Lipase derived from pseudomonas and application of lipase
A technology of Pseudomonas and lipase, which is applied in the field of lipase derived from Pseudomonas and its application, can solve the problems of not meeting the high optical purity requirements of drugs, low optical purity, etc., and achieve the improvement of ee value and high optical purity. The effect of purity
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Embodiment 1
[0024] Example 1: Cloning of wild-type lipase derived from Pseudomonas
[0025] Design a pair of primers psu-F: 5'-CATATGGTTCAGATTCAGGG-3';
[0026] psu-R:5'-CTCGAGTACAGACATGTACC-3';
[0027] The polymerase chain reaction (PCR reaction) was carried out with Pseudomonas species as the starting strain template. Pseudomonas was purchased from the China General Microorganism Culture Collection and Management Center, numbered Pseudomonas CGMCC No.4184. PCR reaction system: 18μL of redistilled water, 2.5μL of PCR buffer, 1μL of magnesium chloride (25mM concentration), 1.5μL of dNTP (2.5mM concentration), 0.5μL of the above two primers, 20ng of the above template, 2 units of Taq enzyme (Shanghai Sangon Company).
[0028]PCR conditions: denaturation at 94°C for 10 minutes, denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute and 15 seconds, a total of 30 cycles, and a final extension at 72°C for 10 minutes.
Embodiment 2
[0029] Example 2: Plasmid construction containing lipase gene
[0030] After the PCR amplification reaction, use 1% agarose gel to perform nucleotide electrophoresis with a voltage of 100 volts. After the band clearly appears, perform gel tapping on the target band with a size of about 1200 bp, and use the gel to recover The target DNA fragment was recovered by the kit (Takara Bio Co., Ltd.), and then the obtained DNA fragment and the vector pET24a+ (Novagen Co., Ltd.) were combined according to the method described in the 2009 version of the product catalogue of Takara Bio Co., Ltd. (website: www.takara.com.cn). Double digestion with restriction enzymes NdeI and XhoI.
[0031] The digested products were again subjected to DNA electrophoresis at a voltage of 100 volts, and fragments and vectors with corresponding sizes of about 1000 bp and 5300 bp were recovered. Finally, after mixing the fragments and vectors in a ratio of 3:1, 1.5 units of T4 ligase were added, and the tempe...
Embodiment 3
[0033] Example 3: Construction of genetically engineered bacteria expressing lipase
[0034] With calcium chloride transformation method (edited by Hao Fuying et al, "Molecular Biology Experimental Technology", Peking University Press, 1998, p. 12-15) the recombinant plasmid finally obtained in Example 2 was transformed into E.coli BL21 ( DE3) (Novagen Company), namely obtain the lipase Escherichia coli expression strain.
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