Open AccessPurification and Characterization of an Aspergillus Oryzae ‐Produced Carboxylesterase that Catalyzes O‐Deacetylation of a Fully Acetylated O‐Glucoside of N ‐Phenylacetohydroxamic Acid
Author(s) -
Yoshioka Tadao,
Ohno Hiroaki,
Uematsu Takayoshi
Publication year - 1997
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1997.00058.x
Subject(s) - chemistry , aspergillus oryzae , carboxylesterase , acetylation , hydrolysis , glucoside , enzyme , diisopropyl fluorophosphate , moiety , diisopropyl ether , stereochemistry , lipase , organic chemistry , biochemistry , ether , medicine , alternative medicine , pathology , gene
A carboxylesterase {23,4,6‐tetra‐ O ‐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 / A 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.