title: Purification and characterization of an Aspergillus oryzae-produced carboxylesterase that catalyzes O-deacetylation of a fully acetylated O-glucoside of N-phenylacetohydroxamic acid.
abstract: A carboxylesterase [2,3,4,6-tetra-O-acetyl-1-[(N-acetyl-N-phenylamino)oxy]-1-deoxy-beta-D-g lucopyranoside (GPA) O-deacetylase] from a culture product of Aspergillus oryzae (Taka diastase) was purified 8500-fold with a yield of 3%. The molecular mass of the purified enzyme was shown to be 35 +/- 1 kDa by SDS/PAGE. The enzyme shows a selective O-deacetylation activity of GPA to give the fully O-deacetylated glucoside. Among the substrates tested, the enzyme did not hydrolyze benzoyl and phenylacetyl esters and acetamides. In the hydrolysis of p-nitrophenyl esters, the acyl preference is acetyl > propionyl > butyryl, judging from the Vmax/Km values. A good correlation between log(Vmax/Km) and the Taft's Es constant of the alkyl group of the acyl moiety was obtained. The optimum pH was around 7.3 at 37 degrees C, and the enzyme was inhibited by mercuric chloride, p-chloromercuribenzoate and diisopropyl fluorophosphate. This enzyme should be useful for the selective removal of acetyl groups that serve to protect hydroxyl groups during carbohydrate synthesis.
authors.affiliation: Department of Chemical Hygiene, Hokkaido Institute of Pharmaceutical Sciences, Otaru, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Yoshioka
authors.forename: H
authors.initials: H
authors.lastname: Ohno
authors.forename: T
authors.initials: T
authors.lastname: Uematsu
mesh_terms: D000107:Acetylation; D001236:Aspergillus oryzae / Q000201:enzymology*; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006877:Hydroxamic Acids / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D006877:Hydroxamic Acids; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase