title: Heterologous expression and biochemical characterization of acetyl xylan esterase from Coprinopsis cinerea.
abstract: Acetyl xylan esterase (AXE) from basidiomycete Coprinopsis cinerea Okayama 7 (#130) was functionally expressed in Pichia pastoris with a C-terminal tag under the alcohol oxidase 1 (AOX1) promoter and secreted into the medium at 1.5 mg l(-1). Its molecular mass was estimated to be 65.5 kDa based on the SDS-PAGE analysis, which is higher than the calculated molecular mass of 40 kDa based on amino acid composition. In-silico analysis of the amino acid sequence predicted two potential N-glycosylation sites. Results from PNGase F deglycosylation and mass spectrum confirmed the presence of N-glycosylation on the recombinant AXE with predominant N-glycans HexNAc2Hex9-16. The recombinant AXE showed best activity at 40 °C and pH 8. It showed not only acetyl esterase activity with a Km of 4.3 mM and a Vmax of 2.15 U mg(-1) for hydrolysis of 4-nitrophenyl acetate but also a butyl esterase activity for hydrolysis of 4-nitrophenyl butyrate with a Km of 0.11 mM and Vmax of 0.78 U mg(-1). The presence of two additional amino acid residues at its native N-terminus was found to help stabilize the enzyme against the protease cleavages without affecting its activity.
authors.affiliation: Institute of Chemical and Engineering Sciences, Agency for Science, Technology and Research (A STAR), 1 Pesek Road, Jurong Island, 627833, Singapore.
authors.forename: Veeresh
authors.initials: V
authors.lastname: Juturu
authors.forename: Christina
authors.initials: C
authors.lastname: Aust
authors.forename: Jin Chuan
authors.initials: JC
authors.lastname: Wu
mesh_terms: D000115:Acetylesterase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000363:Agaricales / Q000201:enzymology* / Q000235:genetics; D002087:Butyrates / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D015870:Gene Expression; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009596:Nitrophenols / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D011499:Protein Processing, Post-Translational; D013696:Temperature
chemical_list: D002087:Butyrates; D009596:Nitrophenols; C033592:4-nitrophenyl butyrate; C008642:4-nitrophenyl acetate; D000115:Acetylesterase; C081851:acetylxylan esterase