Quick and low cost immobilization of proteinases on polyesters: comparison of lactobacilli cell-envelope proteinase and trypsin for protein degradation

Cell-envelope proteinases (CEPs) are a class of proteolytic enzymes produced by lactic acid bacteria and have several industrially relevant applications. However, soluble CEPs are economically unfavorable for such applications due to their poor stability and lack of reusability. In a quest to prepare stable biocatalysts with improved performance, CEP from Lactobacillus delbrueckii subsp. lactis 313 and trypsin (as a model enzyme) were immobilized onto nonwoven polyester fabrics in a three-step protocol including ethylenediamine activation and glutaraldehyde crosslinking. Immobilization gave protein loading yields of 21.9% (CEP) and 67.7% (trypsin) while residual activity yields were 85.6% (CEP) and 4.1% (trypsin). The activity of the immobilized enzymes was dependent on pH, but was retained at elevated temperatures (40-70°C). An increase in Km values was observed for both enzymes after immobilization. After 70 days of storage, the immobilized CEP retained ca. 62% and 96% of initial activity when the samples were stored in a lyophilized form at -20°C or in a buffer at 4°C, respectively. Both immobilized CEP and trypsin were able to hydrolyze proteins such as casein, skimmed milk proteins and bovine serum albumin. This immobilization protocol can be used to prepare immobilized biocatalyst for various protein degradation processes.

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.

Dietary assimilation and the digestive strategy of the omnivorous anomuran land crab Birgus latro (Coenobitidae).

On Christmas Island, Indian Ocean, the diet of robber crabs, Birgus latro (Linnaeus) was generally high in fat, storage polysaccharides or protein and largely comprised fruits, seeds, nuts and animal material. The plant items also contained significant amounts of hemicellulose and cellulose. In laboratory feeding trials, crabs had similar intakes of dry matter when fed artificial diets high in either fat or storage polysaccharide, but intake was lower on a high protein diet. Assimilation coefficients of dry matter (69–74%), carbon (72–81%), nitrogen (76–100%), lipid (71–96%) and storage polysaccharide (89–99%) were high on all three diets. B. latro also assimilated significant amounts of the chitin ingested in the high protein diet ( 93%) and hemicellulose (49.6–65%) and cellulose (16–53%) from the high carbohydrate and high fat diets. This is consistent with the presence of chitinase, hemicellulase and cellulase enzymes in the digestive tract of B. latro. The mean retention time (27.2 h) for a dietary particle marker (⁵⁷Co-labelled microspheres) was longer than measured in leaf-eating land crabs. The feeding strategy of B. latro involves the selection of highly digestible and nutrient-rich plant and animal material and retention of the digesta for a period long enough to allow extensive exploitation of storage carbohydrates, lipids, protein and significant amounts of structural carbohydrates (hemicellulose, cellulose and chitin).

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.

Endogenous production of endo-β-1,4-glucanase by decapod crustaceans

The potential ability to produce cellulase enzymes endogenously was examined in decapods crustaceans including the herbivorous gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes, the amphibious freshwater crab Austrothelphusa transversa, the terrestrial hermit crab, Coenobita variabilis the parastacid crayfish Euastacus, and the crayfish Cherax destructor. The midgut gland of both G. natalis and D. hirtipes contained substantial total cellulase activities and activities of the cellulase enzymes endo-β-1,4-glucanase and β-glucosidase. With the exception of total cellulase and β-glucosidase from D. hirtipes, the enzyme activities within the midgut gland were higher than those within the digestive juice. Hence, the enzyme activities appear to reside predominantly within midgut gland, providing indirect evidence for endogenous synthesis of cellulase enzymes by this tissue. A 900 bp cDNA fragment encoding a portion of the endo-β-1,4-glucanase amino acid sequence was amplified by RT-PCR using RNA isolated from the midgut gland of C. destructor, Euastacus, A. transversa and C. variabilis. This provided direct evidence for the endogenous production of endo-β-1,4-glucanase. The 900 bp fragment was also amplified from genomic DNA isolated from the skeletal muscle of G. natalis and D. hirtipes, clearly indicating that the gene encoding endo-β-1,4-glucanase is also present in these two species. As this group of evolutionary diverse crustacean species possesses and expresses the endo-β-1,4-glucanase gene it is likely that decapod crustaceans generally produce cellulases endogenously and are able to digest cellulose.

In vitro effect of arsenical compounds on glutathione-related enzymes

The mechanism of arsenic toxicity is believed to be due to the ability of arsenite (AsIII) to bind protein thiols. Glutathione (GSH) is the most abundant cellular thiol, and both GSH and GSH-related enzymes are important antioxidants that play an important role in the detoxification of arsenic and other carcinogens. The effect of arsenic on the activity of a variety of enzymes that use GSH has been determined using purified preparations of glutathione reductase (GR) from yeast and bovine glutathione peroxidase (GPx) and equine glutathione S-transferase (GST). The effect on enzyme activity of increasing concentrations (from 1 μM to 100 mM) of commercial sodium arsenite (AsIII) and sodium arsenate (AsV) and a prepared arsenic(III)−glutathione complex [AsIII(GS)3] and methylarsenous diiodide (CH3AsIII) has been examined.

Skeletal muscle htererogeneity in fasting-induced upregulation of genes encoding UCP2, UCP3, PPARĂ and key enzymes of lipid oxidation

The uncoupling protein homologs UCP2 and UCP3 have been proposed as candidate genes for the regulation of lipid metabolism. Within the context of this hypothesis, we have compared, from fed and fasted rats, changes in gene expression of skeletal muscle UCP2 and UCP3 with those of carnitine palmitoyltransferase I and medium-chain acyl-CoA dehydrogenase, two key enzymes regulating lipid flux across the mitochondrial #-oxidation pathway. In addition, changes in gene expression of peroxisome proliferator-activated receptor gamma, a nuclear transcription factor implicated in lipid metabolism, were also investigated. The results indicate that in response to fasting, the mRNA levels of UCP2, UCP3, carnitine palmitoyltransferase I and medium-chain acyl-CoA dehydrogenase are markedly increased, by three- to sevenfold, in the gastrocnemius and tibialis anterior (fast-twitch muscles, predominantly glycolytic or oxidative-glycolytic), but only mildly increased, by less than twofold, in the soleus (slow-twitch muscle, predominantly oxidative). Furthermore, such muscle-type dependency in fasting-induced transcriptional changes in UCP2, UCP3, carnitine palmitoyltransferase and medium-chain acyl-CoA dehydrogenase persists when the increase in circulating levels of free fatty acids during fasting is abolished by the anti-lipolytic agent nicotinic acid - with blunted responses only in the slow-twitch muscle contrasting with unabated increases in fast-twitch muscles. Independently of muscle type, however, the mRNA levels of peroxisome proliferator-activated receptor gamma are not altered during fasting. Taken together, these studies indicate a close association between fasting-induced changes in UCP2 and UCP3 gene expression with those of key regulators of lipid oxidation, and are hence consistent with the hypothesis that these UCP homologs may be involved in the regulation of lipid metabolism. Furthermore, they suggest that in response to fasting, neither the surge of free fatty acids in the circulation nor induction of the peroxisome proliferator-activated receptor gamma gene may be required for the marked upregulation of genes encoding the UCP homologs and key enzymes regulating lipid oxidation in fast-twitch muscles.

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

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 V_max of 42.0 µmol 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 β-1,3-glycosidic bonds. In addition to the hydrolysis of β-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 V_max of 19.6 µmol 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-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 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.

Morphological and biochemical alterations of abalone testicular germ cells and spawned sperm and their fertilizing ability

In this study, we aimed to detect morphological and biochemical changes in developing germ cells (Gc), testicular sperm (Tsp), and spawned sperm (Ssp) using capacitation-associated characteristics. Gradual changes in the profiles of two membrane proteins, namely NaCl- and detergent-extractable proteins, were observed as compared Gc with Tsp and Tsp with Ssp. These membrane modifications were accomplished mostly through the introduction of new protein sets, both peripheral and integral, into Tsp and Ssp membranes. Activation of serine proteases, particularly in Ssp detergent-extracted proteins with the molecular masses of 38–130 kDa was evident and marked a major difference between Ssp and Tsp. An increase in the level of tyrosine phosphorylation of the proteins ranging from 15 to 20 kDa was noted in Tsp and remained constant in Ssp. Specifically, these three capacitation-associated characteristics could be detected in Ssp, possessing full fertilizing capacity. The lack of an activated proteolytic activity in Tsp resulted in a delayed fertilization, but not affected fertilizing ability. We believe that these characteristics should be advantageous in predicting abalone sperm fertilizing capability, particularly in cases when isolated germ cells or purified Tsp are used in place of spawned sperm in abalone aquaculture.

Developmental changes in the expression of creatine synthesizing enzymes and creatine transporter in a precocial rodent, the spiny mouse

Background : Creatine synthesis takes place predominately in the kidney and liver via a two-step process involving AGAT (L-arginine:glycine amidinotransferase) and GAMT (guanidinoacetate methyltransferase). Creatine is taken into cells via the creatine transporter (CrT), where it plays an essential role in energy homeostasis, particularly for tissues with high and fluctuating energy demands. Very little is known of the fetal requirement for creatine and how this may change with advancing pregnancy and into the early neonatal period. Using the spiny mouse as a model of human perinatal development, the purpose of the present study was to comprehensively examine the development of the creatine synthesis and transport systems.

Results : The estimated amount of total creatine in the placenta and brain significantly increased in the second half of pregnancy, coinciding with a significant increase in expression of CrT mRNA. In the fetal brain, mRNA expression of AGAT increased steadily across the second half of pregnancy, although GAMT mRNA expression was relatively low until 34 days gestation (term is 38–39 days). In the fetal kidney and liver, AGAT and GAMT mRNA and protein expression were also relatively low until 34–37 days gestation. Between mid-gestation and term, neither AGAT or GAMT mRNA or protein could be detected in the placenta.

Conclusion : Our results suggest that in the spiny mouse, a species where, like the human, considerable organogenesis occurs before birth, there appears to be a limited capacity for endogenous creatine synthesis until approximately 0.9 of pregnancy. This implies that a maternal source of creatine, transferred across the placenta, may be essential until the creatine synthesis and transport system matures in preparation for birth. If these results also apply to the human, premature birth may increase the risk of creatine deficiency.

Amylase and glucosidase enzyme inhibitory activity of ginger (Zingiber officinale Roscoe) an in vitro study

The prevalence of type 2 diabetes has reached to an epidemic proportion in Sri Lanka. The need for achieving better control of blood glucose level has been evident in diabetes management. However it is not easy to achieve this goal in a large proportion of patients. This is partly due to limitations of currently available pharmacological agents which stimulate research on novel anti-diabetic agents with different mechanisms. Digestive enzymes have been targeted as potential avenues for modulation of blood glucose concentration through inhibition of the enzymatic breakdown of complex carbohydrates to meal derived glucose absorption. Acarbose is a widely used oral anti-diabetic drug which inhibits the α-glucosidase, enzyme responsible for breaking down of disaccharides and polysaccharides into glucose. Many herbal extracts have been found to posses similar inhibitory effects. Ginger (Zingiber officinale Roscoe) has developed a reputation in treatment of several diseases. In vitro enzymic inhibitory effect of ginger was investigated in this study. Enzymes α -amylase and α -glucosidase treated with either Acarbose or ginger extract were allowed to react with cooked rice and percentages of glucose content were measured. The glucosidase and amylase activities on the rice were inhibited by addition of ginger cause significant reduction in glucose percentages (36.86± 1.05 to 26.87± 2.17, P<0.05 and 49.04±0.65 to 35.35±2.22, P<0.05) which showed comparable results with Acarbose on glucosidase activity (36.86± 1.05 to, 27.8±1.32 P<0.05). Results of the study indicates ginger as a potential plant based amylase and glucosidase inhibitor in carbohydrate digestion but usage in glycaemic control in human has to be investigated further.

Comparative effects of the blue green algae Nodularia spumigena and a lysed extract on detoxification and antioxidant enzymes in the green lipped mussel (Perna viridis)

Nodularia spumigena periodically proliferates to cause toxic algal blooms with some aquatic animals enduring and consuming high densities of the blue green algae or toxic lysis. N. spumigena contains toxic compounds such as nodularin and lipopolysaccharides. This current work investigates physiological effects of exposure from bloom conditions of N. spumigena cells and a post-bloom lysis. Biochemical and antioxidative biomarkers were comparatively studied over an acute 3-day exposure. In general, a post-bloom N. spumigena lysis caused opposite physiological responses to bloom densities of N. spumigena. Specifically, increases in glutathione (GSH) and glutathione peroxidase (GPx) and decreases in glutathione S-transferase (GST) were observed from the N. spumigena lysis. In contrast, N. spumigena cell densities decreased GSH and increased GST and lipid peroxidation (LPO) in mussels. Findings also suggest that at different stages of a toxic bloom, exposure may result in toxic stress to specific organs in the mussel.

Modeling of enzyme–substrate complexes for the metalloproteases MMP-3, ADAM-9 and ADAM-10

The matrix metalloproteases (MMPs) and the ADAMs (A Disintegrin And Metalloprotease domain) are proteolytic enzyme families containing a catalytic zinc ion, that are implicated in a variety of normal and pathological processes involving tissue remodeling and cancer. Synthetic MMP inhibitors have been designed for applications in pathological situations. However, a greater understanding of substrate binding and the catalytic mechanism is required so that more effective and selective inhibitors may be developed for both experimental and clinical purposes. By modeling a natural substrate spanning P4-P4‘ in complex with the catalytic domains, we aim to compare substrate-specificities between Stromelysin-1 (MMP-3), ADAM-9 and ADAM–10, with the aid of molecular dynamics simulations. Our results show that the substrate retains a favourable antiparallel beta-sheet conformation on the P-side in addition to the well-known orientation of the P'-region of the scissile bond, and that the primary substrate selectivity is dominated by the sidechains in the S1' pocket and the S2/S3 region. ADAM-9 has a hydrophobic residue as the central determinant in the S1' pocket, while ADAM-10 has an amphiphilic residue, which suggests a different primary specificity. The S2/S3 pocket is largely hydrophobic in all three enzymes. Inspired by our molecular dynamics calculations and supported by a large body of literature, we propose a novel, hypothetical, catalytic mechanism where the Zn-ion polarizes the oxygens from the catalytic glutamate to form a nucleophile, leading to a tetrahedral oxyanion anhydride transition state.

Seasonal variations of total lipid and fatty acid contents in muscle, gonad and digestive glands of farmed jade tiger hybrid abalone in Australia

The lipid and fatty acid (FA) contents of muscle, gonad and digestive glands (DG) of Jade Tiger hybrid abalone were studied over the four seasons. Higher contents of total lipid and saturated fatty acids (SFA) were found in summer from muscle. For gonad the higher total lipid content was found in summer and spring whereas the SFA content peaked in summer only. For DG the higher contents of total lipid and SFA were recorded in all seasons except autumn. Winter samples showed significantly higher content of PUFA in all three types of tissue. High contents of eicosapentaenoic acid (EPA, 20:5 n−3), docosapentaenoic acid (DPA, 22:5 n−3) and docosahexaenoic acid (DHA, 22:6 n−3) were recorded in winter from muscle, although no marked variations were observed from gonad. For DG the high content of DHA was also observed in winter whilst EPA and DPA maintained high levels in all seasons except summer.