Biochemical characterization of recombinant acetyl xylan esterase from Aspergillus awamori expressed in Pichia pastoris: mutational analysis of catalytic residues.
We engineered an acetyl xylan esterase (AwaxeA) gene from Aspergillus awamori into a heterologous expression system in Pichia pastoris. Purified recombinant AwAXEA (rAwAXEA) displayed the greatest hydrolytic activity toward alpha-naphthylacetate (C2), lower activity toward alpha-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. Putative catalytic residues, Ser(119), Ser(146), Asp(168) and Asp(202), were substituted for alanine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the four mutant enzymes were examined. The S119A and D202A mutant enzymes were catalytically inactive, whereas S146A and D168A mutants displayed significant hydrolytic activity. These observations indicate that Ser(119) and Asp(202) are important for catalysis. The S146A mutant enzyme showed lower specific activity toward the C2 substrate and higher thermal stability than wild-type enzyme. The lower activity of S146A was due to a combination of increased K(m) and decreased k(cat). The catalytic efficiency of S146A was 41% lower than that of wild-type enzyme. The synthesis of ethyl acetate was >10-fold than that of ethyl n-hexanoate synthesis for the wild-type, S146A and D168A mutant enzymes. However, the D202A showed greater synthetic activity of ethyl n-hexanoate as compared with the wild-type and other mutants.
Koseki
Takuya
T
National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-hiroshima 739-0046, Japan. tkoseki@nrib.go.jp
Miwa
Yozo
Y
Fushinobu
Shinya
S
Hashizume
Katsumi
K
D000115:Acetylesterase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D019943:Amino Acid Substitution / Q000235:genetics; D001216:Asparagine / Q000235:genetics; D001230:Aspergillus / Q000201:enzymology*; D020134:Catalytic Domain; D004252:DNA Mutational Analysis; D004795:Enzyme Stability; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012694:Serine / Q000235:genetics; D013379:Substrate Specificity
D011994:Recombinant Proteins; D012694:Serine; D001216:Asparagine; D000115:Acetylesterase; C081851:acetylxylan esterase