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.
Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India.
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M
Nisha
Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India. Electronic address: tsnarayana@gmail.com.
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Satyanarayana
D004789:Enzyme Activation; D004795:Enzyme Stability; D014644:Genetic Variation; D056506:Geobacillus / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C082507:amylopullulanase
ANS fluorescence; Amylopullulanase; Circular dichroism; Conformational stability; Thermostability