title: Role of Met93 and Thr96 in the lid hinge region of Rhizopus chinensis lipase.
abstract: We engineered Rhizopus chinensis lipase to study its critical amino acid role in catalytic properties. Based on the amino acid sequence and three-dimensional model of the lipase, residues located in its lid hinge region (Met93 and Thr96) were replaced with corresponding amino acid residues (Ile93 and Asn96) found in the lid hinge region of Rhizopus oryzae lipase. The substitutions in the lid hinge region affected not only substrate specificity but also the thermostability of the lipase. Both lipases preferred p-nitrophenyl laurate and glyceryl trilaurate (C12). However, the variant S4-3O showed a slight decline in activity toward long-chain fatty acid (C16-C18). When enzymes activities decreased by half, the temperature of the variant (45 °C) was 22 °C lower than the parent (67 °C), probably substantially destabilized the structure of the lid region. The interfacial kinetic analysis of S4-3O suggested that the lower catalytic efficiency was due to a higher K m* value. According to the lipase structure investigated, Ile93Met played a role of narrowing the size of the hydrophobic patch, which affected the substrate binding affinity, and Asn96Thr destabilized the structure of the lipase by disrupting the H-bond interaction in the lid region.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
authors.forename: Shan-Shan
authors.initials: SS
authors.lastname: Zhu
authors.forename: Ming
authors.initials: M
authors.lastname: Li
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Yu
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism*; D008969:Molecular Sequence Data; D010168:Palmitates / Q000378:metabolism; D012233:Rhizopus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature; D013912:Threonine / Q000378:metabolism*; D014170:Transformation, Genetic
chemical_list: D005656:Fungal Proteins; D010168:Palmitates; C050015:4-nitrophenyl palmitate; D013912:Threonine; D008715:Methionine; D008049:Lipase