Expression and Characterization of a GH38 α-Mannosidase from the Hyperthermophile Pseudothermotoga thermarum

Abstract

This study focused on the bio-characterization of a GH38 α-mannosidase from the hyperthermophile Pseudothermotoga thermarum DSM 5069. We aimed to successfully express and characterize this thermophilic α-mannosidase and to assess its functional properties. Subsequently, recombinant α-mannosidase PtαMan was expressed in Escherichia coli BL21(DE3) and purified via affinity chromatography, and native protein was verified as a tetramer by size exclusion chromatography. In addition, the activity of α-mannosidase PtαMan was relatively stable at pH 5.0-6.5 and temperatures up to 75 ℃. α-Mannosidase PtαMan was active toward Co²⁺ and had a good catalytic efficiency deduced from the kinetic parameters. However, its activity was strongly inhibited by Cu²⁺, Zn²⁺, SDS, and swainsonine. In summary, this cobalt-required α-mannosidase is putatively involved in the direct modification of glycoproteins.

Keywords: Divalent cobalt; Glycosyl hydrolase; Hyperthermophile; Size exclusion chromatography; α-Mannosidase.