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.

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.

Xylanase covalent binding onto amidated pectin beads: Optimization, thermal, operational and storage stability studies and application.

Amidated pectin-polyethylene imine-glutaraldehyde (AP-PEI-GA) immobilizer was prepared. The ideal protocol that should be adopted during the immobilizer preparation was investigated via Box-Behnken design (BBD), and it comprised processing the AP beads with 3.4 % (w/w) PEI solution of pH 9.65 followed by 5.96 % (v/v) GA solution. The obtained AP-PEI-GA immobilizer was efficient, and it acquired 3.03 U.g-1 of immobilized xylanase (im-xylanase) activity. The computed Km and Vmax values for AP-PEI-GA im-xylanase were 16.67 mg.ml-1 and 20 g.ml-1.min-1, respectively. Through covalent coupling to AP-PEI-GA, Aspergillus niger xylanase thermodynamic properties T1/2 and D-values were increased by 2.05, 3.08, and 1.35 at 40, 50, and 60 °C, respectively. ΔHd and ΔGd for AP-PEI-GA im-xylanase at 40, 50, and 60 °C were higher than those for free form emphasizing more resistance to thermal denaturation. Im-xylanase showed 100 % activity for 20 successive cycles and hydrolyzed different agro-industrial wastes into reducing sugar and xylooligosaccharides (XOS) with more efficiency on pea peel (PP). AP-PEI-GA im-xylanase, PP weight, and hydrolysis time that should be adopted to obtain the highest reducing sugar and XOS yield were optimized through central composite design (CCD). Extracted XOS showed prebiotic and anti-oxidant activities.

Prebiotic-mediated gastroprotective potentials of three bacterial levans through NF-κB-modulation and upregulation of systemic IL-17A.

This study aimed to investigate whether the gastroprotective effects of three types of bacterial levans are correlated with their prebiotic-associated anti-inflammatory/antioxidant potentials. Three levans designated as LevAE, LevP, and LevZ were prepared from bacterial honey isolates; purified, and characterized using TLC, NMR, and FTIR. The anti-inflammatory properties of levan preparations were assessed in LPS-stimulated RAW 264.7 cell lines, while their safety and gastroprotective potentials were assessed in Wistar rats. The three levans significantly reduced ulcer number (22.29-70.05 %) and severity (31.76-80.54 %) in the ethanol-induced gastric ulcer model compared to the control (P < 0.0001/each), with the highest effect observed in LevAE and levZ (200 mg/each) (P < 0.0001). LevZ produced the highest levels of glutathione; catalase activity, and the lowest MDA levels (P = 0.0001/each). The highest anti-inflammatory activity was observed in LevAE and levZ in terms of higher inhibitory effect on IL-1ß and TNF-α production (P < 0.0001 each); COX2, PGE2, and NF-κB gene expression. The three levan preparations also proved safe with no signs of toxicity, with anti-lipidemic properties as well as promising prebiotic activity that directly correlated with their antiulcer effect. This novel study highlights the implication of prebiotic-mediated systemic immunomodulation exhibited by bacterial levans that directly correlated with their gastroprotective activity.

Statistical, physicochemical, and thermodynamic profiles of chitinase production from local agro-industrial wastes employing the honey isolate Aspergillus niger EM77.

Enzyme synthesis from local wastes has a lot of potential because it eliminates the problem of waste accumulating in conjugation while also cutting the cost of these useful products. Plackett-Burman (PBD) and central composite designs (CCD) were used to optimize the manufacturing process utilizing the honey isolate Aspergillus niger EM77, resulting in a 60-fold increase in enzyme productivity using a group of wastes comprising wheat, rice straw, and sawdust. The enzyme had its optimum activity at 60 °C, pH 5, and had high thermo-stability at 60 °C, with Km and Vmax of 0.8 mg mL-1 and 2083.33 µmol mL-1 min-1 respectively. The activation (Ea) and deactivation (Ed) energies of chitinase were 2.78 and 174.46 kJ mol-1, respectively, with the thermodynamic constants ΔH° and ΔG° ensuring enzyme stability. As a result, Aspergillus niger EM77 chitinase has the efficiency to meet the global market demand for chitinase enzyme while also providing a significantly lower price than what is now available on the websites of specialist international companies. The production process is almost costless because it is based primarily on waste and contains traces of minerals.

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.

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.

Characterization of Aspergillus niger MK981235 xylanase with extraction of anti-hepatotoxic, antioxidant, hypocholesterolemic and prebiotic Corchorus olitorius stems xylooligosaccharides.

The object of this study was to utilize agro-industrial waste Corchorus olitorius stems (molokhia stems, MS) as substrate, for Aspergillus niger MK981235 xylanase production and as source of biologically active xylooligosaccharides (XOS). This study succeeded in utilization of Aspergillus niger MK981235 xylanase under different saccharification conditions designed by central composite design (CCD) for extraction of 15 biologically active XOS (anti-hepatotoxic, antioxidant, hypocholesterolemic and prebiotic) with different monosaccharides constituents composition and percent. A. niger MK981235 xylanase showed the highest activity 6.60 U·ml-1 at 50 °C with 1.5% xylan. The kinetics included Km and Vmax were determined to be 6.67 mg·ml-1 and 20 µmol·ml-1·min-1, respectively. Moreover, A. niger MK981235 xylanase thermodynamics Ea (activation energy) and Ed (activation energy of denaturation) were determined to be 21.95 and 39.51 KJ·mol-1, respectively. The highest prebiotic effect (growth promation) was exerted by the central MS XOS on Lactobacillus plantarum and Lactobacillus rhamnosus (125 and 135.3%, respectively). Also, the central MS XOS, exerted the highest cholesterol reduction and antioxidant activities 74.7 and 92%, respectively, showed remarkable in vivo protective role against the hepatic toxicity of lithium carbonate evaluated by changes in body weight, liver function markers (AST, ALT, Alb, total bilirubin) and tissue makers (MDA and GSH).

Functionalized κ-carrageenan/hyperbranched poly(amidoamine)for protease immobilization: Thermodynamics and stability studies.

Hyperbranched polyamidoamine (PAMAM) has a high density of surface amino groups. This feature was exploited in the surface modification of κ-carrageenan gel beads for successful protease immobilization. The optimum PAMAM pH was at 2.1 and 9.3. However, treatment of κ-carrageenan gel beads at the higher pH showed re-usability for more than seven successive times. As a result of central composite design optimization, the maximum immobilization yield was obtained by soaking 8 U for 24 h. The comparative thermodynamics studies showed an enhancement in the thermal stability at high temperature for the immobilized protease as well as increased half-life time from 24.06 min to 79.95 min. Also, the D-values increased from 165.03 min to 548.23 min for free and immobilized enzyme, respectively. Moreover, the enzyme stability enhancement for the immobilized protease catalyst was accompanied with a remarkable increase in the enthalpy and in the free energy. Immobilized protease onto Carr-PAMAM gel beads can retain 89% of its initial activity and lost only 11% after 8 weeks of storage at 4 °C. Furthermore, an effective removal of silver from used X-ray film by the immobilized protease was achieved for six repeated cycles.

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).

Improvement of catalytic, thermodynamics and antifungal activity of constitutive Trichoderma longibrachiatum KT693225 exochitinase by covalent coupling to oxidized polysaccharides.

Our study full filled in two main goals preparation of constitutive exochitinase with low cost, utilizing non-chitin containing agricultural wastes, and improving the thermodynamics of purified Trichoderma longibrachiatum KT693225 exochitinase by covalent coupling to sodium periodate activated agar. Central composite design (CCD) was used to improve the chemical modification of Trichoderma longibrachiatum KT693225 exochitinase. Optimum temperature for conjugated exochitinase 60 °C was higher than native form 40 °C. Covalent coupling to oxidized agar caused 4.32, 2.75 and 2.44-fold increase in half-life values at 50, 55 and 60 °C, respectively. Also, conjugated exochitinase showed higher D-values (decimal reduction time) 1790.49 compared to 733.08 min for native form at 60 °C. Moreover, conjugated form had lower deactivation constant rate (kd) 0.39 × 10-3 min-1at 60 °C than native form 1.7 × 10-3 min-1. Native exochitinase exhibited higher activation energy (Ea) 3.39 Kcal·mol-1 and lower energy for denaturation (Ed) 6.88 Kcal·mol-1 compared to 3.21 and 13.05 Kcal·mol-1, respectively for conjugated form. The values of thermodynamic parameters for inactivation of native and conjugated exochitinase indicated that conjugation significantly decreased entropy (ΔS°) and increased enthalpy (ΔH°) and free energy (ΔG°) of deactivation. Conjugated exochitinase exhibited higher antifungal effect against Alternaria alternata, Fusarium oxysporium and Aspergillus niger than native form.

Purification, physicochemical and thermodynamic studies of antifungal chitinase with production of bioactive chitosan-oligosaccharide from newly isolated Aspergillus griseoaurantiacus KX010988.

In our search for chitinase and chitosanase producer from unconventional sources, the marine-derived fungus Aspergillus griseoaurantiacus KX010988 was obviously the best producer of the highest chitinase and chitosanase activities by solid state fermentation of potato shells. Chitinase was purified in three steps involving ammonium sulphate precipitation, DEAE-cellulose ion-exchange chromatography and Sephacryl S-300 gel chromatography. 12.55 fold increase in purity with a recovery of 17.6 was obtained. The molecular mass of the purified chitinase was found to be 130kDa. It was optimally active at pH 4.5 and 40°C. Km and Vmax values were 0.22mgmL-1 and 19.6µmolemin-1mg-1 respectively. Mn2+ and Zn2+ ions lead to increased chitinase activity. While Fe2+and Cu2+ions strongly inhibited the chitinase activity. The thermodynamics of pure chitinase including activation energy for thermal denaturation (Ea,d), change of free energy (ΔGd), enthalpy(ΔHd), entropy(ΔSd) and half life values (T1/2) at 40, 50 and 60°C were determined. Chitinase showed antifungal activity against pathogenic fungus Fusarium solani. Chitosanase was partially purified by acetone precipitation (50-75%) v/v concentration. The hydrolytic products of moderate molecular weight of chitosan by chitosanase were analyzed by thin layer chromatography (TLC) after 12 and 24h respectively. Chitosan-oligosaccharides showed good antibacterial and antioxidant activities.

Kinetic and thermodynamic characteristic of Aspergillus awamori EM66 levansucrase.

This study is a new trial aimed to solve levansucrase high cost and levan associated problems during the purification process. Also, kinetic and thermodynamic study was done to compare between the partial pure (PP) and purified forms (PF). Within this context, Aspergillus awamori EM66 levansucrase was produced constitutively (5.44 U.mL-1) using rice straw as the sole medium component. The enzyme was partially purified and was eluted as single protein after two purification steps. Its molecular weight was determined to be 44.5 KDa. The optimum temperature recorded 40 °C for both enzyme forms. While, the purification process lowering the enzyme pH from 5.2 to 4.0. The NaCl concentrations (0.5-3.0 M) pointed to the halophilic nature of the enzyme. The PP form retained about 76% of its original activity after 1 h at 55 °C while the other retained about 57% after 45 min. at the same temperature. The kinetic parameters Km and Vmax concluded that the PF was more efficient than the PP. The thermodynamic parameters such as Ea, Ed, T1/2, D-value, also, ∆G*, ∆H* and ∆ S* for activation recorded that the PP had higher stability than the PF.

Optimization of pectinase immobilization on grafted alginate-agar gel beads by 24 full factorial CCD and thermodynamic profiling for evaluating of operational covalent immobilization.

Pectinase produced by a honey derived from the fungus Aspergillus awamori KX943614 was covalently immobilized onto gel beads made of alginate and agar. Polyethyleneimine, glutaraldehyde, loading time and enzyme's units were optimized by 24 full factorial central composite design (CCD). The immobilization process increased the optimal working pH for the free pectinase from 5 to a broader range of pH4.5-5.5 and the optimum operational temperature from 55°C to a higher temperature, of 60°C, which is favored to reduce the enzyme's microbial contamination. The thermodynamics studies showed a thermal stability enhancement against high temperature for the immobilized formula. Moreover, an increase in half-lives and D-values was achieved. The thermodynamic studies proved that immobilization of pectinase made a remarkable increase in enthalpy and free energy because of enzyme stability enhancement. The reusability test revealed that 60% of pectinase's original activity was retained after 8 successive cycles. This gel formula may be convenient for immobilization of other industrial enzymes.

Production, immobilization and thermodynamic studies of free and immobilized Aspergillus awamori amylase.

Enzyme cost, stability and its thermodynamic characteristics are the main criteria for industrial use. In this study, Aspergillus awamori amylase was constitutively produced using various agro-industrial wastes. Olive oil cake gave the highest activity (230U/g). The amylase was partially purified to 2.81-fold purification. Immobilization was achieved using different carriers by covalent binding. The novel carrier Ca+2 alginate (Alg) starch (St)/polyethyleneimine (PEI)/glutaraldehyde (GA), showed the highest operational stability and was selected for further studies. The optimum temperature for the free and immobilized form was 50°C and 55-60°C, respectively. The immobilization process had a major role in improving enzyme thermal stability. In comparison to free enzyme, the immobilized form showed the highest optimum temperature, activation energy (Ea) and deactivation rate constants (kd). Also, t1/2, D-values (decimal reduction time), change in enthalpy (ΔH° kJmol-1), and Gibbs free energy (ΔG°) increased and was higher than the native enzyme within 50-80°C. The magnitude of negative value of entropy (ΔS° kJmol-1) for immobilized enzyme was negative for the free and immobilized enzymes revealing that native form of enzyme was in more ordered state. Km and Vmax values were slightly affected by the temperature variations 40-70°C.