Presence and properties of cellulase and hemicellulase enzymes of the gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes
School of Biological, Earth and Environmental Sciences, The University of NSW, Sydney, NSW 2052, Australia
* Author for correspondence at present address: School of Biological and Chemical Sciences, Faculty of Science and Technology, Deakin University Geelong Campus, Pigdons Road, Geelong, VIC 3217, Australia (e-mail: stuart.linton{at}deakin.edu.au)
Accepted 18 August 2004
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Summary |
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Key words: land crab, Gecarcoidea natalis, Discoplax hirtipes, cellulase, endo-ß-1, 4-glucanase, cellobiohydrolase, ß-1, 4-glucosidase, laminarinase, xylanase, licheninase, fibre digestion
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Introduction |
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Crystalline or native cellulose is hydrolysed to its component glucose
units by the combined activities of endo-ß-1,4-glucanase (EG),
cellobiohydrolase (CBH) and ß-1,4-glucosidase. Endo-ß,1-4-glucanase
randomly hydrolyses internal ß-1,4-glycosidic bonds of cellulose polymers
of four or more glucose units (Scrivener
and Slaytor, 1994; Tokuda et
al., 1997
; Wantanabe
et al., 1997
). It thus shortens cellulose chains,
solubilizes cellulose polymers and provides a substrate for
exo-ß,1-4-glucosidase. However, it can also catalyse transglycosylation
to rejoin the glucose oligomers (Lindner et
al., 1983
). Cellobiohydrolase attacks the non-reducing
ends of cellulose chains and typically cleaves off the glucose-ß-1,4
dimer, cellobiose. Cellobiohydrolase is also thought to disrupt the hydrogen
bonding in crystalline cellulose, thus allowing the EG to endo-depolymerize.
As this enzyme only attacks the ends of chains, its activity will be low
unless endoglucanase is present to provide sufficient substrate. In the
cockroach Panesthia cribrata and the termite Reticulitermes
speratus, CBH activity is also catalyzed by the EG
(Scrivener and Slaytor, 1994
;
Watanabe et al.,
1997
). The third enzyme involved in the complete hydrolysis of
cellulose to glucose is ß-1,4-glucosidase; this enzyme hydrolyses
cellooligosaccharides to glucose.
The term hemicellulose includes a range of alkali-soluble polysaccharides
(Bacic et al., 1988).
More specifically, they are defined as carbohydrate polymers of either xylose,
glucose, mannose or mannose and glucose joined mainly by ß-1,4 and
ß-1,3 glycosidic bonds (Bacic et al.,
1988
). Xylan, lichenin, ß-D-glucan and laminarin
are common hemicelluloses. Xylan, the next most abundant carbohydrate polymer
after cellulose, is a ß-1,4-linked polymer of xylose
(Puls et al., 1988
). Lichenin
is a glucose polymer with the glucose units being joined with mainly
ß-1,4 glycosidic bonds and some ß-1,3 glycosidic bonds in the chain
(Bacic et al., 1988
).
Similar polysaccharides, called mixed linkage ß-glucan, are found in
cereals and grasses (Terra and Ferreira,
1994
). Like lichenin, laminarin (or callose) is a glucose polymer
but with the sugars joined principally by ß-1,3 glycosidic bonds
(Bacic et al., 1988
;
Terra and Ferreira, 1994
).
Laminarin is present in the cell walls of fungi, in phloem and in plant wound
tissue (Terra and Ferreira,
1994
). Laminarin is the chief food reserve of algae
(Vonk and Western, 1984
).
Xylanase, licheninase and laminarinase hydrolyse xylan, lichenin and
laminarin, respectively (reviewed by Terra
and Ferreira, 1994
).
The diet of the gecarcinid land crabs Gecarcoidea natalis and
Discoplax hirtipes mainly consists of green and brown leaves
(Greenaway and Linton, 1995;
Greenaway and Raghaven, 1998
)
of which cellulose and the hemicelluloses are major constituents. Cellulose
makes up 1218% of the dry matter of brown leaves of the fig (Ficus
macrophylla) and 11% of the dry matter of green leaves
(Greenaway and Linton, 1995
;
Greenaway and Raghaven, 1998
),
while hemicelluloses constitute 1826% of the dry matter of brown fig
leaves (Greenaway and Linton,
1995
; Greenaway and Raghaven,
1998
). Both G. natalis and D. hirtipes
assimilate substantial amounts of cellulose and hemicellulose from their leaf
litter diet (Greenaway and Linton,
1995
; Greenaway and Raghaven,
1998
); 43% and 49%, respectively, by G. natalis and 21%
and 20%, respectively, by D. hirtipes fed brown leaf litter of F.
macrophylla (Greenaway and Linton,
1995
; Greenaway and Raghaven,
1998
). As the crabs clearly hydrolyse these fibre components, it
is probable that they possess cellulase and hemicellulase enzymes. In the
present study, we investigated the presence and characteristics of cellulases
and hemicellulases from G. natalis and D. hirtipes. Total
cellulase activity and activities of ß-1,4-glucosidase, EG, licheninase,
laminarinase and xylanase were measured within the digestive juice of both
species. Where possible, the kinetics (Vmax and
Km) of the cellulase enzymes and the inhibitory constant
(Ki) of glucono-D-lactone on
ß-1,4-glucosidase were also determined. It was envisaged that differences
in the activities of the cellulase enzymes may explain reported differences in
the assimilation of cellulose and hemicellulose between these two species.
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Materials and methods |
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Methods
Digestive juice used in measurements was taken from the foreguts of
experimental animals as follows. The crabs were held ventral side up on a
polystyrene board, and a fine polythene tube was inserted into the cardiac
stomach via the mouth and oesophagus. A small plastic wedge was used
to prevent the mandibles from cutting the tube. Up to 2 ml of dark brown
digestive juice could be collected by gentle suction with a 2-ml syringe
attached to the tubing. The procedure did not harm the crabs. Fluid was
centrifuged at 10 000 g for 5 (min, to remove food debris, and
the supernatant was used for analyses. Fluid could be stored at 4°C for
several days without loss of enzyme activity.
Measurement of enzyme activities
Cellulase activities
Total cellulase activity and activities of ß-1,4-glucosidase
(cellobiase; EC 3.2.1.21) and EG (EC 3.2.1.4) were measured in digestive juice
taken from the cardiac stomach of Gecarcoidea natalis and
Discoplax hirtipes using modified versions of the methods of Schulz
et al. (1986) and Hogan et al.
(1988
). Reactions and
incubations were carried out at 40°C in 1.5-ml Eppendorf centrifuge tubes
in an Eppendorf thermomixer. Measurement at 40°C allowed direct comparison
with data for cellulase activities of other invertebrates. Absorption values
of the samples were measured using an LKB Ultraspec II spectrophotometer.
Activities of the enzymes are presented per ml of digestive juice. Expression
per mg of protein is not meaningful in this situation where crude juice is
used since the bulk of the protein does not represent the enzyme of interest,
is highly variable and may even be dietary in origin. It is likely that the
volume of fluid in the foregut remains relatively constant. Hanes plots
derived from the data on cellulase activity at different substrate
concentrations were used to determine if enzyme activity followed
Michaelis-Menten kinetics. Where this was established, the kinetic parameters
(Km, Vmax) were then calculated from
the plots.
ß-1,4-glucosidase. Activity was measured as the rate of production of glucose from cellobiose (Cat. No. C-7252; Sigma Chemical Corp., St Louis, MO, USA). Digestive juice (25 µl) was mixed with 25 µl of 0.1 mol l-1 acetate buffer (pH 5.5) and 50 µl of either 2.92, 14.61, 29.21 or 58.4 mmol l-1 cellobiose in the same buffer, and the mixture was incubated at 40°C for 30 min. The reaction was stopped by the addition of 25 µl of 0.3 mol l-1 tri-chloro acetic acid, and excess acid was neutralized with 5 µl of 2.5 mol l-1 K2CO3. Precipitated protein was pelleted by centrifugation at 10 000 g for 10 min. A blank (75 µl buffer plus 25 µl digestive juice) and a standard (50 µl of 7 mol l-1 glucose in buffer + 25 µl digestive juice + 25 µl buffer) were prepared for each sample analysed. This enabled correction for the background absorption due to the digestive juice at the wavelength measured.
Glucose concentration was measured in 50 µl (G. natalis) or 25 µl (D. hirtipes) samples of the incubation mixture using a commercial glucose assay kit (Sigma Cat No. 510-A). The 50 µl or 25 µl samples were diluted to a total of 100 µl with water in a 1.5-ml micro test tube. 1 ml of the colour reagent supplied with the kit was then added, and the mixture was vortexed and incubated at 37°C for 30 min. After incubation, the absorbance of the samples was read at 445 nm.
Glucono-D-lactone competitively inhibits ß-1,4-glucosidase
(Scrivener and Slaytor, 1994;
Shewale and Sadana, 1981
;
Santos and Terra, 1985
). The
inhibitory constant of glucono-D-lactone on ß-1,4-glucosidase
was also determined by measuring ß-1,4-glucosidase activity in the
presence of 20 mmol l-1 glucono-D-lactone and
058.43 mmol l-1 cellobiose.
Endo-ß-1,4-glucanase. Activity was measured as the
rate of production of reducing sugars from the substrate, carboxymethyl
cellulose (Sigma Cat. No. C-5678). Digestive juice (20 µl) was mixed with
80 µl buffer and 100 µl of 0.1, 0.5, 1 and 2% (w/v) carboxymethyl
cellulose in the same buffer. Blanks contained 20 µl of digestive juice and
180 µl of buffer, while standards contained 20 µl of digestive juice
plus 100 µl glucose (13 mmol l-1) in buffer and 80 µl buffer.
The buffer was 0.1 mol l-1 acetate buffer, pH 5.5, containing 30
mmol l-1 of the ß-1,4-glucosidase inhibitor
glucono-D-lactone. Samples, standards and blanks were incubated at
40°C for 10 min and the reaction stopped by the addition of 25 µl of
0.3 mol l-1 HCl. Excess acid was then neutralized by the addition
of 5 µl of 2.5 mol l-1 K2CO3. The reducing
sugar produced during the incubation was measured as glucose equivalents by
the tetrazolium blue method of Jue and Lipke
(1985) using 5 mmol
l-1 glucose as a standard. Absorption of samples, standards and
blanks was read at 660 nm.
Total cellulase activity. Total cellulase activity was measured as the rate of production of glucose from microcrystalline cellulose (Sigmacell 20). Digestive juice (50 µl) was mixed with 100 µl of either 0.1, 0.5, 1 or 2% (w/v) Sigmacell 20 (Sigma Cat. No. S3504) made up in buffer. Suspension of the cellulose was ensured by vortexing the stock cellulose immediately before pipetting. Blanks contained digestive juice and buffer while standards contained digestive juice, buffer and 7 mmol l-1 glucose. The buffer used was 0.1 mol l-1 acetate, pH 5.5. The mixture was incubated and agitated for 60 min at 40°C in an Eppendorf thermomixer before the reaction was stopped by the addition of 25 µl of 0.3 mol l-1 tri-chloro acetic acid. The excess acid was neutralized with 5 µl of 2.5 mol l-1 K2CO3 before assay of glucose. The incubation mixture was centrifuged (10 000 g for 10 min) and the glucose concentration determined in a 25 µl or 50 µl aliquot of the supernatant as described for ß-1,4-glucosidase.
The Km values for EG and CBH are given as mg substrate ml-1 reaction mixture since the substrates (carboxymethyl cellulose and cellulose) consist of carbohydrate polymers of varying length and do not have a uniform molecular mass.
Hemicellulase activities
Activities of the hemicellulase enzymes laminarinase
[endo-ß-1,3-glucanase (EC 3.2.1.39)], licheninase [endo-ß-1,3; 1,4
glucanase (EC 3.2.1.73)], xylanase [endo-ß-1,4-xylanase (EC 3.2.1.8) and
1,4-ß-D-xylan xylanhydrolase (EC 3.2.1.37)] were measured in
the digestive juice from D. hirtipes and G. natalis.
Laminarinase. Laminarinase activity was measured as the production of reducing sugars from the hydrolysis of laminarin (from Laminaria digitata; Sigma Cat. No. L-9634). Digestive juice (20 µl) was mixed with 50 µl of 1% (w/v) laminarin and 130 µl of 0.1 mol l-1 Na acetate buffer, pH 5.5. Blanks and standards were run at the same time. Blanks consisted of 20 µl of digestive juice and 180 µl of assay buffer, while standards consisted of 20 µl of digestive juice, 100 µl of 13 mmol l-1 glucose and 80 µl of assay buffer. Samples, blanks and standards were incubated with agitation at 40°C for 10 min. The reaction was stopped by the addition of 50 µl of 0.3 mol l-1 HCl and neutralized with 10 µl of 2.5 mol l-1 K2CO3. Reducing sugars were measured in a 10 µl aliquot as described above.
Licheninase. The activity of licheninase was measured by the production of reducing sugars from the hydrolysis of lichenin (from Cetraria islandica; Sigma Cat. No. L-6133). Digestive juice (20 µl) was mixed with 100 µl of 0.1% (w/v) lichenin and 80 µl of 0.1 mol l-1 Na acetate buffer, pH 5.5. To correct for the background absorbance of the digestive juice, blanks and standards were run at the same time. Digestive juice (20 µl) and 180 µl of assay buffer constituted the blank while 20 µl of digestive juice plus 100 µl of 13 mmol l-1 glucose and 80 µl of assay buffer constituted the standard. Samples, blanks and standards were incubated with agitation at 40°C for 10 min. The reaction was stopped with 50 µl of 0.3 mol l-1 HCl and the mixture was then neutralized with 10 µl of 2.5 mol l-1 K2CO3. Reducing sugars were measured in a 10 µl sample as described above.
Xylanase. Xylanase activity was measured as the production of reducing sugars from the hydrolysis of xylan (from birchwood, Betula; Sigma Cat. No. X-0502). Digestive juice (20 µl) was incubated with 100 µl of 1% (w/v) xylan and 80 µl of 0.1 mol l-1 Na acetate buffer, pH 5.5. Blanks (20 µl of digestive juice and 180 µl of buffer) and standards (20 µl of digestive juice, 100 µl of 13 mmol l-1 glucose and 80 µl of assay buffer) were run at the same time. Samples, blanks and standards were incubated with agitation at 40°C for 60 min. After this period, the reaction was stopped by precipitating the protein with 50 µl of 0.3 mol l-1 HCl and was neutralized by the addition of 10 µl of 2.5 mol l-1 K2CO3. Reducing sugars were measured in a 10 µl sample of this reaction mixture as described above.
pH
The activities of ß-1,4-glucosidase, EG and total cellulase were
measured over a pH range of 49. Acetate buffer was used to maintain pH
values of 4 and 5.5, and Tris buffer was used for the higher pH values of
79. The pH of gut fluid was measured anaerobically at 25°C using
freshly drawn samples of fluid from the foregut and a Radiometer G299a
capillary pH electrode and Radiometer PHM 73 meter (Radiometer, Copenhagen,
Denmark).
Protein
The concentrations of protein in samples of digestive juice were measured
using a BioRad protein assay kit and bovine -globulin standard (BioRad,
Hercules, CA, USA).
Statistics
Statistical comparisons (ANCOVA and one- and two-way ANOVA with Tukey's HSD
post hoc tests) were made using the statistical computing package
Systat 7 (Systat Software Inc., Richmond, CA, USA) to calculate the
statistical probabilities.
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Results |
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The presence of EG was confirmed by the production of reducing sugars during the incubation of carboxymethyl cellulose with digestive juice (Fig. 2). Carboxymethyl cellulose is not a substrate for either CBH or ß-1,4-glucosidase. In similar fashion, the presence of ß-1,4-glucosidase in digestive juice was confirmed by the production of significant quantities of glucose when cellobiose was incubated with digestive juice (Fig. 3). This enzyme is generally ineffective against crystalline or carboxymethyl cellulose. The activities of total cellulase, EG and ß-1,4-glucosidase, from both G. natalis and D. hirtipes, initially increased with substrate concentration but levelled out at high concentrations (Figs 1, 2, 3), consistent with saturation of these enzymes at the higher substrate concentrations.
pH and cellulase activities
Foregut fluid from both study species was slightly acidic, with that of
D. hirtipes (6.03±0.04, N=7) significantly more acid
than the juice from G. natalis (6.69±0.03, N=4)
(t-test; P<0.001). The activities of
ß-1,4-glucosidase, EG and total cellulase from both G. natalis
and D. hirtipes were affected by the pH of the incubation buffer
(Table 1) and shared similar pH
maxima and hence pH ranges for optimal activity. For total cellulase from
G. natalis, optimal activity occurred at pH 5.5, but for D.
hirtipes total cellulase activities were low with no obvious pH optimum
(Table 1). For
ß-1,4-glucosidase, optimal activity occurred between pH 4 and 7
(Table 1); high levels of
activity were measured at both pH 4 and pH 5.5 for G. natalis and at
pH 4, 5.5 and 7 for D. hirtipes
(Table 1).
Endo-ß-1,4-glucanase activities were maximal at pH 5.5, 7 and 9 and lower
at pH 4 and 8 in digestive juice from both D. hirtipes and G.
natalis (Table 1).
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Enzyme activities
Total cellulase activity
The total cellulase activity on crystalline cellulose was higher in the
digestive juice from G. natalis than in that from D.
hirtipes (Fig. 1);
2.8x higher at the highest substrate concentration used of 13.3 mg
ml-1 cellulose (Table
2). Total cellulase activity from G. natalis followed
Michaelis-Menten kinetics since the Hanes plot of its activity was linear
(r2=0.624, P=0.001;
Fig. 4). Total cellulase
activity had a Km of 2.43 mg ml-1 cellulose
incubation mixture and a Vmax of 0.117 µmol
min-1 ml-1 digestive juice
(Table 2). By contrast, the
Hanes plot of total cellulase activity from D. hirtipes was not
linear (r2=0.003, P=0.774;
Fig. 4) and did not follow
Michaelis-Menten kinetics (Fig.
4), so its kinetic parameters could not be calculated.
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Endo-ß-1,4-glucanase
Endo-ß-1,4-glucanase activities in digestive juice were slightly, but
consistently, higher in G. natalis than in D. hirtipes
(P<0.05; two-way ANOVA; Fig.
2). The differences in activities between species were maintained
across the range of substrate concentration used. Endo-ß-1,4-glucanase
activity from G. natalis was 1.17x the EG activity from D.
hirtipes at a substrate concentration of 10 mg ml-1 CMC
(Table 2). The Hanes plot of
the EG activity from G. natalis was linear
(r2=0.514, P=0.0001) while that from D.
hirtipes was not (r2=0.041, P=0.2;
Fig. 5). Hence, the EG from
G. natalis followed Michaelis-Menten kinetics
(Fig. 5) with a
Km of 3.03 mg ml-1 CMC incubation mixture and a
Vmax of 6.11 µmol min-1 ml-1
digestive juice (Table 2). Equivalent values could not be calculated for EG from D.
hirtipes.
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ß-1,4-glucosidase
The activity of ß-1,4-glucosidase from D. hirtipes was higher
than that from G. natalis (P<0.05; two-way ANOVA;
Fig. 3). At a cellobiose
concentration of 29.22 mmol l-1, the activities differed by a
factor of 2.8 (Fig. 3;
Table 2).
ß-1,4-Glucosidase from both species followed Michaelis-Menten kinetics
since Hanes plots of ß-glucosidase activities were linear
(Fig. 6). The
Vmax of ß-1,4-glucosidase from D. hirtipes
was 3.4x higher than that from G. natalis (P<0.005;
ANCOVA; Table 2) and the
Km value for ß-1,4-glucosidase from D.
hirtipes was lower than that from G. natalis
(P<0.001; ANCOVA; Table
2). Hence, ß-1,4-glucosidase activity from D.
hirtipes saturated at lower concentrations of cellobiose and was higher
at all substrate concentrations than ß-1,4-glucosidase from G.
natalis.
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Effect of inhibitor on the activity of ß-1,4-glucosidase
Vmax values for inhibited and uninhibited
ß-glucosidase were similar in both G. natalis and D.
hirtipes (Figs 3,
6;
Table 2). The
Km of ß-1,4-glucosidase in the presence of 20 mmol
l-1 glucono-D-lactone was much higher than in its
absence (Figs 3,
6;
Table 2).
This situation (similar Vmax but different
Km values in the presence and absence of an inhibitor) is
indicative of competitive inhibition of ß-1,4-glucosidase by
glucono-D-lactone in both species. Glucono-D-lactone has
also been identified as a competitive inhibitor of ß-1,4-glucosidase in
previous studies (Scrivener and Slaytor,
1994; Shewale and Sadana,
1981
; Santos and Terra,
1985
). The inhibitory constant of glucono-D-lactone on
ß-1,4-glucosidase (Ki) was calculated using
Km values from Table
2 and the equation:
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The Ki of glucono-D-lactone on ß-1,4-glucosidase was 11.12 mmol l-1 for ß-1,4-glucosidase from G. natalis and 4.53 mmol l-1 for ß-1,4-glucosidase from D. hirtipes.
Hemicellulase activities in the digestive juice
Incubation of laminarin, lichenin or xylan with digestive juice from G.
natalis or D. hirtipes yielded reducing sugars
(Table 3), an outcome
consistent with the presence of laminarinase, licheninase and xylanase in the
digestive juice. The activities of the enzymes in the digestive juice of both
G. natalis and D. hirtipes were in the order laminarinase
> licheninase > xylanase (Table
3). Licheninase activity in the digestive juice from D.
hirtipes was 1.73x that of G. natalis
(Table 3) whilst the activities
of laminarinase were similar between species and this was also the case for
xylanase (Table 3).
|
Activities of the individual hemicellulases (licheninase, laminarinase and xylanase) measured were higher than those for total cellulase activity in the two species (Tables 2, 3).Laminarinase activity was much higher than the activity of EG and ß-1,4-glucosidase (Tables 2, 3). For G. natalis, licheninase activity was similar to EG activity but higher than ß-1,4-glucosidase activity (Tables 2, 3). Licheninase activity from D. hirtipes was higher than activities of both EG and ß-1,4-glucosidase (Tables 2, 3). Xylanase activities for both G. natalis and D. hirtipes were lower than the respective EG and ß-glucosidase activities (Tables 2, 3).
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Discussion |
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Cellulase activity
Total cellulase and EG activities from G. natalis were higher than
those from D. hirtipes (Figs
1,
2) and this should allow more
rapid hydrolysis of cellulose by G. natalis, which is consistent with
the earlier reports of higher cellulose assimilation efficiency in the latter
species (Greenaway and Linton,
1995; Greenaway and Raghaven,
1998
).
In G. natalis, the Km for EG was similar to values reported for other invertebrates (Tables 2, 4). The EG activity reported here may well exceed the in vivo value since the experimental substrate (carboxymethyl cellulose), unlike the natural substrate (crystalline cellulose), is in solution and more available for enzymatic hydrolysis.
|
ß-1,4-glucosidase
ß-1,4-glucosidase activity in the digestive juice exhibited a higher
Vmax and substrate affinity in D. hirtipes than
in G. natalis (Fig. 3;
Table 2). This contrasted with
the findings for the other cellulase enzymes EG and total cellulase
activity where activity was higher in G. natalis. This
difference may be explained by the affinity of ß-1,4-glucosidase for the
inhibitor glucono-D-lactone and consequent substrate and product
affinity (discussed below).
Competitive inhibition of ß-1,4-glucosidase with glucono-D-lactone
The inhibition constant (Ki) for ß-1,4-glucosidase
was higher in G. natalis (11.12 mmol l-1) than in D.
hirtipes (4.53 mmol l-1), and hence the ß-1,4-glucosidase
from G. natalis had a lower affinity for the inhibitor than that from
D. hirtipes. This is consistent with the ß-1,4-glucosidase from
G. natalis being resistant to inhibition. The high
Ki of ß-glucosidase from both gecarcinid species
relative to Ki values for insects
(Table 4) indicates that this
enzyme is not particularly sensitive to inhibition by
glucono-D-lactone in gecarcinids.
Glucono-D-lactone inhibits activity of ß-1,4-glucosidase by
mimicking the transitional form involved in the hydrolysis of the
ß-glycosidic bond (Terra and
Ferreira, 1994). A lower affinity for the transitional form, and
hence glucono-D-lactone, means that this form is not stabilized as
well and the reaction would proceed at a slower rate, and this may explain why
the digestive juice of G. natalis had a lower ß-glucosidase
activity than that from D. hirtipes
(Fig. 3). Reduced
ß-1,4-glucosidase activity would not affect the overall rate of cellulose
hydrolysis unless its activity was lower than that of the slowest step,
probably the solubilization and initial depolymerization of the cellulose
chains. A lower affinity for the transitional form would also mean that the
affinity of the enzyme for its substrate and product would be reduced. This
could account for lower substrate affinity exhibited by the
ß-1,4-glucosidase from G. natalis (cf. D. hirtipes;
Fig. 3). In comparison with
insects, the affinity of ß-1,4-glucosidase from G. natalis was
low since its Km was in the upper range of the
Km for these animals (Tables
2,
4).
The inhibition resistance of ß-1,4-glucosidase may counteract product inhibition. This means that the cellulase enzymes of G. natalis can hydrolyse cellulose despite the presence of the end product, glucose. Such a property would contribute to the overall efficiency of the cellulase system of G. natalis. The cellulase enzymes of G. natalis may also be able to hydrolyse cellulose even in the presence of high levels of sugars from other dietary items such as fruits and seeds. High concentrations of glucose may be present in the stomach given that the stomach is the site of mastication and some enzymatic digestion while the midgut gland is the site of absorption.
By contrast, the cellulases of D. hirtipes had low activity and
were susceptible to product inhibition, and overall cellulose hydrolysis in
this species was not as efficient as that in G. natalis. Thus, D.
hirtipes may require a diet that contains a greater amount of more
readily digestible material to satisfy its energy requirements. This could
explain why D. hirtipes prefers green to brown leaves while G.
natalis shows no preference (Greenaway
and Raghaven, 1998). Green leaves contain more digestible
components such as cellular proteins, storage carbohydrates and lipids and
less cellulose than brown leaves (Greenaway
and Raghaven, 1998
).
pH optima for cellulase activity
The activities of the cellulases examined were maintained across quite a
broad range of pH (Table 1) but
with maximal activities in the pH range 5.57. As the measured pH of the
digestive juices of the study species (6.69±0.03 for G.
natalis and 6.03±0.04 for D. hirtipes) fell within this
range, the cellulase enzymes will operate at or near their maximal activity.
EG and ß-1,4-glucosidase from insects and the crustacean Cherax
quadricarinatus had similar pH maxima, ranging between pH 4 and 6
(Watanabe et al.,
1997; Tokuda et
al.,1997
; Table
4). The pH values of the digestive juices of G. natalis
and D. hirtipes were similar to the pH of the gut of the herbivorous
isopod Porcellio scaber (pH 5.56.5;
Zimmer and Topp, 1997
).
Endo-ß-1,4-glucanase from both G. natalis and D.
hirtipes exhibited additional activity peaks at pH 9
(Table 1), however this is
considered to be an artefact of the assay and would not occur in
vivo.
Tannins inhibit digestive enzymes by binding to the protein and
precipitating it. They precipitate more protein under acid than under alkaline
conditions, and certain insects [e.g. the cricket Telleogryllus
commodus, the New Zealand grass grub (Costelytra zealandica) and
the gypsy moth (Lymantria dispar)] have highly alkaline guts (pH
range 811) to counteract the precipitation of enzymes by dietary
tannins (Cooper and Vulcano,
1997; Briggs and McGregor,
1996
; Schultz and Lechowicz,
1986
). The foregut fluid from G. natalis was slightly
more alkaline than that of D. hirtipes, but the small difference is
unlikely to significantly alter the effects of dietary tannins on the
digestive enzymes of the two species as both pH values fell within the acid
range.
Hemicellulase activities
Digestive juices from the gecarcinid land crabs G. natalis and
D. hirtipes hydrolyse laminarin, lichenin and xylan and so contain
activities of the hemicellulases laminarinase, licheninase and xylanase. Given
that cellulose, laminarin, lichenin and xylose are carbohydrate polymers with
sugar units joined by ß-glycosidic bonds, it is possible that only one or
two multicatalytic enzymes may catalyse all the reactions rather than multiple
enzymes each catalysing one reaction. Exactly how many ß-glycohydrolases
are present in the digestive juice of the gecarcinid crabs is unknown and
requires further investigation.
The respective activities of ß-glycohydrolases in the study species
were laminarinase > licheninase > xylanase > total cellulase.
Consequently, assimilation of the different hemicelluloses and cellulose would
be expected to follow a similar rank order, notably laminarin > lichenin
> xylan > cellulose (Tables
2,
3). Thus, in diets containing
equal amounts of hemicellulose and cellulose, both G. natalis and
D. hirtipes would be expected to gain more carbohydrate from
hemicellulose than cellulose. The high activities of laminarinase in the gut
fluid of G. natalis and D. hirtipes indicate that they are
particularly efficient in the hydrolysis of laminarin
(Table 3). Laminarin is the
chief food reserve of algae (Vonk and
Western, 1984), and both species have been observed to scrape
algae from rocks, soil and tree root buttresses (S.M.L., personal
observation). Mixed ß-D-glucans are a major component of
cereals and grasses (Terra and Ferreira,
1994
; McCleary,
1988
). Gecarcinid crabs are able to utilise
ß-D-glucans since both G. natalis and D.
hirtipes possess moderate licheninase activity
(Table 3). D. hirtipes
is better able to assimilate lichenin than G. natalis since its
digestive juice has a higher licheninase activity
(Table 3). G. natalis
and D. hirtipes possess low xylanase activities and will only be able
to hydrolyse xylan slowly.
Differences in assimilation coefficients for hemicellulose reported
previously for the study species (Greenaway
and Linton, 1995; Greenaway and
Raghaven, 1998
) cannot be interpreted in terms of hemicellulase
activities measured in this study, as the types of hemicellulose present in
Ficus and Erythrina leaves are not known.
Differences in cellulase activities and properties may explain differences in cellulase assimilation and dietary preferences
G. natalis had a higher cellulose assimilation efficiency than
D. hirtipes when fed a diet of brown fig leaves (43% and 21%,
respectively; Greenaway and Linton,
1995; Greenaway and Raghaven,
1998
), and the current study confirms that G. natalis
hydrolyses cellulose more effectively than D. hirtipes since it has
higher total cellulase activity. In addition, the ß-1,4-glucosidase from
G. natalis is resistant to product inhibition and consequently it can
hydrolyse cellulose despite the presence of high levels of glucose from
cellulose hydrolysis or simple sugars from other dietary items such as fruit.
This property may also be indicative of resistance of the enzyme to the
effects of dietary tannins.
Origin of the cellulase and hemicellulase enzymes
Cellulase and hemicellulase enzymes may be either produced endogenously or
by microorganisms within the gut. Endogenous production is possible in the
study species, given that other invertebrate groups such as insects, molluscs
and nematodes possess a gene for EG and synthesize this enzyme endogenously
(reviewed by Watanabe and Tokuda,
2001). The origin of cellulases in gecarcinid crabs is
investigated in a subsequent manuscript.
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Acknowledgments |
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References |
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