Purification, physico-chemico-kinetic characterization and thermal inactivation thermodynamics of milk clotting enzyme from Bacillus subtilis MTCC 10422

- Isolated the free (soluble) enzyme from soil-borne bacterium identified as Bacillus subtilis MTCC 10422.
- Purified the crude enzyme extract of bacterial broth through sequential chromatographies.
- Characterized the purified enzyme for kinetic constants and reaction parameters.
- Thermodynamic behaviour of the enzyme was monitored under denaturating conditions.
- Milk processing based industrial usage of this enzyme requires bioreactor or pilot plant level up-gradation.

Extracellular milk clotting enzyme from Bacillus subtilis MTCC 10422, which has the capacity of forming milk curds, was purified 32.9 fold with 20.82% recovery using sequential chromatographic techniques. The molecular weight of the enzyme was found to be 27 kDa which is of monomeric nature. The optimum temperature of the enzyme was found to be 45 °C and it was quite stable in the temperature range of 35-65 °C. The enzyme showed the pH optima of 6.0, and quite stable in broad pH range of 5.0-8.0 and showed a typical hyperbolic velocity saturation curve with Km value of 5 mg mL−1 with skim milk as a substrate. Calcium chloride at the concentration of 10 mM was found to be the most effective stimulator, accelerating the enzyme activity by about 2.8 folds. Various kinetic parameters towards thermo-inactivation of milk clotting enzyme were studied. Thermal inactivation behaviour of the purified enzyme suggested its thermostability and also its sensitivity to duration of heat treatment. After purification, both yield and recovery of purified preparation were found appreciable. Since this enzyme has wider pH stability and thermostability, usage of this high activity microbial enzyme for various cheese preparations can be evaluated at commercial scale.