STUDY OF THE EFFECTS OF NACL CONCENTRATION ON STABILITY OF ?-AMYLASE BMAN1?C USING A MOLECULAR DYNAMICS SIMULATION APPROACH

• Main Author: Akbar Thufail, Muhammad
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/64822
• Institution: Institut Teknologi Bandung

Starch is the main source of carbohydrates for humans and has various industrial uses. Hydrolysis of starch in the wastewater treatment, syrup, and detergent industries requires enzymes that have good resistance to extreme conditions. Such industries needed enzymes that are able to hydrolyze starch in high salt concentrations. ?-Amylases are hydrolase enzymes that act on alpha/?(1?4) glycosides in glycogen, starch, and ?-glucans. ?-Amylases are classified in the glycoside hydrolase family 13 (GH13) having aspartate catalytic residues as nucleophiles, glutamate as proton donors, and a second aspartate as transition state stabilizers. This enzyme is known to have unique adaptability to high temperature and salt concentration environment. Adaptation to these conditions is in the form of many amino acid compositions which are negatively charged and have a secondary structure of alpha helices and beta sheets. Atypical ?-amylase BmaN1 isolated from Bacillus megaterium NL3 strain, bacterial isolate from sea lake anemone Kakaban Island, Derawan Islands, East Kalimantan, had different sustained catalytic residues from GH13 with the first aspartate shifted to the Asp203 position, the second aspartate by His294, while glutamate remained in a sustainable position. This amylase is known to have a C-terminal region which reduces its solubility in air and its activity in hydrolyzing starch. The low solubility in air is due to BmaN1 forming inclusion bodies by the presence of a transmembrane helical structure at its C-terminus. A variant of BmaN1 without a C-terminal region, BmaN1?C, was constructed to increase the hydrolytic activity of starch and its solubility. This study aims to examine the effect of NaCl concentration on the stability of the structure of BmaN1 ?-amylase using a molecular dynamics simulation approach with a simulation time of 100 ns using the AMBER18 program. Molecular dynamics simulation analysis also aims to determine the cause of the stability. This study also aims to determine the concentration of NaCl on starch hydrolysis activity experimentally using the DNS method. Excluded C-terminal BmaN1 variant, BmaN1?C, was used as a comparison for the presence of the C-terminated region of the enzyme. The results of the molecular dynamics simulation show that the structure of BmaN1 and BmaN1?C has good stability at the optimum temperature of 50?C and in the variation of NaCl salt concentration 3.0 M; 2.0 M; 1.0 M; and 0.5 M. The stability was shown based on the analysis of the RMSD and the radius of gyration of the structure. Analysis of residues and secondary protein structures showed that there was a region that played a role in providing stability properties to BmaN1, namely residues 179 – 201. The presence of the C-terminus region would make the structural stability of BmaN1 lower than BmaN1?C. Determining the activity of ?-amylase BmaN1?C was experimentally carried out at various concentrations of NaCl 0.0 – 2.0 M with the DNS method which showed the greatest decrease in activity at NaCl 1.0 M by 38.2% with a specific activity value of 0.047 U/mg.