Baptista R P, Cabral J M, Melo E P
Universidade do Algarve - U.C.T.A., Campus de Gambelas 8000 Faro, Portugal.
Biotechnol Bioeng. 2000 Dec 20;70(6):699-703. doi: 10.1002/1097-0290(20001220)70:6<699::aid-bit13>3.0.co;2-n.
The effect of trehalose (0.5 M) on the thermal stability of cutinase in the alkaline pH range was studied. The thermal unfolding induced by increasing temperature was analyzed in the absence and in the presence of trehalose according to a two-state model (which assumes that only the folded and unfolded states of cutinase were present). Trehalose delays the reversible unfolding. The midpoint temperature of the unfolding transition (Tm) increases by 4.0 degrees C and 2. 6 degrees C at pH 9.2 and 10.5, respectively, in the presence of trehalose. At pH 9.2 the thermal unfolding occurs at higher temperatures (Tm is 52.6 degrees C compared to 42.0 degrees C at pH 10.5) and a refolding yield of around 80% was obtained upon cooling. This pH value was chosen to study the irreversible inactivation (long-term stability) of cutinase. Temperatures in the transition range from folded to unfolded state were selected and the rate constants of irreversible inactivation determined. Inactivation followed first-order kinetics and trehalose reduced the observed rate constants of inactivation, pointing to a stabilizing effect on the irreversible inactivation step of thermal denaturation. However, if the contribution of reversible unfolding on the irreversible inactivation of cutinase was taken into account, i.e., considering the fraction of cutinase molecules in the reversible unfolded conformation, the intrinsic rate constants can be calculated. Based on the intrinsic rate constants it was concluded that trehalose does not delay the irreversible inactivation. This conclusion was further supported by comparing the activation energy of the irreversible inactivation in the absence and in the presence of trehalose. The apparent activation energy in the absence and in the presence of trehalose were 67 and 99 Kcal/mol, respectively. The activation energy calculated from intrinsic rate constants was higher in the absence (30 Kcal/mol) than in the presence of trehalose (16 Kcal/mol), showing that kinetics of the irreversible inactivation step increased in the presence of trehalose. In fact, trehalose stabilized only the reversible step of thermal denaturation of cutinase.
研究了海藻糖（0.5 M）在碱性pH范围内对角质酶热稳定性的影响。根据双态模型（该模型假设角质酶仅存在折叠态和未折叠态），在不存在和存在海藻糖的情况下，分析了温度升高引起的热解折叠过程。海藻糖延迟了可逆解折叠。在pH 9.2和10.5时，存在海藻糖的情况下，解折叠转变的中点温度（Tm）分别升高了4.0℃和2.6℃。在pH 9.2时，热解折叠发生在更高的温度下（Tm为52.6℃，而在pH 10.5时为42.0℃），冷却后可获得约80%的复性产率。选择该pH值来研究角质酶的不可逆失活（长期稳定性）。选择了从折叠态到未折叠态转变范围内的温度，并测定了不可逆失活的速率常数。失活遵循一级动力学，海藻糖降低了观察到的失活速率常数，表明其对热变性的不可逆失活步骤具有稳定作用。然而，如果考虑可逆解折叠对角质酶不可逆失活的贡献，即考虑处于可逆解折叠构象的角质酶分子的比例，则可以计算内在速率常数。基于内在速率常数得出结论，海藻糖不会延迟不可逆失活。通过比较不存在和存在海藻糖时不可逆失活的活化能，进一步支持了这一结论。不存在和存在海藻糖时的表观活化能分别为67和99千卡/摩尔。由内在速率常数计算得到的活化能在不存在海藻糖时（30千卡/摩尔）高于存在海藻糖时（16千卡/摩尔），表明存在海藻糖时不可逆失活步骤的动力学加快。实际上，海藻糖仅稳定了角质酶热变性的可逆步骤。
