Molecular cloning and production of recombinant Pcal_0672, a family GH57 glycoside hydrolase from Pyrobaculum calidifontis

An open reading frame of putative gene (designated as Pcal_0672), that belongs to family GH57 (UniProt acc # A3MTY1 and GenBank # ABO08098.1), was found in Pyrobaculum calidifontis . It consists of 485 amino acid residues and was amplified, cloned, and produced in Escherichia coli using T7 expression vector, pET-22b(+). The BLAST result revealed its low similarity index with characterized amylases of family GH57 i.e., 31–18% with non-specified amylase from thermostable bacterium of De Fuca hydrothermal Vent and maltogenic amylase from P. furiosus , respectively. Moreover, two putative catalytic residues, Glu (E 152 ) and Asp (D 234 ), were found in conserved regions of the enzyme at position 3 and 4, respectively. SDS-PAGE analysis of E. coli BL21-CodonPlus (DE3)-RIL cell lysates revealed production of recombinant Pcal_0672 as insoluble aggregates. Despite of its production as insoluble aggregates, the enzyme was able to hydrolyze soluble starch (0.5%), when analyzed by I 2 /KI based enzyme assay (at 80 ºC) followed by zymogram activity on gel. Zymographic activity assay analysis revealed that these insoluble aggregates were highly thermostable and retained their activity even after autoclaving in the presence of 1% Triton X-100 or 5% SDS. It depicts that putative Pcal_0672 is active on starch, highly thermostable archaeal enzyme.