Design strategies for novel esterase purification processes from Trichoderma harzianum-An insight into kinetic and thermodynamic analyses.

Designing purification strategies for the fermentation derived enzymes is the most challenging task and require a thorough understanding on the physio-chemical properties of the target enzyme. The current investigation aims to establish purification strategies for a novel esterase from Trichoderma harzianum followed by their kinetic and thermodynamic analyses. The highest esterase purification fold of 6.65 was observed with the acetone-based precipitation at 60 % (v/v) saturation level among other precipitation studies. Further, using Taguchi orthogonal design, the optimal composition of an aqueous two-phase system consisting of PEG 6000 (30 % w/w), sodium citrate (15 %, w/w), sodium chloride (10 %, w/w), and pH (7.0) was obtained that resulted in the esterase purification fold of 3.93. A higher esterase purification fold of 20.47 was obtained using size exclusion chromatography technique. The optimal temperature and pH for the maximum activity of purified esterase were found to be 30 °C and 7, respectively. The thermal deactivation of esterase was followed the first order rate kinetics for the temperature range from 323 K to 353 K. The activation energy (Ea), enthalpy (ΔH), and entropy (ΔS) of thermal denaturation of esterase were estimated to be 69 (kJ/mol), 66.29 (kJ/mol), and 1.21(kJ/mol.K), respectively. The addition of solvents such as ethanol, methanol, acetone, and chloroform at 50 % (v/v) and metal ions, such as calcium chloride, sodium chloride, and zinc sulphate at 1 mM enhanced the esterase activity. Esterase had a higher affinity towards p-nitrophenyl acetate as compared to other short and long-chain fatty acid substrates as observed from the turnover number and Michaelis-Menten kinetic constants. Furthermore, the relative molecular weight of esterase was estimated around 75 kDa using native and SDS-PAGE electrophoresis.