Evaluation of lupine seeds (Lupinus albus L.) neutral extract as a texture improver in low-fat yogurt production.

Aqueous lupine seeds (Lupinus albus L.) extracts were evaluated as a natural fat substitute in low-fat yogurt production. Thus, the chemical composition, particle size, molecular weight, total phenolic (TPC), and total flavonoids (TFC) of the selected extract were estimated. Also, the antimicrobial activity and antioxidant capacity of selected extract were investigated. Yogurt with neutral lupine extract (NeLP) had the highest all sensorial attributes compared to other extracts. Also, the incorporation of NeLP during low-fat yogurt processing increased the solid content, and viscosity, as well as improved the textural profile and sensorial attributes without any negative effect on the yogurt's color. SEM micrographs of NeLP-yogurt microstructure showed a matrix characterized by large fused casein micelles clusters with comparatively lower porosity compared to control yogurt (without NeLP). The chemical composition of NeLP indicated that the major sugar constituents are glucose and galactose with different molar fractions. The molecular weight of NeLP is 460.5 kDa with a particle size of 1519.9 nm. Also, IC50 of NeLP is 0.589 mg/ml, while TPC and TFC are 7.17, and 0.0137 g/100 g sample, respectively. Hence, lupine neutral extract (0.25%) could be used as a fat replacer or texture improver ingredient in such low-fat yogurt which led to improved its characteristics without any negative defect during 7 days at 5 °C.

Statistical optimization of enzyme cocktail production using Jew's mallow stalks residues by a new isolate Aspergillus flavus B2 via statistical strategy and enzymes characterization.

This study investigates the production of the enzyme cocktail by the isolated fungi Aspergillus flavus B2 (GenBank accession number OL655454) using agricultural and industrial (AI) residues as the sole substrate. Of all the AI residues tested, Jew's mallow stalk was the best inducer substrate for enzyme cocktail production without adding any nutrients. Statistical optimization using Response Surface Methodology enhanced the production by 5.45, 5.20, and 3.34-fold, respectively for pectinase, xylanase, and CMCase. Optimum temperature, activation energy (Ea), and activation energy for denaturation (Ed) were determined. Michaelis constant (Km) for CMCase, xylanase, and pectinase enzyme was 1.82, 1.23, and 1.05 mg/mL, respectively. Maximum reaction rate (Vmax) was 4.67, 5.29, and 17.13 U/mL, respectively for CMCase, xylanase, and pectinase. Thermal stability revealed that pectinase, CMCase, and xylanase enzymes retained 64.7%, 61.8%, and 53.2% residual activities after incubation for 1 h at 50 °C. Half-life time (t0.5) of pectinase, CMCase, and xylanase at 50 °C were 189.38, 129.8, and 127.89 min, respectively. Thermodynamics of the produced enzymes enthalpy (ΔH*d), free energy (ΔG*d), and entropy (ΔS*d) were determined at 40, 50, and 60 °C. In the presence of EDTA (5 mM), CMCase, xylanase, and pectinase retained 69.5%, 66.2%, and 41.2%, respectively of their activity. This work is significant for the valorization of AI residues and the production of value-added products.

Antioxidant capacity and antitumor activity of the bioactive protein prepared from orange peel residues as a by-product using fungal protease.

Agricultural and industrial residues (AIR) are renewable biomass sources present in large quantities causing pollution. Converting AIR to eco-friendly products (bioactive materials) reduces their quantity and impact on the environment, in addition to reducing production costs. Therefore, orange peel (OP) protein degradation, antioxidant capacity, and antitumor activity were investigated using Aspergillus niger WA 2017 protease. The highest value of the protein hydrolysate with the highest antioxidant using the DPPH method was obtained after 24 h. The single-factor method boosted the protein hydrolysate and the DPPH antioxidant activity by 3.7 and 1.7-fold, respectively. Statistical optimized conditions (Central Composite Method) increased the hydrolysate value and the DPPH antioxidant activity by 1.6 and 1.1-fold, respectively. The central trial samples exhibited the highest DPPH antioxidant activity (62.37 %), while the control sample recorded 20 %. All antioxidant tests in vitro (DPPH, reducing power, ABTS, and FRAP) confirmed the superiority of the potent hydrolysate as a good antioxidant. In vitro antitumor activity, the potent hydrolysate exhibited the highest effect on the Ehrlich Ascites Carcinoma Cells viability as it recorded 60.62 % dead cells. In vivo antitumor activity, the volume of the untreated tumor mice was found to be 1.4-fold bigger than the volume obtained from the potent hydrolysate. The increase in life span (ILS %) for oral treatment and intraperitoneal injection treatment with the potent hydrolysate increased by 13.91 and 19.42 %, respectively, compared to the untreated tumor.

Protease immobilization on a novel activated carrier alginate/dextrose beads: Improved stability and catalytic activity via covalent binding.

Protease from Bacillus thuringiensis strain-MA8 was successfully immobilized onto activated Alginate/dextrose (Alg/dex) beads as a new carrier with immobilization yield 77.6 %. The carrier was characterized using Scanning electron microscopy and Fourier transforms infrared spectrophotometer at every step of the immobilization process. Immobilized protease showed an increase of 10 °C in the optimum temperature compared to the free enzyme. However, the optimum pH for both the free and the Alg/dex/protease was found to be 8. The lower activation energy and deactivation rate constant and the higher half-life time and D-value confirm that the new Alg/dex carrier is suitable for promoting enzyme stability. The raise in thermal stability is also shown by the increased deactivation energy of the Alg/dex/protease compared to its free form by 1.47-fold. Likewise, the enzyme immobilization enhancement of Alg/dex/protease was accompanied by a marked increase in enthalpy and Gibbs free energy. The negative entropy for both free and Alg/dex/protease indicates that the enzyme is more stable in thermal deactivation. The Km and Vmax for the Alg/dex/protease were 2.05 and 1.22-times greater than the free form. Furthermore, Alg/dex/protease displayed good reusability as it retained 92.7 and 52.4 % of its activity after 8 and 12 hydrolysis cycles.

Statistical improvement of protease production from a new isolate Bacillus thuringiensis strain-MA8 and its application in the production of enzyme-modified cheese.

Enzyme-modified cheese (EMC) is a concentrated cheese flavor that is produced enzymatically from dairy substrates to provide an intense source of cheese flavor with broad applications. In this study, EMC was produced by enzymatic biotransformation from a new bacterial isolate described and molecularly identified as Bacillus thuringiensis strain-MA8. Optimization of protease production conditions using one-variable-at-a-time followed by multi-factorial (Plackett-Burman and Box-Behnken) designs increased production by 7-fold. Protease was used at different concentrations (300 and 900 U/100 g curd) as a cost-effective source of concentrated cheese flavor in the EMC preparation. Sensorial evaluation of EMC revealed that the overall acceptability, flavor, and texture were improved from the 2nd day compared to the control, and then decreased on the 4th day without any apparent bitterness. The chemical characteristics of EMC showed that the addition of protease extracts increased the total volatile fatty acids, water-soluble nitrogen, and acidity of EMC significantly (p≤0.05) compared to the control. The amino acids profile revealed that EMC1 which was treated with (300 U/100 g curd) protease had the highest essential amino acids (EAA) and EAA/total amino acids ratio. Nutritional parameters including protein efficiency ratio, biological value, and chemical score of EMC were higher than control based on Val, Met + Cys, Ile, Leu, and Phe + Tyr amino acids. Also, Scanning Electron Microscopy showed significant changes in EMC compared to the control. In conclusion, the addition of (300 U/100g curd) of protease revealed good EMC characteristics without any apparent defect.

Preparation and characterization of sugilite glass from basalt for α-amylase immobilization, statistical optimization of the immobilization process and description of free and immobilized enzyme.

Bacterial α-amylase was immobilized on sugilite from modified basalt rock as a new carrier. A set of glass compositions based on sugilite formula KNa2M2Li3Si12O30 (M = Al or Mn or Fe) were prepared. The glasses were prepared through melting-quenching technique and samples of glass were converted to glass ceramic. Among the tested glasses and glass ceramic only sugilite glass based on M = Fe (BSF) give promising results. The sugilite BSF glass was characterized using DSC analysis, FTIR absorption, and SEM. The sugilite glass revealed high thermal resistant till ∼770 °C. Under optimized conditions of the Central composite design, the immobilization yield improved by 4.7-fold. The affinity to starch increased after enzyme immobilization by 4.3-fold. The lower rate of deactivation constant and the increase of t ½ and D-value confirm the suitability of BSF and immobilization method in enhancing enzyme stability. The improvement in thermostability of immobilized α-amylase was judged by the change in thermodynamic parameters. In conclusion, the prepared sugilite BSF glass can be utilized as a new carrier suitable for stabilization of α-amylase enzyme by immobilization.

Solvent polarity dictates the anti-inflammatory potency and mechanism of two purslane (Portulaca oleracea) seed extracts.

This study aimed to assess the effect of solvent polarity on anti-inflammatory potency and the underlying mechanisms of two purslane seed extracts. Methanol and dichloromethane extracts were prepared using Soxhlet extraction and chromatographically analyzed. Antioxidant activities were assessed by different assays, while the anti-inflammatory potentials were assessed in RAW 264.7 macrophage cells. Methanol extraction yielded 15.5% water-soluble extract while dichloromethane produced 3.74% fixed oil. Nineteen phenolic compounds were chromatographically identified in methanol extract compared with 16 in the fixed oil including omega fatty acids and phytosterols. Methanol extract showed significantly higher capacity in radical scavenging assays (p < .001), but the fixed oil showed higher total antioxidant capacity (p < .001). Both extracts demonstrated anti-inflammatory potentials with different mechanisms, where the phenol-rich methanol extract significantly reduced TNF-α (p = .0371) and IL-1ß (p = .0029) production through an antioxidant-mediated pathway, while the fixed oil inhibited COX1, COX2, and PGE2 gene expression through the upregulation of IL-10. PRACTICAL APPLICATIONS: Both purslane extracts presented herein demonstrated remarkable antioxidant/ anti-inflammatory potentials that could be safely utilized as natural antioxidants and inflammation remedies or as functional food products, particularly that they showed no cytotoxic effects.

Pulicaria crispa mitigates nephrotoxicity induced by carbon tetrachloride in rats via regulation oxidative, inflammatory, tubular and glomerular indices.

CONTEXT: Carbon tetrachloride (CCl4) induces oxidative stress in various tissues by altering antioxidants defense system. Recently, there has been a substantial use of phytotherapy to treat different diseases. OBJECTIVE: This study was designed to evaluate the curative effect of Pulicaria crispa (Forssk.) Benth et Hook (Family Asteraceae) aerial parts ethanol extract against CCl4 induced toxicity in rats kidneys. MATERIALS AND METHODS: Nephrotoxicity was induced by intraperitoneal injection with CCl4 in a dose of 0.5 mL/kg b.wt./twice a week for six consecutive weeks. Serum kidney function tests, oxidative stress markers, inflammatory cytokines, nephrotoxicity biomarkers and histopathological observation were evaluated. RESULTS: CCl4 increased serum kidney function parameters, malondialdehyde level, inflammatory cytokines, and nephrotoxicity markers, while decreased certain oxidative stress indices as superoxide dismutase and glutathione refereeing to the control group (p < 0.0001). Administration of P. crispa ethanol extract to CCl4 injured rats attenuated these changes with variable degrees. The results were confirmed through the observed amelioration of the renal histological architectures. CONCLUSION: P. crispa ethanol extract possesses potent curative effect against CCl4-induced nephropathy through improvement of kidney function, oxidative stress, inflammatory and nephrotoxicity index and the renal histopathological features. To establish the therapeutic and pharmacological applications of the plant, additional researches are required.

Urinary transferrin and proinflammatory markers predict the earliest diabetic nephropathy onset.

AIM: This study aimed to determine the earliest markers of diabetic nephropathy (DN) onset with discriminative potentials from controlled diabetes (CD). METHODS: Sixty male Wistar rats were allocated into three groups (20/group), the two diabetic groups CD and DN received 45 and 65 mg/kg STZ in 0.1 mole/L citrate buffer, respectively, while the control group received only the vehicle. Serum/urinary levels of glomerular, tubular, oxidative and proinflammatory markers were weekly monitored. RESULTS: Each diabetic group showed a different pattern of inflammatory, oxidative and signs of nephropathy along the study period, but none had a discriminative power until the fourth week. At this time point, levels of urinary transferrin, serum/urinary IL-6 and TNF-α as well as urinary IL-18 were significantly higher in DN group compared to CD (p = 0.0217, <0.0001, 0.0005, 0.0004, 0.0006, 0.0019, respectively). Predictive thresholds of these markers were calculated by receiver operating characteristic (ROC) curve that showed area under curve (AUC) of 0.9375 for transferrin with cut-off value of 35.2 mg/dL and 1.000 for serum/urinary IL-6 and TNF-α and urinary IL-18 with cut-of values 224.1, 82.11, 6.596, 125.9 and 21.86 pg/mL, respectively. CONCLUSION: Urinary transferrin and the inflammatory endpoints proposed in this study might represent promising biomarkers for the early DN onset.

Rice straw and orange peel wastes as cheap and eco-friendly substrates: A new approach in ß-galactosidase (lactase) enzyme production by the new isolate L. paracasei MK852178 to produce low-lactose yogurt for lactose-intolerant people.

Converting wastes to valuable products is the main target for many kinds of research nowadays. Wastes represent an environmental problem and getting rid of it is not easy and causes pollution. Accordingly, this study offers production of the valuable enzyme ß-galactosidase using rice straw and orange peel as the main medium constituents. ß-galactosidase converts lactose to glucose and galactose which are simple sugars and can be fermented easily by lactose-intolerant people who represent more than 50% of the world's population. It was produced by Lactobacillus paracasei, a series isolated from fermented milk, identified using 16S ribosomal RNA gene partial sequence and had the accession number MK852178. Plackett-Burman (PB) and Central Composite (CCD) Designs optimized the production scoring 1.683(10)6 U/ml with a difference five times higher than the non-optimized medium. The addition of 0.3 or 0.6% of ß-galactosidase serves as a good fortification for manufacturing nutritional and therapeutic low-lactose yogurt with no significant differences in total protein, total solids, fat, and ash between control and all treatments. The chemical, rheological and sensory properties of the final produced yogurt were evaluated during storage periods up to 9 days at 5 °C. In conclusion, L. paracasei MK852178 ß-galactosidase is a promising additive in manufacturing low lactose yogurt for lactose-intolerant people since it reduces the lactose content and doesn't influence the chemical and sensory properties.

Corrigendum to "Catalytic, kinetic and thermal properties of free andimmobilized Bacillus subtilis -MK1 α-amylase on Chitosan-magnetic nanoparticles" [Biotechnol. Rep. 26 (2020) e00443].

[This corrects the article DOI: 10.1016/j.btre.2020.e00443.].

Catalytic, kinetic and thermal properties of free andimmobilized Bacillus subtilis -MK1 α-amylase on Chitosan-magnetic nanoparticles.

Bacillus subtilis strain-MK1 α-amylase was successfully immobilized on Chitosan-magnetic nanoparticles (Ch-MNP) that had been modified with polyethyleneimine (PEI) and glutaraldehyde (GA). Optimization of Ch-MNP/PEI/GA beads modification by Central Composite design enhanced the immobilization yield (IY %) by 1.5-fold. Ch-MNP/PEI/GA was characterized before and after modification and immobilization by FTIR and SEM. Ch-MNP/PEI/GA/Enzyme showed the same pH optima of free enzyme, while an elevation 10 °C in temperature optima was observed after its immobilization. Ch-MNP/PEI/GA/Enzyme displayed higher Km and Vmax values (2.1 and 1.2-fold) and lower Vmax/Km ratio (1.7-fold), respectively than the free enzyme. Compared to the free enzyme, Ch-MNP/PEI/GA/Enzyme exhibited lower activation energy, lower deactivation constant rate, higher D-values, higher half-life, and higher energy for denaturation. Immobilization of α-amylase increased enthalpy (4.2-fold), free energy (1.1-fold) and decreased entropy (4.6-fold) of thermal inactivation. A significant increase in pH stability of Ch-MNP/PEI/GA/Enzyme was observed especially at alkaline pH values. In addition, Ch-MNP/PEI/GA/Enzyme preserved 83.2 % of its initial activity after 15 consecutive cycles. When storing Ch-MNP/PEI/GA/Enzyme at 4 °C the residual activity was 100 and 86 %, respectively after 21 and 40 days. Finally, immobilization process improved the catalytic properties and stabilities, thus raising the suitability for industrial processes with lower cost and time.

Impact of betanin against paracetamol and diclofenac induced hepato-renal damage in rats.

Context: Paracetamol (PAR) and diclofenac (DF) are the most popular consumed analgesics and anti-inflammatory medications.Objective: This study aimed to explore the protective effect of betanin (Bet) against PAR or DF induced hepato-renal damage in rats.Methods: Rats were randomly divided into five groups: Normal control (NC) group rats were given saline only. PAR group rats received PAR (400 mg/kg). PAR/Bet treated group rats administered PAR (400 mg/kg) plus Bet (25 mg/kg). DF group rats received DF (10 mg/kg). DF/Bet treated group rats administered DF (10 mg/kg) plus Bet (25 mg/kg). All drugs were given by gavage for 28 consecutive days.Results: PAR and DF administration in high dose and long-time induced liver and kidney injury, disrupted serum lipid profile, enhanced serum levels of inflammatory and oxidative stress markers, triggered DNA fragmentation and caused drastic changes in the histopathological pictures of the two organs. Bet supplementation succeeded to ameliorate most of the biochemical changes and protected DNA from damage as obtained from comet assay. Histological features in H&E taken to different groups also mirrors this findings.Conclusion: Bet exerted a potential anti-inflammatory and antioxidant effect against hepato-renal damage induced by PAR or DF overconsumption.

Protective effects of betanin against paracetamol and diclofenac induced neurotoxicity and endocrine disruption in rats.

Context: Overconsumption of paracetamol (PAR) and diclofenac (DF) have been reported to induce neurotoxicity and endocrine disruption. Objective: The current study was designed to explore the protective potential of betanin against PAR and DF inducing neurotoxicity and endocrine disruption in a rat model. Material and Methods: Forty rats were equally divided into five groups: group I served as control, group II received PAR (400 mg/kg), group III received PAR plus betanin (25 mg/kg), group IV received DF (10 mg/kg) and group V received DF plus betanin orally for 28 consecutive days. Thyroid axis hormones, sex hormone, neurotransmitters, paraoxonase-1, hemeoxygenase-1 and nuclear factor-2 were measured by ELISA. While, the oxidative stress markers were colorimetrically estimated. Moreover, DNA damage and histopathological picture of the brains were investigated. Results: A marked reduction in thyroid axis hormones, brain neurotransmitters and serum testosterone as well as enhanced oxidative stress and brain DNA damage accompanied by drastic changes in the brain histopathological picture were recorded in the challenged PAR and DF groups. Betanin supplementation ameliorated most of the biochemical and histopathological changes induced by PAR or DF. Conclusion: The study suggests betanin of potential protective effects against neurotoxicity and endocrine disruption induced by PAR and DF overconsumption.

Catalytic, kinetic and thermodynamic properties of free and immobilized caseinase on mica glass-ceramics.

Bacillus megaterium 314 strain was able to utilize agricultural and industrial wastes for metallo-protease production. Orange peel and wheat bran were found as the most suitable carbon and nitrogen sources, respectively. Optimized production process enhanced the enzyme production by 5.1-folds. Glass and glass-ceramic with different particle sizes based on mica were used as inorganic carrier. Protease enzyme was immobilized by covalent bonding and physical adsorption methods on nanoparticle supports. Enzyme physically adsorbed on glass ceramic (particle size 0.71-1.0 mm) had the highest residual activity and the highest immobilization yield. Glass-ceramic was characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM). Immobilized enzyme exhibited activation energy (E a ) and deactivation rate constant at 60 °C (k d ) about 1.29 and 1.46-times, respectively lower than free enzyme. Moreover, adsorbed enzyme had higher energy for denaturation (E d ), half-life (t 1/2 ), and decimal reduction time (D). The thermodynamic parameters of irreversible thermal denaturation for the protease enzyme indicate that immobilized enzyme had higher enthalpy (ΔH°), free energy (ΔG°), and entropy (ΔS°) than free one. There was a significant improvement in the maximum reaction velocity Vmax (2.5-fold), Michaelis constant Km (1.9-fold), and catalytic efficiency Vmax/Km (4.7-fold) values after immobilization indicating the efficiency and effectiveness of immobilization approach.

Melatonin and/or rowatinex attenuate streptozotocin-induced diabetic renal injury in rats.

The study aimed to explore the prophylactic effect of melatonin, rowatinex; a naturally occurring renal drug, and its combination on diabetic nephropathy in type 2 diabetic rats. Diabetes was induced by intraperitoneal injection of a single dose of streptozotocin (50 mg/g body weight). Three days before diabetes induction, rats were daily treated with melatonin, rowatinex and their combination continuously for 8 weeks. Evaluation was done through measuring blood urea nitrogen (BUN), serum uric acid, serum creatinine, urine creatinine, creatinine clearance, nitric oxide (NO), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), total antioxidant capacity (TAC), kidney injury molecule-1 (KIM-1), heat shock protein-70 (HSP-70), caspase-3, transforming growth factor ß1 (TGFß1), DNA degradation by the comet assay and total protein contents. Histopathologic study was also done for the kidney and the pancreas. Drastic changes in all measured parameters of the diabetic rats were observed. Treatment with melatonin and rowatinex showed amelioration to variable degrees. In conclusion, melatonin showed the most potent effect on protecting rats from deleterious action of diabetic nephropathy followed by its combination with rowatinex.

Response surface methodology for production, characterization and application of solvent, salt and alkali-tolerant alkaline protease from isolated fungal strain Aspergillus niger WA 2017.

Isolated strain Aspergillus niger WA 2017 was selected as potential protease producer and was identified on the basis of 18S rDNA gene homology. Optimization of protease production conditions was performed using statistical methodology. The most significant factors were identified by Plackett-Burman design (PB) and were optimized by Central Composite design (CCD). The enzyme production was increased by 3.6-fold with statistically optimized medium when compared to the basal medium. Based on the protease activity, 25-50% ethanol fraction exhibited the highest specific activity. The partially purified enzyme showed its highest activity (4.7-fold) after 10 min incubation at pH 10.0 and 60 °C. The enzyme was stable over a wide range of pH (7-11) and salt concentration (up to 20%). Kinetic parameters Michaelis constant (Km) and maximum velocity (Vmax) were calculated at varying casein concentrations. Additionally, thermal stability of the enzyme was substantially improved by NaCl. The enzyme showed excellent stability and compatibility in presence of organic solvents and detergents retaining 115.3 and 114.5% of its activity in presence of ethanol and Tide, respectively at 40 °C for 1 h. The results revealed that the produced enzyme was able to recover silver from used X-ray film under optimized condition using statistical methodology (CCD).

Enhancement stability and catalytic activity of immobilized α-amylase using bioactive phospho-silicate glass as a novel inorganic support.

α-Amylase enzyme was immobilized on bioactive phospho-silicate glass (PS-glass) as a novel inorganic support by physical adsorption and covalent binding methods using glutaraldehyde and poly glutaraldehyde as a spacer. Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM) studies confirmed the glass-enzyme linkage. Dissolution of PS-glass in acidic and neutral pH is higher than that of alkaline pH. Some immobilization variables were optimized using statistical factorial design (Central Composite Design). Optimized immobilization variables enhanced the immobilization yield (IY) from 27.9 to 79.9% (2.9-fold). It was found that the immobilized enzyme had higher optimum temperature, higher half-life time (t1/2), lower activation energy (Ea), lower deactivation constant rate (kd) and higher decimal reduction time (D-values) within the temperature range of 40-60°C. Differential scanning calorimetry analysis (DSC) confirmed the thermalstability of the immobilized enzyme. The immobilized enzyme was stable at a wide pH range (5.0-8.0). Kinetic studies of starch hydrolysis demonstrated that immobilized enzyme had lower Michaelis constant (Km), maximum velocity (Vmax) and catalytic efficiency (Vmax/Km) values. The storage stability and reusability of the immobilized enzyme were found to be about 74.7 and 62.5% of its initial activity after 28days and 11cycles, respectively. Enhanced α-amylase stabilities upon immobilization make it suitable for industrial application.

Catalytic, kinetic and thermodynamic properties of stabilized Bacillus stearothermophilus alkaline protease.

Bacillus stearothermophilus alkaline protease was conjugated to several oxidized polysaccharides of different chemical structure. The conjugates were evaluated for the kinetic and thermodynamic stability. The conjugated enzyme with oxidized pectin had the highest retained activity (79.5%) and the highest half-life (T1/2) at 50°C and pH 9.0. Compared to the native protease, the conjugated preparation exhibited lower activation energy (Ea), lower deactivation constant rate (kd), higher T1/2, and higher D values (decimal reduction time) within the temperature range of 50-60°C. The thermodynamic parameters for irreversible inactivation of native and conjugated protease indicated that conjugation significantly decreased entropy (ΔS*) and enthalpy (ΔH*) of deactivation. The calculated value of activation energy for thermal denaturation (Ead) for the conjugated enzyme was 20.4KJmole-1 higher over the native one. The results of thermodynamic analysis for substrate hydrolysis indicated that the enthalpy of activation (ΔH*) and free energy of activation (free energy of substrate binding) ΔG*E-S and (ΔG*), (free energy of transition state) ΔG*E-T values were lower for the modified protease. Similarly, there was significant improvement of kcat, kcat/Km values. The enzyme proved to be metalloprotease and significantly stimulated by Ca2+ and Mg2+ whereas Hg2+, Fe3+ Cu2+ and Zn2+ inhibited the enzyme activity. There was no pronounced effect on substrate specificity after conjugation.

Combination of melatonin and certain drugs for treatment of diabetic nephropathy in streptozotocin-induced diabetes in rats.

Diabetic nephropathy is a major complication of diabetes and a leading cause of end-stage renal failure in many developed countries. The study aimed to evaluate the efficiency of certain drugs and melatonin in the treatment of nephropathy secondary to diabetes. Diabetes was induced in rats by a single intraperitoneal injection of streptozotocin (50 mg/kg body weight). Three days after induction of diabetes (460-500 mg/dl), rats were treated daily for 60 days with Rowatinex, melatonin, Rowatinex + melatonin, Amosar (Losartan Potassium) (LSP) and LSP + melatonin. The evaluations were made by measuring blood urea nitrogen (BUN), serum uric acid, serum creatinine, urine creatinine, creatinine clearance, nitric oxide, malondialdehyde, superoxide dismutase, glutathione, total antioxidant capacity, kidney injury molecule-1, heat shock protein-70, caspase-3, transforming growth factor ß1, and DNA degradation by comet assay and total protein contents. The histopathological picture of the kidneys and pancreases was confirmed in our results. Diabetic rats showed drastic changes in all the measured parameters. Treatment with melatonin and the selected drugs revealed amelioration levels with variable degrees. In conclusion, the combination of LSP and melatonin had the most potent effect on treating the deleterious action of diabetes on rat kidney.