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L-lactic dehydrogenase derived from Lactobacillus casei and application of L-lactic dehydrogenase

A technology of lactate dehydrogenase and phenyllactic acid, applied in the field of L-lactate dehydrogenase, can solve the problems of low PLA concentration, pollution, and difficult separation of by-products

Inactive Publication Date: 2017-09-19
JIANGNAN UNIV
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Problems solved by technology

[0003] PLA can be metabolized by a variety of microorganisms, and it is a by-product in the metabolic pathway of bacteria, such as most lactic acid bacteria such as Lactobacillus plantarum (Lactobacillus plantarum), Pediococcus acidilactici (Pediococcus acidilactici), etc., and some fungi such as Geotrichum candidum ), fluorescent Vickers yeast (Wickerhamiafluorescens) TK1 can produce PLA, but the concentration of PLA produced by existing wild strains is very low
At present, PLA can be synthesized by chemical and biological methods, but due to the harsh reaction conditions of chemical methods, complex technical routes, many by-products are difficult to separate, especially the shortcomings of environmental pollution, biosynthesis is due to its high efficiency and reaction conditions. Mild and other advantages, in recent years by the majority of researchers of all ages
[0004] Studies have shown that a variety of L-lactate dehydrogenases in microorganisms can asymmetrically reduce phenylpyruvate to L-PLA, but there are significant differences in the ability of different L-LDHs to asymmetrically reduce phenylpyruvate. For example, Jia Jianghua and other clones expressed Derived from L1-LDH and L2-LDH genes in Lactobacillus plantarum, the specific activity of recombinant L1-LDH to phenylpyruvate is 71.06 U / mg, while the specific activity of recombinant L2-LDH to phenylpyruvate is only 0.06U / mg, its enantiomeric purity has not been determined

Method used

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  • L-lactic dehydrogenase derived from Lactobacillus casei and application of L-lactic dehydrogenase

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Experimental program
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Effect test

Embodiment 1

[0026] The cloning of embodiment 1 LcLDH1 gene and the construction of expression plasmid

[0027] According to the nucleotide sequence corresponding to L-LDH (CAP07851) of L. casei BL23, the following primers were designed:

[0028] LcLDH1-F: 5' -CATATG GTGGCAAGTATTACGGATAA-3', containing Nde I restriction site.

[0029] LcLDH1-R: 5'- CTCGAG CTGACGAGTTTCGATGTCATT-3', containing Xho I restriction site.

[0030] Extract the total DNA of Lactobacillus casei, use LcLDH1-F and LcLDH1-R as primers for PCR amplification, PCR conditions are: 94°C 4min; 94°C 30s, 51°C 30s, 72°C 1min 10s, 30 cycles; 72°C 10min. The PCR product was analyzed by 1% agarose gel electrophoresis, the target band was recovered by tapping the gel and ligated with the pUCm-T vector (pUCm-T-Lcldh1), transformed into E.coli JM109, and sent to Shanghai after the correct identification by bacterial liquid PCR The nucleotide sequence of LcLDH1 was obtained by biosequencing. The pUCm-T-Lcldh1 and pET-22b(+) p...

Embodiment 2

[0031] Example 2 Construction and Induced Expression of Recombinant Bacteria E.coli / Lcldh1

[0032] Transform pET-22b(+)-Lcldh1 into E.coli BL21(DE3), and after screening on a resistance plate containing Amp, pick positive transformants and place them in 2 mL of resistant LB liquid medium containing Amp, at 37°C 220rpm shaker shaking culture 14 ~ 16h. Transfer to 30mL of the same medium according to the inoculum size of 2%, and culture on a shaker at 220rpm at 37°C until the mid-logarithmic growth phase (OD600 =0.6~0.8), add IPTG to a final concentration of 0.4mmol / L, induce culture at 20°C and 220rpm for 8h. After the induction, the cells were collected by centrifugation and washed several times with phosphate buffer (pH=7.0) for SDS-PAGE analysis. Take the strain containing only empty pET-22b(+) as the control, see the results figure 1 , the recombinant strain E.coli / Lcldh1 (ie E.coli BL21(DE3)pET-22b(+)-Lcldh1) had a protein band at 36.4kDa with the same size as the targe...

Embodiment 3

[0033] Example 3 Whole cells of recombinant bacteria catalyze phenylpyruvate to generate L-PLA

[0034] Add 300 μL of bacterial suspension (30 mg of wet bacteria) and 250 μL of sodium phosphate buffer (pH 7.0) to a 1 mL centrifuge tube, incubate at 37°C for 5 min, then add 50 μL of glucose (final concentration 50 mmol / L) and 400 μL of phenylpyruvate ( Final concentration 10mmol / L), after reacting for 1h, take 200μL reaction solution and add 800μL methanol to terminate the reaction, pass through 0.22μm organic filter membrane, and carry out HPLC analysis; another 200μL reaction solution is extracted with 1mL ethyl acetate, and the upper organic phase is passed through After drying with anhydrous sodium sulfate, pass through a 0.22 μm organic filter membrane for enantioselective analysis. The results showed that after the system reacted for 40min, the concentration of L-PLA was 5.62mmol / L, and the productive rate was 56.2%, ee p >99%, after 80min of reaction, phenylpyruvate was...

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Abstract

The invention discloses L-lactic dehydrogenase derived from Lactobacillus casei and application of L-lactic dehydrogenase, and belongs to the technical field of bioengineering. The invention provides the L-lactic dehydrogenase, and realizes heterologous expression of the gene of the L-lactic dehydrogenase. A recombinant plasmid pET-22b(+)-Lcldh1 containing the gene is constructed, and is converted into escherichia coli to obtain recombinant escherichia coli E.coli / Lcldh1 containing the expression plasmid pET-22b(+)-Lcldh1. By taking 10mmol / L of phenylpyruvic acid as a substrate, whole-cell catalysis is performed on recombinant LcLDH1 engineering bacteria, and reaction lasts for 1h to obtain L-PLA (D-phenyllactic acid), wherein concentration of L-PLA is 8.59mmol / L; the yield is 85.9 percent, and eep is greater than 99 percent. Purified LcLDH1 reaches 294.2U / mg in specific activity; the L-lactic dehydrogenase shows relatively high catalytic activity and enantioselectivity, and has obvious application potentiality and economic value.

Description

technical field [0001] The invention relates to L-lactate dehydrogenase derived from Lactobacillus casei and application thereof, belonging to the technical field of bioengineering. Background technique [0002] Phenyllactate acid (PLA), whose system name is 2-hydroxy-3-phenylpropionic acid, is a high-value organic acid with two enantiomers: L-PLA and D-PLA. The molecular formula of PLA is C 9 h 10 o 3 , the relative molecular mass is 166.17, its properties are stable, and its melting point is 121-125°C; PLA has good solubility in water, and it has good thermal stability in aqueous solution. Both L-PLA and D-PLA have a broad-spectrum antibacterial effect, and can be used as a natural antibacterial agent to replace chemically synthesized preservatives, and can be synthesized into a new polymer material of polyphenyllactic acid through polymerization reaction to replace polylactic acid polymer material. Therefore, phenyllactic acid has broad application prospects in the fi...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N1/21C12N15/70C12P7/42C12R1/19
CPCC12N9/0006C12N15/70C12P7/42C12Y101/01027
Inventor 邬敏辰李雪晴袁风娇刘艳李剑芳
Owner JIANGNAN UNIV
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