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-0-acetyl- 1-[(N-acetyl-N-phenylamino)oxy]-1-deoxy-β-D-glucopyranoside (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 V(max)/K(m) values. A good correlation between log(V(max)/K(m)) and the Taft's E(s) constant of the alkyl group of the acyl moiety was obtained. The optimum pH was around 7.3 at 37°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.