Point mutations enhance catalytic efficiency of Geobacillus stearothermophilus α-glucosidase: A biochemical characterization study

Hekim S., Kaçıran A., Akmehmet A. N., BELDÜZ A. O., Bektaş K. İ., Mamatjan Y., ...More

International Journal of Biological Macromolecules, vol.315, 2025 (SCI-Expanded) identifier identifier

  • Publication Type: Article / Article
  • Volume: 315
  • Publication Date: 2025
  • Doi Number: 10.1016/j.ijbiomac.2025.144379
  • Journal Name: International Journal of Biological Macromolecules
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Academic Search Premier, BIOSIS, Biotechnology Research Abstracts, CAB Abstracts, Chemical Abstracts Core, EMBASE, Food Science & Technology Abstracts, INSPEC, Veterinary Science Database
  • Keywords: Catalytic efficiency, Heterologous expression, Kinetics, Point mutation, α-Glucosidases
  • Karadeniz Technical University Affiliated: Yes

Abstract

α-Glucosidases are important enzymes with a broad spectrum of industrial applications. However, traditional protein engineering often struggles to improve activity without compromising stability and usually demands extensive screening. Here, we targeted the α-glucosidase (AG) from Geobacillus stearothermophilus (Gst), using in silico analyses and literature precedent to select point mutations N61H and N258P. We cloned the mutated gstAG gene into the pET-28a(+) vector and expressed in Escherichia coli. After expressing and purifying both wild-type and mutant enzymes, we performed detailed biochemical assays. Both mutants maintained GstAG's optimum temperature (60 °C) and pH (6.5). However, each displayed enhanced catalytic efficiency: N61H lowered the Michaelis constant (Kₘ) by 1.5-fold and raised the turnover number (kcat) by 1.7-fold relative to the wild type. These results offer a blueprint for engineering α-glucosidases with improved performance, unlocking new commercial and biotechnological applications.