Thrombolytic and anticoagulant efficiencies of purified fibrinolytic enzyme produced from Cochliobolus hawaiiensis under solid-state fermentation.

Cochliobolus hawaiiensis Alcorn Assiut University Mycological Centre 8606 was chosen from the screened 20 fungal species as the potent producer of fibrinolytic enzyme on skimmed-milk agar plates. The greatest enzyme yield was attained when the submerged fermentation (SmF) conditions were optimized, and it was around (39.7 U/mg protein). Moreover, upon optimization of fibrinolytic enzyme production under solid-state fermentation (SSF), the maximum productivity of fibrinolytic enzyme was greatly increased recorded a bout (405 U/mg protein) on sugarcane bagasse, incubation period of 5 days, moisture level of 100%, initial pH of salt basal medium 7.8, incubation temperature at 35°C, and supplementation of the salt basal medium with corn steep liquor (80％, v/v). The yield of fibrinolytic enzyme by C. hawaiiensis under SSF was higher than that of SmF with about 10.20-fold. The purification procedures of fibrinolytic enzyme by ammonium sulfate (70%), gel filtration, and ion-exchange columns chromatography caused a great increase in its specific activity to 2581.6 U/mg protein with an overall yield of 55.89%, 6.37 purification fold and molecular weight of 35 kDa. Maximal activity was recorded at pH 7 and 37°C. Significant pH stability was recorded at pH 6.6-7.2, and thermal stability was recorded at 33-41°C. The enzyme showed the highest affinity toward fibrin, with Vmax of 240 U/mL and an apparent Km value of 47.61 mmol. Mg2+ and Ca2+ moderately induced fibrinolytic activity, whereas Cu2+ and Zn2+ greatly suppressed the enzyme activity. The produced enzyme is categorized as serine protease and non-metalloprotease. The purified fibrinolytic enzyme showed efficient thrombolytic and antiplatelet aggregation activities by completely prevention and dissolution of the blood clot which confirmed by microscopic examination and amelioration of blood coagulation assays. These findings suggested that the produced fibrinolytic enzyme is a promising agent in management of blood coagulation disorders.

Production of antibiotic carbomycin from Streptomyces graminofaciens with high lipid content mutation.

The most important tools in killing and overcoming on the microbes and pathogens that cause diseases in medicine and/or in agriculture are the antibiotics. The discovery and synthesis of the microbial natural products or antibiotics has greatly developed genetically and biotechnologically quickly in the last decades. It is necessary to access this great genetic diversity by finding ways to increase the level of expression of these biosynthetic pathways. In this study, we carried out an improvement in the antibiotic production of weak Streptomyces graminofaciens strain NBR9 that has high lipid content; using Ultra-Violet irradiation mutagenesis. This strain was isolated from the Northern Region in the kingdom of Saudi Arabia and identified biochemically and confirmed genetically by sequencing of the 16S rRNA gene as Streptomyces graminofaciens NBR9; Accession No. (MN640578). The resultant mutant strain showed increasing in their antimicrobial activities. The methods and techniques used for the antibiotic extraction, purification, characterization and identification proved that the obtained antibiotic is same with antibiotic Carbomycin.

Laundry Detergent Compatibility and Dehairing Efficiency of Alkaline Thermostable Protease Produced from Aspergillus terreus under Solid-state Fermentation.

Aspergillus terreus was chosen for production of alkaline protease using solid-state fermentation (SSF). The maximum enzyme yield reached about 34.87 U/mg protein after optimization of fermentation parameters. The produced alkaline protease was purified by precipitation with iso-propanol and then purified through gel filtration and ion exchange column chromatography with a yield of 53.58% and 5.09- fold purification. The enzyme has shown to have a molecular weight of 35 kDa. Optimal pH and temperature for the enzyme activity were 9.5 and 50°C respectively. The highest activity was reported towards casein, with an apparent Km value of 6.66 mg/mL and Vmax was 30 U/mL. The enzyme activity was greatly repressed by phenylmethylsulfonyl fluoride (PMSF). Sodium dodecyl sulfate (SDS) caused activation in enzyme activity. The enzyme retained about 83.8, 70.6, 74.5, 76.4 and 66.4% of its original activity after incubation with Aerial, Leader, Oxi, Persil and Tide, respectively for 8 h at 60°C. Adding of the enzyme in detergents improved the cleansing performance to the blood stains and suggested to be used as a detergent additive. Our outcomes showed that protease could be used as environment green-approach in dehairing process.

Purification, characterization and anticancer efficiency of L-glutaminase from Aspergillus flavus.

The aim of this work was to purify L-glutaminase from Aspergillus flavus. The enzyme was purified 12.47-fold from a cell-free extract with a final specific activity of 613.3 U/mg and the yield was 51.11%. The molecular weight of the enzyme, as estimated by SDS-PAGE, was found to be 69 kDa. The maximal activity of L-glutaminase was recorded at pH 8 and 40°C. The highest activity was reported towards L-glutamine as substrate, with an apparent Km value of 4.5 mmol and Vmax was 20 Uml-1. The enzyme was activated by Na+ and Co2+, while it was greatly suppressed by iodoacetate, NEM, Zn2+ and Hg2+ at 10 mM. L-glutaminase activity increased with a gradual increase of sodium chloride concentration up to 15%. In vivo, the median lethal dose (LD50) was approximately 39.4 mg/kg body weight after intraperitoneal injection in Sprague Dawley rats. Also, L-glutaminase showed no observed changes in liver and kidney functions and hematological parameters on rates. Purified A. flavus L-glutaminase had neither a cognizable effect on human platelet aggregation nor hemolytic activity. In addition, MTT assay showed that the purified L-glutaminase has a high toxic impact on Hela and Hep G2 cell lines with an IC50 value 18 and 12 µg/ml, respectively, and a moderate cytotoxic effect on HCT-116 and MCF7 cells, with an IC50 value 44 and 58 µg/ml, respectively.

Anticancer and antifungal efficiencies of purified chitinase produced from Trichoderma viride under submerged fermentation.

Trichoderma viride AUMC 13021 isolated from Mangrove soil of Ras Mohammed protected area at Sharm El-Sheikh, Egypt, was optimized to promote chitinase activity under submerged fermentation. The maximum enzyme yield (38.33 U/mg protein) was obtained at 1.4% of colloidal chitin, 96 h of incubation, 35°C, pH 6.5 and 125, rpm and using maltose (1%) and yeast extract (1%) as supplementation of salt basal medium. The enzyme has been purified with an overall yield of 73.1% and 5.48 purification fold, and a specific activity of 210.16 U/mg protein. The molecular mass of the purified chitinase was 62 kDa. Maximal activity of chitinase was recorded at pH 6.5 and 40°C. The highest activity was recorded in the case of colloidal chitin, with an apparent Km value of 6.66 mg/ml and Vmax of 90.8 U/ml. The purified chitinase was activated by Ca2+ and Mn2+ while the activity was inhibited by Hg2+, Zn2+, Cu2+, Co2+, dodecyl sulphate and EDTA. In vivo, the median lethal dose (LD50) was approximately 18.43 mg/kg body weight of Sprague Dawley rats. MTT assay showed that the purified chitinase has a toxic effect to MCF7 with an IC50 value 20 µg/ml, and HCT-116 cell lines with an IC50 value 44 µg/ml. Moreover, the purified enzyme showed significant antifungal activity against Fusarium oxysporum f. sp. lycopersici race 3 the causal agent of tomato wilt.

Comparative study of a new alkaline L-methioninase production by Aspergillus ustus AUMC 10151 in submerged and solid-state fermentation

Twenty four fungal species were screened for their ability to produce alkaline L-methioninase on methionine-glucose liquid medium. Aspergillus ustus AUMC 10151 displayed the highest yield of enzyme (10.8 U/mg protein), followed by A. ochraceus and Fusarium proliferatum. Upon optimization of the submerged fermentation (SmF)conditions, the maximum enzyme yield (18.23 U/mg protein) was obtained on a medium containing L-methionine (0.5%), sucrose (0.95%), KH2PO4 (0.1%) and 175 rpm. Seven agro-industrial by-products were screened as substrates for L-methioninase production under solid-state fermentation (SSF). Wheat bran resulted 38.1 U/mg protein, followed by rice bran (27.6 U/mg protein) and soya bean meal (26.6 U/mg protein). Maximum alkaline L-methioninase (99.56U/mg protein) was achieved at initial moisture content of 71.5%, inoculum size of 2.0 mL of spore suspension, initial pH 8.5, incubation period eight days at 30°C and supplementation of the salt basal medium with pyridoxine(100 μg/mL) and beet molasses (20% v/v). The productivity of L-methioninase by A. ustus under SSF was higher than that of SmF about 5.45 fold under optimum conditions.

Enhanced alkaline cellulases production by the thermohalophilic Aspergillus terreus AUMC 10138 mutated by physical and chemical mutagens using corn stover as substrate.

A thermohalophilic fungus, Aspergillus terreus AUMC 10138, isolated from the Wadi El-Natrun soda lakes in northern Egypt was exposed successively to gamma and UV-radiation (physical mutagens) and ethyl methan-sulfonate (EMS; chemical mutagen) to enhance alkaline cellulase production under solid state fermentation (SSF) conditions. The effects of different carbon sources, initial moisture, incubation temperature, initial pH, incubation period, inoculum levels and different concentrations of NaCl on production of alkaline filter paper activity (FPase), carboxymethyl cellulase (CMCase) and ß-glucosidase by the wild-type and mutant strains of A. terreus were evaluated under SSF. The optimum conditions for maximum production of FPase, CMCase and ß-glucosidase were found to be the corn stover: moisture ratio of 1:3(w/v), temperature 45 °C, pH range, 9.0-11.0, and fermentation for 4, 4 and 7 day, respectively. Inoculum levels of 30% for ß-glucosidase and 40% for FPase, CMCase gave the higher cellulase production by the wild-type and mutant strains, respectively. Higher production of all three enzymes was obtained at a 5% NaCl. Under the optimized conditions, the mutant strain A. terreus M-17 produced FPase (729 U/g), CMCase (1,783 U/g), and ß-glucosidase (342 U/g), which is, 1.85, 1.97 and 2.31-fold higher than the wild-type strain. Our results confirmed that mutant strain M-17 could be a promising alkaline cellulase enzyme producer employing lignocellulosics especially corn stover.

Enhanced alkaline cellulases production by the thermohalophilic Aspergillus terreus AUMC 10138 mutated by physical and chemical mutagens using corn stover as substrate

Abstract A thermohalophilic fungus, Aspergillus terreus AUMC 10138, isolated from the Wadi El-Natrun soda lakes in northern Egypt was exposed successively to gamma and UV-radiation (physical mutagens) and ethyl methan-sulfonate (EMS; chemical mutagen) to enhance alkaline cellulase production under solid state fermentation (SSF) conditions. The effects of different carbon sources, initial moisture, incubation temperature, initial pH, incubation period, inoculum levels and different concentrations of NaCl on production of alkaline filter paper activity (FPase), carboxymethyl cellulase (CMCase) and β-glucosidase by the wild-type and mutant strains of A. terreus were evaluated under SSF. The optimum conditions for maximum production of FPase, CMCase and β-glucosidase were found to be the corn stover: moisture ratio of 1:3(w/v), temperature 45 °C, pH range, 9.0–11.0, and fermentation for 4, 4 and 7 day, respectively. Inoculum levels of 30% for β-glucosidase and 40% for FPase, CMCase gave the higher cellulase production by the wild-type and mutant strains, respectively. Higher production of all three enzymes was obtained at a 5% NaCl. Under the optimized conditions, the mutant strain A. terreus M-17 produced FPase (729 U/g), CMCase (1,783 U/g), and β-glucosidase (342 U/g), which is, 1.85, 1.97 and 2.31-fold higher than the wild-type strain. Our results confirmed that mutant strain M-17 could be a promising alkaline cellulase enzyme producer employing lignocellulosics especially corn stover.