Introduction Directory UMM :Data Elmu:jurnal:I:Insect Biochemistry and Molecular Biology:Vol30.Issue12.Dec2000:

Insect Biochemistry and Molecular Biology 30 2000 1139–1146 www.elsevier.comlocateibmb Purification and properties of a β -glycosidase purified from midgut cells of Spodoptera frugiperda Lepidoptera larvae 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 13 December 1999; received in revised form 3 April 2000; accepted 4 April 2000 Abstract Two β -glycosidases BG Mr 47,000 and Mr 50,000 were purified from Spodoptera frugiperda Lepidoptera: Noctuidae midguts. These two polypeptides associate or dissociate depending on the medium ionic strength. The Mr 47,000 BG probably has two active sites. One of the putative active sites cellobiase site hydrolyses p-nitrophenyl β -d-glucoside NP β Glu 79 of the total activity in saturated enzyme, cellobiose, amygdalin and probably also cellotriose, cellotetraose and cellopentaose. The cellobiase site has four subsites for glucose residue binding, as can be deduced from cellodextrin cleavage data. The enzymatic activity in this site is abolished after carbodiimide modification at pH 6.0. Since the inactivation is reduced in the presence of cellobiose, the results suggest the presence of a carboxylate as a catalytic group. The other active site of Mr 47,000 BG galactosidase site hydrolyses p-nitrophenyl β -d-galactoside NP β Gal better than NP β Glu, cleaves glucosylceramide and lactose and is unable to act on cellobiose, cellodextrins and amygdalin. This active site is not modified by carbodiimide at pH 6.0. The Mr 47,000 BG N-terminal sequence has high identity to plant β -glycosidases and to mammalian lactase–phlorizin hydrolase, and contains the QIEGA motif, characteristic of the family of glycosyl hydrolases. The putative physiological role of this enzyme is the digestion of glycolipids galactosidase site and di- and oligosaccharides cellobiase site derived from hemicelluloses, thus resembling mammalian lactase–phlorizin hydrolase.  2000 Elsevier Science Ltd. All rights reserved. Keywords: Intestinal β -glycosidase; Substrate specificity; Toxic glycosides; Glycolipid digestion; Insect β -glucosidase; Spodoptera frugiperda

1. Introduction

β -glycosidases EC 3.2.1 are exoenzymes, removing monosaccharides from the non-reducing end of di- andor oligosaccharides. Depending on the monosacch- aride that is removed, the β -glycosidase is named β -glu- cosidase glucose, β -galactosidase galactose, β -xylosi- dase xylose, and so on. Frequently the same β - glycosidase is able to hydrolyse several different monos- accharide residues from glycosides. In this case, β -gluco- sidase is used to name all enzymes, which remove glu- cose efficiently Terra and Ferreira, 1994. In insects, β -glycosidases play a role in terminal digestion of cellulose and hemicelluloses, in the cleavage of the carbohydrate moieties of glycoproteins see Terra and Ferreira, 1994 and may also hydrolyse glycolipids, Corresponding author. Fax + 55-11-38182186. E-mail address: clfterraiq.usp.br C. Ferreira. 0965-174800 - see front matter  2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 0 0 0 0 0 9 0 - 4 as proposed by Marana et al. 1995. However, the pre- cise function of many β -glycosidases is not clear, since some of them only hydrolyse synthetic substrates Morgan, 1975; Marana et al., 1995. The insect digestive tract has enzymes hydrolysing a variety of β -glycosides. Some insects have three or four digestive β -glycosidases with different substrate speci- ficity. In others we found only one of these enzymes, which is able to hydrolyse as many different β -glycos- ides as the other three or four enzymes together Ferreira et al., 1998. Among the substrates of insect β -glycosid- ases there are toxic β -glycosides, which are produced by plants to avoid insect attack. Some insect species are able to feed on these plants without any harm, whereas others have their growth impaired. These differences in performance may be due to a detoxification mechanism acting after glycoside hydrolysis or may be based on differential β -glycosidase specificity Ferreira et al., 1997. Differences in β -glycosidase specificity are found 1140 S.R. Marana et al. Insect Biochemistry and Molecular Biology 30 2000 1139–1146 even in insects from the same order. To understand the molecular basis and the physiological meaning of those differences, it is necessary to characterise β -glycosidases from different insects, describing their specificity, num- ber of active sites and amino acid residues related to catalysis and substrate binding. In this work, one of the major midgut β -glycosidases from Spodoptera frugiperda larvae was purified and shown to possess two active sites with different speci- ficities, resembling the mammalian dimeric lactase– phlorizin hydrolase.

2. Materials and methods