Search

In this study we report the extraction and characterization of sericin using water alone, from two non-mulberry silks - Antheraea assamensis (A. assamensis) and Philosamia ricini (P. ricini) compar...

Silk nanofibers have been produced and examined in recent years for a range of advanced biomedical and biotechnological applications. Their fabrication involves the canonical approach of dissolving...

We characterised fibres and papers of microfibrillated silk from Bombyx mori produced by mechanical and enzymatic process. Milling increased the specific surface area of fibres from 1.5 to 8.5 m2/g and that enzymatic pre-treatment increased it further to 16.5 m2/g. These fibrils produced a uniform, significantly strong (tenacity 55 Nm/g) and stiff (Young's modulus > 2 GPa) papers. Enzymatic pre-treatment did not reduce molecular weight and tensile strength of papers but significantly improved fibrillation. Silk remained highly crystalline throughout the fibrillation process. Protease biodegradation was more rapid after fibrillation. Biodegradation was impacted by structural change due to enzymatic pre-treatment during the fibrillation. Biodegraded silk had much higher thermal degradation temperature. The unique combination of high strength, slow yet predicable degradation and controllable wicking properties make the materials ideally suited to biomedical and healthcare applications.

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 1383bp 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 47.6-47.7kDa; this closely matches the molecular mass (47.4kDa) 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 1356bp was sequence from M. platycheles, while an incomplete open reading frames of 1384 and 1523bp were respectively sequenced from Coenobita brevimanus and C. perlatus. The midgut gland of M. platycheles contained activity (0.704±0.218μmol 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.

Silver nanoparticles (AgNPs) are in situ synthesized for the first time on microfibrillated silk (MFS) exfoliated from domesticated Philosamia cynthia ricini (eri) and Bombyx mori (mulberry) silkworm silk fibers. The process is rapid (hours time), does not rely on harmful chemicals, and produces robust and flexible AgNPs coated MFS (MFS-AgNPs) protein papers with excellent handling properties. None of these can be achieved by approaches used in the past to fabricate AgNPs silk systems. MFS bonds the AgNPs strongly, providing good support and stabilization for the NPs, leading to strong wash fastness. The mechanical properties of the MFS-AgNPs papers largely do not change compared to the MFS papers without nanoparticles, except for some higher concentration of AgNPs in the case of mulberry silk. The improved tensile properties of eri silk papers with or without AgNPs compared to mulberry silk papers can be attributed to the higher degree of fibrillation achieved in eri silk and its inherent higher ductility. MFS-AgNPs from eri silk also exhibit strong antibacterial activity. This study provides the basis for the development of smart protein papers based on silk fiber and functional nanomaterials.

SUMMARY A 160 kDa enzyme with β-glucosidase 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

Terrestrial decapods consume a wide variety of plant and animal material. The potential adaptations of carnivorous, omnivorous, and herbivorous terrestrial crustaceans were studied by examining the functional morphology of the gastric mill. Two closely related species from each feeding preference group were examined to identify which features of the mill were due to phylogeny and which were due to adaptation. The morphology of the gastric mill matched the diet well; the gastric mills of the carnivorous species (Geograpsus grayi and Geograpsus crinipes) possessed a blunt, rounded medial tooth and flattened lateral teeth with a longitudinal grinding groove. These features make them well suited to a carnivorous diet of soft animal tissue as well as hard material, such as arthropod exoskeleton. In contrast, the mill of the herbivorous gecarcinids (Gecarcoidea natalis and Discoplax hirtipes) consisted of a medial tooth with sharp transverse ridges and lateral teeth with sharp interlocking cusps and ridges and no grinding surface. These features would efficiently shred fibrous plant material. The morphology of the mill of the omnivorous coenobitids (Coenobita perlatus and Birgus latro) was more generalized toward a mixed diet. However, the mill of B. latro was more adapted to deal with highly nutritious food items, such as nuts and heavily calcified decapods. Its mill possessed lateral teeth with extended ridges, which sat close to the calcified cardiopyloric valve to form a flattened floor. Hard items trapped in the mill would be crushed against this surface by the medial tooth.

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.

Both Engaeus sericatus and Cherax destructor are omnivorous crayfishes consuming a variety of food items. Materials identified in the faeces of both E. sericatus and C. destructor consisted of mainly plant material with minor amounts of arthropod animals, algae and fungi. The morphology of the gastric mill of C. destructor suggests that it is mainly involved in crushing of food material while the gastric mill of E. sericatus appears to be better suited to cutting of food material. Given this, the gastric mill of E. sericatus may be better able to cut the cellulose and hemicellulose fibres associated with fibrous plant material. In contrast, the gastric mill of C. destructor appears to be more efficient in grinding soft materials such as animal protein and algae. Both species accumulated high amounts of lipids in their midgut glands (about 60% of the dry mass) which were dominated by triacylglycerols (81-82% of total lipids). The dominating fatty acids were 16:0, 16:1(n-7), 18:1(n-9), 18:2(n-6), and 18:3(n-3). The two latter fatty acids can only be synthesised by plants, and are thus indicative of the consumption of terrestrial plants by the crayfishes. The similarity analysis of the fatty acid patterns showed three distinct clusters of plants and each of the crayfish species. The complement of digestive enzymes, proteinases, total cellulase, endo-beta-1,4-glucanase, beta-glucosidase, laminarinase and xylanase within midgut gland suggests that both C. destructor and E. sericatus are capable of hydrolysing a variety of substrates associated with an omnivorous diet. Higher activities of total cellulase, endo-beta-1,4-glucanase and beta-glucosidase indicate that E. sericatus is better able to hydrolyse cellulose within plant material than C. destructor. In contrast to E. sericatus, higher total protease and N-acetyl-beta-D-glucosaminidase activity in the midgut gland of C. destructor suggests that this species is better able to digest animal materials in the form of arthropods. Differences in total cellulase and gastric mill morphology suggest that E. sericatus is more efficient at digesting plant material than C. destructor. However, the contents of faecal pellets and the fatty acid compositions seem to indicate that both species opportunistically feed on the most abundant and easily accessible food items.

SUMMARY 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 Vmax of 42.0 μmol reducing sugars produced min–1 mg protein–1, a Kmof 0.126% (w/v) and an optimum pH range of 5.5–7, and hydrolysed mainlyβ-1,3-glycosidic bonds. In addition to the hydrolysis ofβ-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 Vmax of 19.6 μmol reducing sugars produced min–1 mg protein–1, a Km 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 hydrolysingβ-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.

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.

This article reviews the current knowledge of cellulose and hemicellulose digestion by herbivorous land crabs using the gecarcinid Gecarcoidea natalis as a model species for this group. Cellulose digestion in the gecarcinids is hypothesised to require mechanical fragmentation and enzymatic hydrolysis. Mechanical fragmentation is achieved by the chelae, mandibles and gastric mill, which reduce the material to particles less than 53 µm. The gastric mill shows adaptations towards a plant diet; in particular, there are transverse ridges on the medial and lateral teeth and ventral cusps on the lateral teeth that complement and interlock to provide efficient cutting surfaces. Enzymatic hydrolysis of cellulose and hemicellulose is achieved through cellulase and hemicellulase enzymes. In the gecarcinids, 2–3 endo-β-1,4-glucanases, one β-glucohydrolase and a laminarinase have been identified. The endo-β-1,4-glucanases are multifunctional, with both endo-β-1,4-glucanase and lichenase activity. Complete cellulose hydrolysis is achieved through the synergistic action of the endo-β-1,4-glucanase and β-glucohydrolase. The evidence for the endogenous production of the cellulase and hemicellulase enzymes, their evolutionary origin and possible evolution in invertebrates as they colonised land is also discussed.