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A d-lactate dehydrogenase mutant and its application

A technology of lactate dehydrogenase and phenyllactate, applied in the field of D-lactate dehydrogenase mutants, can solve the problems of complicated separation and purification, etc.

Active Publication Date: 2019-03-12
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a modified D -Method for efficient conversion of phenylpyruvate to D-phenyllactic acid by lactate dehydrogenase

Method used

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  • A d-lactate dehydrogenase mutant and its application
  • A d-lactate dehydrogenase mutant and its application
  • A d-lactate dehydrogenase mutant and its application

Examples

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Embodiment 1

[0037] Embodiment 1, the preparation of D-lactate dehydrogenase mutant

[0038] According to the simulated three-dimensional structure of the DLDH744 protein containing substrates PPA and NADH, the glutamine (Q) close to the C3 end of the PPA substrate was mutated into smaller hydrophobic amino acids alanine (A), valine acid (V), leucine (L), tyrosine (Y) at position 101 is mutated to phenylalanine (F), phenylalanine (F) at position 298 is mutated to tyrosine (Y ), methionine (M) at position 307 was mutated into leucine (L), and D-lactate dehydrogenase mutants Q51V, Q51L, Q51A, Y101F, F298Y, and M307L mutants were constructed.

[0039] The amino acid sequence of the wild-type D-lactate dehydrogenase is sequence 2, and the nucleotide sequence of its coding gene is sequence 1.

[0040] The amino acid sequence of D-lactate dehydrogenase mutant Q51V is the sequence obtained by mutating the 51st Gln of sequence 2 to Val; the nucleotide sequence of the coding gene of D-lactate dehy...

Embodiment 2

[0064] Embodiment 2, the enzyme activity assay of D-lactate dehydrogenase mutant

[0065] Determination of the enzyme activity of D-lactate dehydrogenase mutants In view of the fact that the coenzyme NADH has a maximum absorption peak at 340nm, the enzyme activity is defined by the decrease speed of the NADH340nm absorbance value, as follows:

[0066] Phenylpyruvate solution: use phenylpyruvate and 100mmol / L phosphate buffer (recipe: weigh 79g NaCl, 2gKCl, 2.4g KH 2 PO 4 and 1.8g K 2 HPO 4 , dissolved in 800ml of distilled water, adjusted the pH value of the solution to 7.4 with HCl, and finally added distilled water to make up to 1L. Store in a refrigerator at 4°C. ) to mix, the solution obtained, and the concentration of phenylpyruvate is 200mM;

[0067] NADH solution: mix NADH and 100mmol / L phosphate buffer to obtain a solution, and the concentration of NADH is NADH.

[0068] 200 μl reaction system: 10 μL phenylpyruvate solution, 10 μL NADH solution and appropriate en...

Embodiment 3

[0078] Example 3, D-lactate dehydrogenase mutant M307L whole cell transformation reaction optimization

[0079] Cultivate the recombinant bacteria JM109(DE3) / M307L to the exponential phase, transfer to fresh LB liquid medium with 1% inoculum, the medium contains kanamycin at a concentration of 40mg / L, cultivate until the OD600nm reaches about 0.6, add the final IPTG with a concentration of 1mM was used to induce culture at 16°C for 16h. 4°C, centrifuge at 5000r / min for 15min to collect the bacteria, wash the bacteria three times with pH 7.0, 50mM phosphate buffer, and resuspend in different pH (5.5-8.5) of phosphate buffer (recipe: weigh 79g NaCl, 2g KCl, 2.4g KH 2 PO 4 and 1.8g K 2 HPO 4 , dissolved in 800ml of distilled water, adjusted the pH value of the solution to 5.5-8.5 with HCl, and finally added distilled water to make up to 1L. ), obtain pH value 5.5 bacterial cell suspension, pH value 6 bacterial cell suspension, pH value 6.5 bacterial cell suspension, pH value...

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Abstract

The invention discloses a D-lactic dehydrogenase mutant and application thereof. Protein is protein shown in the sequence A or is protein which has the same function, is derived from the sequence A and is formed by substituting, and / or deleting and / or adding one or more amino acid residues to the protein shown in the sequence A; the sequence A is a sequence obtained by mutating Met at the <307>th bit into Leu in the sequence 2. It is proved through experiments that D-LDH 744 form Sporolactobacillus inulinus is selected and mutated to obtain the D-lactic dehydrogenase mutant M307L. The mutant has high D-lactic dehydrogenase activity, and compared with D-lactic dehydrogenase without mutation, and enzyme activity of D-LDH with PPA (phenylpropanolamine hydrochloride) as a substrate is improved.

Description

technical field [0001] The present invention relates to the fields of molecular biology and bioengineering preparation, in particular to a D-lactate dehydrogenase mutant and its application, in particular to the transformation of a D-lactate dehydrogenase and the efficient conversion of phenylpyruvate by resting cell method Method for synthesizing D-phenyllactic acid. Background technique [0002] Phenyllactic acid is a small molecular compound, and its second carbon atom is a chiral carbon, so there are two enantiomers, namely D-phenyllactic acid and L-phenyllactic acid. Phenyllactic acid has been shown to inhibit Listeria in culture media, milk and cheese, as well as some Gram-positive and Gram-negative bacteria such as E. coli. Phenyllactic acid is considered to be the third generation of new antibacterial substances produced by lactic acid bacteria (Lactobacillus) and can be used in food systems. It is generally believed that D-phenyllactic acid plays a major role in an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/53C12P7/42
CPCC12N9/0006C12P7/42C12Y101/01027
Inventor 陈晶瑜彭星月韩北忠
Owner CHINA AGRI UNIV
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