title: Biochemical properties of Glu-SH3 as a family 13 glycoside hydrolase with remarkable substrate specificity for trehalose: Implications to sequence-based classification of CAZymes.
abstract: A novel glycoside hydrolase from Exiguobacterium sp. SH3 was characterized. The enzyme, designated as Glu-SH3, was predicted by in silico analysis to have structural similarity with members of oligo-1,6-glucosidase and trehalose-6-phosphate hydrolase subfamilies in the GH-13 family of glycoside hydrolases. The gene was expressed in Escherichia coli and the recombinant enzyme was purified as a His-tagged protein of about 60 kDa. The enzyme was shown to have remarkable substrate specificity for trehalose. The characteristic ability of Glu-SH3 to hydrolyze trehalose was ascertained by zymography, thin layer chromatography, and NMR spectroscopy. The maximum activity of Glu-SH3 was obtained at 35 °C and pH 7, but it was able to exhibit more than 90% of the activity within the pH range of 5-8. The Vmax and Km values were estimated to be 170 U and 4.5 mg ml(-1), respectively. By comparison with trehalases, Glu-SH3 with Kcat and Kcat/Km values of 1552 s(-1) and 119.4 mM(-1) s(-1) can be recognized as a very efficient trehalose-hydrolyzing glycosidase. Given the phylogeny and the substrate specificity of Glu-SH3, it may be assumed that the enzyme shares a common ancestor with oligo-1,6-glucosidases but have evolved distinctly to serve a physiological function in trehalose metabolism.
authors.affiliation: Division of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, 14155-6343, Iran.
authors.forename: Kamran Khalili
authors.initials: KK
authors.lastname: Ghadikolaei
authors.affiliation: Division of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, 14155-6343, Iran.
authors.forename: Maral
authors.initials: M
authors.lastname: Shojaei
authors.affiliation: Division of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, 14155-6343, Iran.
authors.forename: Armin
authors.initials: A
authors.lastname: Ghaderi
authors.affiliation: Division of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, 14155-6343, Iran.
authors.forename: Farzaneh
authors.initials: F
authors.lastname: Hojjati
authors.affiliation: Division of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, 14155-6343, Iran.
authors.forename: Kambiz Akbari
authors.initials: KA
authors.lastname: Noghabi
authors.affiliation: Division of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, 14155-6343, Iran. Electronic address: shahbani@nigeb.ac.ir.
authors.forename: Hossein Shahbani
authors.initials: HS
authors.lastname: Zahiri
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D000068536:Bacillota / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008670:Metals / Q000737:chemistry; D009834:Oligo-1,6-Glucosidase / Q000737:chemistry; D010802:Phylogeny; D000072417:Protein Domains; D013379:Substrate Specificity; D013696:Temperature; D014199:Trehalose / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D007477:Ions; D008670:Metals; D014199:Trehalose; D006026:Glycoside Hydrolases; D009834:Oligo-1,6-Glucosidase
keywords: Exiguobacterium; GH13 family; Glycoside hydrolase; Substrate specificity; Trehalose