BIOCHEMICAL PROPERTIES CHARACTERIZATION OF RECOMBINANT -AMYLASE BMAN1 FROM BACILLUS MEGATERIUM NL3

• Main Author: Akbar Thufail, Muhammad
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/75595
• Institution: Institut Teknologi Bandung

?-Amylase hydrolyzes ?-1,4 glycosidic bonds in starch to produce oligosaccharides. ?-Amylase BmaN1 was isolated from Bacillus megaterium NL3 associated with anemones in Kakaban Atoll, Kalimantan. BmaN1 is classified into the 13 glycoside hydrolase (GH) family, the GH13_45 subfamily based on its constituent amino acid compounds. BmaN1 has different conserved catalytic residues from other members of the GH13_45 subfamily. The difference of its conserved catalytic residues are a shift of nucleophilic aspartate residues to position i+1 (Asp203), replacement of transition state stabilizer aspartate by histidine (His294), while hydrogen bond donor glutamate residue remains in its conserved position (Glu231). BmaN1 has a double tryptophan residue on the ?3 helix which predicted to contribute in raw starch binding. The aims of this study are to determine the stability of protein structure in salt using in silico approach, characterize its biochemical properties, and determine the activity of raw starch hydrolysis by BmaN1. The characterization of structural stability of BmaN1 was carried out by molecular dynamics (MD) method using the AMBER software to study changes in protein structure under various concentration of NaCl. In terms of protein structural stability, the BmaN1 mutant without the C-terminus end (BmaN1?C) was used as a comparison. Characterization of the biochemical properties of BmaN1 determined by soluble starch hydrolysis using the DNS method at various conditions of temperature, pH, and salt concentration. Structural modeling of BmaN1 and BmaN1?C showed the presence of tryptophan residues which are predicted to contribute in binding starch, namely at positions 145, 154, 189, 190, and 278. Based on the RMSD value and radius of gyration, the structures of BmaN1 and BmaN1?C are estimated to be stable at a NaCl concentration of 3.0 M. However, BmaN1 and BmaN1?C have salt sensitive regions at residues of 179 – 201 which are known from the increase in RMSF values when NaCl is added. Biochemical characterization of BmaN1 showed that the kinetic parameters of KM and Vmax were 96.63 mg/mL and 2,699 mg/minute, the turnover number (kcat) was 150,539/minute, and the catalytic efficiency (kcat/KM) was 25.96 mL/mg.s at optimum conditions of 50°C and pH 6.5. BmaN1 is able to hydrolyze raw corn and cassava starch with Degree of Hydrolysis (DH) at 4.00% and 4.22%, respectively. Hydrolysis activity of soluble starch by BmaN1 decreased at 0.5 M; 1.0M; and 2.0 M NaCl concentration, respectively 52.83%, 70.95% and 83.19%. Furthermore, the addition of EDTA and SDS inhibitors in concentration of 10 mM also reduced BmaN1 starch hydrolysis activity by 59.98% and 40.23%, respectively. Conformational changes in the BmaN1 tryptophan residues was observed with decrease in tryptophan fluorescence emission intensity when NaCl was added.