title: Development of thermostable Candida antarctica lipase B through novel in silico design of disulfide bridge.
abstract: Lipase B from Candida antarctica (CalB) is a versatile biocatalyst for various bioconversions. In this study, the thermostability of CalB was improved through the introduction of a new disulfide bridge. Analysis of the B-factors of residue pairs in CalB wild type (CalB-WT) followed by simple flexibility analysis of residues in CalB-WT and its designated mutants using FIRST server were newly proposed to enhance the selective power of two computational tools (MODIP and DbD v1.20) to predict the possible disulfide bonds in proteins for the enhancement of thermostability. Five residue pairs (A162-K308, N169-F304, Q156(-) L163, S50-A273, and S239C-D252C) were chosen and the respective amino acid residues were mutated to cysteine. In the results, CalB A162C-K308C showed greatly improved thermostability while maintaining its catalytic efficiency compared to that of CalB-WT. Remarkably, the temperature at which 50% of its activity remained after 60-min incubation (T⁶⁰₅₀) of CalB A162C_K308C was increased by 8.5°C compared to that of CalB-WT (55 and 46.5°C, respectively). Additionally, the half-life at 50°C of CalB A162C-K308C was 4.5-fold higher than that of CalB-WT (220 and 49 min, respectively). The improvement of thermostability of CalB A162C-K308C was elucidated at the molecular level by molecular dynamics (MD) simulation.
authors.affiliation: Department of Chemical Engineering, Kwangwoon University, Seoul 139-701, Republic of Korea.
authors.forename: Quang Anh Tuan
authors.initials: QA
authors.lastname: Le
authors.forename: Jeong Chan
authors.initials: JC
authors.lastname: Joo
authors.forename: Young Je
authors.initials: YJ
authors.lastname: Yoo
authors.forename: Yong Hwan
authors.initials: YH
authors.lastname: Kim
mesh_terms: D019943:Amino Acid Substitution; D002175:Candida / Q000201:enzymology*; D003553:Cystine / Q000737:chemistry; D015195:Drug Design; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D005813:Genes, Synthetic; D006358:Hot Temperature; D007700:Kinetics; D008049:Lipase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D010843:Pichia; D011487:Protein Conformation; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D016415:Sequence Alignment; D044366:Transition Temperature
chemical_list: D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D003553:Cystine; D008049:Lipase; C431349:lipase B, Candida antarctica