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首页> 外文期刊>Biophysical Chemistry: An International Journal Devoted to the Physical Chemistry of Biological Phenomena >A spectroscopic analysis of thermal stability of the Chromobacterium viscosum lipase
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A spectroscopic analysis of thermal stability of the Chromobacterium viscosum lipase

机译：黏性色杆菌脂肪酶的热稳定性的光谱分析

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摘要

The thermal stability of the lipase from Chromobacterium viscosum was assessed by deactivation (loss of activity), fluorescence, circular dichroism (CD) and static light scattering (SLS) measurements. Lipase fluorescence emission is dominated by the tryptophyl contribution. An increase in the tyrosyl contribution from 2 to 16% was only observed upon prolonged incubation at 60 degreesC. The effect of temperature on the tryptophyl quantum yield was studied and two activation energies were calculated. Tryptophan residues in the native structure have an activation energy of 1.9 kcal mol(-1) for temperature-dependent non-radiative deactivation of the excited state. A structural change occurs at approximately 66.7 degreesC and the activation energy increases to 10.2 kcal mol(-1). This structural change is not characterized by tryptophan exposure on the surface of the protein. The deactivation and the evolution of structural changes with time after lipase incubation at 60 degreesC were assessed by fluorescence, CD and SLS measurements. CD spectra show that both secondary and tertiary structures remain native-like after incubation at 60 degreesC in spite of the fluorescence changes observed (red-shift from 330 to 336 nm on the trytophyl emission). SLS measurements together with the CD data show that deactivation may be due to protein association between native molecules. Deactivation and the decrease on the fraction of non-associated native lipase evaluated by changes in fluorescence intensity with time, show apparent first order kinetics. According to the rate constants, fluorescence changes precede deactivation pointing to an underestimation of the deactivation. Reactivation upon dilution during the activity assay and substrate-induced reactivation due to lipase interfacial adsorption are possible causes for this underestimation. (C) 2000 Elsevier Science S.A. All rights reserved. [References: 34]
机译：通过减活（活性损失），荧光，圆二色性（CD）和静态光散射（SLS）测量来评估粘膜色杆菌中脂肪酶的热稳定性。脂酶的荧光发射主要由色氨酸贡献。仅在60℃下长时间孵育时，才观察到酪氨酰贡献从2％增加到16％。研究了温度对色氨酸量子产率的影响，并计算了两种活化能。天然结构中的色氨酸残基具有1.9 kcal mol（-1）的活化能，用于激发态的温度依赖性非辐射失活。结构变化发生在大约66.7摄氏度，活化能增加到10.2 kcal mol（-1）。这种结构变化的特征不是蛋白质表面色氨酸暴露。通过荧光，CD和SLS测量评估脂肪酶在60℃温育后的失活和结构变化随时间的演变。 CD光谱表明，尽管观察到荧光变化（在叶to发射时从330nm到336nm红移），但是在60℃温育后，二级和三级结构都保持天然状。 SLS测量结果和CD数据表明，失活可能是由于天然分子之间的蛋白质缔合所致。失活和通过荧光强度随时间变化评估的未结合的天然脂肪酶的分数的降低，显示出明显的一级动力学。根据速率常数，荧光在失活之前发生变化，这表明该失活被低估了。活性测定过程中稀释后的重新活化和由于脂肪酶界面吸附引起的底物诱导的重新活化可能是这种低估的原因。 （C）2000 Elsevier Science S.A.保留所有权利。 [参考：34]

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