Vol. 12, Issue 6 (2023)

Lipases are enzymes which catalyze the hydrolysis of triglycerides into fatty acids and glycerol. Lipases of microbial origin are more stable, enjoy greater industrial importance presenting a fascinating field of future research. The methylotropic yeast Pichia pastoris has gained widespread acceptance as a system of choice for heterologous expression. In this study, the lipase gene of 639 bp from Bacillus subtilis was amplified and further ligated into expression vector pPICZαA and transformed into competent E. coli DH5α cells. Additionally, the recombinant plasmid was PCR amplified and double digested using Kpn I and Not I restriction enzymes for confirmation. The confirmed recombinant plasmid pPICZαA::rLP was designated as pYNAPICBL20622, and it was digested with Sac I to obtain a linearized recombinant plasmid. The linearized construct pYNAPICBL20622 was transformed into Pichia pastoris strain X-33 via electroporation. The integration of the lipase gene into the Pichia pastoris genome was confirmed by PCR analysis using gene-specific primers and AOX primers. The lipase gene confirmed Pichia pastoris clones can be utilized for the production of lipase and utilized in various industrial use.