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bioRxiv · 10.1101/2025.09.10.675465

EXTRACTION, ISOLATION, PURIFICATION AND OPTIMIZATION OF AMYLASE AND PROTEASE ENZYMES ISOLATED FROM Bacillus species

Acharya, A.·Subedi, S.
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Abstract

BackgroundAmylase and protease are two of the most demanded and widely used commercial enzymes. Amylases have many industrial uses: starch conversion (food/bakery industry), sizing agent (textile industry and paper industry), degradation of starchy residue from clothes (detergent industry), conversion of starch to fermentable sugar (fuel industry) etc. Their main source is of microbial origin. About two-third of the industrial enzymes (amylase, protease, cellulose, penicillinase, chitinase, nucleases, esterase, lipase etc.) are produced by Bacillus spp. Among bacteria, Bacillus species are specific producers of extracellular enzymes. ObjectiveThe present work comprised the identification of amylase and protease producing Bacillus spp and exposure of the producers to various parameters for the maximum yield of the enzyme. MethodsTo isolate and identify the amylase and protease producing strain, soil samples were collected from different vegetation from the altitude at 4367.35 feet above sea level. The isolates were screened and various biochemical tests and morphological observations were done to identify the isolates. The enzymes were produced by the submerged state fermentation (SmF) from the isolates and purified by dialysis. Effects of temperature, pH, and different carbon and nitrogen sources of the medium using SmF were optimized. ResultsAmong 95 isolates, 36 were identified. Among the identified isolates, Bacillus subtilis and Bacillus thuringiensis were optimized for the amylase and protease production respectively. The maximum amylase production was found at 42C temperature, in fructose as a carbon sugar, peptone as a nitrogen source and at pH 7. Similarly, the maximum protease production was found at 42C temperature, in sucrose as a carbon sugar, ammonium sulphate as a nitrogen source. The producers inhabited the soil of leguminous plant. ConclusionIn the present study, a natural polymer gelatin is used with the nutrient agar medium to help in cell immobilization for maximum production of alkaline protease by strains of Bacillus. More sophisticated process of purification might yield more enzyme compared to the dialysis in our process that yield 42U/ml/min and 52U/ml/min respectively.

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Acharya, A., Subedi, S.. 2025-09-11. EXTRACTION, ISOLATION, PURIFICATION AND OPTIMIZATION OF AMYLASE AND PROTEASE ENZYMES ISOLATED FROM Bacillus species. https://doi.org/10.1101/2025.09.10.675465

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