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Malate dehydrogenase mutant gene and construction method and application thereof

A technology of malate dehydrogenase and mutant genes, which is applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of long time-consuming, cumbersome process, and unfavorable splitting of D/L-malic acid, and achieve high-efficiency splitting Effects of Enzymes for Separation and Purification

Inactive Publication Date: 2019-02-05
ANHUI NORMAL UNIV
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Problems solved by technology

Screening wild-type malate dehydrogenase with high activity in the natural state is time-consuming and cumbersome, which is not conducive to obtaining highly active malate dehydrogenase for the resolution of D / L-malic acid and other synthesis Method for the purification of D-malic acid

Method used

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  • Malate dehydrogenase mutant gene and construction method and application thereof
  • Malate dehydrogenase mutant gene and construction method and application thereof
  • Malate dehydrogenase mutant gene and construction method and application thereof

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Embodiment Construction

[0031] In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be described in further detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0032] It should be noted that in the examples, WT: wild type; T1 indicates a mutant named according to the number of mutation sites.

[0033] 1. Selection of mutation sites

[0034] Select key sites for base mutation by comparing the similarity of the structural domains. In T1, amino acid residue P46 at position 46 in ScMDH was replaced with R (P46→R). As shown in Table 1:

[0035] Table 1 Mutation site selection of ScMDH

[0036]

[0037] 2. According to the gene sequence (NC_003888) of Streptomyces coelicolor A3(2) MDH in GenBank, two pairs of mdh mutation primers (P1 / P2, P3 / P4) were designed with Primer Premier 5.0 software. Mutation process see Figure 4 .

[0038] Two fragments of A (P1 / P2) and B (P3 / P4) we...

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Abstract

The invention discloses a malate dehydrogenase mutant gene and a construction method and application thereof, and belongs to the technical field of biological genetic engineering. The malate dehydrogenase mutant gene is characterized in that streptomyces coelicolor malate dehydrogenase is taken as a template, a gene sequence capable of expressing the malate dehydrogenase is constructed through genetic engineering measures such as primer design, stepwise PCR (polymerase chain reaction), fusion PCR, transformed bacteria resistance screening and nucleotide sequencing, and the constructed malate dehydrogenase is transferred into DE3 strains of escherichia coli to be expressed; the activity of the expressed malate dehydrogenase is two times or above that of malate dehydrogenase under wild-typesample expression, and more efficient resolution and purification enzymes are provided for production of D-malic acid.

Description

technical field [0001] The invention relates to the technical field of biological genetic engineering, in particular to a malate dehydrogenase mutant gene, a construction method and an application thereof. Background technique [0002] In the natural state, malic acid (2-hydroxysuccinic acid) exists in three forms because there is a chiral carbon atom in the molecule, namely D-malic acid, L-malic acid, and DL-malic acid. L-malic acid is one of the components of citric acid cycle metabolism in organisms. Because it is easier to be metabolized and absorbed by the human body, it is widely used in food, pharmaceutical, chemical and other fields. D-malic acid is a rare organic acid in nature. D-malic acid is often used as a chiral source in drugs because of its special chirality, and is mainly used in the fields of chiral drug synthesis, chiral additives, chiral auxiliaries, etc., such as antibiotics, drug pheromones, antiviral Synthesis of drugs, anticancer drugs, antihistamin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/04C12N15/70C12P7/46
CPCC12N9/0006C12N15/70C12P7/46C12Y101/01037
Inventor 葛亚东侯少林蒋璐璐苏凤智
Owner ANHUI NORMAL UNIV
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