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D-lactic dehydrogenase mutant and application thereof

A technology of phenyllactic acid and amino acid, applied in the field of D-lactate dehydrogenase mutants, which can solve the problems of complicated separation and purification

Active Publication Date: 2016-05-18
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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  • D-lactic dehydrogenase mutant and application thereof
  • D-lactic dehydrogenase mutant and application thereof
  • D-lactic dehydrogenase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Preparation of D-lactate dehydrogenase mutant

[0038] According to the simulated three-dimensional structure of the DLDH744 protein containing the 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) and valine, respectively. Acid (V), leucine (L), tyrosine (Y) at position 101 was mutated to phenylalanine (F), and phenylalanine (F) at position 298 was mutated to tyrosine (Y) ), 307-position methionine (M) was mutated to leucine (L), and D-lactate dehydrogenase mutants Q51V, Q51L, Q51A, Y101F, F298Y, and M307L mutants were constructed.

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

[0040] The amino acid sequence of D-lactate dehydrogenase mutant Q51V is the sequence obtained by mutating Gln at position 51 of SEQ ID NO: 2 to Val; - Sequence obtained by mutating CA a...

Embodiment 2

[0064] Example 2. Determination of enzyme activity of D-lactate dehydrogenase mutants

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

[0066] Phenylpyruvic acid solution: use phenylpyruvic acid and 100mmol / L phosphate buffer (recipe: weigh 79gNaCl, 2gKCl, 2.4gKH 2 PO 4 and 1.8gK 2 HPO 4 , dissolve in 800ml of distilled water, adjust the pH of the solution to 7.4 with HCl, and finally add distilled water to dilute to 1L. Store in a 4°C refrigerator. ) mixing, the obtained solution, and the concentration of phenylpyruvate is 200mM;

[0067] NADH solution: mix with 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. Optimization of the whole-cell transformation reaction of D-lactate dehydrogenase mutant M307L

[0079] Cultivate the recombinant strain JM109(DE3) / M307L to the exponential phase, transfer it to fresh LB liquid medium with 1% inoculum, and the medium contains kanamycin with a concentration of 40mg / L, and cultivate until the OD600nm reaches about 0.6, and then add the final IPTG at a concentration of 1 mM was incubated at 16 °C for 16 h. 4 ℃, 5000r / min centrifugation for 15min to collect the bacteria, wash the bacteria three times with pH7.0, 50mM phosphate buffer, and resuspend in different pH (5.5 ~ 8.5) phosphate buffer (recipe: weigh 79gNaCl, 2gKCl ,2.4gKH 2 PO 4 and 1.8gK 2 HPO 4 , dissolve in 800ml of distilled water, adjust the pH of the solution to 5.5-8.5 with HCl, and finally add distilled water to dilute to 1L. ) in, obtain pH value 5.5 bacterial suspension, pH 6 bacterial suspension, pH 6.5 bacterial suspension, pH 7 bacterial suspension, pH 7...

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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 invention relates to the fields of molecular biology and bioengineering preparation, in particular to a D-lactate dehydrogenase mutant and application thereof, 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] Phenyllactate is a small molecule compound whose second carbon atom is a chiral carbon, so there are two enantiomers namely D-phenyllactic acid and L-phenyllactic acid. Phenyl lactic 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 Lactobacillus that can be used in food systems, and it is generally believed that D-phenyllactic acid plays a major role in bacteriostasis. In addition, ph...

Claims

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Application Information

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Patent Type & Authority Applications(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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