RESUMO
The Trichoderma harzianum l-methioninase was purified 7.15-fold with a recovery of 47.9% and the specific activity of 74.4 U/mg of protein. The purified enzyme has an apparent molecular mass of 48 kDa on SDS-PAGE and exhibited maximum activity at pH 8 and 35 °C. The enzyme was catalytically stable below 50 °C and at a pH range of 6.0-8.5. The thermal inactivation of l-methioninase exhibited first-order kinetics with the k value between 5.71 × 10-4 min-1 and 1.83 × 10-2 min-1. The studies on thermodynamic parameters of l-methioninase indicated the compaction and aggregation of the enzyme molecule during denaturation. This is the first report of thermodynamic analysis of thermal inactivation in l-methioninase. The enzyme activity was enhanced by Li+ and inhibited by Cu2+, Co2+, Fe2+, Hydroxylamine and PMSF. The purified enzyme showed K m , V max and k cat value of 1.19 mM, 21.27 U/mg/min and 16.11 s-1, respectively. The l-methioninase inhibited the growth of human cell lines hepatocellular carcinoma (Hep-G2) and breast carcinoma (MCF-7) with IC50 values of 14.12 µg/ml and 20.07 µg/ml, respectively. The in vivo antitumor activity of l-methioninase was evaluated against DAL cell lines bearing in Swiss albino mice. The enzyme effectively reduced tumor volume, packed cell volume, viable cell count and restored hematological parameters, serum enzyme and lipid profile to normal levels compared to DAL control mice. The present study has demonstrated the high efficacy of Trichoderma harzianum l-methioninase against cancer cell lines in vitro and in vivo conditions. The purified l-methioninase has significant thermal stability and better catalytic properties than the enzyme purified from other sources.
RESUMO
The present study reports the optimized production and purification of an extremely active fibrinolytic enzyme from newly isolated marine bacterium Fictibacillus sp. strain SKA27, with a specific activity of 125,107.85 U/mg and an apparent molecular weight of 28 kDa on SDS-PAGE. Wheat bran extract used for submerged production proved to be highly beneficial and enhanced fibrinolytic enzyme production when combined with yeast extract and CaCl2. Optimization of culture media by response surface methodology (RSM) resulted in high root mean square error (RMSE), which led to the training of a back propagation multilayer artificial neural network (ANN) with 3-5-1 topology for better prediction quality. The prediction and optimization capabilities of regression and ANN were critically examined and ANN displayed higher proficiency with R 2 of 0.99 and RMSE of 2.0 compared to 0.98 R 2 and 48.9 RMSE of the regression model. An adept ANN linked genetic algorithm (GA) optimized the medium components to achieve 1.8-fold higher enzyme production (4175.41 U/mL). Further, a new and improved in vitro qualitative analysis displayed high specificity of purified enzyme to fibrin.
