Purification and characterisation of endo-beta-1,4-glucanase and laminarinase enzymes from the gecarcinid land crab Gecarcoidea natalis and the aquatic crayfish Cherax destructor.
Laminarinase and endo-beta-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 micromol 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 beta-1,3-glycosidic bonds. In addition to the hydrolysis of beta-1,3-glycosidic bonds, laminarinase (EC 3.2.1.39) from C. destructor was capable of significant hydrolysis of beta-1,4-glycosidic bonds. It had a V(max) of 19.6 mumol 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-beta-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 beta-1,4-glycosidic bonds, but was also capable of significant hydrolysis of beta-1,3-glycosidic bonds. Two endo-beta-1,4-glucanases, termed 1 and 2, with respective molecular masses of 53+/-3 and 52 kDa, were purified from C. destructor. Endo-beta-1,4-glucanase 1 was only capable of hydrolysing beta-1,4-glycosidic bonds and had an optimum pH of 5.5. Endo-beta-1,4-glucanases from both species produced some glucose, cellobiose and other short oligomers from the hydrolysis of carboxymethyl cellulose.
Allardyce
Benjamin J
BJ
School of Life and Environmental Sciences, Deakin University, Pigdons Road, Geelong, Victoria, 3217, Australia. balla@deakin.edu.au
Linton
Stuart M
SM
D000818:Animals; D003400:Astacoidea / Q000201:enzymology*; D003386:Brachyura / Q000201:enzymology*; D002480:Cellulase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D044602:Cellulases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D002845:Chromatography; D005618:Fresh Water; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D013379:Substrate Specificity
D002482:Cellulose; D044602:Cellulases; D002480:Cellulase