Purification and Molecular Characterization of Protease from Lacticaseibacillus paracasei IN17 Isolated from Fermented Fish (Inasua)

Abstract

Protease is a protein degrading enzyme that is widely used in the food fermentation and protein processing industries. An example of a fermented fish product known as inasua, which originates from Maluku in Indonesia, is a proteolytic bacterial source. The aims of this study were to purify, characterize, and analyze the molecular characteristics of the protease produced by Lacticaseibacillus paracasei IN17. Proteases from extracellular and intracellular fractions were partially purified using dialysis and ammonium sulfate precipitation. Metal ion effects, kinetic parameters, electrophoretic analysis, and the ideal pH and temperature were all used to assess the enzymatic properties. The extracellular protease enzyme showed an increase in specific activity from 0.079 to 0.182 U mg-¹ with a yield of 18.9%. Maximum activity was observed at pH 6.0 for the extracellular enzyme and pH 7.0 for the intracellular fraction at 37 °C. Mn²⁺ enhanced the activity, and the kinetics analysis showed higher catalytic efficiency in the extracellular enzyme. An active protein band ~26 kDa was detected, and structural modeling revealed a conserved Ser–His–Asp catalytic triad. This work seeks to expand current knowledge of LAB proteolytic systems derived from non-dairy fermented foods by integrating purification strategies, enzymatic profiling, and gene analysis. This research supports SDGs 3 and SDGs 12 by promoting functional food development and responsible utilization of local microbial resources.