Partial purification and characterization of protease extracted from kinema

Dambar Bahadur Khadka; Tikaram Pahadi; Sunil Aryal; Dhan Bahadur Karki

doi:10.1016/j.heliyon.2024.e27173

Partial purification and characterization of protease extracted from kinema

Heliyon. 2024 Feb 27;10(5):e27173. doi: 10.1016/j.heliyon.2024.e27173. eCollection 2024 Mar 15.

Authors

Dambar Bahadur Khadka^{1

2}, Tikaram Pahadi², Sunil Aryal¹, Dhan Bahadur Karki¹

Affiliations

¹ Central Department of Food Technology, Tribhuvan University, Dharan, Nepal.
² Central Campus of Technology, Tribhuvan University, Dharan, Nepal.

Abstract

Proteases are large group of highly demanded enzymes having huge application in food and pharmaceutical industries. Numerous sources, including plants, microorganisms, and animals, can be used to obtain protease. Due to its affordability and safety consideration, fermented foods have recently attracted more attention as a source of microbial protease. The present study aimed to extract protease from kinema, partially purify the extracted protease following dialysis after precipitation with ammonium sulfate, and determine general characteristics of protease. The kinema having highest proteolysis activity after three days of control fermentation (Temperature 30±2 °C, RH 66 ± 2%) was taken for the study. About 2.45 fold of purification with overall recovery of 63.21% was achieved after precipitation with ammonium sulfate at 30-70% saturation level followed by dialysis of crude extracted protease. The dialysed kinema protease had specific activity of 7.90 U/mg. The enzyme remained actively functional across a wider pH (5-9) and temperature (40-60 °C) range. SDS-PAGE and Zymogram confirmed the presence of three major active bands respectively of 29.04 kDa, 36.09 kDa and 46.35 kDa in the kinema protease extract. The enzyme kinetics data on casein, fitted to Mechaelis Mentens' plots showed the protease had V_max of 1.001 U/ml with corresponding K_m value of 0.825 mg/ml. Metal ions such as iron, mercury and aluminium showed the inhibition effect whereas presence of sodium, zinc, and calcium shows the activation effect on protease performance. The enzyme was active over various natural substrates; showing maximal activity on casein, and subsequent to bovine serum albumin, gelatin, hemoglobin and whey protein respectively. Furthermore, molecular weight distribution of the protease extract and activity inhibition with ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride, suggesting the protease from kinema could be a metal dependent serine protease or mixture of them.

Keywords: Characterization; Enzyme extraction; Kinema; Partial purification; Protease.