Characterization of an isozyme of beta-glucosidase from sweet almond

Abstract

A sweet almond beta-glucosidase (EC 3.2.1.21) isozyme was purified from commercial crude product. The process of purification consisted of a Protein-Pak Q anion exchange chromatography following by a Superdex 75 HR gel filtration separation. The purified enzyme is a monomeric glycoprotein with molecular weight of 58 kDa and pI = 4.55 which is distinguished from reported isozymes. The enzyme has a pH optimum in the range of 5.2-5.6 when p-nitrophenyl-beta-D-glycopyranosides are used as substrate and is stable up to 50 degrees C at that pH range. The purified protein also exhibits profound beta-galactosidase and alpha-L-arabinosidase activity. The study of substrate specificity revealed that lacking of hydroxymethyl group at C-5 of glycosides resulted in higher affinity for substrate binding to enzyme, whereas the chemical step of hydrolysis (k(cat)) was prevented significantly. The pH activity profile displayed a bell-shaped curve for all measured p-nitrophenyl-beta-D-glycopyranosides with apparent pK(1) and pK(2) values of 4.4-4.7 and 6.2-6.4, respectively. This isozyme was strongly inhibited by delta-gluconolactone (K-i = 160 mu M) and 4-phenylimidazole (K-i = 17.8 mu M) reversibly at pH 6.2. Among the tested glycoses, the binding affinity of N-acetyl-beta-D-glucosamine to the enzyme (K-i = 52 mM) was 6 times stronger than that of glucose and its epimers.