Modeling and evaluation of the sucrose-degrading activity of recombinantly produced oligo-1,6-glucosidase from A. gonensis

Prep Biochem Biotechnol. 2024;54(3):273-281. doi: 10.1080/10826068.2023.2227883. Epub 2023 Jun 28.

Abstract

Fructose is the most preferred sugar to provide benefits for sweetening and health. As many industrial enzymes are used to produce High Fructose Syrup (HFS), it is vital to explore alternative enzymes for fructose production. Oligo-α-1,6-glucosidase (O-1-6-glucosidase) hydrolyzes non-reducing ends of isomaltooligosaccharides, panose, palatinose, and an a-limit dextrin by breaking α-1,6-glucoside bonds, although it generally has no activity on α-1,4-glucoside bonds of maltooligosaccharides. In this study, sucrose-hydrolyzing activity of O-1-6-glucosidase of thermophilic A. gonensis was evaluated. For this purpose, O-1-6-glucosidase gene region of A. gonensis was cloned in the pET28(a)+ expression vector, the expression product was purified, modeled, and biochemically characterized. The optimal activity of the enzyme was to be at pH 7.0 and 60 °C. The enzyme activity was halved at the end of the 276th h at 60 °C. The enzyme maintained its activity even after 300 h at pH 6.0-10.0. The values of Km, Vmax, kcat, and kcat/Km were determined as 44.69 ± 1.27 mM, 6.28 ± 0.05 µmoL/min/mg protein, 6.70 1/s and 0.15 1/mMs-1, respectively. While Zn2+, Cu2+, Pb2+, Ag2+, Fe3+, Hg2+, and Al2+ metal ions inhibited O-1-6-glucosidase, Mn2+, Fe2+, and Mg2+ ions activated the enzyme. Consequently, A. gonensis O-1-6-glucosidase (rAgoSuc2) has interesting properties, especially for HFS production.

Keywords: Anoxybacillus gonensis; High Fructose Syrup; oligo-α-16-glucosidase; sucrose.

MeSH terms

  • Fructose
  • Glucosidases*
  • Glucosides
  • Ions
  • Magnesium*
  • Oligo-1,6-Glucosidase
  • Radioisotopes*
  • Sucrose*

Substances

  • Glucosidases
  • Sucrose
  • Magnesium-28
  • Fructose
  • Glucosides
  • Ions
  • Oligo-1,6-Glucosidase
  • Magnesium
  • Radioisotopes