Characterization of an Isozyme of β‐Glucosidase from Sweet Almond

Abstract

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 p I =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 (k cst ) was prevented significantly. The pH activity profile displayed a bell‐shaped curve for all measured p ‐nitrophenyl‐β‐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 δ‐gluconolactone (K i = 160 μM) and 4‐phenylimidazole (K i = 17.8 μM) reversibly at pH 6.2. Among the tested glycoses, the binding affinity of N‐acetyl‐β‐D‐glucosamine to the enzyme (K l = 52 mM) was 6 times stronger than that of glucose and its epimers.