Construction of leucine dehydrogenase mutants and application thereof

A technology of leucine dehydrogenase and mutants, which can be applied in the application, enzyme, oxidoreductase and other directions, can solve the problems such as the influence of catalytic efficiency, and achieve the effect of efficient preparation

Active Publication Date: 2018-09-21
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no research report on the modification of the substrate channel of leucine dehydrogenase,

Method used

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  • Construction of leucine dehydrogenase mutants and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Construction of Leucine Dehydrogenase Mutants

[0039] The pET-28a recombinant plasmid containing the leucine dehydrogenase gene derived from Bacillus cereus was used as a template.

[0040] The oligonucleotide fragment containing the mutation point is used as the upstream primer, and the oligonucleotide fragment near 2254 on the pET-28a plasmid is used as the downstream primer. The specific primers are as follows (bold and underlined are the mutation sites):

[0041] PF-T45M: 5'-CCGGCTCTTGGTGGA ATG AGAATGTGGACATAT-3'

[0042] PF-E116V: 5'-CGTTACATTACAGCT GTT GATGTTGGTACAACA-3'

[0043] PR-28a2254: 5'-GCCTTACTGGTTAGCAGAATG-3'

[0044] The mutant plasmid was constructed by two-step PCR method of the whole plasmid (Sanchis, J., Fernández, L., Carballeira, J.D., Drone, J., Gumulya, Y., & H., et al. Appl Microbiol Biotechnol, 2008, 81, 387-397.). PCR amplification system: template 0.5 μL, upstream and downstream primers 0.2 μL, dNTP Mix 2 μL, HS DNA Po...

Embodiment 2

[0046] Example 2: Induced expression of leucine dehydrogenase mutants

[0047] The leucine dehydrogenase mutant engineering bacteria constructed in Example 1 were inoculated into LB liquid medium containing 50 μg / mL kanamycin, cultured overnight at 37° C. and 160 r / min, and then transferred to 2 L of LB base. The inoculum size is 8%, the culture temperature is 37°C, the rotation speed is 300r / min, and the ventilation rate is 1.0vvm. After culturing for 2-3 hours, add IPTG with a final concentration of 0.5mM, and reduce the induction temperature to 28°C. After 16 hours of induction, collect the bacteria by centrifugation at 8,000 rpm at 4°C for 10 minutes, and store them in a -70°C refrigerator for later use.

Embodiment 3

[0048] Embodiment 3: Separation and purification of leucine dehydrogenase mutant

[0049] Take 0.5 g of the wet bacterial cells collected in Example 2, wash twice with 10 mL of 50 mM PB buffer solution of pH 7.5, resuspend in 10 mL of 50 mM PB buffer solution of pH 7.5, oscillate and shake, and then crush under ultrasonic waves. Break for 1s, stop for 3s, the total time is 15min. The cell lysate was centrifuged at 12,000 rpm for 20 min to remove cell debris, and the supernatant, namely the crude enzyme solution, was collected and filtered with a 0.22 μm filter membrane for subsequent separation and purification of the enzyme. The purification column is a Ni-NTA column with a packing volume of 5mL. First equilibrate the Ni-NTA column with loading equilibration buffer M20 (20mM sodium phosphate, 500mM NaCl and 20mM imidazole, pH 7.4), and load the column at a rate of 0.5mL / min. Sample crude enzyme solution, eluted with loading equilibration buffer M20 to remove unadsorbed prote...

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Abstract

The invention provides construction of leucine dehydrogenase mutants and application thereof and belongs to the field of genetic engineering. The invention provides three leucine dehydrogenase mutantsof SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8, and application of the mutants and genetically engineered bacteria producing the mutants in preparation of optical pure chiral L-alpha-amino acid throughammoniation reduction of alpha-ketoacid. The invention has the advantages that the L-alpha-amino acid prepared by ammoniation reduction from the leucine dehydrogenase mutants or the genetically engineered bacteria has high catalytic activity and stability, high-optical purity L-alpha-amino acids can be synthesized (ee is greater than 99%), the conversion rate of L-phenylglycine catalyzed by mutantenzymes is improved by 2.62 times, and a practical and effective strategy is provided for industrial production.

Description

technical field [0001] The invention relates to the construction and application of a leucine dehydrogenase mutant, which belongs to the field of genetic engineering. Background technique [0002] Optically pure L-α-amino acid is an important chemical and pharmaceutical raw material and has a broad application market. For example, L-α-aminobutyric acid can be used as the synthesis of anti-tuberculosis drug ethambutol hydrochloride and anti-epileptic drug levetiracetam. L-tert-leucine is the central amino acid in the drug structure of the tyrosine kinase JAK3 inhibitor developed by Roche, Switzerland. Leucine dehydrogenase (LeuDH, EC1.4.1.9) has been widely used in the preparation of L-α-amino acids (Krix, G., Bommarius, A.S., Drauz, K., Kottenhahn, M., [0003] Schwarm, M., Kula, M.R.. Journal of Biotechnology, 1997, 53, 29-39.). And some studies have carried out site-directed mutagenesis on leucine dehydrogenase to modify its properties, but they all focused on modifying ...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12N15/53C12P13/04
CPCC12N9/0016C12P13/04C12Y104/01009
Inventor 饶志明周俊平王雅玲陈佳杰杨套伟徐美娟张显
Owner JIANGNAN UNIV
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