RESUMO
Two putative α-mannosidase genes isolated from the rather unexplored soil bacterium Solitalea canadensis were cloned and biochemically characterised. Both recombinant enzymes were highly selective in releasing α-linked mannose but no other sugars. The α-mannosidases were designated Sca2/3Man2693 and Sca6Man4191, and showed the following biochemical properties: the temperature optimum for both enzymes was 37 °C, and their pH optima lay at 5.0 and 5.5, respectively. The activity of Sca2/3Man2693 was found to be dependent on Ca(2+) ions, whereas Cu(2+) and Zn(2+) ions almost completely inhibited both α-mannosidases. Specificity screens with various substrates revealed that Sca2/3Man2693 could release both α1-2- and α1-3-linked mannose, whereas Sca6Man4191 only released α1-6-linked mannose. The combined enzymatic action of both recombinant α-mannosidases allowed the sequential degradation of high-mannose-type N-glycans. The facile expression and purification procedures in combination with strict substrate specificities make α-mannosidases from S. canadensis promising candidates for bioanalytical applications.
Assuntos
Proteínas de Bactérias/química , Bacteroidetes/enzimologia , Manose/química , alfa-Manosidase/química , Bacteroidetes/genética , Especificidade por Substrato , alfa-Manosidase/genéticaRESUMO
We present a generic method for screening small molecule kinases for their acceptor specificity. The release of the reaction byproduct adenosine diphosphate (ADP) triggers a concentration-dependent formation of amylose from sucrose, by using the combined enzymatic action of sucrose synthase and glycogen synthase. Kinase activities could be quantified photometrically after the formation of a dark-blue amylose-polyiodide complex. We demonstrate that this method can be used to profile both known and novel nucleotide- and sugar-kinases for their substrate specificity. Using a facile and widely available methodology, the amylose-polyiodide small-molecule kinase assay presented herein has the potential to perform substrate screenings of small molecule kinases in a high-throughput manner.