Cloning, purification, and functional characterization of recombinant pullulanase from Bacillus cereus ATCC 14579 for improved detergent performance


Zafar A., Masood A., Rahman Z., HAMID A., Javed M. H., Zulfiqar A., ...More

Journal of Surfactants and Detergents, 2024 (SCI-Expanded) identifier

  • Publication Type: Article / Article
  • Publication Date: 2024
  • Doi Number: 10.1002/jsde.12796
  • Journal Name: Journal of Surfactants and Detergents
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Academic Search Premier, Chemical Abstracts Core, Chimica, Compendex, INSPEC
  • Keywords: Bacillus cereus, bioinformatics analysis, cloning, detergent, Escherichia coli, pullulanase
  • Yozgat Bozok University Affiliated: No

Abstract

The present study outlines the approach that was employed for cloning, expression, and characterization of the recombinant pullulanase enzyme from Bacillus cereus ATCC 14579 into Escherichia coli BL21(DE3) using pET-25b (+) expression vector. The recombinant pullulanase enzyme was purified using ammonium sulfate precipitation and immobilized metal ion affinity chromatography (IMAC). The molecular mass of the purified pullulanase enzyme was measured using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as 95 kDa. The purified recombinant pullulanase enzyme demonstrated significant thermal stability, maintaining its structural integrity and functionality at temperatures as high as 90°C over a period of 4 h. The inclusion of divalent metal ions, specifically Ca2+ and Mg2+, had a positive effect on the activity of the pullulanase enzyme. Conversely, the presence of Co2+ and EDTA (Ethylene Diamine Tetra Acetic acid) resulted in suppression of the enzyme activity. The purified pullulanase enzyme demonstrated remarkable resistance when exposed to organic solvents. The enzyme activity was notably decreased in the presence of SDS (Sodium Dodecyl Sulfate) while β-mercaptoethanol and tween-60 did not substantially affect the enzyme activity and stability which suggest its potential applicability in the detergent sector. This discovery indicates a potential approach to improve the effectiveness of currently available detergents in the marketplace.