The activity of different digestive b-glycosidases from D. saccharalis change when amygdalin (a cyanogenic b-glucoside produced by plants) is added to the diet (Ferreira et al., 1997, Insect. Biochem. Molec. Biol., 27, 55-59). Preliminary work had indicated that one of those enzymes can hydrolyse both O- and S-glycosides, a specificity that has never been reported before. To study this enzyme and uncover the details, two lines of investigation were started: (A) to cloning and expressing the b-glycosidases from D. saccharalis; (B) further characterizing the enzyme with putative new specificity. By using general primers based on conserved sequences from family 1 of glycoside hydrolases and an expression library from D. saccharalis midgut epithelium, we sequenced and cloned a cDNA with 1587 bp that possibly encodes for a b-glycosidase with 508 amino acids, which has more identity and similarity (71 and 83%, respectively) to Spodoptera frugiperda (Lepidoptera) digestive b -glucosidase. The enzyme has a signal peptide of 16 amino acids, one glycosylation site and catalytical groups, homologous to other b-glycosidases.
The putative digestive O- plus S- glycosidase enzyme was purified from insect midgut and incubated with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC, a reagent specific for carboxylate groups) for different periods of time. Using methylumbelliferyl-b-glucoside or sinigrin (S-glucoside) as substrates we determined, in both cases, a kobs of inactivation of 0.006s-1. Besides, the hydrolysis of p-nitrophenyl-b-galactoside (NPbGal) was inhibited by sinigrin and the corresponding Ki value was compared to the Km of sinigrin as substrate. The same was done using sinigrin as inhibitor and NPbGal as substrate. Km and Ki values determined are (mM): for sinigrin, 1.38 and 1.23 and for NPbGal 0.3 and 0.2, respectively. As Ki and Km values are very similar, the results indicate that both substrates are processed at the same active site.
Supported by CNPq and FAPESP.