THE ROLE OF Y101 IN BINDING SUBSTRATE ON ?- AMILASE BMAN2

• Main Author: Restyani, Inda
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/55115
• Institution: Institut Teknologi Bandung

?-Amylase (E.C 3.2.1.1) is an enzyme that hydrolyze ?- (1,4) glycosidic bonds in starch to produce linear and branched oligosaccharides. ?Amylase have a broad spectrum of industrial application such as in the food, pharmaceutical, detergent, textile, and bioethanol manufacturing industries. In addition, ?-amylase can be used as a pollutant removal agent and produce paper with a firmer and smoother surface. ?-Amylase can be obtained from plants, animals, and microorganism. Bacillus megaterium NL3 produces ?-amylase BmaN2 which can degrade starch. Bioinformatics study shows that BmaN2 was predicted to have three surface binding site (SBS) residues, namely Y101; H141; W179 which plays a role in substrate binding. The purpose of this experiment was to determine the role of Y101 residue in substrate binding of BmaN2. Therefore, to determine the role of Y101, mutations were carried out in the TAT codon into TGG to produce Y101W and TAT into TCT to produce Y101S. Site directed mutagenesis were performed by PCR technique using a recombinant plasmid template pET30a-bmaN2 which carrying BmaN2 gene. BmaN2 WT, Y101W, and Y101S were expressed in Escherichia coli BL21 (DE3) with the addition of 0,1 mM IPTG at 25oC for 4 hours. SDS-PAGE analysis showed the presence of a protein with a molecular mass of 61,5 kDA. The specific activities for BmaN2 WT, Y101W, and Y101S were 33,53 U/mg, 1,21 U/mg. and 0,73 U/mg, respectively. These results indicate that Y101 from BmaN2 has an important role in subtrate binding.