title: Site-Directed Mutagenesis of a Hyperthermophilic Endoglucanase Cel12B from Thermotoga maritima Based on Rational Design.
abstract: To meet the demand for the application of high activity and thermostable cellulases in the production of new-generation bioethanol from nongrain-cellulose sources, a hyperthermostable β-1,4-endoglucase Cel12B from Thermotoga maritima was selected for further modification by gene site-directed mutagenesis method in the present study, based on homology modeling and rational design. As a result, two recombinant enzymes showed significant improvement in enzyme activity by 77% and 87%, respectively, higher than the parental enzyme TmCel12B. Furthermore, the two mutants could retain 80% and 90.5% of their initial activity after incubation at 80°C for 8 h, while only 45% for 5 h to TmCel12B. The Km and Vmax of the two recombinant enzymes were 1.97±0.05 mM, 4.23±0.15 μmol·mg(-1)·min(-1) of TmCel12B-E225H-K207G-D37V, and 2.97±0.12 mM, 3.15±0.21 μmol·mg(-1)·min(-1) of TmCel12B-E225H-K207G, respectively, when using CMC-Na as the substrate. The roles of the mutation sites were also analyzed and evaluated in terms of electron density, hydrophobicity of the modeled protein structures. The recombinant enzymes may be used in the hydrolysis of cellulose at higher temperature in the future. It was concluded that the gene mutagenesis approach of a certain active residues may effectively improve the performance of cellulases for the industrial applications and contribute to the study the thermostable mechanism of thermophilic enzymes.
authors.affiliation: Jiangsu Provincial Engineering Laboratory for Biomass Conversion and Process Integration, Huaiyin Institute of Technology, Huaian, Jiangsu 223003, P. R. China; School of Life Science and Chemical Engineering, Huaiyin Institute of Technology, Huaian, Jiangsu 223003, P. R. China.
authors.forename: Jinfeng
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Jiangsu Provincial Engineering Laboratory for Biomass Conversion and Process Integration, Huaiyin Institute of Technology, Huaian, Jiangsu 223003, P. R. China.
authors.forename: Hao
authors.initials: H
authors.lastname: Shi
authors.affiliation: Jiangsu Provincial Engineering Laboratory for Biomass Conversion and Process Integration, Huaiyin Institute of Technology, Huaian, Jiangsu 223003, P. R. China; School of Life Science and Chemical Engineering, Huaiyin Institute of Technology, Huaian, Jiangsu 223003, P. R. China.
authors.forename: Linyu
authors.initials: L
authors.lastname: Xu
authors.affiliation: Jiangsu Provincial Engineering Laboratory for Biomass Conversion and Process Integration, Huaiyin Institute of Technology, Huaian, Jiangsu 223003, P. R. China; School of Life Science and Chemical Engineering, Huaiyin Institute of Technology, Huaian, Jiangsu 223003, P. R. China; Jiangsu Key Laboratory for Biomass-based Energy and Enzyme Technology, Huaiyin Normal University, Huaian, Jiangsu 223300, P. R. China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Jiangsu Provincial Engineering Laboratory for Biomass Conversion and Process Integration, Huaiyin Institute of Technology, Huaian, Jiangsu 223003, P. R. China; School of Life Science and Chemical Engineering, Huaiyin Institute of Technology, Huaian, Jiangsu 223003, P. R. China.
authors.forename: Xiangqian
authors.initials: X
authors.lastname: Li
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002480:Cellulase / Q000737:chemistry / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D002480:Cellulase