title: Improving the thermostability of Geobacillus stearothermophilus xylanase XT6 by directed evolution and site-directed mutagenesis.
abstract: Protein engineering of the thermostable xylanase XT6 from Geobacillus stearothermophilus was performed to obtain enzymes with improved thermal tolerance. Mutants producing such enzymes were obtained after several rounds of directed evolution using error-prone PCR and sequence family shuffling, in combination with a consensus-based semi-rational approach. The most thermostable mutant enzyme contained 13 amino acid substitutions and its half-life of inactivation was 52-fold of that of the wild-type. Its reaction temperature for maximum activity increased from 77 degrees C to 87 degrees C, and catalytic efficiency (k(cat)/K(m)) increased by 90%. The mutant is of potential interest for industrial applications.
authors.affiliation: Chengdu Institute of Biology, Chinese Academy of Sciences, P.O. Box 416, Chengdu 610041, China.
authors.forename: Zhi-Gang
authors.initials: ZG
authors.lastname: Zhang
authors.forename: Zhuo-Lin
authors.initials: ZL
authors.lastname: Yi
authors.forename: Xiao-Qiong
authors.initials: XQ
authors.lastname: Pei
authors.forename: Zhong-Liu
authors.initials: ZL
authors.lastname: Wu
mesh_terms: D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D032621:DNA Shuffling; D019020:Directed Molecular Evolution / Q000379:methods*; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D015723:Gene Library; D005820:Genetic Testing; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics*; D006207:Half-Life; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D017354:Point Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D013696:Temperature
chemical_list: D043364:Endo-1,4-beta Xylanases