Biochemical characterization of recombinant acetyl xylan esterase from Aspergillus awamori expressed in Pichia pastoris: Mutational analysis of catalytic residues

• Published in: Biochimica et biophysica acta 2005-05, Vol.1749 (1), p.7-13
• Main Authors: Koseki, Takuya, Miwa, Yozo, Fushinobu, Shinya, Hashizume, Katsumi
• Format: Article
• Language: English
• Subjects: Acetyl xylan esterase; Acetylesterase - chemistry; Acetylesterase - genetics; Acetylesterase - isolation & purification; Amino Acid Substitution - genetics; Asparagine - genetics; Aspergillus - enzymology; Aspergillus awamori; Catalytic Domain; Catalytic residue; DNA Mutational Analysis; Enzyme Stability; Mutagenesis, Site-Directed; Pichia - genetics; Pichia pastoris; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Serine - genetics; Site-directed mutagenesis; Substrate Specificity
• ISSN: 1570-9639; 0006-3002; 1878-1454
• DOI: 10.1016/j.bbapap.2005.01.009

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 α-naphthylacetate (C2), lower activity toward α-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.