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Method for enzymatic synthesis of D-leucine

An enzymatic synthesis, leucine technology, applied in the field of enzyme catalysis, can solve the problems of cumbersome product extraction and purification steps, economic unfeasibility, long production cycle, etc., and achieve the effect of good industrial development and application prospects

Pending Publication Date: 2021-05-25
洛阳华荣生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The production cycle of the fermentation method is long and the extraction and purification steps of the fermented product are cumbersome. Whether it is a chemical resolution method, an enzymatic method or an induced crystallization method, leucine, which is relatively expensive, must be used as a raw material for chiral resolution, which is economically unfeasible. Row

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  • Method for enzymatic synthesis of D-leucine

Examples

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Effect test

Embodiment 1

[0045] Embodiment 1 Construction of wild-type D-amino acid dehydrogenase expression strain

[0046] For the wild-type D-amino acid dehydrogenase SEQ ID NO:1 derived from Ureibacillus thermosphaericus, codon optimization was performed, and the coding gene sequence SEQ ID NO:2 was synthesized from the whole gene, and restrictions were designed at both ends of the gene The endonuclease sites Nde I and XhoI were subcloned into the corresponding sites of the vector pET24a (Novagen) to obtain the recombinant plasmid pET24a-utDAADH. The recombinant plasmid pET24a-utDAADH was transformed into the expression host Escherichia coli BL21(DE3) to obtain the recombinant Escherichia coli utDAADH expressing the wild enzyme.

Embodiment 2

[0047] The construction of embodiment 2 glucose dehydrogenase expression bacterial strain

[0048] For the glucose dehydrogenase SEQ ID NO: 6 derived from Bacillus cereus, the whole gene synthesis coding gene sequence SEQ ID NO: 7, and restriction endonuclease sites Nde I and XhoI were designed at both ends of the gene , subcloned into the corresponding site of the vector pET24a (Novagen) to obtain the recombinant plasmid pET24a-bcGDH. The recombinant plasmid pET24a-bcGDH was transformed into the expression host Escherichia coli BL21(DE3) to obtain the recombinant Escherichia coli bcGDH expressing glucose dehydrogenase.

Embodiment 3

[0049] Embodiment 3 construction (D94A, R199V, H249T) mutant strain

[0050] According to the method reported in Example 3 of patent document CN202110108232.9, construct (D94A, R199V, H249T) mutant SEQ ID NO: 3 expression strains, including the following steps:

[0051] 3.1 Using the plasmid pET24a-utDAADH of the utDAADH strain as a template, aiming at the 94th, 199th, and 249th positions in wild-type D-amino acid dehydrogenation SEQ ID NO: 1, it was transformed into D94A, R199V, H249T, and according to the method in Example 1, a mutant expression strain utDAADH-M containing these three mutations was constructed. The primers used in the construction process are as follows: utDAADH-94F: 5'-CAACACCATCGACTCTTTC GCC ACCCACGCTCGTATC-3', utDAADH-199F: 5'-CTACCCGTGAAAAACACGCTGTTGAATGCTTCGTTGTTC-3', utDAADH-199R: 5'-GAACAACGAAGCATTCAACAGCGTGTTTTTCACGGGTAG-3', utDAADH-249R: 5'-GATAACGAAGCCGCCGGTGGGCATACCAGTG-3'.

[0052] Using the pET24a-utDAADH plasmid as a template and utDAADH-94F...

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Abstract

The invention discloses a method for enzymatic synthesis of D-leucine, alpha-ketoisocaproic acid is used as a substrate, D-amino acid dehydrogenase is used for catalyzing the substrate to carry out dehydrogenation reaction and amino transfer reaction, D-leucine is obtained, the ee value of the product exceeds 99%, and the method has wide industrial application prospects.

Description

technical field [0001] The invention belongs to the technical field of enzyme catalysis, and in particular relates to a method for enzymatically synthesizing D-leucine and a D-amino acid dehydrogenase mutant used in the method. Background technique [0002] D-leucine is an unnatural amino acid, also known as D-2-amino-4-methylpentanoic acid, with a CAS number of 328-38-1, and it is a white scaly powder. It is a branched-chain amino acid that can be used as a nutritional supplement to help muscles recover after training, control blood sugar, provide energy to body tissues, and enhance immunity. D-leucine is also widely used in the fields of medicine and chemical industry. [0003] D-leucine can be obtained by fermentation (CN110330441A); or obtained by chiral resolution of DL-leucine racemate (CN103981248A). Both methods have their own advantages and disadvantages, and their common feature is higher cost . The production cycle of the fermentation method is long and the ext...

Claims

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Application Information

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IPC IPC(8): C12P13/06C12N9/04C12N9/06C12N15/53
CPCC12P13/06C12N9/0014C12N9/0006C12Y104/99001C12Y101/9901
Inventor 范文超高书良丁鹏
Owner 洛阳华荣生物技术有限公司
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