A single amino acid substitution enhances the catalytic activity of family 11 xylanase at alkaline pH.
Random mutagenesis of the gene encoding family 11 xylanase was used to obtain alkalophilic mutants. The catalytic domain of the chimeric enzyme Stx15, which was constructed from Streptomyces lividans xylanase B and Thermobifida fusca xylanase A, was mutated using error-prone PCR and screened for halo formation on dye-linked xylan plates and activity toward soluble xylan. A positive mutant, M1011, was isolated, and it was found that mutation A49V was responsible for the alkalophilicity of the mutant. Mutation A49V increased the specific activity at pH 9.1 and the stability of mutant A49V was not significantly different from that of Stx15 at 60 degrees C. Both enzymes retained more than 90% of their relative activity from pH 4.7 to 9.1 after 1 h of incubation at 60 degrees C. Analysis of the kinetic parameters at various pH values showed that the A49V mutation reduced the Km in the alkaline pH range, resulting in the higher specific activity of the A49V mutant enzyme.
Shibuya
Hajime
H
Forestry and Forest Products Research Institute, Ibaraki, Japan. bushi@ffpri.affrc.go.jp
Kaneko
Satoshi
S
Hayashi
Kiyoshi
K
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data
D017931:DNA Primers; D043364:Endo-1,4-beta Xylanases