CONSTRUCTION AND EXPRESSION OF ?-AMILASE BMAN1 MUTANT

• Main Author: Husnul Huluq, Ramdani
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/33675
• Institution: Institut Teknologi Bandung

?-Amylase catalyzes hydrolysis of ?-1,4-glycosidic linkage in oligasaccharides and polysaccharides. In general, ?-amylase has three conserved catalytic residues, namely two aspartate and one glutamate residues. However, in silico study shows that BmaN1 ?- amylase from Bacillus megaterium NL3, has only one glutamate out of the three conserved catalytic residues. Interestingly, in spite of having only one conserved catalytic residue, BmaN1 is capable of hydrolysing both soluble and raw starch. Hence, it is predicted that there could be other amino acid residue which is involved in the catalytic reaction. Amino acid sequence alignment analysis showed the presence of Aspartate 203 in the catalytic region, but its position is shifted from the conserved catalytic residu location. Therefore, the aims of this research were to construct BmaN1 mutants and to study the role of Asp203 in starch hydrolysis. Two BmaN1 mutants, namely Asp203Ala mutant (GAT?GCG) and Asp203Asn mutant (GAT?AAT), have been constructed employing PCR-based-site directed mutagenesis. The wild type BmaN1, Asp203Ala mutant (D203A), and Asp203Asn mutant (D203N) were then expressed in Escherichia coli ArcticExpress (DE3). The SDS PAGE analysis results showed that BmaN1, D203A and D203N were expressed as proteins with molecular weight of ~56 kDa. The BmaN1 wild type degrades soluble starch with activity of 45.8 U/mg. A decrease of activity was observed in the D203N mutant (30.4 U/mg), while the activity of D203A (1.8 U/mg) was nearly abolished. Taken together, this research demonstrates that Asp203 residue of BmaN1 plays an important role in starch hydrolysis.