Heterologous expression and characterization of thermostable lipase (Lk1) in Pichia pastoris GS115

A gene encoding thermostable lipase namely LK1 was successfully sub-cloned into expression vector pPICZ alpha A and integrated into a chromosomal fungal host, Pichia pastoris GS115. The recombinant mut(+) clones selected on zeocine-YPD plates were heterologically expressed into Pichia pastoris GS115 using 1% methanol as inducer at 30 degrees C during 120 h in BMMY medium. The protein was expressed at molecular mass 35.5 kDa following SDS-PAGE analysis. The enzyme still showed lipase activity on pNP as substrate. Thermostable lipase was purified by affinity chromatography using Ni-NTA column with a purification fold of 11.6 and yield of 31.75%. Further characterization of the enzyme showed that the enzyme has highest spesicificity on pNP-laurate as substrate with optimum temperature at 60 degrees C, pH 8. In addition, the enzyme still maintained the activity up to 4 h in incubation at 60 degrees C and maintained 50% activity at 3 h incubation. All of the above data suggested that the enzyme is an alkaline tolerance and thermostable lipase.