Department of Microbiology, Faculty of Biological Science, Alzahra University, Tehran, Iran.
Department of Biotechnology, Faculty of Biological Science, Alzahra University, Tehran, Iran.
Int J Biol Macromol. 2019 Feb 1;122:914-923. doi: 10.1016/j.ijbiomac.2018.10.172. Epub 2018 Oct 26.
Biocatalysis in presence of organic solvents has numerous industrially attractive advantages in comparison to traditional aqueous solvents. In some cases, the presence of organic molecules such as methanol in the processes such as enzymatic production of biodiesel is inevitable. However, enzyme inactivation and/or instability in organic solvents limits such biotechnological processes. Although it was found that some enzymes are more and others are less tolerant against organic solvents, the structural basis of such differences is relatively unknown. In this work, using molecular dynamics simulations, we have investigated the structural behavior of enzymes with completely different structural architecture including lipase, laccase and lysozyme in the presence of methanol as polar and hexane as non-polar organic solvents. In agreement with the previous experimental observations, simulations showed that lipase is more tolerant against both polar and non-polar organic solvents. It is found that lipase has high stability in pure hexane even higher than that obtained in the aqueous solvent. In contrast, laccase shows better stability in the aqueous conditions. To obtain general mechanism of enzyme inactivation in the presence of methanol and hexane, we have treated lysozyme as model enzyme in the different percentages of these solvents in long MD simulations. It is found that lysozyme is completely denatured at high concentration- of methanol, but it remains native at low concentration of this solvent. Interestingly, the concentration-dependence structural behavior of enzyme was completely different in the presence of hexane. It was obtained that low concentrations of hexane may impose more instability on the enzyme conformation than higher percentages. Results also showed that presence of water is determining factor in the enzyme stability at high concentrations of hexane. Pure hexane may also lead to the surface denaturation of the enzymes. Both methanol and hexane denaturation mechanisms were initiated by diffusion of organic solvent in hydrophobic core. However, enzyme denaturation in hexane was continued by a collapse of hydrophobic core and entering hexane molecules to the core, but in methanol it was completed by decomposition of the secondary structures. In both cases it was found that beta structures are more prone to destabilize than helix structures. This may be a reason for obtained results about lower stability of laccase with β-barrel architecture than lipase with multiple helixes at it surface. In total, by our extensive structural data, it was found that the forces which stabilize tertiary structure have pivotal role in enzyme tolerance against both polar and non-polar organic solvents.
相比传统的水溶液溶剂，有机溶剂中生物催化具有许多工业上有吸引力的优势。在某些情况下，有机分子如甲醇的存在是不可避免的，例如在酶法生产生物柴油的过程中。然而，酶在有机溶剂中的失活和/或不稳定性限制了这些生物技术过程。尽管已经发现一些酶对有机溶剂的耐受性更高，而另一些酶的耐受性更低，但这种差异的结构基础相对未知。在这项工作中，我们使用分子动力学模拟研究了具有完全不同结构架构的酶，包括脂肪酶、漆酶和溶菌酶，在甲醇作为极性溶剂和己烷作为非极性有机溶剂的存在下的结构行为。与先前的实验观察结果一致，模拟表明脂肪酶对极性和非极性有机溶剂的耐受性更高。结果表明，脂肪酶在纯己烷中的稳定性甚至比在水溶液中的稳定性更高。相比之下，漆酶在水相条件下显示出更好的稳定性。为了获得甲醇和己烷存在下酶失活的一般机制，我们将溶菌酶作为模型酶，在这些溶剂的不同百分比下进行长 MD 模拟。结果表明，在高浓度甲醇中，溶菌酶完全变性，但在低浓度甲醇中保持天然状态。有趣的是，在存在己烷的情况下，酶的浓度依赖性结构行为完全不同。结果表明，低浓度的己烷可能比高浓度对酶构象施加更大的不稳定性。结果还表明，水的存在是在高浓度己烷中酶稳定性的决定因素。纯己烷也可能导致酶表面变性。甲醇和己烷的变性机制都是由有机溶剂在疏水区扩散引发的。然而，在己烷中，酶的变性是通过疏水区的崩塌和己烷分子进入核心来继续的，但在甲醇中，它是通过二级结构的分解来完成的。在这两种情况下，都发现β结构比螺旋结构更容易失稳。这可能是获得的结果的原因之一，即具有β桶结构的漆酶的稳定性低于表面具有多个螺旋的脂肪酶。总的来说，通过我们广泛的结构数据，发现稳定三级结构的力在酶对极性和非极性有机溶剂的耐受性方面起着关键作用。
