Lipase LIP, gene and application thereof
A lipase gene and lipase technology, applied in the field of lipase and its gene and application, can solve the problems of high production cost and low enzyme activity
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Embodiment 1
[0063] Example 1. Cloning and sequence analysis of Aspergillus niger lipase gene lip
[0064] Using RNA extraction kit, extract the total RNA of Aspergillus niger according to the reverse transcriptase SuperScript TM III Reverse Transcriptase instructions for synthesizing the first strand cDNA. Using cDNA as template, design primers to amplify the lipase gene for PCR amplification. The PCR product was double digested with restriction enzymes EcoRI and NotI, and then ligated with the same digested Pichia expression vector pPICzalphaA , The ligation product was transformed into E. coli Topl0 competent cells, after screening with the antibiotic Zeocin, a positive clone was obtained. Extract the plasmid of the positive clone. The samples were sent to Shanghai Yingjun Biological Company for sequencing. The sequencing results showed that the obtained cloned DNA insert contained a complete open reading frame of the lipase gene. The lipase gene lip has a total length of 894 bp (SEQ I...
Embodiment 2
[0069] Example 2. Construction of Pichia pastoris engineering bacteria containing lipase gene lip
[0070] The above recombinant expression vector pPICzαA-lip was linearized with SacI, and the linearized recombinant vector was electroporated to transform Pichia pastoris X33 to obtain Pichia pastoris recombinant strain transformant. Transfer the transformant to the YPDZ medium plate with a toothpick. Incubate at 28°C for 2 days. Use toothpicks to transfer the colonies on the YPDZ medium plate to the lipase activity detection function plate, add methanol dropwise to induce culture, and observe the changes of the lipase activity detection function plate regularly. The experimental results are as figure 1 As shown, there are obvious color circles around transformants 1 and 2, indicating that the lipase gene introduced into Pichia pastoris X33 has achieved functional expression and is secreted outside the cell.
Embodiment 3
[0071] Example 3. Highly efficient expression of lipase recombinant strain
[0072] The single colony of the recombinant bacteria is subjected to high-density fermentation culture. Prepare 20L basic salt medium, sterilize it in a 50L automatic control fermentation tank, and cool to normal temperature for later use. Adjust the pH value of the fermentation broth to 4.8 with ammonia and phosphoric acid, and control the dissolved oxygen to be greater than 30% by adjusting the speed and air flow, and the fermentation temperature is 30°C. The whole fermentation process is divided into 3 stages: The first stage is the cell culture stage. The recombinant bacteria are inoculated into the fermentor at 10% of the inoculum amount, and 4L of 50% glucose that has been sterilized is fed in for 24 to 30 hours. Take the supplement of glucose as a sign; the second stage is the starvation stage. When the glucose is supplemented, no carbon source is added. When the dissolved oxygen rises to more th...
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