Galactofuranosidase from JHA 19 Streptomyces sp.: subcloning and biochemical characterization.

Affiliation

ICOA UMR CRNS 7311, Université d'Orléans, Rue de Chartres, BP 6759, 45067, Orléans Cedex 2, France. Electronic address: [Email]

Abstract

Despite the crucial role of the rare galactofuranose (Galf) in many pathogenic micro-organisms and our increased knowledge of its metabolism, there is still a lack of recombinant and efficient galactofuranoside hydrolase available for chemo-enzymatic synthetic purposes of specific galactofuranosyl-conjugates. Subcloning of the Galf-ase from JHA 19 Streptomyces sp. and its further overexpression lead us to the production of this enzyme with a yield of 0.5 mg/L of culture. It exhibits substrate specificity exclusively towards pNP β-d-Galf, giving a KM value of 250 μM, and the highest enzymatic efficiency ever observed of 14 mM-1  s-1. It proved to be stable to temperature up to 60 °C and to at least 4 freeze-thaw's cycles. Thus, Galf-ase demonstrated to be an efficient and stable biocatalyst with greatly improved specificity toward the galactofuranosyl entity, thus paving the way to the further development of transglycosylation and thioligation reactions.

Keywords

Glycoside hydrolase,Subcloning,Thio-β-D-galactofuranoside inhibition,β-D-galactofuranosidase,

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