Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India.
Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India.
Int J Biol Macromol. 2015 May;76:279-91. doi: 10.1016/j.ijbiomac.2015.02.046. Epub 2015 Mar 5.
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.
远紫外圆二色光谱分析表明，在 30 至 90°C 的温度范围内，极端嗜热芽孢杆菌 NP33 （gt-apuΔC）的 C 端截断直链淀粉普鲁兰酶具有紧凑且热稳定的结构，其熔点[58°C]高于极端嗜热芽孢杆菌 NP33 直链淀粉普鲁兰酶（gt-apu）[50°C]和来自极端嗜热芽孢杆菌 NP33 的 N 端截断直链淀粉普鲁兰酶（gt-apuΔN）[55°C]。gt-apuΔC 和 gt-apuΔN 中无规卷曲的显著减少表明构象稳定性得到改善，从而提高了它们的热稳定性。gt-apuΔC 的热稳定性的提高得到了酶失活动力学参数的证实。gt-apuΔC 的色氨酸荧光发射（335nm）和丙烯酰胺猝灭常数（22.69M（-1））表明，C 端截断增加了蛋白质的构象稳定性，使深埋的色氨酸残基更加稳定。8-苯胺基萘磺酸（ANS）荧光实验表明，gt-apu 的展开使其疏水面暴露的程度大于 gt-apuΔC 和 gt-apuΔN。
