Green extraction of milk-clotting proteases from sweet Indrajao (Wrightia tinctoria) stem utilizing an ethanol-phosphate based aqueous two-phase system - optimization and scalability.

Aqueous two-phase system (ATPS) is emerging as a green or an environmentally friendly strategy for extracting enzymes of biotechnological importance. Purification of milk-clotting proteases from Wrightia tinctoria R.Br. stem using ethanol - dipotassium phosphate based ATPS was optimized using central composite design of response surface methodology. The partitioned enzyme (PE) from the system with 20.00% (w/w) ethanol and 28.49% (w/w) dipotassium phosphate was found to exhibit high milk clotting index (210.64 ± 18.61), yield% (136.40 ± 4.54) and purification factor (3.57 ± 1.01). Scale-up at different laboratory scales (10× and 20×) demonstrated the feasibility resulting with comparable milk clotting index, yield% and purification factor between the levels. Subsequently, ethanol from 20× (200 g) was recovered by rotary evaporation and reused for a new cycle of ATPS (100 g) successfully. The green certificate score of the process (>90 in 1 g, 100 g and 200 g) classified it under category "A", signifying its ecological sustainability. Further analysis revealed the comparable nature of casein (whole and κ) hydrolysis between PE and commercially available vegetable coagulant, Cynzime®. Prolonged stability of the crude enzyme extract and PE was observed when stored at -20 °C. The cheese prepared using PE was found to be comparable in terms of its yield% and physico-chemical characteristics to that of Cynzime® demonstrating the commercial prospects of PE as a vegetable milk coagulant in cheese preparation. Findings from the study collectively illustrate the commercial potential of single-step green purification technique (ATPS) for extraction of milk-clotting proteases from W. tinctoria stem.