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A kind of maleate hydratase mutant and its application

A technology of acid hydratase mutation and mutant, applied in the field of enzyme catalysis, can solve the problem of low catalytic efficiency and achieve the effect of improving enzyme activity

Active Publication Date: 2021-03-09
ZHEJIANG HUARUI BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Studies have found that some fumarate hydratases (fumC) can also catalyze the hydration reaction of maleic acid to produce D-malic acid. Inspired by this, we screened and compared fumarate hydratases from various sources and found that The fumarate hydratase fumC derived from the photosynthetic bacterium Rhodopseudomonas capsulata has the above-mentioned catalytic function, but the catalytic efficiency is not high, which is judged to be caused by low enzyme activity. Therefore, it is necessary to modify the enzyme in order to Mutants with high enzymatic activity

Method used

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  • A kind of maleate hydratase mutant and its application
  • A kind of maleate hydratase mutant and its application
  • A kind of maleate hydratase mutant and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] The construction of embodiment 1 wild-type maleate hydratase expression strain

[0054] 1.1 For the fumarate hydratase fumC derived from Rhodopseudomonas capsulata, according to the reported amino acid sequence (UniProtKB-A0A507ZEG8), through the codon frequency optimization of Escherichia coli, Suzhou Jinweizhi Biotechnology Co., Ltd. was entrusted to synthesize the whole gene sequence. Sequence SEQ ID NO: 1. Subcloning into pSH plasmid (Zhejiang Huarui Biotechnology Co., Ltd.) to obtain the expression vector pSH-fumC expressing the wild-type enzyme SEQ ID NO:1.

[0055] Forward primer FumC-F: 5'- CATATG ACCGCGACCCGCACCGA-3',

[0056] Reverse primer FumC-R: 5'- CTCGAG TTAGCCTTTTGGGCTGATCA-3'.

[0057] PCR amplifies about 1.4kb fragment, PCR reaction system includes: forward primer, reverse primer 0.3μM each, template 50ng, 1xKOD Neo plus buffer, 0.2mM dNTP, 1.5mM MgSO 4 , KOD neo plus 1U, add double distilled water to make the total system 50μl. PCR conditions: ...

Embodiment 2

[0060] Example 2 Construction of fumC random mutation point library and screening by error-prone PCR method

[0061] 2.1 Construction of fumC random mutation point library by error-prone PCR method

[0062] Using the sequence SEQ ID NO:1 as a template, an error-prone PCR technique was used to construct a random mutant library. The forward primer fumC-Nde1-F is 5’-ATGACCGCGACCCGCACCGA-3’, and the reverse primer fumC-Xho1-R is 5’- CTCGAG TTAGCCTTTTGGGCTGATCA-3'.

[0063] 50μL error-prone PCR reaction system includes: 50ng plasmid template pSH-fumC, 30pmol pair of primers fumC-Nde1-F and fumC-Xho1-R, 1X Taq buffer, 0.2mM dGTP, 0.2mM dATP, 1mM dCTP, 1mM dTTP, 7mMMgCl 2 , (0mM, 0.05mM, 0.1mM, 0.15mM, 0.2mM) MnCl 2 , 2.5 units of Taq enzyme (Fermentas).

[0064]The PCR reaction conditions were: 95°C for 5 minutes; 94°C for 30s, 55°C for 30s, 72°C for 2min / kbp; 30 cycles; 72°C for 10min.

[0065] The 2.0kb random mutation fragment was recovered from the gel as a large primer, a...

Embodiment 3

[0079] Embodiment 3 Mutant bacterial strain construction

[0080] We focused on the mutant fumC-1480 to investigate its ability to catalyze the conversion of maleic acid to D-malic acid through a one-step hydration reaction. To this end, the fumC-1480 mutant gene SEQ ID NO:4 was cloned into the pSH plasmid to obtain the expression vector pSH-fumC-1480 expressing the maleate hydratase mutant SEQ ID NO:3.

[0081] Using the plasmid pSH-fumC-1480, it can be constructed into different expression systems, such as Escherichia coli, Pichia pastoris, Bacillus subtilis, etc. For example, transform the plasmid pSH-fumC-1480 into BL21(DE3) competent cells, spread on kan+LB plates, culture at 37°C overnight, pick 10 single colonies, inoculate them into test tubes containing LB liquid medium, and culture at 37°C Overnight, the bacteria were collected by centrifugation, the plasmid was extracted, and the gene sequencing confirmed that the mutation was correct, and the engineering bacteria ...

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Abstract

The invention discloses a maleate hydratase mutant with an amino acid sequence shown in SEQ ID NO: 3. Compared with wild maleate hydratase, the maleate hydratase mutant has the advantage that the enzyme activity of catalyzing one-step hydration reaction of maleic acid to generate D-malic acid is remarkably improved. When whole cells expressing the maleate hydratase mutant are adopted to catalyze reaction of a 100g / L maleic acid substrate to generate D-malic acid, the optical purity of the product exceeds 99%, and the maleate hydratase has industrial development and application prospects.

Description

technical field [0001] The invention belongs to the technical field of enzyme catalysis, and in particular relates to a mutant of maleic acid hydratase and its use in enzymatically catalyzing the hydration reaction of maleic acid to produce D-malic acid. Background technique [0002] D-malic acid, CAS 636-61-3, molecular weight 134, also known as 2-hydroxysuccinic acid, is a rare organic acid in nature, which is widely used in the pharmaceutical industry. D-malic acid is used as a chiral precursor The body is mainly used in the synthesis of chiral drugs, and can be used as a raw material for the synthesis of antibiotics, antiviral drugs, antihistamines, etc. The synthesis method of D-malic acid can generally be synthesized by asymmetric synthesis method, racemate physical resolution method and biological method. Existing biological methods are mainly natural microorganism splitting method and whole cell transformation method. Both methods have problems such as difficult hig...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P7/46C12R1/19
CPCC12N9/88C12N15/70C12P7/46C12Y402/01002
Inventor 范文超高书良王金刚袁圣伦
Owner ZHEJIANG HUARUI BIOTECHNOLOGY CO LTD
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