INVESTIGATING THE ROLE OF TRYPTOPHAN 179 OF ?- AMYLASE BMAN2 IN STARCH BINDING

• Main Author: Aqib Hanif, Muhammad
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/62716
• Institution: Institut Teknologi Bandung

Plants typically use starch as a carbon and energy source. Seeds, roots, tubers, and legumes are the most common sources of starch. Enzymatic hydrolysis of starch can be accomplished with the aid of ? -amylase. Bacillus megatrium NL3 from Lake Kakaban in Indonesia produces ? -amylase BmaN2 which belongs to the GH13 family. BmaN2 can hydrolyze starch into linear and branched oligosaccharides. The In silico study of BmaN2 revealed that Tyr101, His141, and Trp179 are predicted to play role as SBS(Surface Binding Substrate). The purpose of this research was to determine the role of W179 residue in substrate binding. Therefore, to determine the role of W179, two variants of BmaN2 designated as BmaN2 W179F and BmaN2 W179A were generated by mutation of TGG into TTC to produce BmaN2 W179F and TGG into GCT to produce BmaN2 W179A. Site directed mutagenesis were performed by PCR using a recombinant plasmid template pET30a- bmaN2 carrying BmaN2 gene. BmaN2 WT, BmaN2 W179F, and BmaN2 W179A expression in Escherichia coli BL21(DE3) were induced by addition of 0.1 mM IPTG at 25 ºC 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, BmaN2 W179F, and BmaN2 W179A were 1.612 U/mg, 1.431 U/mg, and 0.6842 U/mg, respectively. These results indicate that W179 of BmaN2 has an important role in subtrate binding.