Abstract

This study aimed to better characterize a recently purified stable extracellular alkaline peptidase produced by Penicillium aurantiogriseum (URM 4622) through fluorescence spectroscopy, far-UV circular dichroism, kinetic and thermodynamic models to understand its’ structure-activity and denaturation. Fluorescence data showed that changing pH leads to tryptophan residues exposure to more hydrophilic environments at optimum activity pH 9.0 and 10.0. When thermally treated, it displayed less unfolding at these pH values, along with 4-fold less photoproducts formation than at neutral pH. Different pH CD spectra showed more β-sheet (21.5–43.0%) than α-helix (1–6.2%). At pH9.0, more than 2-fold higher α-helix content than any other pH. The melting temperature (T_m) was observed between 50 and 60 °C at all pH studied, with lower T_m at pH 9.0–11.0 (54.9–50.3 °C). The protease displayed two phase transition, with two energies of denaturation, and a 4-fold higher thermal stability (ΔH°_m) than reports for other microorganism’s proteases. An irreversible folding transition occurs between 50 and 60 °C. It displayed energies of denaturation suggesting higher thermal stability than reported for other microorganism’s proteases. These results help elucidating the applicability of this new stable protease.

摘要

本研究旨在通过荧光光谱、远紫外圆二色性、动力学和热力学模型更好地表征最近纯化的由金黄色青霉（URM 4622）产生的稳定细胞外碱性肽酶，以了解其结构活性和变性。荧光数据表明，在最佳活性 pH 9.0 和 10.0 时，改变 pH 会导致色氨酸残基暴露于更亲水的环境中。当热处理时，它在这些 pH 值下表现出较少的展开，与中性 pH 值相比，光产物的形成少 4 倍。不同的 pH CD 光谱显示更多的 β-折叠 (21.5-43.0%) 比 α-螺旋 (1-6.2%)。在 pH9.0 时，α-螺旋含量比任何其他 pH 值高 2 倍以上。熔化温度_（Tm) 在 50 到 60 °C 之间观察到所有研究的 pH 值，在 pH 9.0–11.0 (54.9–50.3 °C) 时T _{m较低。该蛋白酶显示出两个相变，具有两种变性能量，并且热稳定性 (ΔH° m} ) 比其他微生物蛋白酶报道的高 4 倍。在 50 和 60 °C 之间发生不可逆的折叠转变。它显示出的变性能量表明比其他微生物蛋白酶报道的更高的热稳定性。这些结果有助于阐明这种新的稳定蛋白酶的适用性。