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A 160kDa enzyme with β-gIucosidase activity was purified from the midgut gland of the land crab Gecarcoidea natalis. The enzyme was capable of releasing glucose progressively from cellobiose, cellotriose or cellotetraose. Although β-glucosidases (EC 3.2.1 .21) have some activity towards substrates longer than cellobiose, the enzyme was classified as a glucohydrolase (EC 3.2.1.74) as it had a preference for larger substrates (cellobiose

This study aimed to sequence and identify a glycosyl hydrolase family 9 (GHF9) endo-β-1,4-glucanase expressed in the midgut gland of the herbivorous gecarcinid land crab, Gecarcoidea natalis . Hence this would explain the gene responsible for the production of previously purified and characterised endo-β-1,4-glucanases. Three different transcripts, two complete and one partial were sequenced from cDNA and an open reading frame of 1383 bp was produced. Translated, this would produce a putative protein of 460 amino acid residues, including a 16 amino acid residue signal peptide. The mature protein (without signal peptide) is predicted to have a molecular mass of 47.6–47.7 kDa; this closely matches the molecular mass (47.4 kDa) of one of the three endo-β-1,4-glucanase/lichenase enzymes purified previously from G . natalis . It is therefore proposed that the gene described here encodes one of the previously characterised enzymes. The presence of multiple transcripts suggests gene duplication. To confirm that the gene is widely expressed within the Crustacea, cDNA encoding a GHF9 endo-β-1,4-glucanase was also sequenced in diverse crustaceans, the deposit feeding soldier crab, Mictyris platycheles and the terrestrial hermit crabs, Coenobita purlatus and C . brevimanus . An open reading frame of 1356 bp was sequence from M . platycheles , while an incomplete open reading frames of 1384 and 1523 bp were respectively sequenced from Coenobita brevimanus and C . perlatus . The midgut gland of M . platycheles contained activity (0.704 ± 0.218 μmol reducing sugars produced. min − 1 ·mg − 1 tissue wet weight) of a 26.3 ± 0.3(5) endo-β-1,4-glucanase isozyme (determined from activity staining). These species, particularly M . platycheles does not consume and digest significant amounts of plant cellulose. This implies that the ancestral enzyme is not a cellulase, but rather it may be involved in hydrolysing cellulose like polysaccharides within other organisms such as algae. [ABSTRACT FROM AUTHOR]

This study investigated synergism between endo-β-1,4-glucanase and β-glucohydrolase enzymes from Gecarcoidea natalis. Together, these enzymes efficiently hydrolyse the cellulose-like polymer, carboxymethyl cellulose, to glucose. Endo-β-1,4-glucanase and β-glucohydrolase, isolated previously from G. natalis, were incubated in vitro using a ratio of the measured activities that matches that found in their digestive juice (5.4 : 1). Their combined activity, measured as the release of glucose from carboxymethyl cellulose, was greater than the sum of their separate activities. Hence they synergistically released glucose from carboxymethyl cellulose (degree of synergy: 1.27). This may be due to the complementary nature of the products of endo-β-1,4-glucanase activity and the preferred substrates of the β-glucohydrolase. β-glucohydrolase may also enhance cellulose hydrolysis by removing cellobiose, a potential competitive inhibitor of endo-β-1,4-glucanase. The synergistic interaction of these two enzymes further supports the previous suggestion that this species possesses a novel two-enzyme cellulase system that differs from the traditional three-enzyme fungal model. [ABSTRACT FROM AUTHOR]

To identify the gene responsible for the production of a β-1,3-glucanase (laminarinase) within crustacea, a glycosyl hydrolase family 16 (GHF16) gene was sequenced from the midgut glands of the gecarcinid land crab, Gecarcoidea natalis and the freshwater crayfish, Cherax destructor . An open reading frame of 1098 bp for G. natalis and 1095 bp for C. destructor was sequenced from cDNA. For G. natalis and C. destructor respectively, this encoded putative proteins of 365 and 364 amino acids with molecular masses of 41.4 and 41.5 kDa. mRNA for an identical GHF16 protein was also expressed in the haemolymph of C. destructor . These putative proteins contained binding and catalytic domains that are characteristic of a β-1,3-glucanase from glycosyl hydrolase family 16. The amino acid sequences of two short 8–9 amino acid residue peptides from a previously purified β-1,3-glucanase from G. natalis matched exactly that of the putative protein sequence. This plus the molecular masses of the putative proteins matching that of the purified proteins strongly suggests that the sequences obtained encode for a catalytically active β-1,3-glucanase. A glycosyl hydrolase family 16 cDNA was also partially sequenced from the midgut glands of other amphibious ( Mictyris platycheles and Paragrapsus laevis ) and terrestrial decapod species ( Coenobita rugosus , Coenobita perlatus , Coenobita brevimanus and Birgus latro ) to confirm that the gene is widely expressed within this group. There are three possible hypothesised functions and thus evolutionary routes for the β-1,3-glucanase: 1) a digestive enzyme which hydrolyses β-1,3-glucans, 2) an enzyme which cleaves β-1,3-glycosidic bonds within cell walls to release cell contents or 3) an immune protein which can hydrolyse the cell walls of potentially pathogenic micro-organisms. [ABSTRACT FROM AUTHOR]