Optimization of Production Conditions, Partial Purification, and Characterization of Alkaline Protease Enzymes from Bacillus thuringiensis for Industrial Applications
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Alkaline proteases have been substantially used in diverse industrial sectors due to their remarkable pH tolerance, thermal stability and ability to substitute harsh chemical processes. The persistent demand for protease enzymes with improved characteristics has led to search for alternative enzymes. The main objective of this research was to produce and characterize protease enzyme, and evaluate its potential industrial applications. An effective protease-producing bacterium was isolated from soil of tannery waste. Enzyme activity was confirmed by the clear zone of casein hydrolysis on Skim Milk Agar Medium and identified as Bacillus thuringiensis using 16S rRNA sequencing. Optimization of cultural and physicochemical parameters, including incubation time, temperature, inoculum size, pH, and substrate, yielded maximal enzyme production under the following conditions: pH 9.0, temperature 37℃, incubation period of 36 h, agitation speed 150 rpm, and 4% inoculum (1.35 × 108 CFU/ml), resulting in a peak activity of 145.6 U/ml. The enzyme showed highest activity at pH 10.0, and showed thermostability by maintaining maximum activity at 35-65℃. SDS-PAGE analysis revealed an apparent molecular mass of 45 kDa. The enzyme exhibited a Vmax of 173.41 Unit/mg with casein and a Km of 4.381 mg/ml, showing high catalytic performance as well as high substrate affinity. Finally, as part of the industrial application, the enzyme effectively dehaired goat and cow hides, removed blood stain from cotton, and hydrolyzed gelatin on X-ray films. Collectively, these findings underscore the potential of alkaline protease as a sustainable biocatalyst for eco-friendly industrial applications and environmentally benign waste management.
Abstract
Alkaline proteases have been substantially used in diverse industrial sectors due to their remarkable pH tolerance, thermal stability and ability to substitute harsh chemical processes. The persistent demand for protease enzymes with improved characteristics has led to search for alternative enzymes. The main objective of this research was to produce and characterize protease enzyme, and evaluate its potential industrial applications. An effective protease-producing bacterium was isolated from soil of tannery waste. Enzyme activity was confirmed by the clear zone of casein hydrolysis on Skim Milk Agar Medium and identified as Bacillus thuringiensis using 16S rRNA sequencing. Optimization of cultural and physicochemical parameters, including incubation time, temperature, inoculum size, pH, and substrate, yielded maximal enzyme production under the following conditions: pH 9.0, temperature 37℃, incubation period of 36 h, agitation speed 150 rpm, and 4% inoculum (1.35 × 108 CFU/ml), resulting in a peak activity of 145.6 U/ml. The enzyme showed highest activity at pH 10.0, and showed thermostability by maintaining maximum activity at 35-65℃. SDS-PAGE analysis revealed an apparent molecular mass of 45 kDa. The enzyme exhibited a Vmax of 173.41 Unit/mg with casein and a Km of 4.381 mg/ml, showing high catalytic performance as well as high substrate affinity. Finally, as part of the industrial application, the enzyme effectively dehaired goat and cow hides, removed blood stain from cotton, and hydrolyzed gelatin on X-ray films. Collectively, these findings underscore the potential of alkaline protease as a sustainable biocatalyst for eco-friendly industrial applications and environmentally benign waste management.