Molecular dynamics study to improve the substrate adsorption of Saccharomycopsis fibuligera R64 alpha-amylase by designing a new surface binding site.

Background: 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, Saccharomycopsis fibuligera 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. Methods: 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. Results: 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. Conclusion: 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.