1141 S.R. Marana et al. Insect Biochemistry and Molecular Biology 30 2000 1139–1146
2.8. Protein determination and hydrolase assays Protein was determined according to Bradford 1976
using ovoalbumin as a standard. β
-glycosidase activity was determined by measuring the release of p-nitrophenolate Terra et al., 1979 from
NP β
Glu p-nitrophenyl-
β -d-glucopiranoside;
and NP
β Gal p-nitrophenyl-
β -d-galactopiranoside, reducing
groups Noelting and Bernfeld, 1948 from laminarin and
CMC carboxymethyl
cellulose or
glucose Dahlqvist, 1968 from different alkyl
β -glucosides, cel-
lobiose, cellotriose, cellopentaose, gentiobiose, prunasin, amygdalin, phlorizin, lactose, laminaribiose and glucos-
ylceramide. In the last case, the solubilization was achi- eved according to Dinur et al. 1984.
All substrates were assayed in 50 mM citrate–sodium phosphate pH 6.0 at 30
° C under conditions such that
activity was proportional to protein concentration and to time. Controls without enzyme or without substrate were
included. One unit of enzyme U is defined as the amount that hydrolyses 1
µ mol of substratemin.
2.9. Chemical modification studies Purified
β -glycosidase was incubated at 30
° C with 6
mM EDC
1-ethyl-3-3-dimethylaminopropyl carbodiimide, 40 mM glycine ethyl ester and 100 mM
TemedN,N,N 9,N9-tetramethyl-ethylenediamineHCl buffer
pH 5.2 or 6.0. When the substrate used to follow the inactivation was cellobiose, the incubation with EDC
was done with or without 25 mM NP β
Gal or 14 mM cellobiose. When the substrate was NP
β Gal, the incu-
bation with EDC was done with or without 14 mM cello- biose. Samples were collected at different periods of
time and the reaction was stopped by a two-fold dilution with 400 mM citrate–sodium phosphate buffer pH 6.0.
When the substrate used to follow the inactivation was cellobiose, after this initial dilution, samples were sub-
mitted to two cycles of five-fold dilution followed by concentration in Microcon centrifuge filters YM-10
Amicon. This procedure is necessary to avoid cello- biose hydrolysis inhibition by NP
β Gal present in the
reaction media. 2.10. Kinetic studies
The effect of substrate concentration on purified β
- glycosidase was determined using at least 10 different
substrate concentrations. K
m
and V
m
values mean and SEM were determined by linear regression using the
software Enzfitter Elsevier, Biosoft. When the inhibition of the hydrolysis of one substrate
NP β
Glu or NP β
Gal by another substrate NP β
Glu, NP
β Gal or cellobiose was studied,
β -glycosidase was
incubated with at least five different concentrations of substrate in each of at least five different concentrations
of the substrate used as inhibitor. In these studies, two samples were taken from each reaction medium at the
end of incubation. In one sample, p-nitrophenolate was determined to calculate the amount of NP
β Glu or
NP β
Gal that was hydrolysed; in the other sample, glu- cose was measured according to Dahlqvist 1968, with
the final addition of sulfuric acid to change p-nitrophen- olate into the colourless p-nitrophenol. In the medium
with NP β
Glu, after allowance for the amount of glucose originating from it, it is possible to calculate the activity
on cellobiose or NP β
Gal. K
i
values were determined from replots of slopes of Linewaver–Burk plots against
inhibitor concentration Segel, 1975, using the software Enzfitter Elsevier, Biosoft.
3. Results
3.1. Purification of a Mr 47,000 b-glycosidase
S. frugiperda β
-glycosidase activity is almost restric- ted to midgut epithelium and probably located at the cell
glycocalix Ferreira et al., 1994. When a sample of sol- uble midgut cellular fraction was applied to a Superose
12 column at low and high ionic strength, a single β
- glycosidase activity is recovered with Mr of 120,000 and
66,000, respectively. The reapplication of the high ionic strength eluate into a low ionic strength chromatography,
gave one β
-glycosidase activity peak, with a Mr of 125,000, indicating that there are two polypeptides that
can associate one with the other, depending on the ionic strength Fig. 1.
To purify the β
-glycosidases, samples from soluble midgut cellular fraction were applied to a Superose col-
umn in low and subsequently in high ionic strength. The eluted active fractions of the second run were pooled
and then submitted to ion-exchange chromatography, resulting in two partially resolved
β -glycosidases. Active
Fig. 1. Activity on NP
β Glu after gel filtration of midgut
β -glycosid-
ases on Superose 12 HR 1030 FPLC system. The column was equi- librated and eluted with 20 mM triethanolamine pH 6.0 with
h or without
r 1 M NaCl. Active fractions eluted from the chromato- graphy with NaCl were applied in the same column with buffer lacking
NaCl G.
1142 S.R. Marana et al. Insect Biochemistry and Molecular Biology 30 2000 1139–1146
Fig. 2. Electrophoresis in SDS-7.5 polyacrylamide gel slab. Lane
H, midgut soluble cellular fraction; S1, low and S2 high ionic strength Superose eluates; Q1 and Q2, fractions 21–24 and 31–35 eluted from
Mono Q; A1 and A2, more active fractions pooled after elution from hydrophobic chromatography of Q1 and Q2 materials.
fractions of each peak were pooled, trying to avoid cross contamination. Two
β -glycosidases were purified to
homogeneity submitting these pools to hydrophobic chromatography.
The Mr of the β
-glycosidases obtained by SDS-PAGE are 47,000 and 50,000 Fig. 2. The recovery and enrich-
ment of β
-glycosidase activities are shown in Table 1. Taking into account that there are two activities in the
initial sample, the recovery of each β
-glycosidase is higher than the figures presented in Table 1.
3.2. N-terminal amino acids The N-terminal sequencing of the purified Mr 47,000
β -glycosidase resulted in the following sequence:
YTKFPNGFTFGVATASHQIEGAWNxxK, where
x denotes unidentified amino acid residues. The presence
of the motif QIEGA near the amino terminal is a charac- teristic of the family of the glycosyl hydrolases Rojas
et al., 1995.
Table 1 Purification of
β -glycosidases from S. frugiperda larval midgut
Fraction Specific
Yield Purification
activity factor
mUmg Cellular supernatant
14.6 100
1 Superose low ionic
47.4 85
3.2 strength eluate
Superose high ionic 172
57 11.8
strength eluate Mono Q eluate Q1
1441 19
98.7 Mono Q eluate Q2
4500 36
308 Alkyl Superose eluate A1 1041
12 71
Alkyl Superose eluate A2 5770 30
395
The N-terminal sequence of S. frugiperda β
-glycosid- ase has highest identity with plant
β -glycosidases Table
2. High identity is also seen with mammalian lactase– phlorizin hydrolase, which is located in the intestinal
epithelium and is responsible for lactose and glycosyl- ceramide digestion.
3.3. Specificity of purified Mr 47,000 b-glycosidase
The Mr 47,000 β
-glycosidase has a broad specificity, hydrolysing aryl-
β -glycosides NP
β Glu and NP
β Gal,
di- cellobiose, gentiobiose and lactose and oligo- saccharides cellotriose, cellotetraose and cellopentaose
and the cyanogenic glucoside amygdalin Table 3. The enzyme is also able to hydrolyse glucosylceramides 0.3
mUmg. As far as we know, this is the first time that the hydrolysis of glycolipids by an insect
β -glycosidase
is described. The enzyme is unable to hydrolyse the alkyl
β -gluco-
sides pentyl-, octyl- and decyl- β
-d-glucosides 2 mM, the disaccharide laminariobiose 7 mM, the polysac-
charides CMC 0.25 pv and laminarin 0.25 pv, and the plant glucoside phlorizin 1 mM. Prunasin, the
cyanogenic glucoside that arises after removing one glu- cosyl residue from amygdalin is poorly hydrolysed by
this enzyme, with a K
m
of more than 50 mM. Cellobiose is an enzyme substrate but is unable to
inhibit NP β
Gal hydrolysis Table 4. The simplest expla- nation for this result is that cellobiose and NP
β Gal are
hydrolysed at different sites in the β
-glycosidase called
Table 2 Percentage of identity and similarity of the N-terminal sequence from
S. frugiperda β
-glycosidase in relation to other β
-glycosidases
a
Enzyme Source
Identity Similarity
β -glycosidase
Caldocellum 76
85 saccharoliticum
β -glycosidase
Zea mays 71
90 β
-glycosidase Brassica napus
71 71
Prunasina hydrolase Prunus serotina
68 89
precursor β
-glycosidase Avena sativa
66 80
Dhurrinase Sorghum bicolor
66 80
Linamarase Manihot esculenta
61 83
Mirosinase Brasica napus
61 83
β -glycosidase
Manihot esculenta 61
80 Lactase-phloridzin
Oryctolagus cuniculus 60 78
hydrolase Lactase-phloridzin
Rattus rattus 56
78 hydrolase
Lactase-phloridzin Homo sapiens
56 78
hydrolase Cyanogenic precursor
Trifolium repens 47
69 β
-glycosidase
a
The amino acid sequence from Mr 47,000 β
-glycosidase was com- pared with sequences from data banks with the help of Blast software
www.ncbi.nlm.nih.gov.
1143 S.R. Marana et al. Insect Biochemistry and Molecular Biology 30 2000 1139–1146
Table 3 Substrate specificities of purified Mr 47,000
β -glycosidase. Data correspond to site 1 cellobiase site, except when otherwise specified site 2,
glycosylceramidase site Substrate
K
m
mM k
cat
s
21
k
cat
K
m
s
21
mM
21
Relative k
cat
K
m
NP β
Glu
a
0.32 ±
0.02 0.39
± 0.03
1.21 ±
0.05 100
Cellobiose 3.6
± 0.7
0.061 ±
0.004 0.017
± 0.004
1.3 Cellotriose
0.50 ±
0.07 0.0222
± 0.0004
0.044 ±
0.006 3.6
Cellotretraose 0.31
± 0.06
0.0135 ±
0.0003 0.044
± 0.008
3.4 Cellopentaose
0.29 ±
0.05 0.0135
± 0.0003
0.047 ±
0.007 3.6
Gentiobiose 0.6
± 0.1
0.098 ±
0.16 0.16
± 0.03
13 Amygdalin
0.3 ±
0.1 0.059
± 0.19
0.19 ±
0.07 16
NP β
Glu
a
site 2 1.2
± 0.1
0.11 ±
0.01 0.09
± 0.09
7 NP
β Gal site 2
2.4 ±
0.1 3.03
± 0.03
1.26 ±
0.06 100
Lactose site 2 50
± 10
0.44 ±
0.06 0.008
± 0.002
0.6
a
K
m
and V
m
for NP β
Glu were calculated from data obtained before and after inactivation of the cellobiase site details in Section 2. Activity on lactose is assigned to site 2 based on EDC modification see text. Figures are means and SEM.
Table 4 Inhibition of Mr 47,000
β -glycosidase
a
Substrate Inhibitor
K
i
mM Cellobiose
NP β
Gal 3.4
± 0.3
NP β
Gal NP
β Glu
1.0 ±
0.2 NP
β Gal
Cellobiose N.I.
NP β
Glu NP
β Gal
6.6 ±
0.3 NP
β Glu
Cellobiose 1.2
± 0.1
a
Substrates acting as inhibitors were simple linear competitive inhibitors. N.I., no inhibition. Figures are means and SEM. Details in
Section 2.
cellobiase and galactosidase sites in this study. NP β
Glu could be hydrolysed by both active sites, since neither
the K
i
of NP β
Gal nor the K
i
of cellobiose are equal to their corresponding K
m
values when NP β
Glu is used as substrate compare Tables 3 and 4. Amygdalin is hydro-
lysed only at the cellobiase site, since even a concen- tration of amygdalin as high as 15-fold its K
m
was unable to inhibit the hydrolysis of NP
β Gal at a concentration
equal to one K
m
. The presence of two different active sites in Mr
47,000 β
-glycosidase is also indicated by chemical modification experiments. EDC modifies carboxylates
that have been demonstrated to be catalytic groups in many glycosidases see White and Rose, 1997. The
β -
glycosidase inactivation by EDC plus glycine ethyl ester follows pseudo first-order kinetics. When the modifi-
cation reaction is done at pH 6.0, the activity upon cello- biose decreases, while the activity upon NP
β Gal remains
constant. The cellobiase activity inactivation is protected by the presence of 14 mM cellobiose or 12 mM NP
β Gal
in the reaction media Fig. 3. The results given above indicate that NP
β Gal can bind but is not hydrolysed at
the cellobiose site. The activity towards lactose is not affected by EDC
modification at pH 6 not shown. This indicates that this substrate is hydrolysed at the same site as NP
β Gal
galactosidase site. NP
β Glu is hydrolysed mainly at the cellobiase site,
although the galactoside site also displays some activity on this substrate. The K
m
and k
cat
for NP β
Glu were cal- culated in both active sites as followed: after 100 inac-
tivation of cellobiose activity by EDC at pH 6.0, the K
m
and k
cat
for NP β
Glu hydrolysis are, 1.2 mM and 0.11 s
21
, respectively. Taking into account the activity upon NP
β Glu before and after complete modification of the
cellobiase site, the kinetic parameters of the cellobiase site in relation to this substrate were estimated as:
K
m
= 0.32 mM, k
cat
= 0.39 s
21
.
4. Discussion
