RESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) is among the common cancers, but difficult to diagnose and treat. L-asparaginase has been introduced in the treatment protocol of pediatric acute lymphoblastic leukemia (ALL) since the 1960s with a good outcome and increased survival rates to nearly 90%. Moreover, it has been found to have therapeutic potential in solid tumors. Production of glutaminase-free-L-asparaginase is of interest to avoid glutaminase-related toxicity and hypersensitivity. In the current study, an extracellular L-asparaginase that is free of L-glutaminase was purified from the culture filtrate of an endophytic fungus Trichoderma viride. The cytotoxic effect of the purified enzyme was evaluated in vitro against a panel of human tumor cell lines and in vivo against male Wister albino mice intraperitoneally injected with diethyl nitrosamine (200 mg/kg bw), followed by (after 2 weeks) oral administration of carbon tetrachloride (2 mL/kg bw). This dose was repeated for 2 months, and after that, the blood samples were collected to estimate hepatic and renal injury markers, lipid profiles, and oxidative stress parameters. RESULTS: L-asparaginase was purified from T. viride culture filtrate with 36 purification folds, 688.1 U/mg specific activity, and 38.9% yield. The highest antiproliferative activity of the purified enzyme was observed against the hepatocellular carcinoma (Hep-G2) cell line, with an IC50 of 21.2 g/mL, which was higher than that observed for MCF-7 (IC50 34.2 g/mL). Comparing the DENA-intoxicated group to the negative control group, it can be demonstrated that L-asparaginase adjusted the levels of the liver function enzymes and the hepatic injury markers that had previously changed with DENA intoxication. DENA causes kidney dysfunction and altered serum albumin and creatinine levels as well. Administration of L-asparaginase was found to improve the levels of the tested biomarkers including kidney and liver function tests. L-asparaginase treatment of the DENA-intoxicated group resulted in a significant improvement in the liver and kidney tissues to near normal similar to the healthy control group. CONCLUSION: The results suggest that this purified T. viride L-asparaginase may be able to delay the development of liver cancer and may be used as a potential candidate for future application in medicine as an anticancer medication.
RESUMO
BACKGROUND: The increasing demand and the continuous depletion in fossil fuels have persuaded researchers to investigate new sources of renewable energy. Bioethanol produced from cellulose could be a cost-effective and a viable alternative to petroleum. It is worth note that ß-glucosidase plays a key role in the hydrolysis of cellulose and therefore in the production of bioethanol. This study aims to investigate a simple and standardized method for maximization of extracellular ß-glucosidase production from a novel fungal isolate under solid-state fermentation using agro-industrial residues as the sole source of carbon and nitrogen. Furthermore, purification and characterization of ß-glucosidase were performed to determine the conditions under which the enzyme displayed the highest performance. RESULTS: A fungus identified genetically as a new Aspergillus sp. DHE7 was found to exhibit the highest extracellular ß-glucosidase production among the sixty fungal isolates tested. Optimization of culture conditions improved the enzyme biosynthesis by 2.1-fold (174.6 ± 5.8 U/g of dry substrate) when the fungus grown for 72 h at 35 °C on jojoba meal with 60% of initial substrate moisture, pH 6.0, and an inoculum size of 2.54 × 107 spores/mL. The enzyme was purified to homogeneity through a multi-step purification process. The purified ß-glucosidase is monomeric with a molecular mass of 135 kDa as revealed by the SDS-PAGE analysis. Optimum activity was observed at 60 °C and pH of 6.0, with a remarkable pH and thermal stability. The enzyme retained about 79% and 53% of its activity, after 1 h at 70 °C and 80 °C, respectively. The purified ß-glucosidase hydrolysed a wide range of substrates but displaying its greater activity on p-nitrophenyl-ß-D-glucopyranoside and cellobiose. The values of Km and Vmax on p-nitrophenyl ß-D-glucopyranoside were 0.4 mM and 232.6 U/mL, respectively. Purified ß-glucosidase displayed high catalytic activity (improved by 25%) in solutions contained ethanol up to 15%. CONCLUSION: ß-glucosidase characteristics associated with its ability to hydrolyse cellobiose, underscore its utilization in improving the quality of food and beverages. In addition, taking into consideration that the final concentration of ethanol produced by the conventional methods is about 10%, suggests its use in ethanol-containing industrial processes and in the saccharification processes for bioethanol production.
RESUMO
The influences of nutritional components affecting lipase production from the new Aspergillus niger using wheat bran as substrate were studied by employing Plackett-Burman and central composite statistical designs. Out of the 11 medium components tested, sucrose, KH2PO4 and MgSO4 at final concentrations of 3.0, 1.0 and 0.5 g/L, respectively, were reported to contribute positively to enzyme production (20.09 ± 0.98 U/g ds). The enzyme was purified through ammonium sulfate precipitation followed by Sephadex G-100 gel filtration. Molecular mass of the purified lipase was 57 kDa as evident on SDS-PAGE. Different methods of immobilization were studied and the highest immobilization yield of 81.7 ± 2.18% was reported with agarose (2%) and the optimum temperature was raised from 45 to 50 °C. Immobilized lipase could retain 80% of its original activity at 60 °C after 1 hr of incubation, and was stable at pH values between neutral and alkaline pH. Lipase-catalyzed transesterification process of fungal oil resulted in a fatty acid methyl ester yield consisting of a high percentage of polyunsaturated fatty acids (83.6%), making it appropriate to be used as winter-grade biodiesel. The operational stability studies revealed that the immobilized lipase could keep 70% of its total activity after 5 cycles of the transesterification process.
