Abstract
A thermostable acid protease from M. indicus leaves was purified 10-fold using a 4-step protocol. We were able to isolate a purified protease fraction with a molecular weight of 50 kDa and exhibited maximal protease activity at pH 4.0 and 40 °C. Structural analysis revealed that the protease is monomeric and non-glycosylated. The addition of epoxy monocarboxylic acid, iodoacetic acid, and dimethyl sulfoxide significantly reduced protease activity while dramatically increasing the inhibition of Mn2+, Fe2+, and Cu2+. The activation energy of the hydrolysis reaction (33.33 kJ mol−1) and activation energy (Ed = 105 kJ mol−1), the standard enthalpy variation of reversible protease unfolding (2.58 kJ/mol) were calculated after activity measurements at various temperatures. Thermal inactivation of the pure enzyme followed first-order kinetics. The half-life (t1/2) of the pure enzyme at 50 °C, 60 °C, and 70 °C was 385, 231, and 154 min, respectively. Thermodynamic parameters (entropy and enthalpy) suggested that the protease was highly thermostable. This is the first report on the thermodynamic parameters of proteases produced by M. indicus. The novel protease appears to be particularly thermostable and may be important for industrial applications based on these thermodynamic properties.
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•The novel acid protease was purified from Melilotus indicus leaves.•MALDI-TOF and LC-MS/MS mass fragmentation pattern of thermolysis showed that acid protease was a monomer with molecular mass of 50 kDa.•The optimum temperature and pH of the enzyme were 40 °C and 4.0 respectively.•The acid protease was purified from M. indicus was highly thermostable (D-values and Z-values) and followed first order kinetics.•The inhibitory effects of metal ions on the enzyme activity were insignificant.