PURIFICATION AND CHARACTERIZATION OF BMAN1?C RECOMBINANT ?-AMYLASE FROM BACILLUS MEGATERIUM NL3

• Main Author: Novia Madhani, Indri
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/75141
• Institution: Institut Teknologi Bandung

?-Amylase (1,4-?-D-glucanohydrolase, E.C 3.2.1.1) hydrolyzes ?-1,4-glycosidic bonds in polysaccharides, resulting in the formation of oligosaccharides. BmaN1 is an ?-amylase from B ac illus me gate rium NL3, which is isolated from a sea anemone living in Lake Kakaban, East Kalimantan, Indonesia. Based on the similarity of its amino acids sequence, BmaN1 is classified into the glycoside hydrolase family 13 (GH13) and subfamily GH13_45. BmaN1 has distinct catalytic residue differences compared to the conserved catalytic residues of other GH13_45 members. Specifically Asp203 has shifted to the i+1 position from the conserved position, and His294 occupies the conserved aspartate position, while Glu231 is in the conserved position. The C-terminal amino acid residues of BmaN1 contain a hydrophobic region in the form of a transmembrane helix, which is believed to affect the solubility of BmaN1. This study aims to produce and purify BmaN1?C, determine kinetic parameters, physicochemical characteristics, and hydrolytic activity against soluble and raw starch, and perform spectroscopic analysis of BmaN1?C. BmaN1?C protein was expressed in E. coli ArcticExpress(DE3) as a soluble protein approximately 49 kDa. BmaN1?C has a KM values of 52,05 mg/mL and a Vmax value of 296,4 µg/min, a turn-over number (kcat) of 13.792 min-1, and a catalytic efficiency (kcat / KM) of 4,4 mg.mL-1.s-1. BmaN1?C loses 34,9% and 44,2% of its activity upon the addition of 10 mM EDTA and SDS, respectively. The relative activity of BmaN1?C on amylopectin is 70,7%, while BmaN1?C does not show any hydrolysis activity on pullulan and ?-cyclodextrin. BmaN1?C can hydrolyze raw cassava and corn starch with degree of hydrolysis of 0,243 and 0,626, respectively. Trp fluorescence spectroscopy analysis indicates a decrease in fluorescence intensity for BmaN1?C, suggesting its ability to interact with soluble starch. Analysis of the secondary structure composition of BmaN1?C using circular dichroism (CD) spectroscopy revealed an ?-helix content of 25,7%, ?-sheet content of 24,5%, and loop content of 49,8%. This indicates that the composition of BmaN1?C is close to the modelled composition, which consists of an ?-helix content of 31,7%, ?-sheet content of 18,9%, and loop content of 49,4%.