title: Improving catalytic efficiency of endo-β-1, 4-xylanase from Geobacillus stearothermophilus by directed evolution and H179 saturation mutagenesis.
abstract: Endo-β-1, 4-xylanase was cloned from Geobacillus stearothermophilus 1A05583 by PCR. Enzymes with improved catalytic efficiency were obtained using error-prone PCR and a 96-well plate high-throughout screening system. Two variants 1-B8 and 2-H6 were screened from the mutant library containing 9000 colonies, which, when compared with the wild-type enzyme increased the catalytic efficiency (kcat/Km) by 25% and 89%, respectively, acting on beechwood xylan. By sequencing 1-B8 and 2-H6, an identical mutation point H179Y was detected and found to overlap in the active site cleft. Following the introduction of the remaining 19 amino acids into position 179 by site-saturation mutagenesis, the catalytic efficiency of H179F was found to be 3.46-fold that of the wild-type. When Whistidine was substituted by tryptophan, arginine, methionine or proline, the enzyme lost activity. Therefore, the position 179 site may play an important role in regulating the catalytic efficiency.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University,Wuhan 430070,China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Shiyu
authors.initials: S
authors.lastname: Feng
authors.forename: Tao
authors.initials: T
authors.lastname: Zhan
authors.forename: Zongqing
authors.initials: Z
authors.lastname: Huang
authors.forename: Guojie
authors.initials: G
authors.lastname: Wu
authors.forename: Ziduo
authors.initials: Z
authors.lastname: Liu
mesh_terms: D019943:Amino Acid Substitution; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D019020:Directed Molecular Evolution; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001411:Geobacillus stearothermophilus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D016296:Mutagenesis; D009842:Oligopeptides / Q000378:metabolism
chemical_list: C086651:H 179; D009842:Oligopeptides; D006639:Histidine; D043364:Endo-1,4-beta Xylanases
keywords: Catalytic efficiency; Directed evolution; Error-prone PCR; Site-saturation mutagenesis; Xylanase