초록
Expression of a thermo-alkaline lipase gene from Talaromyces thermophilus in Pichia pastoris was researched to enhance its production. The lipase gene (TTL) was genetically optimized and inserted into the downstream of AOX1 promoter and α-factor to construct recombinant plasmid pPIC9K-TTL, which was then transformed into P. pastoris via electroporation, producing 220 positive transformants. Stable integration of lipase gene into chromosomal DNA of transformants was confirmed through PCR analysis. Lipase production was performed at lab scale, and an obvious protein band about 39kDa was detected using SDS-PAGE, which suggested lipase gene in recombinant P. pastoris was successfully extracellularly expressed. This lipase worked efficiently at pH range from 8.9 to 10.5, and showed the maximum activity at pH 9.5. After treating at pH 11 for one hour, 75% of its activity could be remained. It was active above 40<SUP>o</SUP>C up to 70<SUP>o</SUP>C, and the optimal temperature for reaction was 60<SUP>o</SUP>C. High thermostability was observed, and more than 70% activity could be kept after one hour treatment at temperatures up to 80<SUP>o</SUP>C. Lipase activity was promoted by Ca<SUP>2+</SUP> and inhibited by Zn<SUP>2+</SUP> and Cu<SUP>2+</SUP>. This research showed a bright prospect for industrial application of thermo- and alkaline-stable lipase.