A kind of Penicillium expanses cis-epoxysuccinate hydrolase gene and its application
A technology of epoxysuccinic acid and Penicillium expansica, applied in the field of Penicillium cis-epoxysuccinate hydrolase gene, which can solve complex, low production efficiency of L(+)-tartaric acid, hydrolysis of cis-epoxysuccinic acid Enzyme expression is not high
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Embodiment 1
[0027] Example 1: Separation and purification of hydrolase of Penicillus epoxy succinate
[0028] The invention uses strains to expand piaciosis. Penicillium Expansum ) WH-3, the strain is deposited in the General Micro Biological Center (CGMCC) of China Microbial Square Safety Management Committee, and the deposit registration number is CGMCC No.16798, named expanded pupil ( Penicillium Expansum ) WH-3, Major Day: December 03, 2018, Waitsu Address: No.1, Beichen West Road, Chaoyang District, Beijing (zip code: 100101).
[0029] Extending piaciacosis Penicillium Expansum WH-3 is stored in PDA bevel medium, the configuration method of the bevel medium is: weigh 200g peeled fresh potatoes, put potatoes into small pieces into the pot, add 1000ml to the water, heating to boiling, maintained for 30 min. Then use a double gauze, heat filter on the amount cup, leave the filtrate. 20 g of glucose and 15 to 20 g of agar were added, and the filtrate was supplemented to 1000 mL, and 20 min a...
Embodiment 2
[0047] Example 2: Sequencing the N-terminus and C-terminus of the expanded pyrolytic epoxy succinate hydrolase
[0048] Extending the Penic Cushion Epoxy Succinate Hydrozyst N-terminal amino acid sequence and C-terminal amino acid sequence detects by conventional methods, the method is as follows: SDS is carried out by the above-mentioned cryptogenic hydrolase hydrolase. After the polyacrylamide gel is electrophoresed, transferred to the PVDF film by Western hybridization. The membrane was stained with a tab or blue staining, cutting the strip of the ciscloated succinate hydrolase and recovered, and then 10 amino acid sequences of the N-terminus and the C-terminus were performed with the protein sequencer. The sequencing results showed that the N-terminal 10 amino acid sequence of the cisclophydroxy succinate hydrolase is MsrDEPSML (SEQ ID NO: 3), and the C-terminal 10 amino acid sequence is Aryfgiesel (SEQ ID NO: 4).
Embodiment 3
[0049] Example 3: Design a simple primer, cloning of Popular WH-3 ciscloated epoxy succinate hydrolase gene
[0050] According to the N-terminal sequence (SEQ ID NO: 3), a C-terminal sequence (SEQ ID NO: 4), a C-terminal sequence (SEQ ID NO: 4), and a TAA / TGA, and a termination codon sequence (TAA / TGA), C-terminal sequence (SEQ ID NO: 4), and a TAA / TGA. / TAG) Design two simple primers are as follows:
[0051] Primers 1: 5'-atgwsnxgngaygarccccc-3 ';
[0052] Primers 2: 5'-yyanayytcnysytcnatncc -3;
[0053] Among them, R: A / G, Y: C / T, W: A / T, S: g / c, x: A / C, N: A / g / c / t.
[0054] The steps of cloning the epoxy succinate hydrolase gene with a degenerate primer were as follows:
[0055] (1) Inoculation of spore fluid in the self-made PDB medium, 25 ° C 150 rpm oscillated culture 48 h, collecting pellets, and expands Popular WH in accordance with the method described in EZUP Column Fungi Genomic Dna Purification Kit (Sangon Biotech). -3 genome.
[0056] (2) The PC...
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