Xylanase XYL1p from Scytalidium acidophilum: site-directed mutagenesis and acidophilic adaptation.
The role of residues Asp60, Tyr35 and Glu141 in the pH-dependent activity of xylanase XYL1p from Scytalidium acidophilum was investigated by site-directed mutagenesis. These amino acids are highly conserved among the acidophilic family 11 xylanases and located near the catalytic site. XYL1p and its single mutants D60N, Y35W and E141A and three combined mutants DN/YW, DN/EA and YW/EA were over-expressed in Pichia pastoris and purified. Xylanase activities at different pH's and temperatures were determined. All mutations increased the pH optimum by 0.5-1.5 pH units. All mutants have lower specific activities except the E141A mutant that exhibited a 50% increase in specific activity at pH 4.0 and had an overall catalytic efficiency higher than the wild-type enzyme. Thermal unfolding experiments show that both the wild-type and E141A mutant proteins have a T(m) maximum at pH 3.5, the E141A mutant being slightly less stable than the wild-type enzyme. These mutations confirm the importance of these amino acids in the pH adaptation. Mutant E141A with its enhanced specific activity at pH 4.0 and improved overall catalytic efficiency is of possible interest for biotechnological applications.
Al Balaa
Bassam
B
Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria, Damascus, P.O. Box 6091, Syria.
Brijs
Kristof
K
Gebruers
Kurt
K
Vandenhaute
Jean
J
Wouters
Johan
J
Housen
Isabelle
I
D000143:Acids; D000222:Adaptation, Physiological; D001203:Ascomycota / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000302:isolation & purification*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D013696:Temperature
D000143:Acids; D050505:Mutant Proteins; D043364:Endo-1,4-beta Xylanases; C121995:xyl1 xylanase