Insect Biochemistry and Molecular Biology 31 (2001) 1065–1076 www.elsevier.com/locate/ibmb Purification, molecular cloning, and properties of a β-glycosidase isolated from midgut lumen of Tenebrio molitor (Coleoptera) larvae Alexandre H.P. Ferreira, Sandro R. Marana, Walter R. Terra, Cle ´lia Ferreira * Departamento de Bioquı ´mica, Instituto de Quı ´mica, Universidade de Sa ˜o Paulo, C.P 26077, 05513-970 Sa ˜o Paulo, Brazil Received 2 November 2000; received in revised form 12 January 2001; accepted 30 January 2001 Abstract Two β-glycosidases (M r 59k) were purified from midgut contents of larvae of the yellow mealworm, Tenebrio molitor (Coleoptera: Tenebrionidae). The two enzymes (βGly1 and βGly2) have identical kinetic properties, but differ in hydrophobicity. The two glycosidases were cloned and their sequences differ by only four amino acids. The T. molitor glycosidases are family 1 glycoside hydrolases and have the E 379 (nucleophile) and E 169 (proton donor) as catalytic amino acids based on sequence alignments. The enzymes share high homology and similarity with other insect, mammalian and plant β-glycosidases. The two enzymes may hydrolyze several substrates, such as disaccharides, arylglucosides, natural occurring plant glucosides, alkylglucosides, oligocellod- extrins and the polymer laminarin. The enzymes have only one catalytic site, as inferred from experiments of competition between substrates and sequence alignments. The observed inhibition by high concentrations of the plant glucoside amygdalin, used as substrate, is an artifact generated by transglucosylation. The active site of each purified β-glycosidase has four subsites, of which subsites +1 and +2 bind glucose with more affinity. Subsite +2 has more affinity for hydrophobic groups, binding with increasing affinities: glucose, mandelonitrile and nitrophenyl moieties. Subsite +3 has more affinity for glucose than butylene moieties. The intrinsic catalytic constant calculated for hydrolysis of the glucose β-1,4-glucosidic bond is 21.2 s -1 M -1 . The putative physiological role of these enzymes is the digestion of di- and oligosaccharides derived from hemicelluloses.  2001 Elsevier Science Ltd. All rights reserved. Keywords: Intestinal β-glycosidase; Substrate specificity; Subsite affinity; Toxic glycosides; Insect β-glucosidase; T. molitor 1. Introduction β-Glycosidases are exoenzymes that hydrolyze glyco- sidic linkages, removing monosaccharides from the non- reducing terminal of different di- and oligosaccharides. Depending on the monosaccharide that is removed, the β-glycosidase is named β-glucosidase (glucose), β- galactosidase (galactose), β-xylosidase (xylose), and so on. When the enzyme can act upon several substrates it is generally called a β-glycosidase. The β-glycosidase active sites may have several monosaccharide residue-binding subsites. The subsite that binds the residue present at the non-reducing end is called -1. The other subsites are sequentially named +1, * Corresponding author. Fax: +55-11-3818 2186. E-mail address: clfterra@iq.usp.br (Cle ´lia Ferreira). 0965-1748/01/$ - see front matter  2001 Elsevier Science Ltd. All rights reserved. PII:S0965-1748(01)00054-6 +2, +3, etc. The glycosidic linkage to be cleaved is the linkage between -1 and +1 subsite (Davies et al., 1997). Subsite -1 is also called glycone subsite, and all the others form the aglycone subsite. β-Glycosidases have an important role in insect–plant interactions. Diatraea saccharalis, the sugar cane borer, reduces the level of a β-glycosidase that can liberate a cyanogenic compound from amygdalin, when this subst- ance is added to its diet (Ferreira et al., 1997). Another example of the role of β-glycosidase in this interaction is the response of cabbage plants to Pieris brassica her- bivory. The insect β-glycosidase elicites liberation, by the plant, of a compound that attracts a wasp that preys upon the insect (Mattiacci et al., 1995). Evolutionary trends among insect β-glycosidases seem to be from multiple enzymes with different sub- strate specificities to a single enzyme able to hydrolyze all β-glycosides at the same active site, although diet