Presence and properties of cellulase and hemicellulase enzymes of the gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes.

Digestive juice from the herbivorous gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes exhibited total cellulase activity and activities of two cellulase enzymes; endo-ß-1,4-glucanase and ß-1,4-glucosidase. These enzymes hydrolysed native cellulose to glucose. The digestive juice of both species also contained laminarinase, licheninase and xylanase, which hydrolysed laminarin, lichenin and xylan, respectively, to component sugars. The pH optima of ß-1,4-glucosidase, endo-ß-1,4-glucanase and total cellulase from G. natalis were 4–5.5, 5.5 and 5.5–7, respectively. In the digestive juice from D. hirtipes, the corresponding values were 4–7, 5.5–7 and 4–9, respectively. The pH of the digestive juice was 6.69±0.03 for G. natalis and 6.03±0.04 for D. hirtipes and it is likely that the cellulases operate near maximally in vivo. In G. natalis, total cellulase activity and endo-ß-1,4-glucanase activity were higher than in D. hirtipes, and the former species can thus hydrolyse cellulose more rapidly. ß-1,4-glucosidase from G. natalis was inhibited less by glucono-D-lactone (Ki=11.12 mmol l-1) than was the ß-1,4-glucosidase from D. hirtipes (Ki=4.53 mmol l-1). The greater resistance to inhibition by the ß-1,4-glucosidase from G. natalis may contribute to the efficiency of the cellulase system in vivo by counteracting the effects of product inhibition and possibly dietary tannins. The activity of ß-1,4-glucosidase in the digestive juice of D. hirtipes was higher than that of G. natalis.

Food utilisation and digestive ability of aquatic and semi-terrestrial crayfishes, Cherax destructor and Engaeus sericatus (Astacidae, Parastacidae)

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-β-1,4-glucanase, β-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-β-1,4-glucanase and β-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-β-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.

One-step purification and immobilization of his-tagged rhamnosidase for naringin hydrolysis

α-l-Rhamnosidase (EC 3.2.1.40) is an enzyme that catalyzes the cleavage of terminal rhamnoside groups from naringin to prunin and rhamnose. In this study, a His-tag was genetically attached to the rhamnosidase gene ramA from Clostridium stercorarium to facilitate its purification from Escherichia coli BL21 (DE3) cells containing the pET-21d/ramA plasmid. Immobilized metal-chelate affinity chromatography (IMAC) resulted in one-step purification of N-terminally His-tagged recombinant rhamnosidase (N-His-CsRamA) which was immobilized in Ca²⁺ alginate (3%) beads. The optimum pH levels of the free and immobilized recombinant rhamnosidase were found to be 6.0 and 7.5, and the optimum temperature 55 and 60 °C respectively. At 50 °C, the free enzyme was relatively stable and exhibited a less than 50% reduction in residual activity after 180 min of incubation. The free and immobilized enzymes achieved 76% and 67% hydrolysis of the naringin in Kinnow juice respectively. Immobilization of recombinant rhamnosidase enabled its reutilization up to 9 hydrolysis batches without an appreciable loss in activity. This result indicated that the His-tagged thermostable rhamnosidase could be prepared as described and may serve to illustrate an economical and commercially viable process for industrial application.

Cloning and characterisation of the hint homologue of the Thermophile Thermus thermophilus

Screening of a genomic library of the thermophile Thermus thermophilus revealed a novel thermophilic hint gene, homologues of which are highly conserved in genera from archaea to mammals. Hint belongs to the HIT protein super-family, which contains two broad groups, Fhit, associated with tumour suppression in eukaryotes and Hint with putatitive protein kinase C inhibitory activity. In T. thermophilus the 321bp gene has a GC content of 67% overall and 94.4% in the third nucleotide position, with unusually no thymine as a wobble base. The gene product, a small highly conserved 11996Da predicted soluble cytoplasmic protein, offers an ideal opportunity to investigate thermostabilising amino acid substitutions. Here we report on the characterisation of the novel hint sequence.

Xylanolytic modification in wheat flour and its effect on dough rheological characteristics and bread quality attributes

Effects of various xylanase treatments applied at different stages of bread making process on dough rheological characteristics and bread quality attributes were investigated. Different doses (200, 400, 600, 800, and 1000 IU) of purified enzyme were applied at two stages (tempering and mixing). In milling and dough making processes, both types of flour (subjected to enzyme treatment during tempering and flour mixing) exhibited decreasing trend in water absorption, dough development time, dough stability, softening of dough, dough mixing time, viscosity peak, set back, and increasing tendency in peak height and pasting temperature. Treatments during tempering resulted in more significant effects as compared to applications during flour mixing. The dough rising during proofing resulted in enhancement from 137±3.21% (control) to maximum value (192.33±2.90%), when 600 IU of xylanases were applied to 1 kg of wheat grains during tempering. The bread sensory attributes also exhibited significant improvement in response to various doses of purified enzymes.