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Leucine dehydrogenase mutant and application thereof

A technology for leucine dehydrogenase and mutants, which is applied to leucine dehydrogenase mutants and their application fields, can solve the problems of high downstream purification cost, difficult engineering amplification, low enzyme activity and the like

Active Publication Date: 2019-05-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the enzyme conversion process often faces problems such as low enzyme activity, low product concentration, high downstream purification costs, and difficulty in engineering scale-up.

Method used

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  • Leucine dehydrogenase mutant and application thereof
  • Leucine dehydrogenase mutant and application thereof
  • Leucine dehydrogenase mutant and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Embodiment 1: Construction of engineering strains

[0026] Bacillus thuringiensis (the strain Bacillus thuringiensis serovarkurstaki YBT-1520 preserved in our laboratory) was inoculated in LB medium, cultured at 37°C for 12 hours, collected by centrifugation, and the genomic DNA of Bacillus thuringiensis was extracted using a bacterial genome extraction kit. Using primers BtLeuDH-1 and BtLeuDH-2 to clone Bacillus thuringiensis genomic DNA as a template to obtain Bacillus thuringiensis leucine dehydrogenase gene BtLeuDH, its amino acid sequence is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID Shown in NO.2.

[0027] The BtLeuDH gene and expression vector pET28a were digested with restriction endonucleases BamHI and XhoI at 37°C for 4 hours, and then ligated with T4 ligase; the constructed recombinant plasmid pET28a-BtLDH was introduced into E.coli BL21(DE3), and contained Kanamycin (Kan) was cultured overnight on LB plates to obtain engineering ba...

Embodiment 2

[0030] Example 2: Leucine dehydrogenase protein engineering

[0031] (1) Acquisition of mutant library

[0032] Error-prone PCR amplification of BtLeuDH gene fragments, error-prone PCR reaction system: 50 μL system containing 1×Taq DNA polymerase buffer, 0.2mmol / L dATP and dGTP, 1mmol / L dCTP and dTTP, 2-5mmol / L Mg 2+ , 0.2~0.4mmol / L Mn 2+ , 2pmol / μL upstream and downstream primers (see primers BtLeuDH-1 and BtLeuDH-2 in Example 1), using the plasmid pET28a-BtLDH in Example 1 as a template. Error-prone PCR cycle conditions: 94°C for 1.5min, 60°C for 1min, 72°C for 1min, 29 cycles; 72°C for 10min. Among them, by adjusting Mg 2+ and Mn 2+ Libraries with different mutation frequencies can be obtained. The purified mutant BtLeuDH gene fragment was double-digested with BamHI and XhoI, and connected with the same double-digested plasmid pET28a to obtain a mutant recombinant plasmid. The recombinant plasmid containing the mutant gene was transformed into E.coli BL21(DE3), and th...

Embodiment 3

[0036] Embodiment 3: Enzyme activity determination of leucine dehydrogenase mutant BtLDH007

[0037] Seed medium formula: LB medium, yeast powder 5g / L, tryptone 10g / L, NaCl 10g / L.

[0038] Fermentation medium formula: glycerin 8g / L, yeast powder 8g / L, soybean peptone 10g / L, K 2 HPO 4 12H 2 O6.0g / L, KH 2 PO 4 10.0g / L.

[0039] Components of feed medium: glycerol 500g / L, MgSO 4 ·7H 2 O 10g / L.

[0040] Coenzyme regeneration system: Since leucine dehydrogenase catalyzes the synthesis of L-2-aminobutyric acid from 2-ketobutyric acid, coenzyme NADH is required, and the coenzyme is expensive, so the coenzyme NADH regeneration system is coupled to increase the number of cycles of NADH , thereby saving costs and improving conversion efficiency. The system consists of: ammonium formate concentration is 20g / L, NAD + The concentration is 0.6g / L, the enzyme activity of formate dehydrogenase is 1000U / L, with ammonium formate as substrate, NAD + Convert to NADH to realize coenzym...

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Abstract

The invention discloses a leucine dehydrogenase mutant and application thereof and belongs to the technical field of biological engineering. On the basis of the amino acid sequence, shown in SEQ ID NO.1, of the leucine dehydrogenase, an amino acid residue M at the 47th site is mutated into V, an amino acid residue N at the 109th site is mutated into I, and correspondingly the leucine dehydrogenasemutant with mutational sites M47V and N109I is obtained, wherein the amino acid sequence of the leucine dehydrogenase mutant is shown in SEQ ID NO.3, and the leucine dehydrogenase enzyme activity ofa unit strain of recombinant bacteria E.coli BL21-pET28a-BtLDH007 expressing the mutant reaches 170.9 U / g. 2-keto-butyric acid is adopted as a substrate for producing L-2-aminobutyric acid, wherein the highest yield of the L-2-aminobutyric acid reaches 77.6 g / L, and the conversion rate is 96.0%.

Description

technical field [0001] The invention relates to a leucine dehydrogenase mutant and application thereof, belonging to the technical field of bioengineering. Background technique [0002] L-2-aminobutyric acid is a non-natural chiral amino acid with the molecular formula C 4 h 9 NO 2 . L-2-aminobutyric acid has the functions of inhibiting the transmission of human nerve information, enhancing the activity of glucose phosphatase and promoting the metabolism of brain cells. At the same time, L-2-aminobutyric acid is also an important chemical raw material and pharmaceutical intermediate, which has been widely used in the synthesis of drugs, such as the synthesis of anti-tuberculosis drug ethambutol hydrochloride and anti-epileptic drug levetiracetam. [0003] At present, the synthesis methods of L-2-aminobutyric acid include chemical methods and biological methods. The reaction conditions of the chemical method are harsh, easy to generate by-products, and the cost is high, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/70C12N1/21C12P13/04C12R1/19
Inventor 刘立明张君轩付妍刘佳宋伟张灿
Owner JIANGNAN UNIV
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