Coding gene of cis-form epoxy succinic acid hydrolase, polypeptides coded thereby and related applications

A technology for epoxysuccinic acid and encoding gene, which is applied in the field of preparing L-tartaric acid or its salt, and can solve the problems of low expression level, low production efficiency of L(+)-tartaric acid and the like

Active Publication Date: 2014-03-05
HANGZHOU BIOKING BIOCHEM ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The main purpose of the present invention is to overcome the existing cis-epoxysuccinic acid hydrolase expression level is not high, resulting in the defects of low production efficiency of L (+)-tartaric acid, and provide a new...

Method used

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  • Coding gene of cis-form epoxy succinic acid hydrolase, polypeptides coded thereby and related applications
  • Coding gene of cis-form epoxy succinic acid hydrolase, polypeptides coded thereby and related applications
  • Coding gene of cis-form epoxy succinic acid hydrolase, polypeptides coded thereby and related applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1: Separation and purification of cis-epoxysuccinate hydrolase

[0046] Klebsiella sp.BK-58 (Klebsiella sp.BK-58) is stored in LB slant medium, the composition of which is: 1% peptone, 1% sodium chloride, 0.5% yeast extract, 1.5% agar , pH7.0.

[0047] 1: Determination of cis-epoxysuccinate hydrolase activity

[0048] Take 1.0 g of wet cells, wash twice with normal saline, suspend the wet cells with 10 mL of 1 mol / L sodium cis-epoxysuccinate (pH 8.0), react at 37°C for 1 h, and measure the content of tartaric acid in the reaction solution.

[0049] The detection method of tartaric acid content in the reaction solution is as follows: take 2.5mL of 1% ammonium metavanadate solution, place it in a 25mL volumetric flask, add an appropriate amount of the above reaction solution, add 1mL of 1mol / L sulfuric acid, and use distilled water to determine the content of tartaric acid. Make up to 25ml, take a part after mixing and measure the absorbance value at 480nm wi...

Embodiment 2

[0064] Example 2: Sequencing of the N-terminal and C-terminal ends of cis-epoxysuccinate hydrolase

[0065]The N-terminal amino acid sequence and C-terminal amino acid sequence of cis-epoxysuccinate hydrolase are detected by common methods, and the method is as follows: the cis-epoxysuccinate hydrolase obtained after the above separation and purification is subjected to SDS-polyacrylamide After gel electrophoresis, transfer to PVDF membrane by Western blotting. The membrane was stained with Coomassie Brilliant Blue staining solution, the band corresponding to cis-epoxysuccinate hydrolase was cut and recovered, and then the 10 amino acid sequences at the N-terminal were measured with a protein sequencer ABI PROCISETM494cLC, and the protein sequencer ABI PROCISETM491cLC was used to measure Measure its C-terminal 10 amino acid sequence. Sequencing results showed that the N-terminal 10 amino acid sequence of the cis-epoxysuccinate hydrolase is MKFSGASLFA (SEQ ID NO: 3), and the C...

Embodiment 3

[0066] Example 3: Design of degenerate primers to clone cis-epoxysuccinate hydrolase gene

[0067] According to the N-terminal sequence (SEQ ID NO:3), C-terminal sequence (SEQ ID NO:4) and the stop codon sequence (TAA / TGA / TAG) of the above-mentioned cis-epoxysuccinate hydrolase Design two demerge primers as follows:

[0068] Primer 1: 5'-ATGAARTTYWSNGGNGCNWSNYTNTTYGCN-3' (SEQ ID NO: 5);

[0069] Primer 2: 5'-YYANGCNCCNARCATNCCNCCNARYTCNACNAC-3' (SEQ ID NO: 6);

[0070] Among them, R: A / G, Y: C / T, W: A / T, S: G / C, V: A / G / C, N: A / G / C / T.

[0071] The steps for cloning the cis-epoxysuccinate hydrolase gene with degenerate primers are as follows:

[0072] (1) Use liquid LB medium to culture Klebsiella BK-58 at 200rpm on a shaker at 30°C, collect cells after 12h, and extract according to the method described in AxyPrep Bacterial Genomic DNA MiniPrep Kit (AXYGEN) Genome of Klebsiella BK-58.

[0073] (2) Using the above-mentioned genome as a template and primer 1 and primer 2 as p...

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Abstract

The invention relates to a coding gene of cis-form epoxy succinic acid hydrolase, polypeptides coded thereby and related applications. The coding gene of cis-form epoxy succinic acid hydrolase has a nucleotide sequence shown in SEQ ID NO:1. The cis-form epoxy succinic acid hydrolase coded by the gene has an amino acid sequence shown in SEQ ID NO:2. Colibacillus is converted by utilization of prokaryotic expression vectors constructed by the cis-form epoxy succinic acid hydrolase gene and genetically engineered bacteria are constructed. After being induced, the genetically engineered bacteria can convert cis-form epoxy succinic acid or salts thereof into L(+)-tartaric acid or salts thereof efficiently. The cis-form epoxy succinic acid hydrolase obtained from the colibacillus has the capability of converting cis-form epoxy succinic acid or salts thereof into L(+)-tartaric acid or salts thereof efficiently.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering, and provides a novel cis-epoxysuccinate hydrolase encoding gene and its encoded polypeptide, and further provides a polynucleotide for encoding the polypeptide, for expressing the Polypeptide recombinant expression vector, recombinant microbial strain and method for preparing L(+)-tartaric acid or its salt. Background technique [0002] L(+)-tartaric acid [(2R,3R)-2,3-dihydroxybutane-1,4 dihydroxy acid] is an organic acid with natural structure, which is found in certain plant fruits such as grapes and tamarind fruits It has high content and is widely used in food, medicine, construction, chemical industry, textile, electroplating and other industries, and its demand is increasing year by year. In the past, the main method of producing L(+)-tartaric acid was to extract it from tartar, a by-product of wine. At present, the cis-epoxysuccinate hydrolase secreted and expressed by microo...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/14C12N15/63C12N1/21C12N1/15C12N1/19C12P7/46
CPCC12N9/14C12P7/46C12Y303/02
Inventor 张建国谢志鹏程永青潘海峰鲍文娜
Owner HANGZHOU BIOKING BIOCHEM ENG
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