title: Modulation of the thermostability and substrate specificity of Candida rugosa lipase1 by altering the acyl-binding residue Gly414 at the α-helix-connecting bend.
abstract: Candida rugosa Lipase1 (LIP1) is widely used in industrial applications. Optimizing its catalytic performance is still a challenging goal for protein engineers. Mutagenesis of key residues in the active site of the enzyme may provide an effective strategy for enhancing stability and altering substrate specificity. In this study, multiple sequence alignment and structural analysis revealed that the acyl-binding residue, Gly414, of LIP1, which is located at a bend connecting α-helixes, was the non-conserved residue in five other isoenzymes. Using saturation mutagenesis, four mutants with improved stability (G414A, G414M, G414H and G414W) were obtained. Compared to the wild type, the best mutant (G414W) exhibited a remarkable 6.5-fold enhancement in half-life at 60 °C and a 14 °C higher T50(15). Its optimum temperature was increased by 15 °C. Simultaneously, G414W displayed a shift in substrate preference from medium-chain to short-chain pNP-ester. Modeling analysis showed that the multiple interactions formed by hydrophobic clusters and hydrogen bonds in the acyl-binding tunnel might lead to the observed thermostability improvement. Additionally, the bulky tryptophan substitution formed a strong steric hindrance to the accommodation of long-chain substrates in the tunnel. These results indicate that the key acyl-binding residues at the α-helix-connecting bend could mediate enzyme stability and catalytic substrate spectra.
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Xiaofei
authors.initials: X
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China; Shanghai Collaborative Innovation Center for Biomanufacturing (SCICB), East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.
authors.forename: Guangyu
authors.initials: G
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Xie
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Lishi
authors.initials: L
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China; Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130021, China.
authors.forename: Jiao
authors.initials: J
authors.lastname: An
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Li
authors.initials: L
authors.lastname: Cui
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China. Electronic address: yfeng2009@sjtu.edu.cn.
authors.forename: Yan
authors.initials: Y
authors.lastname: Feng
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002175:Candida / Q000201:enzymology* / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D006207:Half-Life; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry
chemical_list: D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D014364:Tryptophan; D002265:Carboxylic Ester Hydrolases; D005998:Glycine
keywords: Bend; Candida rugosa lipases; Saturation mutagenesis; Substrate specificity; Thermostability