Heterologous Overexpression And Molecule Evolution Of Lipase LipY2 From Yarrowia Lipolytica

Lipase is a kind of hydrolytic enzyme which can catalyze the hydrolysis of triglycerides producing glycerol and long chain fatty acids.The extracellular lipase from Yarrowia lipolytica(LipY2)displays high stability at low pH values and is not repressed by bile salts,making it an ideal candidate for pancreatic lipase replacement therapy of pancreatic insufficiency.However,the practical use of this enzyme is often hampered by its sensitivity to trypsin digestion.Therefore,molecular modification of lipY2 is critical to improve its resistance to trypsin by means of protein engineering.In this research,LipY2 eneoding gene was cloned and introduced into the yeast integrative plasmid pPIC9K and then transformed into Pichia pastoris GS115 to heterologous expression.The activity of the recombinant lipase LipY2 was up to 1033 U/mL after 60 h of methanol induction in shake flasks.Simultaneously,susceptible peptide residues of LipY2 were found to be related to the resistance to trypsin through multiple sequence alignment and homology modeling based on the mass spectrometry of trypsin hydrolysis product and its three dimensional structure.Seven mutants,named as M36,M63,M215,M36-63,M36-215,M63-215,and M36-63-215 were constructed via site-directed mutagenesis,according to the mution sites.The effects on lipase activity and enzymatic characteristics of LipY2,such as proteolytic stability and acid stability due to the mutation of different sites were studied as follwed.The results demonstrate that M36 whose mutation sites were K36 and K39 had decreased optimum pH of one unit,increased half-time against trypsin by 8.1%,compared with LipY2.The optimum pH and half-time against trypsin of M63 whose mutation site was R63 were one unit lower and 16.6%higher than that of the LipY2.The mutation sites of M36-63 were K36,K39 and R63 which also had decreased optimum pH of one unit compared with LipY2.The half-time against trypsin of M36-63 were 22.3%higher than LipY2.In sunmmary,the mutants M36,M63 and M36-63 still retained basically equal activities to LipY2 towards similar substrates.Compared with LipY2,they all showed improved acid stability at low pH 1-3 and better resistance to the same concentration trypsin degradation,especially those of the mutant M36-63.The mutation sites of M215 were K215,K218 and R220 which showed quite low lipase activity as well as the mutants M36-215,M63-215 and M36-36-215 whose mutation sites were K36,K39,K215,K218,R220 and R63,K215,K218,R220 and K36,K39,R63,K215,K218,R220.