Heterologous production, purification, and characterization of three starch-degrading enzymes from Geobacillus spp
TICONA, Alonso Roberto Poma; RAMIREZ-ARUA, Heber Esau; CABRERA, Roberto Castellanos; SILVA, Jéssica Pinheiro; STRANZ, Artur Carvalho; ZAVALETA, Amparo Iris; POLIKARPOV, Igor; NORONHA, Eliane Ferreira; HAMANN, Pedro Ricardo Vieira.
TICONA, Alonso Roberto Poma; RAMIREZ-ARUA, Heber Esau; CABRERA, Roberto Castellanos; SILVA, Jéssica Pinheiro; STRANZ, Artur Carvalho; ZAVALETA, Amparo Iris; POLIKARPOV, Igor; NORONHA, Eliane Ferreira; HAMANN, Pedro Ricardo Vieira.
Abstract: Starch-degrading enzymes are key biocatalysts in industrial applications, particularly when derived from thermophilic microorganisms with potential to operate under elevated temperatures. In this study, three recombinant starch-degrading enzymes were heterologously produced, purified, and biochemically characterized: an a-amylase from Geobacillus kaustophilus, and an a-glucosidase and a type I pullulanase from Geobacillus sp. G4, a thermophilic strain isolated from a geothermal field in southern Peru. The three enzymes were successfully expressed in soluble form in Escherichia coli and purified by one-step affinity chromatography. Biochemical characterization showed that a-glucosidase and a-amylase displayed optimum activity at pH 6-7, whereas pullulanase exhibited a broader pH profile, retaining high activity up to pH 9. All three enzymes reached maximum activity at 60 °C, although their thermal stability profiles differed markedly, with pullulanase showing the highest thermostability. Metal ion assays revealed enzyme-dependent effects, with pullulanase being stimulated by Ca2+ and Mg2+, while a-amylase and a-glucosidase showed limited responses to divalent ions. Kinetic analysis using soluble potato starch indicated that a-amylase had the most favorable catalytic profile, with the lowest Km and the highest catalytic efficiency among the three enzymes. Functional hydrolysis assays demonstrated that all enzymes were active on soluble starch and pretreated potato peel, while the enzymatic mixture consistently released the highest concentration of reducing sugars. These results expand the biochemical knowledge of thermophilic amylolytic enzymes from Geobacillus and support their potential use in future enzymatic systems for the conversion of starch-rich residues.
@article={003307437,author = {TICONA, Alonso Roberto Poma; RAMIREZ-ARUA, Heber Esau; CABRERA, Roberto Castellanos; SILVA, Jéssica Pinheiro; STRANZ, Artur Carvalho; ZAVALETA, Amparo Iris; POLIKARPOV, Igor; NORONHA, Eliane Ferreira; HAMANN, Pedro Ricardo Vieira.},title={Heterologous production, purification, and characterization of three starch-degrading enzymes from Geobacillus sp},journal={Fermentation},note={v. 12, n. 6, p. 269-1-269-18 + Supplementary Materials},year={2026}}