Cloning And Expression Of Thermoresistant Lipase Gene In Bacillus Subtilis

A bacterial strain producing thermotolerant lipase was screened from soil polluted by oil with initial screening medium as follows: (NH₄)₂SO₄ 0.1%, K₂HPO₄ 0.1%, MgSO₄ · 7H₂O 0.01%, KCl 0.05%, olive oil 1%, PVA 0.1%, pH 7.5. It was identified as Pseudomonas aeruginosa by physiological biochemical tests, whose lipase catalyzed olive oil most efficiently in 55℃ and pH 7.5, and its activity was 7.3U/mL, so it was a neutral and slightly alkaline lipase.Strain that had high activity was obtained by UV mutagenesis, lipase activity was 8.7U/mL, 19% higher than the original strain after three times.Lipase gene was amplified and ligated to pUCm-T to generate a new vector pUCm-T-LipA for sequencing and analysis, and multiple alignments were performed with other lipase genes, the result showed that there were mutations of nucleotides(2～8) and amino acids(0～2) with the other reported genes.Signal peptide sequence was removed lest it couldn't be recognized by Bacillus subtilis, gene coding for mature peptide was ligated to downstream of sacB signal peptide sequence of pWB980 to form a new ORF and generate pWB980-LipA, gene coding for its chaperone was also amplified and ligated to the downstream of LipA to generate a secretion/expression vector pWB980-LipAB.thechaperone gene was sequenced and analyzed by multiple alignments, resulted showed that there were mutations of nucleotides(1～7) and amino acids (1～2) with the other reported genes.Bacillus subtilisDB104, a two extracellular protease deficient strain, containing pWB980-LipAB was cultured in LB medium, the thalli and supernatant were assayed by SDS-PAGE after being processed, results showed lipase and its chaperone were expressed and lipase was successfully secreted into medium, the recombinant lipase activity was 15.4U/mL and the other characteristics were almost the same as the wild lipase.