Intidhar Bkhairia

Biochemical and molecular characterisation of a new alkaline trypsin from Liza aurata: Structural features explaining thermal stability

This study investigated the fine structure and biochemical characterization of trypsin from the viscera of Liza aurata. The purified enzyme displayed an apparent molecular weight of 23 kDa as determined by sodium dodecyl sulphate-polyaccrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature for the proteolytic activity were 10.0 and 50 °C, respectively. Trypsin was strongly inhibited by serine protease inhibitor. The cDNA of the mature trypsin was cloned and sequenced. It encodes a protein of 222 amino acids, having only 86% of identity with its most homologous trypsin II of the Salmo salar. A phylogenic analysis showed that L. aurata trypsin (LAT) is close to fish enzymes. Given the high amino acid sequence homology between fish enzymes, a 3-D structure model was built using the structure of S. salar as a template. According to this model, structural features common with warm-active trypsins would explain why LAT acts at high temperatures, unlike cold adapted enzymes.

Octopus vulgaris protein hydrolysates: characterization, antioxidant and functional properties

Composition, functional properties and in vitro antioxidant activities of octopus (Octopus vulgaris) protein hydrolysates (OPHs) were evaluated. OPHs were prepared by treatment with commercial Esperase (OPH-Esp), alkaline protease extract from Zebra blenny (Salaria basilica) (OPH-ZB) and enzyme preparation from Bacillus subtilis A26 (OPH-A26). OPHs showed different degrees of hydrolysis (DH from 17.6 to 21%), and hydrophobic/hydrophilic peptide ratio. The amino acid profiles of OPHs showed a high level of essential amino acids, and Lys was the most abundant amino acid. Enzymatic hydrolysis improved solubility significantly as well as emulsifying and foaming properties of octopus proteins. The emulsifying activity index of OPHs decreased with increasing concentrations. Conversely, the foaming abilities increased as the hydrolysate concentrations increased. For the antioxidant activities, five different in vitro assay systems were investigated. All hydrolysates displayed various degrees and dose dependant antioxidant activities. The highest DPPH scavenging activity and reducing power were achieved by OPH-A26. OPH-Esp displayed the highest ability to prevent the bleaching of β-carotene, whereas OPH-ZB exhibited the highest protection against hydroxyl radical induced DNA breakage. The results suggested that OPHs could be used, as a promising source of functional peptides with antioxidant activities, to formulate functional foods.

Evaluation of the biotechnological potential of a novel purified protease BS1 from Bacillus safensis S406 on the chitin extraction and detergent formulation

An extracellular alkaline stable protease BS1 from a new bacteria strain, Bacillus safensis S406, isolated from the Sfax solar saltern, was purified and characterized. The enzyme was purified to homogeneity by ammonium sulfate precipitation, Sephadex G-75 gel filtration, Mono-Q anion-exchange chromatography and ultrafiltration, with a 12.70-fold increase in specific activity and 20.29% recovery. The enzyme has a molecular weight of 29kDa and appeared as a single band on native-PAGE. The optimum pH and temperature values of its proteolytic activity were pH 11.0 and 60°C, respectively. BS1 was tested for the deproteinization of shrimp wastes to extract chitin. An enzyme-protein ratio of 10U/mg of proteins allows to eliminate 93% of protein linked to the chitin after 3h hydrolysis at 45°C. Being very active in alkaline conditions, the potential application of BS1 in laundry formulation was investigated. The enzyme showed high stability in the presence of non-ionic surfactants and some commercial liquid and solid detergents, suggesting its eventual use in detergent formulations.

Antioxidant and hemolytic activities, and effects in rat cutaneous wound healing of a novel polysaccharide from fenugreek (Trigonella foenum-graecum) seeds.

The aim of this work was to evaluate the antioxidant and hemolytic activities as well as the in vivo wound healing performance of a novel polysaccharide (FWEP) extracted from fenugreek (Trigonella foenum-graecum) seeds. The antioxidant activity was evaluated in vivo and in vitro using various assays. Results showed that FWEP exhibited strong antioxidant activities but no hemolytic activity was observed towards bovine erythrocytes. The application of FWEP hydrogel on the wound site in a rat model enhanced significantly wound healing activity and accelerated the wound closure after 14days of wound induction. Histological examination also demonstrated fully re-epithelialized wound with a complete epidermal regeneration. Altogether, these evidences demonstrated that FWEP had strong wound healing potential presumably achieved through its antioxidant activities.

Golden Grey Mullet (Liza aurata) Alkaline Proteases: Biochemical Characterization, Application in the Shrimp Wastes Deproteinization, Laundry Commercial Detergents, and Preparation of Antioxidant Protein Hydrolysate

Digestive alkaline proteinases from golden grey mullet (Liza aurata) were extracted and characterized. The crude alkaline protease showed optimum activity at pH 8.0 and 60°C, and it was highly stable over a wide range of pH from 4.0 to 10.0, retaining more than 80% activity after incubation for 1 h at 4°C. The alkaline proteases showed extreme stability toward nonionic and anionic surfactants after preincubation for 1 h at 25°C and relative stability toward oxidizing agents. Additionally, the crude enzyme showed excellent stability and compatibility with various solid and liquid detergents. Further, proteases from golden grey mullet viscera were found to be effective in the deproteinization of shrimp wastes. The protein removal after 3 h at 45°C with an enzyme/substrate (E/S) ratio of 10 U/mg protein was about 76%. The golden grey mullet proteases were also shown to be efficient in the production of antioxidant protein hydrolysate.

Increasing maneb doses induces reactive oxygen species overproduction and nephrotoxicity in adult mice

Abstract Background and purpose: The aim of this study was to elucidate the biochemical, molecular and histopathological aspects of the kidney injuries as well as the hematological perturbations induced after adult mice exposure to increasing doses of maneb (MB). Material and method: Adult mice were intraperitoneally treated for seven days with four graded doses of MB, corresponding to 1/8, 1/6, 1/4 and 1/2 of its lethal dose (LD50=1500 mg/kg body weight). Results: Hematological analysis revealed a significant disruption in total white blood cells and platelets and a significant decrease in the plasmatic levels of ferrozine in mice treated with 1/8, 1/6 and 1/4 of MB LD50. However, the ferrozine levels increased significantly in the group treated with 1/2 of MB LD50. Evenly, our results showed a significant increase in the levels of malondialdehyde, lipid hydroperoxides, hydrogen peroxide and advanced oxidation protein products in all treated groups. The activities of catalase and glutathione peroxidase decreased significantly in all MB treated mice. Additionally, all treated groups exhibited strong nephrotoxicity signs, including increases in plasma urea, creatinine and albumin levels and lactate dehydrogenase activity, as well as a significant decrease in uric acid levels. Electrophoresis analysis revealed nucleic acid degradation, testifying the genotoxicity of MB. Moreover, the histopathological observations showed severe renal injuries, which could be related to the above mentioned data. Conclusions: Our data showed, for the first time, that the MB tested doses led to oxidative stress installation causing renal cell damages and lowering all defense systems capacities.

New acidic proteases from Liza aurata viscera: Characterization and application in gelatin production.

The present study reports the extraction and characterisation of acidic protease from the viscera of golden grey mullet (LACAP), and its use in gelatin preparation. The optimum pH for the crude extract activity was 3.0, with high stability over a pH range from 3.0 to 7.0. The enzymatic extract lost about 79% of its activity by pepstatin A. Due to its high activity under acidic conditions, gelatin was extracted from the skin of L. aurata using different levels of LACAP (0, 5 and 10U/g of skin, named GGSG0, GGSG5 and GGSG10). The extraction yield of GGSG0 was only 3.3% and the addition of acidic proteases increased the yields, which reached 5% and 8.2% at 5 and 10U/g of skin, respectively. In addition, Fourier transform infrared (FT-IR) and SDS-page profiles of gelatins indicated that the structure was affected by enzymatic pre-treatment. The results of functional properties showed that the emulsion stability and activity indexes (ESI and EAI) of GGSG0 were higher than those of GGSG5 and GGSG10. Furthermore, foam expansion (FE) and foam stability (FS) increased as the concentration of gelatin increased. The results showed that L. aurata by-products can be a potential source of gelatin and protease.

In vitro and in vivo anti-diabetic and anti-hyperlipidemic effects of protein hydrolysates from Octopus vulgaris in alloxanic rats

This study aims to examine the effects of non-hydrolyzed octopus (Octopus vulgaris) muscle proteins (NHOPs) and their hydrolysates (OPHs) on alloxan induced diabetes in Wistar rats (AIDR). Animals were allocated into seven groups of six rats each: control group (C), diabetic group (D) and diabetic rats treated with acarbose (D + Acar), non-hydrolyzed octopus proteins (D + NHOPs) and octopus proteins hydrolysates (D + OPHs) groups. The diabetic rats presented a significant increase in glycemic status such as α-amylase activity (in plasma, pancreas and intestine), hepatic glycogen, blood glucose and glycated hemoglobin (HbA1c) levels, as well as a significant decrease in the levels of plasma insulin and total hemoglobin compared to control group. In addition, plasma and liver contents in total cholesterol, triglycerides and LDL-cholesterol significantly increased in AIDR compared to control group. However, the daily administration of OPHs for 30 days improved the glucose tolerance test, the glycemic status of diabetic rats and corrected the lipid profiles. Further, a significant increase in the activities of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and gamma-glutamyl transpeptidase as well as in the level of plasma bilirubin on diabetic status was observed, indicating considerable hepatocellular injury. OPHs treatment was found to attenuate the increased activities of the plasma enzymes produced by diabetes and caused a subsequent recovery towards normalization compared to the control group. By contrast, the NHOPs treatment was found to increase the glucose metabolic disorders in AIDR. These beneficial effects of OPHs were confirmed by histological findings in the hepatic and pancreatic tissues of diabetic treated rats. Indeed, they avoid lipid accumulation in the hepatocytes and protect the pancreatic β-cells from degeneration. Our results thus suggest that OPHs may be helpful in the preventing from diabetic complications by reversing hepatotoxicity.

Protective effect of Sargussum vulgare sulfated polysaccharide against molecular, biochemical and histopathological damage caused by alloxan in experimental diabetic rats

Diabetes is a serious condition that is linked to the development of oxidative stress. In the context of enhancing the biodiversity of Tunisias flora, this study aimed to evaluate the effect of Sargussum vulgare sulfated polysaccharide (SVSP) on hyperglycemia and diabetes complications in the alloxan-induced diabetic rats. Our results showed a disturbance of carbohydrate, lipid, hematological and histopathological parameters, an increase in the α-amylase enzyme activity and damage to the pancreatic, hepatic and renal tissues in rats rendered diabetic by alloxan. In contrast, treatment with SVSP resulted in a correction of fasting and postprandial blood glucose and HbA1c through inhibition of pancreatic α-amylase. We also noticed an improvement in hemogram parameters and an attenuation of the pancreatic oxidative stress markers as well as histological protections. On the other hand, the administration of SVSP to diabetic rats caused, besides the correction of glycemic and lipid parameters, a good defense against hepatotoxicity and nephrotoxicity of diabetes as well as good antioxidant status and histological protections.

Zebra blenny protein hydrolysates as a source of bioactive peptides with prevention effect against oxidative dysfunctions and DNA damage in heart tissues of rats fed a cholesterol-rich diet

High-cholesterol diet (HCD) is suspected to produce in excess free radicals having adverse effects on human health and causing atherosclerosis damage in heart tissues. In our study, the effects of zebra blenny protein hydrolysates (ZBPHs) were investigated on cardiac oxidant/antioxidant status as well as DNA damage and histopathological disorders in rats, fed with a hypercholesterolemic diet. The molecular weight distribution of the hydrolysates was determined by size exclusion chromatography, which analyzed a representative hydrolysate type with a weight range of 3-20kDa. ZBPHs effectively protected heart genomic DNA against oxidative damage induced by Fentons reagent. HCD promoted oxidative stress with a rise in serum aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) activities and thiobarbituric acid reactive substance (TBARS), advanced oxidation protein product (AOPP) and hydrogen peroxide (H2O2) levels in heart tissues. An increase in glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) activities as well as a fall in ATPase activities and glutathione (GSH) level was also noted in heart of hypercholesterolemic rats. Treatment with ZBPHs ameliorated the biochemical parameters cited above. In addition, pre-treatment with ZBPHs prevented DNA fragmentation. The histopathological findings confirmed the biochemical results and the potential antioxidant activities of ZBPHs which can help the cure and management of cardiovascular diseases induced by high-cholesterol levels.