Isolation, purification and biochemical characterization of alkaline α-amylase from Bacillus subtilis strain W3SFR5 isolated from kitchen Wastes
نویسندگان
1 Department of Molecular Biology and Biotechnology, Pan African University Institute of Basic Sciences Technology and Innovation P.O. Box 62000-00200 Nairobi, Kenya
2 Department of food science and Technology Jomo Kenyatta University of Agriculture and Technology. P.O. Box 62000-00200 Nairobi, Kenya
3 -Department of food science and Technology Jomo Kenyatta University of Agriculture and Technology. P.O. Box 62000-00200 Nairobi, Kenya
4 Department of Molecular Biology and Biotechnology, Pan African University Institute of Basic Sciences Technology and Innovation P.O. Box 62000-00200 Nairobi, Kenya -Department of Biochemistry, Jomo Kenyatta University of Agriculture and Technology. P.O. box 62000-00200 Nairobi, Kenya
doi
10.22037/afb.v10i1.39495چکیده
Background and Objective: Amylase is a hydrolytic enzyme that breaks starch into simple sugars. This enzyme includes uses in starch production, brewery, detergent formulation, paper production and pharmaceuticals as a digest aid. The aim of the present study was to isolate, identify and characterize an alkaline amylase from bacteria of food wastes. Material and Methods: Bacteria were isolated using serial dilution, screened on agar plates and characterized through biochemical assessments and 16S rRNA sequencing. After optimizing the bacterial growth conditions using one factor at a time method, the alkaline amylase was extracted from the culture broth and partially purified using Sephadex G-75 chromatography. Enzyme activity generated by submerged fermentation was assessed using 3,5-dinitrosalicylic and recorded as the mean of three replicates. Results and Conclusion: The bacterial isolate W3SFR5 showed high amylolytic activity in agar culture. Biochemical analysis and sequencing of the 16S rRNA verified the bacterial isolate as Bacillus subtilis (GenBank accession number: OM258620). Bacillus subtilis W3SFR5 was propagated within 30–50 ℃ and pH 6-9. The partially purified Bacillus subtilis W3SFR5 amylase included a molecular weight of 65 kDa and demonstrated a maximum specific activity of 216.02U mg-1. The optimum temperature for the enzyme was 60 °C and the pH was 9. The W3SFR5 amylase was actively stable under temperatures of 50–70 °C and pH of 7-9. Furthermore, 5 mM Fe2+ increased W3SFR5 amylase activity. The enzyme was more resistant to organic solvents, surfactants, inhibitors and oxidizing agents than that most amylases were. Additionally, results showed that W3SFR5 amylase was compatible with most commercial detergents, indicating that it could be used as a detergent additive.
