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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
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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

Benjamin J. Allardyce* and Stuart M. Linton

School of Life and Environmental Sciences, Deakin University, Pigdons Road, Geelong, Victoria, 3217, Australia


Figure 1
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  		Fig. 1. DEAE anion exchange chromatography of the dialysed protein fraction derived from the midgut gland of G. natalis that was precipitated by ammonium sulphate at concentrations between 30 and 60% saturation. Protein content ({square}; mg protein ml–1) and endo-β-1,4-glucanase (•) and laminarinase ({Delta}) activities (µmol reducing sugars produced min–1 ml–1) in the collected fractions.

 

Figure 2
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  		Fig. 2. Hydrophobic interaction chromatography (HIC) of the combined and concentrated fractions 37–55 from anion exchange chromatography described in Fig. 1. The concentrate contained endo-β-1,4-glucanase and laminarinase activities and was derived from the midgut gland of G. natalis. Protein content ({square}, mg protein ml–1) and endo-β-1,4-glucanase (•) and laminarinase ({Delta}; µmol reducing sugars or glucose produced min–1 ml–1) activities of the collected fractions.

 

Figure 3
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  		Fig. 3. Elution profile of protein ({square}; mg protein ml–1) and laminarinase activity ({blacktriangleup}; 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 Fig. 2 were combined, concentrated and loaded onto the Bio-Rad P-100 column. This concentrate was derived from the midgut gland of G. natalis.

 

Figure 4
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  		Fig. 4. SDS polyacrylamide gel electrophoresis of purified and partially purified endo-β-1,4-glucanase and laminarinase from the midgut gland of C. destructor and G. natalis. Gels were silver stained. (A) Lane 1, molecular mass standards with their sizes indicated in kDa. Lane 2, endo-β-1,4-glucanase purified from the midgut gland of G. natalis. Lane 3, endo-β-1,4-glucanase 2 purified from the midgut gland of C. destructor. Lane 4, laminarinase purified from the midgut gland of G. natalis. Lanes 5 and 6, laminarinases partially purified from midgut gland of C. destructor using either Bio-Rad P-100 gel filtration chromatography (Lane 5) or Mono-Q strong anion chromatography (Lane 6) as the third purification step. (B) Lane 1, endo-β-1,4-glucanase 1 purified from the midgut gland of C. destructor. Lane 2, molecular mass standards with their masses indicated in kDa.

 

Figure 5
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  		Fig. 5. Size exclusion chromatography of endo-β-1,4-glucanase derived from the midgut gland of G. natalis. Combined and concentrated fractions 56–70 containing endo-β-1,4-glucanase activity from the DEAE anion exchange chromatography described in Fig. 1 were loaded onto a Bio-Rad P-100 column. Protein ({square}, mg protein ml–1) and endo-β-1,4-glucanase activity ({blacktriangleup}, µmol reducing sugars produced min–1 ml–1) were measured in the fractions collected.

 

Figure 6
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  		Fig. 6. Anion exchange chromatography of the dialysed protein fraction from the midgut gland homogenate of C. destructor that was precipitated by ammonium sulphate at concentrations between 30 and 60% of saturation. Protein sample was loaded onto a Bio-Rad DEAE column and protein concentration (mg protein ml–1, {square}) and endo-β-1,4-glucanase (•) and laminarinase ({Delta}) activities (µmol reducing sugars produced min–1 ml–1) were measured in the eluted fractions.

 

Figure 7
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  		Fig. 7. Hydrophobic interaction chromatography of combined and concentrated fractions 27–36 from the DEAE anion exchange chromatography described in Fig. 5. This concentrate contained laminarinase 1 activity and was derived from the midgut gland of C. destructor. Protein concentration (mg ml–1, {square}) and laminarinase activity (µmol reducing sugars produced min–1 ml–1, {blacktriangleup}) in the collected fractions.

 

Figure 8
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  		Fig. 8. Strong anion exchange chromatography of the laminarinase from C. destructor on a Bio-Rad Mono-Q column. Combined and concentrated fractions 60–62 from the HIC described in Fig. 6 and containing laminarinase 1 derived from the midgut gland of C. destructor were loaded onto the Mono-Q column. Protein concentration (mg protein ml–1, {square}) and laminarinase activity (µmol reducing sugars produced min–1 ml–1, {blacktriangleup}) in the collected fractions.

 

Figure 9
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  		Fig. 9. Gel filtration chromatography of laminarinase derived from C. destructor and after sequential anion exchange chromatography and HIC. Elution profiles of protein (mg protein ml–1, {square}) and laminarinase activity (µmol reducing sugars produced min–1 ml–1, {blacktriangleup}) when combined and concentrated fractions containing laminarinase activity were applied to a Bio-Rad P-100 gel filtration column.

 

Figure 10
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  		Fig. 10. Gel filtration chromatography of endo-β-1,4-glucanases derived from the midgut gland of C. destructor. Samples were loaded onto a Bio-Rad P-100 gel filtration column and the protein concentration (mg protein ml–1, {square}) and endo-β-1,4-glucanase activity (µmol reducing sugars produced min–1 ml–1, {blacktriangleup}) measured. (A) Chromatography of combined and concentrated fractions 1–6 from DEAE anion exchange chromatography described in Fig. 5 containing endo-β-1,4-glucanase 1. (B) Size exclusion chromatography of combined and concentrated fractions 16–22 from DEAE chromatography described in Fig. 5 containing endo-β-1,4-glucanase 2.

 

Figure 11
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  		Fig. 11. Activity of laminarinase (µmol reducing sugars produced min–1 ml–1) purified from the midgut glands of G. natalis ({blacksquare}) and C. destructor ({square}) with increasing concentrations of laminarin. Lines running through the data have been created after the data were fitted to the Michaelis–Menten enzyme kinetics model. Laminarinase from G. natalis had a Vmax of 42.04 µmol reducing sugars produced min–1 mg protein–1 and a Km of 0.1259% (w/v). Laminarinase from C. destructor had a Vmax of 19.6 µmol reducing sugars produced min–1 mg protein–1 and a Km of 0.059% (w/v).

 

Figure 12
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  		Fig. 12. Activity (µmol reducing sugars produced min–1 mg protein–1) of laminarinase (A,C) and endo-β-1,4-glucanase (B,D) purified from the midgut glands of G. natalis (A,B) and C. destructor (C,D) at different pH values. Similar symbols indicate statistically similar means.

 

Figure 13
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  		Fig. 13. Carbohydrates produced by the hydrolysis of laminarin when incubated with laminarinase from G. natalis (A) and C. destructor (B), and by the hydrolysis of carboxymethyl cellulose when incubated with endo-β-1,4-glucanase purified from G. natalis (C) and C. destructor (D). Hydrolysate and standards (glucose and cellobiose) were run on a silica thin layer chromatography (TLC) plate with a mobile phase of n-butanol/acetic acid/water (2:1:1, v/v/v) (Nishida et al., 2007Go). Carbohydrate products were visualised by spraying the TLC plate with 10% (v/v) sulphuric acid in ethanol and incubating at 120°C for 15 min (Nishida et al., 2007Go).

 

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© The Company of Biologists Ltd 2008