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

Abstract

SUMMARY 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.

A glycosyl hydrolase family 16 gene is responsible for the endogenous production of β-1,3-glucanases within decapod crustaceans

“…Sequences of short peptides were determined from a β-1,3-glucanase purified previously from the midgut gland of G. natalis (Allardyce and Linton, 2008). These sequences were compared to the same sequences from the putative amino acid sequence to indicate if the putative amino sequence derived from the cDNA could potentially match that of a previously purified β-1,3-glucanase.…”

“…To do this, a β-1,3-glucanase sample, previously purified from the midgut gland of G. natalis, was run on a 12% polyacrylamide gel and stained with Coomassie blue as per the method of Allardyce and Linton(2008) and Linton and Shirley(2011). A protein band corresponding to the β-1,3-glucanase was excised from the gel and processed by in-gel tryptic digestion.…”

“…Within the decapods, β-1,3-glucanase has been purified and characterised from the midgut glands of the gecarcinid land crab, Gecarcoidea natalis, and the freshwater crayfish, Cherax destructor (Allardyce and Linton, 2008). G. natalis is an herbivorous species that consumes mainly leaf litter (Greenaway and Linton, 1995).…”

“…The β-1,3-glucanase, which is active against laminarin, is thought to exist as a dimer of two 41 kDa subunits (Table 1). It is assumed that it is synthesised endogenously within the midgut gland since high β-1,3-glucanase activities are present in this tissue (Allardyce and Linton, 2008); however, the gene responsible for the endogenous production of this enzyme has yet to be identified in Crustacea. It is likely that the β-1,3-glucanase is the product of a glycosyl hydrolase family 16 (GHF16) gene given that this gene has been confirmed to produce a similar enzyme (of 30-50 kDa) in other invertebrates (Table 1).…”

“…To confirm this, the GHF16 cDNA derived from the midgut gland of G. natalis and C. destructor was sequenced. This sequence was then matched to the characteristics of a β-1,3-glucanase that had been previously purified and characterised from these species (Allardyce and Linton, 2008). To establish the link between the gene and protein, short peptides of β-1,3-glucanase purified from G. natalis were sequenced and compared to the putative amino acid sequences.…”