title: [Improvement of catalytic capability of Paecilomyces thermophila J18 thermostable beta-1,3-1,4-glucanase under acidic condition by directed evolution].
abstract: Directed evolution was used to improve the performance of beta-1,3-1,4-glucanase (designated as PtLicl6A) from Paecilomyces thermophila J18 under acidic condition. A mutant library was constructed by error-prone PCR and DNA shuffling, and positive clones were screened by Congo red staining. More than 1 500 mutants were selected. One mutant (PtLic16AM1) exhibited an optimal activity at pH 5.5, while the optimal pH of the wild-type enzyme was 7.0. The mutant PtLic16AM1 kept the high specific activity and thermotolerence of the wild-type enzyme. Sequence analysis revealed that the mutant enzyme has four sense substitutions which caused four amino acid substitutions - namely T58S, Y110N, G195E and D221G.. Homology modeling showed that among the four amino acid substitutions, Y110N was near the active site of the enzyme, while the other three was distant. T58S and G195E may play key roles in the change of optimal pH. This study provided a new perspective of obtaining applicable 3-1,3-1,4-glucanase for industrial use.
authors.affiliation: College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
authors.forename: Yinan
authors.initials: Y
authors.lastname: Li
authors.forename: Huiyong
authors.initials: H
authors.lastname: Jia
authors.forename: Qiaojuan
authors.initials: Q
authors.lastname: Yan
authors.forename: Zhengqiang
authors.initials: Z
authors.lastname: Jiang
authors.forename: Shaoqing
authors.initials: S
authors.lastname: Yang
mesh_terms: D002384:Catalysis; D019020:Directed Molecular Evolution / Q000379:methods*; D044644:Endo-1,3(4)-beta-Glucanase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D050505:Mutant Proteins / Q000378:metabolism*; D009154:Mutation; D010143:Paecilomyces / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods
chemical_list: D050505:Mutant Proteins; D044644:Endo-1,3(4)-beta-Glucanase