Purification and characterization of a beta-glucosidase from Citrus sinensis var. Valencia fruit tissue

A preliminary survey demonstrated activity for alpha -D-glucosidase, a-D-mannosidase, alpha -L-arabinosidase, beta -D-glucosidase, beta -D-xylosidase, and beta -D-galactosidase in orange fruit flavedo and albedo tissue. alpha -L-Rhamnosidase was not detected. Subsequently, a P-glucosidase was purified from mature fruit rag tissue (composed of intersegmental septa, squeezed juice sacs, and fruit core tissue) of Citrus sinensis var. Valencia. The P-glucosidase exhibited low levels of activity against p-nitrophenyl-beta -D-fucopyranoside (13.5%) and p-nitrophenyl-alpha -D-glucopyranoside (7.0%), compared to its activity against p-nitroplienyl-beta -D-glucopyranoside (pNPG, 100%). The enzyme was purified by a combination of ion exchange (anion and cation) and gel filtration (Superdex and Toyopearl HW-55S) chromatography. It has an apparent molecular mass of 64 kDa by denaturing electrophoresis or 55 kDa by gel filtration chromatography (BioGel P-100). Hydrolysis of pNPG demonstrated a pH optimum between 4.5 and 5.5. At pH 5.0 the temperature optimum was 40 degreesC. At pH 5.0 and 40 degreesC the KM for pNPG was 0.1146 mM and it had a V-max of 5.2792 nkatal.mg(-1) protein (katal = 0.06 International Units = the amount of enzyme that produces, under standard conditions, one mu mol of product per min). Of the substrates tested, the enzyme was most active against the disaccharide cellobiose (1 double right arrow4), but was not active against p-nitrophenyl-beta -D-cellobioside. High levels of activity also were observed with the disaccharides laminaribiose (1 double right arrow3), gentiobiose (1 double right arrow6), and sophorose (1 double right arrow2). Activity greater than that observed with pNPG was obtained with the flavonoids hesperetin-7-glucoside and prunin (naringenin-7-glucoside), salicin, mandelonitrile-beta -D-glucoside (a cyanogenic substrate), and sinigrin (a glucosinolate). The enzyme was not active against amygdalin, coniferin, or limonin glucoside.