Characteristics of thermostable amylopullulanase of Geobacillus thermoleovorans and its truncated variants.

The far-UV CD spectroscopic analysis of the secondary structure in the temperature range between 30 and 90°C revealed a compact and thermally stable structure of C-terminal truncated amylopullulanase of Geobacillus thermoleovorans NP33 (gt-apuΔC) with a higher melting temperature [58°C] than G. thermoleovorans NP33 amylopullulanase (gt-apu) [50°C] and the N-terminal truncated amylopullulanase from G. thermoleovorans NP33 (gt-apuΔN) [55°C]. A significant decline in random coils in gt-apuΔC and gt-apuΔN suggested an improvement in conformational stability, and thus, an enhancement in their thermal stability. The improvement in the thermostability of gt-apuΔC was corroborated by the thermodynamic parameters for enzyme inactivation. The Trp fluorescence emission (335 nm) and the acrylamide quenching constant (22.69 M(-1)) of gt-apuΔC indicated that the C-terminal truncation increases the conformational stability of the protein with the deeply buried tryptophan residues. The 8-Anilino Naphthalene Sulfonic acid (ANS) fluorescence experiments indicated the unfolding of gt-apu to expose its hydrophobic surface to a greater extent than the gt-apuΔC and gt-apuΔN.