RESUMO
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.
