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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

^* Author for correspondence (e-mail: balla{at}deakin.edu.au)

Accepted 1 May 2008

Laminarinase and endo-β-1,4-glucanase were purified and characterised from the midgut gland of the herbivorous land crab Gecarcoidea natalis and the crayfish Cherax destructor. The laminarinase isolated from G. natalis was estimated to have a molecular mass of 41 kDa by SDS-PAGE and 71 kDa by gel filtration chromatography. A similar discrepancy was noted for C. destructor. Possible reasons for this are discussed. Laminarinase (EC 3.2.1.6) from G. natalis had a V_max of 42.0 µmol reducing sugars produced min^–1 mg protein^–1, a K_m of 0.126% (w/v) and an optimum pH range of 5.5–7, and hydrolysed mainly β-1,3-glycosidic bonds. In addition to the hydrolysis of β-1,3-glycosidic bonds, laminarinase (EC 3.2.1.39) from C. destructor was capable of significant hydrolysis of β-1,4-glycosidic bonds. It had a V_max of 19.6 µmol reducing sugars produced min^–1 mg protein^–1, a K_m of 0.059% (w/v) and an optimum pH of 5.5. Laminarinase from both species produced glucose and other short oligomers from the hydrolysis of laminarin. Endo-β-1,4-glucanase (EC 3.2.1.4) from G. natalis had a molecular mass of 52 kDa and an optimum pH of 4–7. It mainly hydrolysed β-1,4-glycosidic bonds, but was also capable of significant hydrolysis of β-1,3-glycosidic bonds. Two endo-β-1,4-glucanases, termed 1 and 2, with respective molecular masses of 53±3 and 52 kDa, were purified from C. destructor. Endo-β-1,4-glucanase 1 was only capable of hydrolysing β-1,4-glycosidic bonds and had an optimum pH of 5.5. Endo-β-1,4-glucanases from both species produced some glucose, cellobiose and other short oligomers from the hydrolysis of carboxymethyl cellulose.

Key words: land crab, Gecarcoidea natalis, Cherax destructor, laminarinase, endo-β-1,4-glucanase