HETEROLOGOUS EXPRESSION, PURIFICATION AND CHARACTERIZATION OF THERMOSTABLE LIP11 FROM Yarrowia lipolytica in Pichia pastoris X33

摘要

A gene encoding Lip 11 from Yarrowia lipolytica MSR80 was cloned and expressed into methanolinducible expression vector pPICZ??A and successfully transformed into Pichia pastoris X33. The recombinantmut+ clones were selected on zeocine-YPD plates and high-yield clones was identified by tributyrin agar platescreening. The clone produced 32110 U/L of enzyme after 48 h expression in BMMY medium following inductionwith 0.5% methanol. Lip 11 was purified by affinity chromatography using Ni+ - NTA column with a purificationfold of 27 and yield of 59%. It was expressed as glycosylated protein of molecular mass 47 kDa. Biochemicalcharacterization revealed that it was more thermostable with improved Kd value of 5.5 x 10–3 at 70 °C and bettercatalytic efficiency than the previously expressed E.coli recombinant enzyme.