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L-malate dehydrogenase mutant and application thereof

A malate dehydrogenase and mutant technology, applied in the field of enzyme catalysis, can solve the problems of complex culture conditions, inability to meet market demands, and high cost of industrialized production

Pending Publication Date: 2021-07-02
洛阳华荣生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation of D-malic acid by biological method is mainly through the microbial self-induced expression of L-malate dehydrogenase to prepare D-malic acid from maleic acid; in addition, see CN110747190A, there is also E. The whole cell catalyzes the preparation of D-malic acid, and the product concentration can reach more than 200g / L. The microorganisms that express L-malate dehydrogenase through their own induction are complicated in culture conditions, and the growth concentration of microorganisms is not high. At the same time, the product will contain a small amount of L-malic acid, DL-malic acid, malic acid dimer and other difficult-to-separate substances are difficult to purify, resulting in high industrial production costs and unable to meet market demand

Method used

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  • L-malate dehydrogenase mutant and application thereof
  • L-malate dehydrogenase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Directed mutation of wild-type L-malate dehydrogenase

[0053] The molecular biology experiments in the examples include plasmid construction, enzyme digestion, connection, competent cell preparation, transformation, medium preparation, etc., mainly referring to "Molecular Cloning Experiment Guide" (third edition), J. Sambrook, Edited by D.W. Russell (US), translated by Huang Peitang et al., Science Press, Beijing, 2002). The specific experimental conditions can be determined by simple experiments if necessary.

[0054] PCR amplification experiments were carried out according to the reaction conditions or kit instructions provided by the plasmid or DNA template suppliers. It can be adjusted by simple experiment if necessary.

[0055] Using the sequence of wild-type L-malate dehydrogenase ffMDH (Genbank: AB161423) as a template, E. coli codon optimization was performed, and the optimized nucleotide sequence was SEQ ID NO:2. Perform whole gene synthesis. The...

Embodiment 2

[0060] Example 2 High-throughput screening of highly active mutant library

[0061] Pick single-clonal transformants on each LB plate obtained in Example 1, inoculate into 500 μL of 96-well deep-well culture plate containing 50 μg / mL kanamycin LB liquid medium, cultivate overnight, and then take 80 μl of overnight culture , transferred to 800μl LB liquid medium containing 50μg / mL kanamycin, cultured at 37°C for 3h, added a final concentration of 0.5mM IPTG, cooled to 30°C, continued to cultivate for 16h, centrifuged at 4000rpm for 5min, used for sedimentation Add 150 ul of 50 mM Glycine-NaOH (pH 7.0) buffer with a final concentration of 1 mg / ml lysozyme to suspend, and place at 30° C. for 30 min. After the cells were lysed, add L-malic acid at a final concentration of 10mM, 2mM NAD + , Judging the size of the activity by measuring the change of the absorbance value at 340nm.

[0062] Enzyme activity assay method:

[0063] Enzyme activity (U / mg) = ΔOD340×Vt×df / (ε×b×Vs×c)

...

Embodiment 3

[0068] Embodiment 3 malate dehydrogenase mutant and catalytic performance identification

[0069] From the mutant transformant obtained in embodiment 2, select 5 high-energy bacterial strains detected by 96-orifice plate, and express bacterial strain (transferring into the transformant of plasmid pET28a-ffMDH) with wild-type L-malate dehydrogenase ffMDH As a control strain, culture in LB shake flasks was carried out according to the method for preparing bacterial cells on a 96-well plate described in Example 2. Collect bacterial cells for enzyme catalytic performance identification. The enzyme activity parameter determination method is the same as above. Before the enzyme activity measurement, the enzyme solution was incubated at 30°C for 2 hours; at the same time, the plasmid was extracted (Axygen small amount plasmid preparation kit) and sent to Nanjing Jinweizhi Biotechnology Co., Ltd. for sequencing Validation of ffMDH mutation targets. The enzyme activity test and sequen...

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Abstract

The invention discloses an L-malate dehydrogenase mutant, wherein the amino acid sequence of the L-malate dehydrogenase mutant is SEQ ID NO: 3 or SEQ ID NO: 4, and compared with wild type L-malate dehydrogenase, the L-malate dehydrogenase mutant has the advantage that the enzyme activity is obviously improved. The L-malate dehydrogenase mutant can be used for producing D-malic acid and oxaloacetic acid with high optical purity through enzymatic resolution of DL-malic acid.

Description

technical field [0001] The invention belongs to the technical field of enzyme catalysis, and in particular relates to an L-malate dehydrogenase mutant and its application for producing D-malate and oxaloacetate. Background technique [0002] Malic acid, also known as 2-hydroxysuccinic acid, has two stereoisomers, L-malic acid and D-malic acid, because there is an asymmetric carbon atom in the molecule. Naturally occurring malic acid is L-type, which exists in almost all fruits, most of which are pome fruits, and can also be produced from fumaric acid through biological fermentation. It is an important intermediate product of the internal circulation of the human body and is easily absorbed by the human body. Therefore, it is widely used as a food additive and functional food with excellent performance in the fields of food, cosmetics, medical and health products. DL-malic acid can be prepared from fumaric acid or maleic acid under the action of a catalyst under high tempera...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P7/46C12R1/19
CPCC12N9/0006C12N15/70C12Y101/01037C12P7/46
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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