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Streptococcus zooepidemicus lactic dehydrogenase gene mutant and application thereof

A Streptococcus lactate dehydrogenase and zooepidemic technology, applied in the field of genetic engineering, can solve the problems of reducing the quality yield of hyaluronic acid, bacterial toxicity, reducing the final concentration of hyaluronic acid, etc. Accumulation, the effect of reducing the amount of lactic acid accumulation

Active Publication Date: 2018-06-22
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the one hand, the accumulation of lactic acid greatly reduces the quality yield of hyaluronic acid, on the other hand, lactic acid will have a toxic effect on the bacteria, reducing the final concentration of hyaluronic acid

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Discovery of Streptococcus zooepidemicus lactate dehydrogenase gene mutant capable of reducing lactic acid production by random mutation

[0021] Streptococcus zooepidemicus ATCC 39920 was cultured overnight on a fresh LB solid plate at 37°C. The next day, add 5ml of sterile water to the plate to elute the bacteria. The eluate was centrifuged in a centrifuge at 4°C and 5000rpm for 10 minutes, the supernatant was removed, the cells were washed twice with 5ml of PBS buffer (0.05M KH2PO4, pH7.0), and then resuspended in 5ml of PBS buffer. Add 100 mg / ml of nitrosoguanidine (NTG) to the resuspension, shake and incubate at 37°C for 40 minutes for mutagenesis, then add 5ml of BHI solution (aqueous solution containing 37g / L brain heart infusion) to resuscitate 6 cells at 30°C Hour. The resuscitated cells were diluted 10-10000 times and spread on a blood agar plate (the components include 10g / L of peptone, 3g / L of beef powder, 5g / L of sodium chloride, 50ml of defibr...

Embodiment 2

[0025] Example 2 Verifying that the ldhA mutant of the lactate dehydrogenase gene can lead to reduced lactic acid production

[0026] The entire genome of the mutated Streptococcus zooepidemicus Sz01 screened in the embodiment was sequenced (completed by BGI Corporation), and it was found that the whole genome of Streptococcus zooepidemicus Sz01 contained 27 mutations in total. Among them, the lactate dehydrogenase gene ldhA of Sz01 has a mutation of one base, and the 697th base is mutated from A to G (A697G) (sequence shown in SEQ ID No.1), resulting in the 233rd amino acid residue of its amino acid sequence The base is mutated from threonine to alanine (T233A) (sequence shown in SEQ ID No.2).

[0027] The lactate dehydrogenase activity of Sz01 was further detected, and its enzyme activity was 0.34U / mg, which was 82% lower than that of the control strain ATCC39920 (1.89U / mg). The detection method of lactate dehydrogenase enzymatic activity is as follows: 1ml of reaction syst...

Embodiment 3

[0034] Example 3 Constructing other recombinant Streptococcus zooepidemicus comprising a mutant of lactate dehydrogenase T233A and verifying its function

[0035] In order to test whether the lactate dehydrogenase mutation T233A can also increase the hyaluronic acid production of other Streptococcus zooepidemicus, this example further mutates the lactate dehydrogenase of Streptococcus zooepidemicus ATCC 35246.

[0036] Using an electroporator (Bio-Rad), pSET4s-sacB-ldhA was transferred into Streptococcus zooepidemicus ATCC35246 by electroporation. The electric shock conditions were voltage 2.5KV, resistance 200Ω, capacitance 25μF (the width of the electric shock cup was 2mm). The recombinant bacteria were screened on a THY plate containing 100 mg / L of spectinomycin. The recombinant bacteria were further cultured overnight in liquid THY medium, and then carried out secondary screening on THY plates containing 100 g / L sucrose. Colony PCR was performed using ldhA-F and ldhA-R as...

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Abstract

The invention provides a streptococcus zooepidemicus lactic dehydrogenase gene mutant and application thereof, and belongs to the technical field of microorganism gene engineering. The streptococcus zooepidemicus causes mutation of one basic group of a lactic dehydrogenase group ldhA through NTG mutagenesis, a 697-bit basic group mutates from A to G (A697G), causing that a 233-bit amino acid residue of the amino acid sequence mutates from threonine into alanine (T233A); the nucleotide sequence of the gene mutant is shown as SEQ ID No.1, so that the enzyme activity of lactic dehydrogenase can be lowered by 50 percent or more, thereby further proving that the streptococcus zooepidemicus containing the gene mutant can remarkably reduce the accumulation of lactic acid during production of hyaluronic acid through fermentation of the streptococcus zooepidemicus containing the gene mutant, the accumulation amount of lactic acid is lowered by 60 percent or more, and the yield of the hyaluronicacid is increased by 50 percent or more. The streptococcus zooepidemicus lactic dehydrogenase gene mutant has an important industrial application prospect.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a mutant of the lactate dehydrogenase gene of Streptococcus zooepidemicus and its application. Background technique [0002] Hyaluronic acid is a polymer polysaccharide composed of D-glucuronic acid and N-acetylglucosamine connected by β-1,3 and β-1,4 glycosidic bonds, also known as hyaluronic acid. Hyaluronic acid widely exists in various parts of the human body and has extremely important physiological functions, such as assisting the diffusion and transport of water and electrolytes, lubricating joints, regulating the permeability of blood vessel walls, and promoting wound healing. In addition, hyaluronic acid has a strong moisturizing effect and is known as an ideal natural moisturizing factor. It is the substance with the best moisturizing properties for cosmetics found in nature. Hyaluronic acid has different molecular weights, and its distribution ranges fr...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/04C12N15/74C12N1/21C12P19/26
CPCC12N9/0006C12N15/746C12P19/26C12Y101/01
Inventor 陈振刘德华
Owner TSINGHUA UNIV
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