title: Engineering a thermostable β-1,3-1,4-glucanase from Paecilomyces thermophila to improve catalytic efficiency at acidic pH.
abstract: To fulfill the need for acid-tolerant and thermostable β-1,3-1,4-glucanases, an error-prone PCR and DNA-shuffling approach was employed to enhance the activity of thermostable β-1,3-1,4-glucanases from Paecilomyces thermophila (PtLic16A) at acidic pH. Mutant PtLic16AM2 was selected and characterized, and showed optimal activity at pH 5.0, corresponding to an acidic shift of 2.0 pH units relative to the wild-type enzyme. Other properties of PtLic16A such as temperature optimum and substrate specificity that are beneficial for industrial applications did not change. Based on the substituted residues of PtLic16AM2, three site-directed mutations, D56G, D221G and C263S, were designed to study these residues' roles. The amino acid residues at positions 56 and 263 were found to be important in determining optimal pH activity. Activity of the D221G variant showed no significant difference from the wild-type. Thus, it appears that the change in optimal pH for PtLic16AM2 was mainly caused by the combination of substitutions D56G and C263S. This study provides a β-1,3-1,4-glucanase (PtLic16AM2) with high potential for industrial applications.
authors.affiliation: Department of Biotechnology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China.
authors.forename: Huiyong
authors.initials: H
authors.lastname: Jia
authors.forename: Yinan
authors.initials: Y
authors.lastname: Li
authors.forename: Yuchun
authors.initials: Y
authors.lastname: Liu
authors.forename: Qiaojuan
authors.initials: Q
authors.lastname: Yan
authors.forename: Shaoqing
authors.initials: S
authors.lastname: Yang
authors.forename: Zhengqiang
authors.initials: Z
authors.lastname: Jiang
mesh_terms: D019020:Directed Molecular Evolution; D044644:Endo-1,3(4)-beta-Glucanase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010143:Paecilomyces / Q000201:enzymology* / Q000235:genetics*; D013696:Temperature
chemical_list: D044644:Endo-1,3(4)-beta-Glucanase