title: Effect of acidic amino acids engineered into the active site cleft of Thermopolyspora flexuosa GH11 xylanase.
abstract: Thermopolyspora flexuosa GH11 xylanase (XYN11A) shows optimal activity at pH 6-7 and 75-80 °C. We studied how mutation to aspartic acid (N46D and V48D) in the vicinity of the catalytic acid/base affects the pH activity of highly thermophilic GH11 xylanase. Both mutations shifted the pH activity profile toward acidic pH. In general, the Km values were lower at pH 4-5 than at pH 6, and in line with this, the rate of hydrolysis of xylotetraose was slightly faster at pH 4 than at pH 6. The N46D mutation and also lower pH in XYN11A increased the hydrolysis of xylotriose. The Km value increased remarkably (from 2.5 to 11.6 mg/mL) because of V48D, which indicates the weakening of binding affinity of the substrate to the active site. Xylotetraose functioned well as a substrate for other enzymes, but with lowered reaction rate for V48D. Both N46D and V48D increased the enzyme inactivation by ionic liquid [emim]OAc. In conclusion, the pH activity profile could be shifted to acidic pH due to an effect from two different directions, but the tightly packed GH11 active site can cause steric problems for the mutations.
authors.affiliation: Department of Biotechnology and Chemical Technology, School of Chemical Technology, Aalto University, Aalto, 00076, Finland.
authors.forename: He
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Biotechnology and Chemical Technology, School of Chemical Technology, Aalto University, Aalto, 00076, Finland.
authors.forename: Ossi
authors.initials: O
authors.lastname: Turunen
mesh_terms: D039903:Actinobacteria / Q000201:enzymology* / Q000235:genetics; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D020125:Mutation, Missense; D014312:Trisaccharides / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D014312:Trisaccharides; C515044:xylotriose; D043364:Endo-1,4-beta Xylanases
keywords: GH11 xylanase; Thermopolyspora flexuosa; active site mutations; bioengineering; protein modeling