ISOLASI, PEMURNIAN DAN KARAKTERISASI ENZIM PROTEASE TERMOSTABIL DARI BAKTERI ISOLAT LOKAL Bacillus subtilis ITBCCB148

Abstract

Protease catalyzes the hydrolysis of peptide bond to produce amino acid and peptide.  This enzyme was used in many industrial processes, especially in the detergent industry.  The aims of this research are to isolate, purify and characterize the extracellular protease from a local bacteria Bacillus subtilis ITBCCB148To achieve these aims, the determination of an optimum condition to produce protease with high activity, production, isolation, and purification of enzyme.  Determination of optimum condition was carried out by various pH and temperatures of fermentation. The purification of the enzyme was conducted by few steps which fractionation with ammonium sulphate, CM- Sephadex C-50 cation exchange column chromatography, and molecular sieves column chromatography using Sephadex G-100. The purified enzyme was characterized by determining the temperature, pH, and kinetic data.  The results showed that the protease which was produced by Bacillus subtilis ITBCCB148 has the optimum activity at the fermentation temperature of 35°C, the fermentation media at pH 7.0; and the duration time of fermentation was 60 hours with unit activity of 0.24 U/mL.  The specific activity of the purified enzyme was 65.8 U/mg; increasing by 263 times than that of the crude enzyme extract with specific activity of only 0.25 U/mg.  The optimum pH value of the purified enzyme was 7.5, however this enzyme was active at a pH range of 6.0-8.0. The optimum temperature value of the pure enzyme was 60°C, while the K_m and V_max value were 6.48 mg mL^-1 and 3.26 mmol mL^-1 min^-1, respectively.

Keywords:  protease, Bacillus subtilis ITBCCB148, isolation and purification