ISSN print edition: 0366-6352 ISSN electronic edition: 1336-9075 Registr. No.: MK SR 9/7 Published monthly

Optimization of three-phase partitioning system for the purification of protease from Calotropis gigantea latex using response surface methodology

Bunty Maskey, Dhan Bahadur Karki, and Nabindra Kumar Shrestha

Central Department of Food Technology, Tribhuvan University, Dharan, Nepal

E-mail: bunty.maskey@cdft.tu.edu.np

Received: 18 July 2023  Accepted: 4 December 2023

Abstract:

The response surface methodology (RSM) was utilized to optimize three-phase partitioning (TPP) parameters for the purification of Calotropis gigantea latex protease. The limits of purification parameters namely saturation of ammonium sulfate salt, volume ratio of tert-butanol to crude latex extract and pH value were selected from single-factor experiments. A Box-Behnken design was employed to determine the effects of TPP parameters on protease purification. From the optimization study, the degree of purification and recovery percentage reached maximum values of 6.7 and 126.12%, respectively, under optimized parameters of 52% saturation of (NH₄)₂SO₄ (w/v), 1.0:1.5 ratio of crude latex extract to tert-butanol (v/v) and pH 6. The concentrated protease in the interfacial phase (IP) demonstrated higher enzyme activity when compared to the lower aqueous phase (AP). The molecular weight of recovered protease was 24 kDa from electrophoretic analysis, and the substrate zymography revealed the presence of protease in IP fraction. The findings of this study demonstrated that TPP is an effective and economic protocol for quick recovery of latex protease in comparison with expensive and time-consuming chromatographic techniques.

Keywords: Latex protease; TPP purification; RSM optimization; SDS-PAGE; Enzyme recovery

Full paper is available at www.springerlink.com.

DOI: 10.1007/s11696-023-03259-4

Chemical Papers 78 (4) 2549–2559 (2024)