ALPHA AMYLASE PRODUCTION, PURIFICATION AND CHARACTERIZATION USING INDIGENOUS BACILLUS SUBTILIS

IQRA BIBI; MUHAMMAD IMRAN; MUHAMMAD TAYYAB; MUHAMMAD ASAD ALI; RAHAT NASEER; SHAHZAD ALI; SAQIB HUSSAIN HADRI

doi:10.30848/PJB2025-6(19

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ALPHA AMYLASE PRODUCTION, PURIFICATION AND CHARACTERIZATION USING INDIGENOUS BACILLUS SUBTILIS

Author(s)

IQRA BIBI Institute of Biochemistry and Biotechnology, Faculty of Biosciences, University of Veterinary and Animal Sciences,Lahore, Pakistan 54000,

MUHAMMAD IMRAN Institute of Biochemistry and Biotechnology, Faculty of Biosciences, University of Veterinary and Animal Sciences,Lahore, Pakistan 54000,

MUHAMMAD TAYYAB Institute of Biochemistry and Biotechnology, Faculty of Biosciences, University of Veterinary and Animal Sciences,Lahore, Pakistan 54000,

MUHAMMAD ASAD ALI Institute of Microbiology, Faculty of Veterinary Sciences, University of Veterinary and Animal Sciences,Lahore,Pakistan

RAHAT NASEER Institute of Biochemistry and Biotechnology, Faculty of Biosciences, University of Veterinary and Animal Sciences,Lahore, Pakistan 54000,

SHAHZAD ALI Department of Wildlife and Ecology, Faculty of Fisheries and Wildlife, University of Veterinary and Animal Sciences,Lahore, Pakistan 54000,

SAQIB HUSSAIN HADRI Department of Biochemistry and Biotechnology, University of Gujrat,,Pakistan

Abstract

The a-amylase enzyme hydrolyzes starch and glycogen into maltose, isomaltose and glucose. It is produced from plants, animals and microbes but microbial a-amylase has the highest commercial and industrial value. Bacillus subtilis was identified on the basis of 16S rRNA. Further, characterization of Bacillus subtilis was done using BLAST tool and phylogenetic tree with MEGA X. Different carbon and nitrogen sources were supplemented to the LB medium. The supplemented LB medium was sterilized and used for the growth of Bacillus subtilis. High yield of a-amylase was produced at 3% molasses and 3% peptone. Alpha Amylase was precipitated at 60% ammonium sulphate concentration then partially purified a-amylase was further purified through column chromatography. The optimum pH and temperature for alpha amylase was found to be 9 and 55°C respectively. Kinetic studies of alpha amylase were performed to check Km and Vmax. values. Impact of different metal ions were observed on a-amylase activity and high a-amylase yield was recorded in the presence of Fe2+ ions. SDS-PAGE technique revealed the molecular weight of a-amylase which was ~46kDa. The purified a-amylase utilized for characterization studies. The role of a-amylase was analyzed in detergent industry with different company detergents. Enzyme activity was estimated by checking release of reducing sugars which had application in textile industry. The enzyme capability with detergents and release of reducing sugars in de-sizing step in textile industry makes alpha amylase suitable for local industries in Pakistan.

Publication Details

Page(s) 2351-2359

DOI 10.30848/PJB2025-6(19

Published Journal: Pakistan Journal of Botany, Volume: 57, Issue: 6, Year: 2025

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Keywords

Optimization, Characterization, Bacillus subtilis, Phylogenetic tree, alpha amylase, Detergent industry, Metal catalysts

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