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Method for producing D(-)-tartaric acid or salt thereof by using gene engineering bacteria

A technology of tartaric acid and amino acids, applied in genetic engineering, plant genetic improvement, microorganism-based methods, etc., can solve the problems of low production efficiency of D(-)-tartaric acid, low expression of ESH enzyme, and complexity.

Active Publication Date: 2009-07-15
HANGZHOU BIOKING BIOCHEM ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complex enzyme system inherent in microorganisms, the expression level of ESH enzyme is not high, and its activity is also restricted by various extracellular factors, resulting in low production efficiency of D(-)-tartaric acid

Method used

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  • Method for producing D(-)-tartaric acid or salt thereof by using gene engineering bacteria
  • Method for producing D(-)-tartaric acid or salt thereof by using gene engineering bacteria
  • Method for producing D(-)-tartaric acid or salt thereof by using gene engineering bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Separation and purification of cis-epoxysuccinate hydrolase

[0062] 1. Separation and purification of cis-epoxysuccinate hydrolase activity

[0063] Bordetella sp.BK-52 (Bordetella sp.BK-52) is stored in BK-1 slant medium, and the medium formula is: 0.5% glucose, 0.5% peptone, 0.5% sodium chloride, 0.25% yeast extract, 2% agar, pH 7.0. Inoculate Bordetella BK-52 from the slant medium into the BK-2 seed medium, shake culture at 30°C for 24 hours, transfer to the BK-3 fermentation medium, and use cis-epoxysuccinate 30 ℃ shaking induction for 36h to produce cis-epoxysuccinate hydrolase. The formula of the BK-2 seed medium is: 0.2% yeast extract, 1% glucose, 0.2% ammonium sulfate, 0.1% dipotassium hydrogen phosphate trihydrate, 0.05% magnesium sulfate heptahydrate, 0.001% ferrous sulfate heptahydrate, pH7 .0. The formula of the BK-3 fermentation medium is: 1% sodium cis-epoxysuccinate, 0.2% yeast extract, 1% glucose, 0.2% ammonium sulfate, 0.1% dipotassium hy...

Embodiment 2

[0071] Example 2 Detection of N-terminal amino acid sequence of cis-epoxysuccinate hydrolase

[0072] The N-terminal amino acid sequence of cis-epoxysuccinate hydrolase is detected by a commonly used method, and the method is as follows: the cis-epoxysuccinate hydrolase obtained after the above separation and purification is run on sodium dodecyl sulfate polyacrylamide gel electrophoresis Afterwards, transfer to PVDF membrane after Western blot. The membrane was stained with Coomassie Brilliant Blue, and the band corresponding to cis-epoxysuccinate hydrolase was cut and recovered, and then its N-terminal sequence was determined with a protein sequencer (Applied Biosystems Procise 492 cLC). Sequencing results showed that its N-terminal amino acid sequence was MTRTKLILEARI (SEQ ID NO: 1).

Embodiment 3

[0073] Example 3 Acquisition of cis-epoxysuccinate hydrolase gene

[0074] The following primers were designed according to the N-terminal sequence of cis-epoxysuccinate hydrolase (SEQ ID NO: 1) and GenBank accession number E50984: 5'-ATGACNYGNACNAARXTN-3' (SEQ ID NO: 2) and 5' - CTAGTTGCTAATACCCAG-3' (SEQ ID NO: 3), wherein Y represents A or C, R represents A or G, X represents T or C, and N represents any one of the four bases A, C, G, T.

[0075] Bordetella BK-52 was cultured at 30°C overnight, the cells were collected by centrifugation, and the genomic DNA of Bordetella BK-52 was extracted with EZ-10 Column Genomic DNA Extraction Kit (Bio Basic Inc.). Using genomic DNA as a template and SEQ ID NO: 2 and SEQ ID NO: 3 as primers, a fragment of about 0.9 kb is obtained by a common PCR technique. The PCR program is: 94°C preheating for 5 minutes, 94°C for 50s, 40°C for 30s, 72°C for 1min, 30 cycles, and finally 72°C for 10min. The PCR product was recovered and purified with ...

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Abstract

The invention relates to a nucleotide sequence from microorganism for coding cis-epoxy succinate hydrase, a cloning vector containing the nucleotide sequence, a recombined host bacterium obtained by transforming the nucleotide sequence and a cis-epoxy succinate hydrase amino acid sequence coded by the recombined host bacterium, and further relates to a method of using the cis-epoxy succinate hydrase to hydrolyze cis-epoxy succinate or salts thereof to prepare D(-)-tartaric acid or salts thereof.

Description

technical field [0001] The present invention relates to an amino acid sequence of a cis-epoxysuccinate hydrolase, a nucleotide sequence encoding the enzyme, a recombinant microorganism expressing the enzyme, and using the enzyme and / or the recombinant microorganism A method for producing D(-)-tartaric acid or a salt thereof from oxysuccinic acid or a salt thereof. Background technique [0002] D(-)-tartaric acid, also known as (2S,3S)-2,3-dihydroxybutane-1,4-dicarboxylic acid, is the isomer of natural L(+)-tartaric acid, which is very common in nature. It rarely exists, and it is mainly used as a chiral source and resolution agent for chiral synthesis in the pharmaceutical industry. At present, the demand for D(-)-tartaric acid is increasing year by year, and there is an urgent need to increase production to meet the needs of domestic and foreign markets. [0003] So far, there are three main production methods for D(-)-tartaric acid: (1) the chemical resolution of DL-tart...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/14C12N15/55C12N15/63C12N1/15C12N1/21C12P7/46C12R1/01C12R1/645
Inventor 张建国谢志鹏鲍文娜潘海峰
Owner HANGZHOU BIOKING BIOCHEM ENG
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