| Abstract: | A sweet almond β-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 apH optimum in the range of 5.2-5.6 when p-nitrophenyl-β-D-glycopyranosides are used as substrate and is stable up to 50 °C at that pH range. The purified protein also exhibits profound β-galactosidase and σ-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 (kcst) was prevented significantly. The pH activity profile displayed a bell-shaped curve for all measured p-nitrophenyl-β-D-glycopyranosides with apparent pK1 and pK2 values of 4.4-4.7 and 6.2-6.4, respectively. This isozyme was strongly inhibited by δ-gluconolactone (Ki = 160 μM) and 4-phenylimidazole (Ki = 17.8 μM) reversibly at pH 6.2. Among the tested glycoses, the binding affinity of N-acetyl-β-D-glucosamine to the enzyme (Kl = 52 mM) was 6 times stronger than that of glucose and its epimers. |
