Pcal_0976, a pullulanase homologue from Pyrobaculum calidifontis , displays a glycoside hydrolase activity but no pullulanase activity

In this study, we have cloned and characterized a novel protein, Pcal_0976, annotated as pullulanase in the genome sequence of hyperthermophilic archaeon Pyrobaculum calidifontis . Motif search showed two glucodextran_C like domains and a domain of unknown function (DUF4134) in Pcal_0976. Multiple alignment with close homologues demonstrated six stretches of conserved regions. When the gene encoding Pcal_0976 was expressed in Escherichia coli , the recombinant protein was produced in insoluble and inactive form, which was solubilized using guanidine hydrochloride and refolded in an active form in the presence of arginine. Refolded Pcal_0976 displayed hydrolysis of glycogen, dextran, dextrin and starch. No hydrolytic activity was detected against pullulan. These results indicate that Pcal_0976 may not be a pullulanase but a novel glycoside hydrolase. Further studies are needed to establish the role of Pcal_0976 in carbohydrate metabolism in this archaeon.