Study On The Structure-function Relationship Of Yarrowia Lipolytica Lipase 2

Yarrowia lipolytica lipase YLLip2 is a common lipase which has been widely used in various fields.It possesses unique property of true lipases-interfacial activation.The crystal structure of YLLip2 in its closed conformation has been resolved.However,YLLip2 is in its open conformation when it is in its functional state.So far,little information was reported on the structure of YLLip2 in its open conformation.Studies on structure-function relationship of YLLip2 is insufficient,especially the mechanism of its interfacial activation.In this study,in order to further elucidate the structure-function relation of YLLip2,the structural model of YLLip2 in its open conformation was constructed via homology modelling,then a series of mutants were constructed,their enzymatic properties and interfacial activation were consequently assayed.The main work and results were summarized below.1.The expression vector of Y.lipolytica was improved.The oleic-acid induced promoter pPOX2 was replaced by the hp4d promoter and the nucleotide sequence encoding signal peptides was introduced into the vector.The improved vector was used for YLLip2 expression and purification.The enzymatic properties and interfacial behavior of YLLip2 was detected.The results showed that the optimum substrate of YLLip2 is p-NPC12 and it preferred to hydrolyze p-NP esters with middle and long chain length,its optimum temperature was 40 0C,YLLip2 retained 17.3%residual activity after incubation 4h at 40 ?.Its optimum pH was 8.0.YLLip2 exhibited notable interfacial activation which indicated that it is a true lipase.2.The structural model of YLLip2 in its open conformation(YLLip2-oc)was constructed via homology modelling.Structural analysis showed that the structure of YLLip2 in its open conformation exhibited high similarity with that of Aspergillus niger feruloyl esterase A(AnFaeA).The most difference is that there are more hydrophobic residues in the substrate binding pocket of YLLip2 than that of AnFaeA..The lid region of YLLip2 superposed well with the cap region of AnFaeA.Moreover,there are more hydrophobic residues in the lid region than in the cap region.It is supposed that hydrophobic residues in substrate binding pocket and lid region have great influence on lipase function.3.Mutants I100Y,F129Y and I100YF129Y were constructed,the target residues are in the substrate binding pocket of YLLip2.Their enzymatic properties and interfacial behavior were characterized.Their optimum substrates are p-NPC10,p-NPC4 and p-NPC4,optimum temperatures are 40 ?,45 ? and 45 ?.All mutants exhibited enhanced thermostability than YLLip2.Their optimum pH are 7.5,7.0 and 6.0.Interfacial assay indicated that the degree of interfacial activation of the mutant enzymes reduced compared with YLLip2.Combining the structural information of AnFaeA,it is concluded that 1100 and F129 are responsible for substrate discrimination and interfacial activation.From an evolutionary point of view,YLLip2 and AnFaeA originated from the same ancestral protein,the two residues are cut-off for divergent evolution.4.Mutants L91D,I95Q and I98T were constructed,the target residues are in the lid region of YLLip2.Their enzymatic properties and interfacial behavior were characterized.Their optimum substrates are p-NPC 12,p-NPC 12 and p-NPC 10,optimum temperatures are all 40 0C.All mutant enzymes exhibited enhanced thermostability than YLLip2.Their optimum pH are 7.5,7.5 and 8.0.Interfacial activation assay showed that the degree of interfacial activation of the mutant enzymes reduced.It is concluded that the hydrophobicity of lid affects lipase activity and interfacial activation significantly.5.Mutants V94W,I100F and V94WI100F were constructed,they are mutants with the introduction of aromatic amino acid residues in the substrate binding pocket,their enzymatic properties and interfacial behavior were characterized,Their optimum substrates of V94W,I100F and V94WI100F are p-NPC12,p-NPC10 and p-NPC10,their optimum temperatures are all 35 ?,the mutants showed enhanced thermostability.Their optimum pH are 7.5,8.0 and 6.0.Their interfacial activation behavior was enhanced compared YLLip2.It is supposed that the introduction of aromatic residues indeed affects enzymatic characteristics and interfacial activation.