Selective purification of protease from ginger and sodom apple by ultrasound assisted liquid-liquid microextraction using natural deep eutectic solvent

Abstract

Ultrasonic assisted liquid-liquid microextraction (UA-LLME) of cysteine proteases from ginger and sodom apple were accomplished with natural deep eutectic solvents (NADES). Five imidazole-based NADES with carbohydrates were synthesised and their thermophysical properties were investigated. Selective partitioning of NADES assisted UA-LLME yielded more protease in NADES enriched top phase. Choice of phase forming compounds and their influence in the concentration of protease was evaluated by calculating the partition coefficient. The linearity of detection, LOD and LOQ for protease were found to be 10–50 μg/ml, 15 and 60 μg/ml respectively. Influential variables towards the extraction were optimised with response surface methodology and it was noted that maximum yield was achieved with 25% (v/v) of NADES, 15% (w/v) of source concentration and ultrasound temperature and time as 35 °C and 10 min respectively. The concentrated extract was purified by gel filtration chromatography was performed followed by affinity chromatography and purity fold with %yield of the enzyme was observed.