Lipase coding gene and engineering strain thereof
A technology of lipase coding and engineering strains, which is applied in the field of lipase coding genes and engineering strains, can solve the problems of low reaction yield, low stability, low activity, etc., and achieve the effect of high yield
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Embodiment 1
[0018] Embodiment 1, Geotrichum candidum lipase gene cloning and sequence analysis
[0019] Geotrichum candidum is preserved by our laboratory. Inoculate into YPD liquid medium, culture overnight at 28°C with shaking, and collect bacteria by centrifugation at 12000rpm. Total DNA was extracted with a total DNA extraction kit. Primers were designed according to the lipase gene sequence reported by GenBank (Lip-F: 5'-CGGAATTCCAGGCCCCCACGGCCGTT-3' and Lip-R: 5'-GCTCTAGATTAACCGTAGAGATTAAGG-3'). Using the total DNA of Geotrichum candidum as a template, the lipase gene sequence was amplified by PCR. The amplification conditions were: 95°C for 3 minutes, 40 cycles (94°C for 30s, 58°C for 30s, 72°C for 1min), and finally 70°C for 5 minutes. After the PCR product was sequenced, the sequence was analyzed by DNAMAN4.0 software.
Embodiment 2
[0020] Embodiment 2, optimal design of lipase gene sequence
[0021] According to the codon preference of Pichia pastoris, the Geotrichum candidum lipase gene was redesigned to replace low-frequency codons with high-frequency codons, keeping the amino acid sequence of the mature protein unchanged. The designed sequence was fully synthesized by Beijing Qingke Biotechnology Co., Ltd. To facilitate cloning, EcoRI and XbaI restriction sites were designed at the 5' and 3' ends of the sequence, respectively.
Embodiment 3
[0022] Embodiment 3, construction and transformation of Pichia pastoris expression vector
[0023] After double digestion of the gene synthesized in Example 2 with restriction endonucleases EcoRI and XbaI, it was ligated into the downstream of the α factor sequence in the vector pPICZαA (Invitrogen, USA) that had been cut with the same restriction enzymes, and the ligated product was transformed into Escherichia coli For Top10 competent cells, positive clones were screened with LB resistance plates containing 25 μg / mL antibiotic Zeocin, and plasmids were extracted to obtain the Pichia pastoris expression vector of lipase, which was named X-33 / pPIC-lip-opt.
[0024] Take 10 μL Pichia pastoris expression vector X-33 / pPIC-lip-opt, and digest it with restriction endonuclease Sac I at 37°C for 24 hours. Add 80 μL of Pichia pastoris X-33 competent cells, mix well, place in a 0.2 cm electric shock cup, ice bath for 5 min, and electric shock at 2000 volts (25 μF), quickly add 1 mL of ...
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