Multi-immobilization of viscozyme, alcalase and flavourzyme by nanomagnetic combi-CLEAs method for oil and protein hydrolysates extraction from rice bran in aqueous phase.

Edible oil isolation from oil seeds by hexane faces pivotal challenges for human health and the environment. The aqueous enzymatic extraction procedure has been remarked as a preferable technique for concurrent oil and protein extraction. A one-pot combined cross-linked enzyme aggregates of Viscozyme, Alcalase and Flavourzyme in nanoscale for coinciding isolation of oil and protein hydrolysates is presented. The ideal parameters for immobilization were 1:1:1 ratio of enzymes in ethanol as aggregator, 0.75:1 ratio of lysine/enzyme mixture, 15 mM glutaraldehyde at temp = 2-3 °C, ultrasonic waves (300 Hz) for 10 min, then holding at 2-3 °C for 2.5 h. The optimum performance of NM-CLEAs was at pH 6.0, temp = 60 °C with particle size 65.5-414 nm, PDI = 0.428, and Ȥ-potential = -41.4 mV, sequentially. The thermo robustness of NM-Combi-CLEAs was approximately 3.6 times elevated than free enzymes at 85 °C in terms of carbohydrase activity and about 2.3 folds in protease performance. The kinetic assessment demonstrated 1.7 folds in Vmax and 2.0 folds increment in catalytic effectiveness (kcat/Km) while 1.1-2.0 folds reduction in Km value. CD and FTIR analyses demonstrated a 9.8 % and 21 % increment in α-helix and random coil configurations, while turn and β-sheet composition diminished by approximately 27.6 % and 12.4 %, sequentially. The oil extraction yield was about 92 % with 86 % protein efficiency after 10 consecutive cycles of CLEAs use. Physicochemical assessment of extracted oil and protein hydrolysates compared to commercial rice bran oil, was represent quality enhancement. Hence, Combi-CLEAs introduces as a biocatalyst with ease of operation, elevated effectiveness, and eco-friendly catalytic approach.