Purification, molecular cloning, and properties of a beta-glycosidase isolated from midgut lumen of Tenebrio molitor (Coleoptera) larvae

Two beta -glycosidases (M-r 59k) were purified from midgut contents of larv ae of the yellow mealworm, Tenebrio molitor (Coleoptera: Tenebrionidae). Th e two enzymes (beta Gly1 and beta Gly2) have identical kinetic properties, but differ in hydrophobicity. The two glycosidases were cloned and their se quences differ by only four amino acids. The T. molitor glycosidases are fa mily 1 glycoside hydrolases and have the E-379 (nucleophile) and E-169 (pro ton donor) as catalytic amino acids based on sequence alignments. The enzym es share high homology and similarity with other insect, mammalian and plan t beta -glycosidases. The two enzymes may hydrolyze several substrates, suc h as disaccharides, arylglucosides, natural occurring plant glucosides, alk ylglucosides, oligocellodextrins and the polymer laminarin. The enzymes hav e only one catalytic site, as inferred from experiments of competition betw een substrates and sequence alignments. The observed inhibition by high con centrations of the plant glucoside amygdalin, used as substrate, is an arti fact generated by transglucosylation. The active site of each purified beta -glycosidase has four subsites, of which subsites +1 and +2 bind glucose w ith more affinity. Subsite +2 has more affinity for hydrophobic groups, bin ding with increasing affinities: glucose, mandelonitrile and nitrophenyl mo ieties. Subsite +3 has more affinity for glucose than butylene moieties. Th e intrinsic catalytic constant calculated for hydrolysis of the glucose bet a -1,4-glucosidic bond is 21.2 s(-1) M-1. The putative physiological role o f these enzymes is the digestion of di- and oligosaccharides derived from h emicelluloses. (C) 2001 Elsevier Science Ltd. All rights reserved.