Production, optimization and characterization of cellulase produced from Aspergillus niger in submerged fermentation...

Cellulase is considered as the most significant enzymes, widely studied owing to its extensive industrial applications and the increasing worldwide research focus on its production. In this research, mango peels were used as a source of carbon to produce cellulase by Aspergillus niger. The enzyme was produced, characterized under different parameters to optimize its activity, and its application potential was also evaluated. Initially, OFAT (one-factor-at-a-time) optimization maximized cellulase yield. The peak activity was noted after a 72-h incubation, when both the substrate concentration and the inoculum were set to 3.5 %, a 3.8-fold increase as compared to the unoptimized conditions (0.5 % substrate and 24 h). Different types of nutritional components, were screened through Plackett-Burman Design (PBD) and selected parameters were optimized by Central Composite Design (CCD) of Response Surface Methodology (RSM). Among these nutritional components, the combination that yielded the highest enzyme activity included 0.1 % yeast extract, 0.5 % MgSO₄, 0.275 % KH₂PO₄, and 0.5 % (NH₄)₂SO₄. The CMCase (carboxymethyl cellulase) activity increased 1.18-fold (17.9 %), FPase (filter paper cellulase) activity showed a more substantial improvement, corresponding to a 1.87-fold (86.8 %) enhancement. Characterization of enzyme showed that maximum activity and stability of CMCase was at temperature 60 °C and 50 °C for FPase and pH 5. Enzyme showed peak activity in the presence of NH₄Cl, KCl, FeSO₄, and CuSO₄. Solvents like SDS, Tween-80, butanol, n-hexane, ethyl alcohol, and isopropanol enhanced the stability of enzyme. Using Arrhenius plot, the values of activation energy (Ea) were calculated as -26.48 kJ/mol and - 20.54 kJ/mol for CMCase and FPase respectively. Enthalpy (∆H) and entropy (∆S) of the system were also calculated and evaluation revealed that the enthalpy for CMCase and FPase were 23.5 kJ/mol and 17.4 kJ/mol whereas the values of entropy were - 19.37 kJ/mol and - 16.3 kJ/mol respectively. Using the Lineweaver-Burk plot the values of K_m and V_max were estimated as K_m = 7.45 mM and V_max = 81.7 U/ml/min for CMCase, and K_m = 1.168 mM and V_max = 158.07 U/ml/min for FPase. Additionally, this study also indicated that Aspergillus niger could be useful for saccharification processes. Maximum level (11 %) of saccharification was observed at duration of 28 h.