Purification And Characterization Of Lipase From Pseudomonas Aeruginosa And Its Hetero-expression

Lipases(EC 3.1.1.3) are a group of enzymes which catalyze the hydrolysis of triacyglycerols at oil and water interfaces.They are found in animals,plants and microorganisms like bacteria,yeasts or archeae.They play an important role in ester synthesis,hydrolysis of several esters and optical resolution. The industrial needs in food,leather,pharmacy and detergent boost lipase's application due to its unique characteristics like mild reaction conditions,high enantioselectivity,environmental friendliness and so on.With increasing studies on non-aqueous enzymes and recognitions of lipase-induced biochemistry phenomenon,a higher level of lipase research is expected to be reached soon.In this research,a high lipase-yield species LIP-YUN was isolated by tributyrin plates.It was identified as Pseudomonas aeruginosa by 16S rDNA.The extracellular secretion lipase LIP-Y from LIP-YUN was isolated and purified.It is 34kDa.The enzyme properties determination reveals that its optional reactive temperature is 60℃,optional pH is at 10.0,and it is stable when pH is ranged from 4 to 12.As to temperature stability,it is stable at 50℃,but the enzymic activity declines dramaticlly at 60℃and 70℃.Most of metal ions and surfactants prompt its enzymic activity.One percent of 6501 detergent even increases its enzymic activity by tenfold.Only few metal ions,such as Mn²⁺,Cr³⁺ and Pb²⁺ were slightly inhibited on the enzyme activity.According to the lipase gene sequences of the same genus reported in Genbank,two flanked specific primers were designed.The full-length lipase gene was amplified by PCR.It is 936bp long, comprising one open reading frame encording a polypeptide of 311 amino acids.Its protein sequence showed highest similarity of 99%with the reported lipases from Pseudomonas aeruginosa.The prokaryotic expression vector Lipase-pET-30 was constructed.About 37kDa recombinant lipase with His-Tag was expressed by IPTG inducing.The expressed product bored normal bioactivity.