the same double enzymes, and obtained recombinant plasmid pET-28a(+)-Lcldh.

[0026] Using the pET-28a(+)-Lcldh recombinant plasmid as a template, using F1primer (sequence shown in SEQ ID NO.5) and R1primer (sequence shown in SEQ ID NO.6) as primers, site-directed mutagenesis was performed by PCR to obtain A recombinant plasmid carrying the mutant gene, named pET-22b(+)-Lcldh I229A .

Embodiment 2

[0027] Embodiment 2: L-lactate dehydrogenase-producing Escherichia coli engineering bacteria construction

[0028] The recombinant plasmid pET-22b(+)-Lcldh that embodiment 1 obtains I229A To transform E.coli BL21 competent cells, the specific method is as follows:

[0029] 1) Inoculate activated E.coli BL21 on LB plate in 2 mL LB medium, culture overnight at 37°C, 220r / min; inoculate 2% of the above culture solution in 5mL LB medium, culture at 37°C, 220r / min for 4h;

[0030] 2) Take 1.4mL of the above bacterial solution and put it into a 1.5mLEP tube, bathe in ice for 10min, centrifuge at 4000r / min for 2min, and collect the bacteria;

[0031] 3) Add 1mL of pre-cooled 0.1M CaCl 2 The above-mentioned cells were resuspended in the solution, placed in an ice bath for 10 minutes, centrifuged at 4000 r / min for 2 minutes, and the bacteria were collected;

[0032] 4) Add 100 μL of pre-cooled 0.1M CaCl 2 Suspend the above cells in the solution and store at 4°C for 30 minutes to tr...

Embodiment 3

[0038] Example 3: Recombinant Escherichia coli induced expression

[0039] Activate the recombinant bacteria LcLdhBL21 constructed in Example 2 on the LB plate, pick a single colony into 2mL LB medium, culture at 37°C and 220r / min for 12h, and inoculate 100mL / 500mL LB medium according to 2% inoculum size, After culturing at 220r / min for 2 hours at 37°C, add IPTG to a final concentration of 0.4mM, induce expression at 20°C for 8h, centrifuge at 8000r / min for 5min to collect bacteria, and obtain crude enzyme solution after ultrasonic crushing, and purify the crude enzyme solution with a nickel column. Methods as below:

[0040] 1) Wash the column with ultrapure water for more than 3 times;

[0041] 2) Wash the column 3 times with the newly prepared binding solution;

[0042]3) Sample loading: filter the fermentation broth with a water membrane. If the amount is large, it can be repeatedly passed through the column; the sample volume is <= 8mL, generally about 4mL; the lower le...

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Abstract

The invention discloses an L-lactate dehydrogenase mutant with the improved catalysis efficiency and a building method thereof, and belongs to the technical field of genetic engineering. On the basisof L-lactate dehydrogenase, the L-lactate dehydrogenase molecular structures are modified through a site-specific mutagenesis biological technology; one strain of L-lactate dehydrogenase escherichia coli engineering bacterium is obtained; the mutant enzyme specific enzyme activity is improved by 6.1 times; the catalytic activity is improved by 4.2 times. The limitation problem of low catalysis activity of the L-lactate dehydrogenase is solved; a novel idea is provided for the L-lactate dehydrogenase molecular modification.

Description

technical field [0001] The invention relates to an L-lactate dehydrogenase mutant with improved catalytic efficiency and a construction method thereof, belonging to the technical field of genetic engineering. Background technique [0002] NADH-dependent L-lactate dehydrogenase (L-lactate dehydrogenase, L-LDH) can asymmetrically reduce α-keto acids to generate chiral α-hydroxy acids, which are widely used in biopharmaceuticals, materials and food industries. For example, L-phenyllactic acid (L-phenyllactic acid) is a natural antibacterial agent, which can also be catalyzed by the asymmetric reduction of phenylpyruvic acid (PPA) by L-LDH, which can replace the antibacterial agent in food additives and animal feed. It can also be used as a precursor for the synthesis of various drugs, and can also be used as a potential substitute for polylactic acid to synthesize new polymer materials from polyphenyllactic acid. [0003] Some chemical and biological methods can be used to syn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/70C12N1/21A23L29/00A61K38/44C12R1/19
CPCA23L29/06A61K38/00C12N9/0006C12N15/70C12Y101/01027
Inventor 李剑芳刘艳张婷李雪晴袁风娇邬敏辰
Owner JIANGNAN UNIV
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