title: Enhancement of the activity and alkaline pH stability of Thermobifida fusca xylanase A by directed evolution.
abstract: Directed evolution has been used to enhance the catalytic activity and alkaline pH stability of Thermobifida fusca xylanase A, which is one of the most thermostable xylanases. Under triple screened traits of activity, alkaline pH stability and thermostability, through two rounds of random mutagenesis using DNA shuffling, a mutant 2TfxA98 with approximately 12-fold increased k(cat)/K(m) and 4.5-fold decreased K(m) compared with its parent was obtained. Moreover, the alkaline pH stability of 2TfxA98 is increased significantly, with a thermostability slightly lower than that of its parent. Five amino acid substitutions (T21A, G25P, V87P, I91T, and G217L), three of them are near the catalytic active site, were identified by sequencing the genes encoding this evolved enzyme. The activity and stabilizing effects of each amino acid mutation in the evolved enzyme were evaluated by site-directed mutagenesis. This study shows a useful approach to improve the catalytic activity and alkaline pH stability of T. fusca xylanase A toward the hydrolysis of xylan.
authors.affiliation: College of Life Science and Biotechnology, Shanghai Jiaotong University, 800 Rm. 211, No. 3 Biopharmacology Building, 800 Dongchuan Road, Shanghai, 200240, P.R. China. qwangsm@sjth.edu.cn
authors.forename: Qin
authors.initials: Q
authors.lastname: Wang
authors.forename: Tao
authors.initials: T
authors.lastname: Xia
mesh_terms: D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D019943:Amino Acid Substitution; D002384:Catalysis; D032621:DNA Shuffling; D019020:Directed Molecular Evolution; D043364:Endo-1,4-beta Xylanases / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017422:Sequence Analysis, DNA; D014990:Xylans / Q000378:metabolism
chemical_list: D014990:Xylans; D043364:Endo-1,4-beta Xylanases