Baroroh Umi, Yusuf Muhammad, Rachman Saadah Diana, Ishmayana Safri, Hasan Khomaini, Subroto Toto
Biotechnology Master Program, Postgraduate School, Universitas Padjadjaran, Bandung 40132, West Java, Indonesia.
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Sumedang 45363, West Java, Indonesia.
Adv Appl Bioinform Chem. 2019 Jun 7;12:1-13. doi: 10.2147/AABC.S198110. eCollection 2019.
Carbohydrate binding module (CBM) and surface binding site (SBS) are two important parts of amylase which respond to the raw starch digestion. They are related to the enzyme ability to adsorb and to catalyze the starch hydrolysis. However, starch processing is still expensive due to the high temperature in the gelatinization step. Therefore, direct starch digestion is more favorable. One of the solutions is to use α-amylase with high starch adsorptivity, which is expected to be capable of digesting starch below the gelatinization temperature. In Indonesia, R64 α-amylase (Sfamy R64) is one of the enzymes with the highest activity on starch. However, its raw starch adsorptivity was low. The aim of this study was to propose an in-silico model of Sfamy R64 mutant by introducing a new SBS using molecular dynamics (MD) simulation. The structural behavior of Sfamy R64 and positive control were studied using MD simulation. Furthermore, the mutants of Sfamy R64 were designed to have a stable SBS which mimics the positive control. The substrate affinity in all systems was evaluated using the molecular mechanics generalized Born surface area (MM/GBSA) method. The stability of a new SBS constructed by seven substitutions and a loop insertion was improved throughout MD simulation. The substrate was consistently bound to the SBS over 55 ns of simulation, as compared to 14 ns in wild-type. The structural behavior of SBS in mutant and positive control was similar. The interaction energies of the positive control, wild-type, and mutant were -17.6, -5.2, and -8.2 kcal/mol, respectively. The enhanced substrate binding in the mutant, due to the existence of a new SBS, suggests the potential of improving starch adsorptivity of Sfamy R64. This result should be useful in developing an enzyme with better substrate adsorption based on the rational computer-aided molecular design approach.
碳水化合物结合模块（CBM）和表面结合位点（SBS）是淀粉酶中响应生淀粉消化的两个重要部分。它们与酶吸附和催化淀粉水解的能力有关。然而，由于糊化步骤中的高温，淀粉加工仍然成本高昂。因此，直接淀粉消化更为有利。解决方案之一是使用具有高淀粉吸附性的α-淀粉酶，预计其能够在糊化温度以下消化淀粉。在印度尼西亚，R64α-淀粉酶（Sfamy R64）是对淀粉活性最高的酶之一。然而，其生淀粉吸附性较低。本研究的目的是通过分子动力学（MD）模拟引入新的SBS，提出Sfamy R64突变体的计算机模拟模型。使用MD模拟研究了Sfamy R64和阳性对照的结构行为。此外，设计Sfamy R64的突变体以具有模仿阳性对照的稳定SBS。使用分子力学广义玻恩表面积（MM/GBSA）方法评估所有系统中的底物亲和力。通过七次取代和环插入构建的新SBS的稳定性在整个MD模拟过程中得到了改善。与野生型的14 ns相比，在55 ns的模拟过程中底物始终与SBS结合。突变体和阳性对照中SBS的结构行为相似。阳性对照、野生型和突变体的相互作用能分别为-17.6、-5.2和-8.2 kcal/mol。由于新SBS的存在，突变体中底物结合增强，表明提高Sfamy R64淀粉吸附性的潜力。该结果对于基于合理的计算机辅助分子设计方法开发具有更好底物吸附性的酶应该是有用的。
