Accelerated molecular dynamics study to compare the thermostability of Bacillus licheniformis and Aspergillus niger α-amylase.

The thermostability of enzymes plays a significant role in the starch hydrolysis process in the industry. The structural difference between thermostable Bacillus licheniformis α-amylase (BLA) and thermolabile Aspergillus niger α-amylase (ANA) is interesting to be explored. This work aimed to study the thermostability-determining factor of BLA as compared to a non-thermostable enzyme, ANA, using molecular dynamics (MD) simulation at a high temperature. A 100 ns of classical MD, which was followed by 200 ns accelerated MD was conducted to explore the conformational changes of the enzyme. It is revealed that the intramolecular interactions through salt bridge interactions and the presence of calcium ions dominates the stability effect of BLA, despite the absence of a disulfide bond in the structure. These results should be useful in designing a thermostable enzyme that can be used in industrial processes.Communicated by Ramaswamy H. Sarma.

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.

Computational Model of the Effect of a Surface-Binding Site on the Saccharomycopsis fibuligera R64 α-Amylase to the Substrate Adsorption.

α-Amylase is one of the important enzymes in the starch-processing industry. However, starch processing requires high temperature, thus resulting in high cost. The high adsorptivity of α-amylase to the substrate allows this enzyme to digest the starch at a lower temperature. α-Amylase from Saccharomycopsis fibuligera R64 (Sfamy R64), a locally sourced enzyme from Indonesia, has a high amylolytic activity but low starch adsorptivity. The objective of this study was to design a computational model of Sfamy R64 with increased starch adsorptivity using bioinformatics method. The model structure of Sfamy R64 was compared with the positive control, ie, Aspergillus niger α-amylase. The structural comparison showed that Sfamy R64 lacks the surface-binding site (SBS). An SBS was introduced to the structure of Sfamy R64 by S383Y/S386W mutations. The dynamics and binding affinity of the SBS of mutant to the substrate were also improved and comparable with that of the positive control.

The Importance of Surface-Binding Site towards Starch-Adsorptivity Level in α-Amylase: A Review on Structural Point of View.

Starch is a polymeric carbohydrate composed of glucose. As a source of energy, starch can be degraded by various amylolytic enzymes, including α-amylase. In a large-scale industry, starch processing cost is still expensive due to the requirement of high temperature during the gelatinization step. Therefore, α-amylase with raw starch digesting ability could decrease the energy cost by avoiding the high gelatinization temperature. It is known that the carbohydrate-binding module (CBM) and the surface-binding site (SBS) of α-amylase could facilitate the substrate binding to the enzyme's active site to enhance the starch digestion. These sites are a noncatalytic module, which could interact with a lengthy substrate such as insoluble starch. The major interaction between these sites and the substrate is the CH/pi-stacking interaction with the glucose ring. Several mutation studies on the Halothermothrix orenii, SusG Bacteroides thetaiotamicron, Barley, Aspergillus niger, and Saccharomycopsis fibuligera α-amylases have revealed that the stacking interaction through the aromatic residues at the SBS is essential to the starch adsorption. In this review, the SBS in various α-amylases is also presented. Therefore, based on the structural point of view, SBS is suggested as an essential site in α-amylase to increase its catalytic activity, especially towards the insoluble starch.