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First published online June 27, 2008
Journal of Experimental Biology 211, 2275-2287 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.017657
Purification and characterisation of endo-β-1,4-glucanase and laminarinase enzymes from the gecarcinid land crab Gecarcoidea natalis and the aquatic crayfish Cherax destructor
School of Life and Environmental Sciences, Deakin University, Pigdons Road, Geelong, Victoria, 3217, Australia
* Author for correspondence (e-mail: balla{at}deakin.edu.au)
Accepted 1 May 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: land crab, Gecarcoidea natalis, Cherax destructor, laminarinase, endo-β-1,4-glucanase
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). These animals are capable of significant cellulose and
hemicellulose assimilation; these compounds represent a significant source of
energy (Linton and Greenaway,
2007). For example, gecarcinid land crabs such as the Christmas
Island red crab, Gecarcoidea natalis, and the Christmas Island blue
crab, Discoplax hirtipes, are able to assimilate 26–43% of
cellulose and 14–49% of hemicellulose from a diet of brown leaf litter
(Greenaway and Linton, 1995;
Greenaway and Raghaven, 1998).
This can be attributed to the activities of the enzymes cellulase (e.g.
endo-β-1,4-glucanase and β-1,4-glucosidase) and hemicellulase (e.g.
xylanase, lichenase and laminarinase), which hydrolyse cellulose and
hemicellulose, respectively, to their component sugars
(Linton and Greenaway,
2007).
Cellulose hydrolysis involves the combined action of
endo-β-1,4-glucanase, cellobiohydrase and β-1,4-glucosidase
(Watanabe and Tokuda, 2001).
Endo-β-1,4-glucanase randomly cleaves β-1,4-glycosidic bonds within
cellulose chains, releasing smaller fragments of random length
(Watanabe and Tokuda, 2001).
These fragments are then processed by cellobiohydrase, progressively releasing
cellobiose units from the non-reducing end of the molecule
(Watanabe and Tokuda, 2001).
Finally, the cellobiose disaccharides are hydrolysed by β-1,4-glucosidase
to release two free glucose molecules
(Genta et al., 2003;
Wang et al., 2004;
Watanabe and Tokuda, 2001).
Crustaceans, like other arthropods, are purported to lack cellobiohydrase and
the combined action of endo-β-1,4-glucanase and β-1,4-glucosidase
may hydrolyse cellulose completely in arthropods
(Scrivener and Slaytor, 1994).
Endo-β-1,4-glucanase (EC 3.2.1.4) is a key enzyme involved in cellulose
hydrolysis since it is capable of hydrolysing β-1,4-glycosidic bonds and
in arthropods it may function as a cellobiohydrase
(Scrivener and Slaytor,
1994).
Crustaceans, like arthropods generally, are able to synthesise
endo-β-1,4-glucanase endogenously
(Byrne et al., 1999;
Davison and Blaxter, 2005;
Linton et al., 2006); the
enzyme is a GHF9 glycosyl hydrolase, which in crustaceans is produced by F
cells of the midgut gland (Byrne et al.,
1999). Zymograms suggest that it consists of a 30 or 40 kDa
protein and is capable of hydrolysing both β-1,3- and
β-1,4-glycosidic bonds (Xue et al.,
1999). While endo-β-1,4-glucanase activity has been readily
measured in crustaceans, the enzyme is yet to be purified and characterised in
terms of molecular mass, substrate specificity, reaction catalysed and kinetic
parameters. Such characterisation would allow a better understanding of the
enzymatic mechanism of cellulose hydrolysis in crustaceans and more generally
in other invertebrates that consume plant material.
In addition to cellulose, crustaceans consuming plant material and algae
will also encounter large amounts of hemicellulose
(Linton and Greenaway, 2007).
Laminarin, the major storage polysaccharide of algae, is one such
hemicellulose consisting of glucose monomers joined together by mainly
β-1,3-glycosidic bonds with some β-1,6-glycosidic bonds
(Bacic et al., 1988). The
molecule, also known by its alternative name callose, is also present in the
cell walls of fungi, plant wound tissue and transient structures such as
pollen mother walls, sieve plates and cotton seed hairs
(Bacic et al., 1988;
Ruiz-Herrera, 1992;
Terra and Ferreira, 1994).
Laminarinase is a hemicellulase enzyme that is capable of hydrolysing
laminarin or callose. To date, two laminarinases have been identified,
endo-β-1,3(4)-glucanase (EC 3.2.1.6) and endo-β-1,3-glucanase (EC
3.2.1.39); endo-β-1,3(4)-glucanase (EC 3.2.1.6) is capable of hydrolysing
both β-1,3- and β-1,4-glycosidic bonds, while
endo-β-1,3-glucanase (EC 3.2.1.39) is capable of hydrolysing mainly
β-1,3-glycosidic bonds (Boeckmann et
al., 2003; Terra and Ferreira,
1994). Laminarinase may also work synergistically with cellulases
such as endo-β-1,4-glucanase to hydrolyse structural polysaccharides
within the plant cell wall (Mansfield et
al., 1999). Although laminarinase activity has been detected in a
range of herbivorous crustacean species
(Linton and Greenaway, 2007),
it is unclear which laminarinase is present, what the catalytic properties of
the enzyme are, and its corresponding function.
Gecarcinid land crabs such as G. natalis and D. hirtipes
are specialist herbivores, whose ancestors adopted a mainly leaf litter diet
during the colonisation of land (Greenaway
and Linton, 1995; Greenaway
and Raghaven, 1998). During this adoption, cellulase and
hemicellulase enzymes, which appear to be present in their aquatic ancestors,
must have become adapted to efficiently hydrolyse the increasing amounts of
cellulose and hemicellulose associated with the intake of terrestrial plant
material. The nature of these adaptations is unknown, but they may have
included alteration of substrate specificity and kinetic parameters
(Km and Vmax). The substrate
specificity may have broadened to allow the utilisation of various plant
structural compounds. Conversely it may have become more specific, increasing
the efficiency of cellulose hydrolysis. Purifying and characterising cellulase
and hemicellulase enzymes from a herbivorous land crab and a distant aquatic
relative such as a freshwater crayfish may help to elucidate these
adaptations.
In this study we have purified and characterised the key enzymes endo-β-1,4-glucanase and laminarinase from the gecarcinid land crab G. natalis, and the freshwater crayfish Cherax destructor. To elucidate possible adaptations towards the hydrolysis of cellulose in a terrestrial plant diet, the characteristics of the purified enzymes from G. natalis and C. destructor were compared.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Preparation of midgut gland homogenate
Prior to dissection, animals were anaesthetised on ice for at least 30 min,
and killed by removing the carapace and heart. Midgut glands from three G.
natalis and between five and eight C. destructor were removed,
pooled and homogenised using a Tissumizer homogeniser (Tekmar, Cincinnati, OH,
USA) in two volumes of homogenisation buffer (100 mmol l–1
sodium acetate, pH 5.5, containing 1 mmol l–1
dithioerythritol and 0.2 mmol l–1 phenylmethylsulphonyl
fluoride). The homogenate was filtered through two layers of wet cheesecloth
and spun at 2060 g for 30 min. The supernatant was then
removed, its volume measured, and kept for ammonium sulphate
precipitation.
Protein separation
Ammonium sulphate precipitation
Ammonium sulphate was slowly added to the supernatant until its
concentration reached 30% of saturation. This solution was then incubated with
stirring for 3.5 h at 4°C to allow protein precipitation. The suspension
was centrifuged at 2060 g for 30 min at 4°C using a
Beckman AllegraTM 21R centrifuge (Beckman Coulter, Fullerton, CA, USA),
the supernatant removed, its volume measured and the protein pellet kept. This
procedure was then repeated with an ammonium sulphate concentration of 60% and
80% of saturation.
The precipitated protein in the 0–30%, 30–60% and 60–80% ammonium sulphate precipitates was re-dissolved in one volume of 100 mmol l–1 sodium acetate buffer (pH 5.5). These re-dissolved precipitates and the supernatant containing ammonium sulphate at 80% of saturation were assayed for protein content and endo-β-1,4-glucanase, laminarinase and β-1,4-glucosidase activities. Enzymes of interest were purified from the re-dissolved 30–60% precipitate using a combination of anion exchange, hydrophobic interaction and gel filtration chromatography. All chromatography was carried out at 4°C.
Liquid chromatography steps
Anion exchange chromatography
Before anion exchange chromatography, the ammonium sulphate was removed and
the buffer changed in the re-dissolved 30–60% ammonium sulphate
precipitate by dialysing it against two changes of 20 mmol
l–1 PIPES buffer (pH 5.5). The dialysate was centrifuged at
3300 g for 30 min to remove any fine particles and then
applied to a 2.5 cmx60 cm Macro-Prep DEAE column (Bio-Rad, Hercules, CA,
USA) for anion exchange chromatography. Proteins were eluted from the DEAE
column using an Econo Gradient pump (Bio-Rad) at a flow rate of 0.3 ml
min–1 and the following conditions: 0–220 min,
isocratic elution with 20 mmol l–1 PIPES buffer (pH 5.5);
220–860 min, sodium chloride concentration in the 20 mmol
l–1 PIPES buffer (pH 5.5) was increased linearly from 0 to
1000 mmol l–1; 860 to 1060 min, sodium chloride concentration
was held at 1000 mmol l–1; 1060–1260 min, sodium
chloride concentration was decreased linearly from 1000 to 0 mmol
l–1; 1260–1460 min, column was re-equilibrated with 20
mmol l–1 PIPES buffer (pH 5.5). Over the 1460min of the
chromatography run, 5.5ml fractions were collected every 18.25 min using a
model 2110 fraction collector (Bio-Rad). Collected fractions were stored at
–20°C until analysis. Every third fraction was analysed for protein
content and endo-β-1,4-glucanase and laminarinase activity. Fractions
containing the enzyme of interest were combined and concentrated by
centrifugation at 2060g for 15min at 4°C in a 10000
nominal molecular weight limit (NMWL) Ultra-15, Ultracel-10K filter (Amicon,
Houston, TX, USA).
Hydrophobic interaction chromatography
Prior to hydrophobic interaction chromatography (HIC), the buffer in the
protein concentrate was changed by adding four volumes of 20 mmol
l–1 PIPES buffer (pH 5.5) to the concentrate and
re-concentrating by centrifugation in a 10 000 NMWL ultrafilter as described
above.
Before chromatography a Macro-Prep methyl HIC (Bio-Rad) column (1.5 cmx50 cm) was equilibrated with 20 mmol l–1 PIPES buffer (pH 5.5), containing 1.5 mol l–1 ammonium sulphate at a flow rate of 0.3 ml min–1. Samples were then loaded and the protein eluted using a linear gradient of 1.5 to 0 mol l–1 ammonium sulphate at a flow rate of 0.3 ml min–1 as follows: 0–220 min, isocratic elution with 20 mmol l–1 PIPES buffer (pH 5.5) containing 1.5 mol l–1 ammonium sulphate; 220–860 min, ammonium sulphate concentration in the PIPES buffer was reduced linearly from 1.5 to 0 mol l–1; 860–1060 min, isocratic elution with 20 mmol l–1 PIPES buffer (pH 5.5) containing 0 mol l–1 ammonium sulphate; 1060–1260 min, ammonium sulphate concentration in the 20 mmol l–1 PIPES buffer (pH 5.5) was increased linearly to 1.5 mol l–1; 1260–1460 min, re-equilibration of the column with 20 mmol l–1 PIPES buffer (pH 5.5) containing 1.5 mol l–1 ammonium sulphate; 5.5 ml fractions were collected every 18.25 min for the entire 1460 min of the chromatography run and stored at –20°C until analysis. Every third fraction was analysed for protein and endo-β-1,4-glucanase and laminarinase activity. Fractions containing activity of the enzyme of interest were combined and concentrated by centrifugation in a 10 000 NMWL ultrafilter as described above.
Gel filtration chromatography
Protein concentrates from either the anion exchange chromatography step or
the HIC step were applied to a 1.5 cmx50 cm P-100 medium bead size gel
filtration column (Bio-Rad). Isocratic elution of proteins was achieved with
0.1 mol l–1 sodium acetate buffer (pH 5.5) at a flow rate of
0.1mlmin–1; 1ml fractions were collected every 10 min from
120 to 920 min.
Strong anion exchange chromatography
Protein concentrates were applied to a 1.0 cmx30 cm Mono-Q column
(Bio-Rad) for strong anion exchange chromatography. Prior to strong anion
exchange chromatography, the buffer of the protein concentrate was changed to
the starting buffer by adding 4 volumes of 20 mmol l–1 PIPES
buffer (pH 5.5) and reconcentrated in a 10 000 NMWL ultrafilter as described
above. The sample was then applied to the column and the proteins eluted with
a 0–1000 mmol l–1 sodium chloride gradient at a flow
rate of 0.4 ml min–1 as follows: 0–40 min, isocratic
elution with 20mmoll–1 PIPES buffer (pH 5.5);
40–160min, sodium chloride concentration in the buffer was increased
linearly from 0 to 1000 mmol l–1; 160–190 min,
isocratic elution with 20 mmol l–1 PIPES buffer (pH 5.5)
containing 1000 mmol l–1 sodium chloride; 190–210 min,
sodium chloride concentration in the PIPES buffer was reduced from 1000 to 0
mmol l–1; 210–240 min, column was re-equilibrated with
isocratic flow of the starting buffer, 20 mmol l–1 PIPES
buffer (pH 5.5); 1.25 ml fractions eluted from the Mono-Q column were
collected every 3 min for the entire 240 min of the chromatography run and
stored at –20°C until analysis. Protein and activity of the enzyme
of interest were measured in every third fraction.
Protein size determination using gel filtration chromatography
The molecular mass of the purified enzymes was calculated by eluting a
250µl aliquot on a 1.0cmx30cm Superdex 200 gel filtration column (GE
Healthcare, Chalfon St Giles, Bucks, UK) and eluted isocratically using 0.1
mmol l–1 sodium acetate buffer (pH 5.5) containing 0.05 mmol
l–1 sodium chloride at a flow rate of 0.3 ml
min–1. Protein eluting from the column was detected with an
EM-1 Econo UV monitor (Bio-Rad). The molecular mass of the enzyme was
estimated by comparing its retention time with that of the log of the
molecular mass of the standards cytochrome c (12.4 kDa), carbonic
anhydrase (29 kDa), albumin (66 kDa), alcohol dehydrogenase (150 kDa) and
β-amylase (200 kDa; Sigma, St Louis, MO, USA). Enzyme activity was
measured in every third fraction in order to confirm that the protein peak
corresponded to the elution of the purified enzyme.
Analysis of fractions and protein concentrates arising form the various chromatography steps
Eluted fractions and the concentrates arising from the various
chromatography steps were analysed for total protein and the enzymes
endo-β-1,4-glucanase or laminarinase. Endo-β-1,4-glucanase and
laminarinase activities were measured as the rate of production of reducing
sugars from the respective hydrolysis of carboxymethyl cellulose (Sigma,
catalogue no. C-5678) and laminarin (from Laminaria digitata; Sigma,
catalogue no. L-9634). Protocols used for the enzyme assays have been
described previously (Linton and
Greenaway, 2004). Protein concentrations were determined using the
Bradford protein assay (Bio-Rad), as per the manufacturer's instructions, and
bovine serum albumin was used as the standard (MP Biomedicals, Aurora, OH,
USA).
SDS-PAGE
The purity of endo-β-1,4-glucanase and laminarinase after liquid
chromatography was analysed by SDS-PAGE electrophoresis using a Mini Protean
system (Bio-Rad), and run as previously described
(Laemmli, 1970). Samples and
HiMarkTM unstained high molecular mass protein standards (Invitrogen,
Carlsbad, CA, USA) were run on pre-cast 10% polyacrylamide gels (Bio-Rad) at
200 V. Samples were prepared for SDS-PAGE using methods described previously
(Deutscher, 1990). Protein
bands were visualised using a silver staining kit (Bio-Rad). Protein size was
determined by plotting log molecular mass against relative migration distance
of the standards with a similar mass range to the proteins of interest. All
molecular mass are expressed as means ± s.d.
Enzyme characterisation
Substrate specificity
Purified enzymes were incubated with carboxymethyl cellulose, laminarin,
lichenan and cellobiose and the specific activity of the enzyme was expressed
as a percentage of the activity of the enzyme using its native substrate.
Endo-β-1,4-glucanase, laminarinase, lichenase and β-1,4-glucosidase
activities were measured as previously described
(Linton and Greenaway,
2004).
Enzyme kinetics and pH optimum
The activity of the purified enzymes was determined at substrate
concentration ranges of 1–7% w/v for endo-β-1,4-glucanase and
0.125–3.6% w/v for laminarinase. The kinetic properties
Vmax and Km were then calculated from
the resulting Michaelis–Menten plot using the graphing software
Prism® v.5 (Graphpad Software, San Diego, CA, USA). The optimum pH was
determined by measuring enzyme activity in the following buffers: acetate
buffer (pH 4.0), sodium acetate buffer (pH 5.5), phosphate buffer (pH 7.0),
Tris buffer (pH 8.0) and glycine buffer (pH 9.0). All
endo-β-1,4-glucanase reactions were carried out with a final substrate
concentration of 1% w/v, while the final concentration of substrate in
laminarinase reactions was 0.25% w/v. The means at each pH were compared using
a one-way ANOVA followed by least significant difference (LSD) analysis.
Statistical probabilities were determined using SPSS v.15 (SPSS Inc., Chicago,
IL, USA).
Thin layer chromatography
A 100 µl aliquot of purified endo-β-1,4-glucanase from either
G. natalis or C. destructor was incubated overnight at
40°C with carboxymethyl cellulose at a final concentration of 2% w/v (pH
5.5). Purified laminarinase was similarly incubated overnight with laminarin
(final concentration 1% w/v, pH 5.5). Incubated samples were separated on a
silica thin layer chromatography (TLC) plate and stained as previously
described (Nishida et al.,
2007).
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Final step in the purification of laminarinase – Bio-Rad P-100 gel filtration column
Size exclusion chromatography of the combined and concentrated fractions
52–58 from HIC revealed one early eluting laminarinase activity peak
(Fig. 3). Laminarinase activity
was detected in fractions 10–19 with fraction 13 containing the largest
enzyme activity (Fig. 3). This
laminarinase activity peak corresponded to a single well-resolved protein peak
(Fig. 3). Fractions 7–21
containing laminarinase activity were combined and concentrated. This
concentrate had a laminarinase specific activity of 7.80 µmol reducing
sugars produced min–1 mg protein–1
(Table 1). When run on an
SDS-PAGE gel, this concentrate contained a single protein band with an
estimated molecular mass of 41 kDa (Fig.
4). However, the molecular mass estimated from the calibration of
the Superdex 200 gel filtration column was 71kDa (data not shown). Since a
single protein band was demonstrated by SDS-PAGE, the laminarinase was deemed
to be purified to homogeneity. After this chromatography step laminarinase had
been purified 80.9 times with a yield of 4.96%
(Table 1).
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Final step in the purification of endo-β-1,4-glucanase – Bio-Rad P-100 gel filtration chromatography
The second endo-β-1,4-glucanase peak resolved by anion exchange
chromatography (fractions 56–70) was applied to a P-100 gel filtration
column (Fig. 5).
Endo-β-1,4-glucanase activity was present in fractions 22–43 with
fraction 34 containing the highest enzyme activity
(Fig. 5). Combined and
concentrated fractions 22–43 had an endo-β-1,4-glucanase specific
activity of 12.74 µmol reducing sugars produced min–1 mg
protein–1 and contained a single 52 kDa protein
(Fig. 4A;
Table 2). Hence
endo-β-1,4-glucanase was purified to homogeneity; a purification factor
of 552 times had been achieved with a yield of 10.2%
(Table 2).
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Purification of laminarinases and endo-β-1,4-glucanases from the aquatic crayfish C. destructor
DEAE chromatography
DEAE anion exchange chromatography of the re-dissolved 30–60%
ammonium sulphate precipitate revealed five large endo-β-1,4-glucanase
activity peaks. Fractions 1, 19, 34, 40 and 52 contained the highest
endo-β-1,4-glucanase activity and were respectively designated as
endo-β-1,4-glucanase peaks 1, 2, 3, 4 and 5
(Fig. 6).
Endo-β-1,4-glucanase 1 and 2 were clearly resolved from laminarinase and
were purified separately (Fig.
6). Endo-β-1,4-glucanase 3 co-eluted with laminarinase. Given
that laminarinase possesses some endo-β-1,4-glucanase activity (see
below) it was thought that endo-β-1,4-glucanase peak 3 may represent this
residual activity. Endo-β-1,4-glucanase 4 and 5 were not purified
further.
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A large laminarinase peak was eluted from the DEAE column (Fig. 6). It was eluted in fractions 27–36 with fraction 34 containing the highest laminarinase activity (Fig. 6). Fractions 27–36 containing laminarinase were combined, concentrated and then applied to a HIC column.
Comparison with DEAE chromatography of the 30–60% ammonium sulphate fractions from C. destructor and G. natalis
The elution profiles of laminarinase from the DEAE column were similar for
C. destructor and G. natalis. For both species there was one
large late-eluting laminarinase peak (Figs
1,
6). The elution profile of
endo-β-1,4-glucanase from the DEAE column differed between species. For
C. destructor there were six endo-β-1,4-glucanase peaks while
there were only two endo-β-1,4-glucanase peaks for G. natalis
(Figs 1,
6). An early eluting
endo-β-1,4-glucanase peak was observed for C. destructor;
however, no such peak was observed for G. natalis. For both G.
natalis and C. destructor there was a large
endo-β-1,4-glucanase peak that co-eluted with laminarinase (Figs
1,
6).
Purification of laminarinase
Hydrophobic interaction chromatography
HIC of the laminarinase concentrate from the anion exchange chromatography
step described above revealed one large, late-eluting laminarinase activity
peak (Fig. 7). Laminarinase
activity was contained in fractions 60–62 with fraction 61 containing
the highest enzyme activity (Fig.
7). Fractions 60–62 were combined, concentrated and then
loaded onto a Bio-Rad Mono-Q column for strong anion chromatography. The
elution profile from the HIC column was similar to the elution profile of
laminarinase from G. natalis eluted from the HIC column, i.e. a
single late-eluting laminarinase peak (Figs
2,
7).
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Mono-Q chromatography
Mono-Q chromatography of the combined and concentrated fractions
60–62 from HIC revealed a large late-eluting laminarinase activity peak
(Fig. 8). Laminarinase was
eluted in fractions 49–52 with fraction 49 containing the highest enzyme
activity (Fig. 8). This
laminarinase peak corresponded to a single protein peak indicating substantial
protein purification had been achieved
(Fig. 8). Fractions 47–53
containing the purified laminarinase were combined and concentrated to a final
volume of 2 ml. This concentrate contained 4.47 units of laminarinase and
three proteins, 41, 49 and 62 kDa in size
(Table 3,
Fig. 4). After this
chromatography step the laminarinase was purified 104.94 times and a 14.82%
yield had been achieved (Table
3). Hence laminarinase has been partially purified from C.
destructor. Given that a laminarinase of 44 kDa has been purified from
G. natalis the 41 kDa protein from C. destructor is probably
laminarinase 1.
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Gel filtration chromatography
During another purification run, fractions from HIC containing laminarinase
activity from C. destructor were combined, concentrated and applied
to a Bio-Rad P-100 gel filtration column for size exclusion chromatography.
This ensured that laminarinase was purified from G. natalis and
C. destructor using a consistent purification method. One large
laminarinase activity peak was eluted from this column
(Fig. 9). Fractions 7–25
contained laminarinase activity with fraction 16 containing the highest
activity (Fig. 9). By
comparison with the elution volumes of protein molecular mass standards from
the gel filtration column, this laminarinase was estimated to be 62 kDa in
size. This is likely to be an underestimate given the laminarinase eluted very
near the void volume and more protein was loaded onto the column compared with
that of protein molecular mass standards (elution volume:void volume=1.21).
However the concentrate of combined and concentrated fractions 7–25
contained two proteins of 41 and 62 kDa in size when it was run on an SDS-PAGE
gel (Fig. 4).
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Gel filtration chromatography of endo-β-1,4-glucanase 1 and endo-β-1,4-glucanase 2
Combined and concentrated fractions containing endo-β-1,4-glucanase 1
(combined fractions 1–6) and endo-β-1,4-glucanase 2 (combined
fractions 16–22) from DEAE anion exchange chromatography were loaded
onto a Bio-Rad P-100 gel filtration column for size exclusion chromatography.
Separate runs of these endo-β-1,4-glucanase 1 and 2 concentrates revealed
that both endo-β-1,4-glucanases were eluted as a single activity peak
(Fig. 10).
Endo-β-1,4-glucanase 1 was eluted in fractions 22–31 with fraction
28 containing the highest enzyme activity
(Fig. 10A) while
endo-β-1,4-glucanase 2 was eluted in fractions 13–28 with the
largest activity being present in fraction 19
(Fig. 10B). Accordingly,
fractions 22–31 eluted from the P-100 column and containing
endo-β-1,4-glucanase 1 were combined and concentrated. Similarly,
fractions 13–28 containing endo-β-1,4-glucanase 2 were combined and
concentrated. The concentrates of both endo-β-1,4-glucanase 1 and 2
contained a single protein (Fig.
4); hence endo-β-1,4-glucanase 1 and 2 were purified to
homogeneity. The molecular mass of endo-β-1,4-glucanase 1 was estimated
to be 53±2 kDa (N=3 purification runs) while the molecular
mass of endo-β-1,4-glucanase 2 was estimated to be 51±0 kDa
(N=2 purification runs). Endo-β-1,4-glucanase 1 had a specific
activity of 19.09 µmol min–1 mg protein–1
with a purification factor of 83.18 times and a percentage yield of 13.24%
(Table 4).
Endo-β-1,4-glucanase 2 had a specific activity of 7.43 µmol
min–1 mg protein–1 with a purification
factor of 32.36 times and a yield of 10.23%
(Table 5).
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Characterisation of the purified enzymes from G. natalis
Laminarinase
Out of all of the substrates tested, laminarinase purified from the midgut
gland of G. natalis hydrolysed laminarin the fastest
(Table 6). This laminarinase
was also capable of limited hydrolysis of carboxymethyl cellulose and hence
possessed a low endo-β-1,4-glucanase activity
(Table 6). Hydrolysis of
lichenan and cellobiose was negligible
(Table 6). Thus laminarinase
from G. natalis is capable of hydrolysing mainly
β-1,3-glycosidic bonds with very limited ability to hydrolyse
β-1,4-glycosidic bonds; it is not capable of hydrolysing mixed
β-D-glucans such as lichenan
(Table 6), so can be assigned
the EC number 3.2.1.39 (Boeckmann et al.,
2003). Laminarinase purified from G. natalis had a
Vmax of 42.0 µmol reducing sugars produced
min–1 mg protein–1 and a
Km of 0.126% (w/v)
(Fig. 11). The enzyme was
active over a broad pH range of 4–8, but was highest at pH 5.5–7
and lower at pH 4 and 9 (Fig.
12A). Incubation of purified laminarinase with 1% (final
concentration) laminarin for 24 h produced glucose, laminaribose and short
oligomers of the laminarin polymer (Fig.
13A).
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Endo-β-1,4-glucanase
Endo-β-1,4-glucanase purified from the midgut gland of G.
natalis hydrolysed carboxymethyl cellulose the fastest
(Table 6). It also possessed
significant activity of laminarinase (20.4% of endo-β-1,4-glucanase
activity) and very low activities of lichenase (4.6% of
endo-β-1,4-glucanase activity) and β-1,4-glucosidase (1.6% of
endo-β-1,4-glucanase activity; Table
6). Given that the endo-β-1,4-glucanase was mainly active
against carboxymethyl cellulose, it was allocated the EC number 3.2.1.4
(Boeckmann et al., 2003).
Endo-β-1,4-glucanase purified from the midgut gland of G. natalis had the highest activity at pH 4–7. At more alkaline pH values (8 and 9) its activity was lower (Fig. 12B). The kinetic parameters for endo-β-1,4-glucanase could not be determined given the enzyme activity did not saturate at the highest workable carboxymethyl cellulose concentrations tested in this study. Carboxymethyl cellulose solutions at concentrations greater than 8% w/v are too viscous to work with. Incubation of the purified endo-β-1,4-glucanase with carboxymethyl cellulose produced cellobiose and short oligomers of glucose, most likely three and four residues long. A very small of amount of glucose was also produced (Fig. 13C).
Characterisation of the purified enzymes from C. destructor
Laminarinase
In addition to laminarin, laminarinase purified form C. destructor
was able to hydrolyse carboxymethyl cellulose and lichenan
(Table 6). Hence it possessed
substantial endo-β-1,4-glucanase activity (28.9% of laminarinase
activity) and significant amounts of lichenase activity (7.49% of laminarinase
activity). Cellobiose was hydrolysed at very low levels by the laminarinase
(Table 6); thus it did not
possess significant β-1,4-glucosidase activity. Given that it can
hydrolyse both β-1,3- and β-1,4-glycosidic bonds it can be assigned
the EC number 3.2.1.6 (Boeckmann et al.,
2003). The purified enzyme had a Vmax of 19.6
µmol reducing sugars produced min–1 mg
protein–1 and a Km of 0.0593% (w/v)
(Fig. 11). Laminarinase
activity was maximal at pH 5.5 (Fig.
12C). Incubation of the purified enzyme with laminarin produced
mainly glucose, and while other short oligomers were again present, their
production appeared to be negligible compared with the amount of glucose
formed (Fig. 13B).
Endo-β-1,4-glucanase
Endo-β-1,4-glucanase purified from C. destructor was able to
hydrolyse mainly carboxymethyl cellulose, but possessed very low activities of
laminarinase (2.46% of endo-β-1,4-glucanase activity) and
β-1,4-glucosidase (0.71% of endo-β-1,4-glucanase activity)
(Table 6). Thus,
endo-β-1,4-glucanase hydrolyses mainly β-1,4-glycosidic bonds and
can be assigned the cellulase EC number 3.2.1.4
(Boeckmann et al., 2003).
Endo-β-1,4-glucanase activity was maximal at pH 5.5 and decreased
moderately at higher pH values. At pH 4 endo-β-1,4-glucanase activity was
low compared with the other pH values
(Fig. 12D). From the
hydrolysis of carboxymethyl cellulose, endo-β-1,4-glucanase from C.
destructor is capable of producing cellobiose, small amounts of glucose
and other short oligomers. Hence it possesses both endo-β-1,4-glucanase
and cellobiohydrase activity (Fig.
13D).
|
DISCUSSION |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Laminarinase from G. natalis produced glucose and oligomers of two
and three glucose residues from the hydrolysis of laminarin, confirming it is
capable of significant glucose production and suggesting that it may also work
in conjunction with β-1,4-glucosidase to hydrolyse laminarin completely.
Laminarin is the main storage polysaccharide in algae; it is also produced in
plants in response to damage or infection and is the main structural component
in fungal cell walls (Bacic et al.,
1988). Large activities of substrate-specific laminarinase within
the midgut gland of G. natalis suggest it is an important energy
source and would allow G. natalis to utilise fungi and algae growing
on detritus within the rainforest. Corroborating evidence comes from
observations that G. natalis scrape off and consume algae from rocks
and tree buttresses (Linton and Greenaway,
2007). In contrast to G. natalis, the laminarinase from
C. destructor produced predominantly glucose, with only small amounts
of short oligomers being detected. This suggests that sources of laminarin
such as algae may be a more important component of the diet of C.
destructor when compared with G. natalis.
While the laminarinase from G. natalis is specialised to hydrolyse only β-1,3-glycosidic bonds, the enzyme from C. destructor is more generalised since it was also capable of limited hydrolysis of β-1,4-glycosidic bonds. The difference between these two distantly related species is unclear. However, the specialisation of the enzyme in G. natalis may be a result of the development of a more efficient enzyme, or it may indicate that the laminarinase from C. destructor aids the endo-β-1,4-glucanase in hydrolysing cellulose, thus making the cellulase system more efficient.
The molecular mass of the laminarinase isolated from the midgut gland of
G. natalis was estimated to be 41 kDa by SDS-PAGE and 71 kDa from gel
filtration chromatography (Figs
3,
4). This discrepancy suggests
that laminarinase may exist as a dimer, joined by either a disulphide bond or
van der Waals forces. Given that β-1,6-glycosidic bonds within laminarin
cause the molecule to be branched
(Lépagnol-Descamps et al.,
1998), the laminarinase dimer may fit in between these branches.
Simultaneous hydrolysis of the two branches may make the laminarinase more
efficient. Alternatively, the laminarinase may contain a laminarin-binding
domain, which is a similar size to the catalytic component. A similar
discrepancy was observed for C. destructor; a 41 kDa band as well as
a second, larger band of 62 kDa was detected by SDS-PAGE
(Fig. 4). The molecular mass of
the laminarinase was estimated to be 62 kDa from gel filtration
chromatography. The 41 kDa band corresponds to the 41 kDa laminarinase from
G. natalis, while the larger band may either be a minor impurity or
represent a native laminarinase dimer.
The size range of laminarinases isolated from various invertebrates is
22–146 kDa (Table 7). The
41 kDa laminarinase isolated from G. natalis and C.
destructor is similar in size to that of the cockroach Periplaneta
americana (Genta et al.,
2003), suggesting that the enzyme may be inherited from a common
ancestral arthropod. Interestingly, the molecular mass of laminarinase
estimated from gel filtration chromatography is similar in size to that of
abalone Haliotis tuberculata
(Lépagnol-Descamps et al.,
1998), krill Euphausia superba
(Suzuki et al., 1987) and
termite Rhagium inquisitor
(Chipoulet and Chararas, 1984)
(Table 7). The variation in the
size estimation of laminarinase may be due to the laminarinase monomer being
observed in some studies and the dimer in others.
|
The laminarinase is most likely to be endogenously produced given its high
activity within the midgut gland of both G. natalis and C.
destructor, and the endogenous production of endo-β-1,4-glucanase by
arthropods (Byrne et al., 1999;
Crawford et al., 2004;
Davison and Blaxter, 2005;
Linton and Greenaway, 2004;
Linton et al., 2006). However,
the gene for the crustacean laminarinase remains to be sequenced and the
midgut gland would be the most logical tissue in which to look for expression
of this gene.
Endo-β-1,4-glucanase characteristics
The major endo-β-1,4-glucanase purified from G. natalis had a
broader substrate specificity than that from C. destructor. The
endo-β-1,4-glucanase from G. natalis hydrolysed mainly
β-1,4-glycosidic bonds but was also capable of significant hydrolysis of
β-1,3-glycosidic bonds. In contrast the endo-β-1,4-glucanase from
C. destructor only hydrolysed β-1,4-glycosidic bonds
(Table 6). The broader
substrate specificity of endo-β-1,4-glucanase from G. natalis
suggests that this species encounters β-1,3- and β-1,4-glycosidic
bonds within its diet and requires an enzyme to break these bonds. This is in
contrast to C. destructor, which has a typical arthropod
endo-β-1,4-glucanase that is only capable of hydrolysing
β-1,4-glycosidic bonds. Broader substrate specificity suggests that the
enzyme from G. natalis should be allocated a different EC number that
reflects this. However, given this glycosyl hydrolase has a preference for
β-1,4-glycosidic bonds and is most likely to be a GHF9 glycosyl
hydrolase, it should retain the classic endo-β-1,4-glucanase EC number of
3.2.1.4 (Linton et al., 2006).
This example highlights the need for caution when trying to classify glycosyl
hydrolases neatly into EC groups.
Endo-β-1,4-glucanase from G. natalis hydrolysed internal
β-1,4-glycosidic bonds within the glucose polymer carboxymethyl cellulose
to produce short glucose oligomers, cellobiose and a very small amount of
glucose. Production of small oligomers of three and four glucose residues is
characteristic of endo-β-1,4-glucanase enzymes and production of
cellobiose is indicative of cellobiohydrase activity
(Linton et al., 2006). Thus,
the combined action of endo-β-1,4-glucanase and β-1,4-glucosidase
may be sufficient to hydrolyse cellulose completely to glucose. Like other
arthropods, G. natalis may lack a cellobiohydrase
(Scrivener and Slaytor, 1994);
however, it remains to be tested whether the combined action of
endo-β-1,4-glucanase and β-1,4-glucosidase in an in vitro
system can explain the rate of cellulose hydrolysis observed in vivo.
The endo-β-1,4-glucanase from C. destructor also produced short
oligomers as well as glucose from carboxymethyl cellulose. The retention of
this similar activity between two distantly related species of crustaceans
further reinforces the importance of cellulose as a source of glucose in
herbivorous crustaceans.
The endo-β-1,4-glucanases purified from G. natalis and C.
destructor are most likely to be GHF9 endo-β-1,4-glucanases given
they have similar molecular masses of 52 and 53±2 kDa, respectively,
when compared with other GHF9 cellulases from arthropod species
(Table 7). This combined with
the large activity within the midgut gland suggests they are likely to be the
product of the endogenous GHF9 gene present in crustaceans, and arthropods in
general, and hence are synthesised endogenously within this tissue
(Byrne et al., 1999;
Crawford et al., 2004;
Linton et al., 2006).
Endo-β-1,4-glucanases from both G. natalis and C.
destructor are also most probably glycosylated given that glycosylation
is a common feature of GHF9 endo-β-1,4-glucanases from other arthropods
(Table 7)
(Lee et al., 2005;
Nishida et al., 2007;
Zhou et al., 2007).
Glycosylation may prevent proteolytic degradation and thus may explain why the
endo-β-1,4-glucanases are relatively stable
(Langsford et al., 1987).
Enzyme activity at different pH
Both the laminarinase and the endo-β-1,4-glucanase from G.
natalis had substantial activity over a pH range of 5.5 to 8
(Fig. 12). While both enzymes
had high activity at both pH 5.5 and pH 7, the laminarinase had maximal
activity at pH 7, while endo-β-1,4-glucanase activity was slightly higher
at pH 5.5. Since the pH of the digestive juice of G. natalis is
6.69±0.3 (Linton and Greenaway,
2004), both enzymes would be working maximally in vivo.
This is also true of the enzymes from C. destructor, both of which
had maximal activity over a pH range of 5.5 to 8. The pH of the gastric juice
of C. destructor is assumed to be similar to that of the closely
related C. quadricarinatus, which is pH 5.8
(Figueiredo et al., 2001).
Other endo-β-1,4-glucanase activity peaks
Four other endo-β-1,4-glucanase peaks were resolved from G.
natalis from HIC (Fig. 2).
The elution profile for two of these endo-β-1,4-glucanases from the
subsequent gel filtration chromatography suggests that the enzymes had a
similar molecular mass to that already purified (52 kDa). Similarly, in C.
destructor two endo-β-1,4-glucanases of similar molecular mass were
purified to homogeneity. The most likely explanation for this is that both
species produce more than one endo-β-1,4-glucanase. Given the similar
molecular masses observed, they are most likely to be isomers. Two
endo-β-1,4-glucanases have been purified from the termite
Reticulitermes speratus (Watanabe
et al., 1998) and the cockroach Panesthia cribrata
(Lo et al., 2003;
Scrivener and Slaytor, 1994).
There may be more than one copy of the GHF9 gene in crustaceans since two
separate studies (Byrne et al.,
1999; Crawford et al.,
2004) contain discrepancies in the sites of introns identified for
the endo-β-1,4-glucanase gene in C. quadricarinatus.
Function of the endo-β-1,4-glucanase and laminarinase within G. natalis and C. destructor
Herbivorous crustaceans such as G. natalis possess a range of
cellulase and hemicellulase enzymes such as the endo-β-1,4-glucanase and
laminarinase examined in this study. Similar enzymes are also present in
distantly related aquatic species such as C. destructor. This
suggests that the aquatic ancestors of the herbivorous gecarcinid land crabs
may have possessed these enzymes that have then evolved to efficiently cope
with the adoption of a leaf litter diet. Given the complex nature of the
structural compounds present within the cell walls of plants, carbohydrate
polymers with mixed β-1,4-, β-1,3- and β-1,6-glycosidic bonds,
hemicellulase and cellulase enzymes may work synergistically to achieve total
hydrolysis of carbohydrate polymers. The possible role of synergism in
cellulase systems remains largely unknown; however, the isolation and
purification of the enzymes described here will enable this to be investigated
in an in vitro system. The enzymes may also interact with the
function of the gastric mill to achieve efficient cellulose hydrolysis.
|
Acknowledgments |
|---|
|
References |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
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; mg protein ml–1) and endo-β-1,4-glucanase
(•) and laminarinase (
) activities (µmol reducing sugars
produced min–1 ml–1) in the collected
fractions.
; µmol reducing sugars or glucose produced
min–1 ml–1) for fractions from a Bio-Rad
P-100 gel filtration column. Fractions 52–58 from the HIC described in
) and C. destructor (
