Encoding gene of cis-epoxysuccinate hydrolase, its encoded polypeptide and related applications
A technology of epoxysuccinic acid and coding genes, which is applied in the field of preparing L-tartaric acid or its salts, and can solve the problems of low expression level and low production efficiency of L(+)-tartaric acid, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0045] Embodiment 1: Separation and purification of cis-epoxysuccinate hydrolase
[0046] Klebsiella sp. BK-58 (Klebsiellasp.BK-58) is stored in LB slant medium, the composition of the slant medium 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, put it in a 25mL volumetric flask, add an appropriate amount of the above reaction solution, add 1mL of 1mol / L sulfuric acid, and distilled water to volume to 25ml, get a part after mixing and measure the absorbance value at 480nm with a spectrophotometer, and calculate ...
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, and 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 ABIPROCISETM494cLC, and the sequence of 10 amino acids at the N-terminal was measured with a protein sequencer ABIPROCISETM491cLC. C-terminal 10 amino acid sequence. Sequencing results showed that the N-terminal 10 amino acid sequence of the cis-epoxysuccinate hydrolase is ...
Embodiment 3
[0066] Example 3: Design of degenerate primers to clone cis-epoxysuccinate hydrolase gene
[0067] According to the N-terminal sequence (SEQIDNO:3), C-terminal sequence (SEQIDNO:4) and the stop codon sequence (TAA / TGA / TAG) of the above-mentioned cis-epoxysuccinate hydrolase, two mergers are designed Primers are 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 12 hours, and extract Klebsiella according to the method described in AxyPrepBacterialGenomicDNAMiniPrepKit (AXYGEN) The genome of BK-58.
[0073] (2) Using the above-mentioned genome as a template and primer 1 and pr...
PUM
| Property | Measurement | Unit |
|---|---|---|
| molecular weight | aaaaa | aaaaa |
| molecular weight | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More