CONSTRUCTION AND CHARACTERIZATION TRP201PHE MUTANT OF Α-AMILASE BAQA

• Main Author: ISLAMI SYOVHIA (NIM :10510043) ; Pembimbing Dr. Dessy Natalia, ZYAHRA
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/19777
• Institution: Institut Teknologi Bandung

α-Amylase (E.C.3.2.1.1) hydrolyzes α-D-(1,4)-glycosidic bond in starch producing dextrins and linear oligosaccharides. This enzyme is widely used in industries, such as textiles, detergents, sweeteners, and papers industries. α-Amylase from Bacillus aquimaris MKSC 6.2 (BaqA) has two consecutive tryptophan residues at positions 201 (Trp201) and 202 (Trp202) which are predicted to have a role in sugar binding. The aims of this research were to construct a BaqA Trp201Phe mutant and to study the function of Trp201 residue in starch hydrolysis. BaqA mutant was generated by site directed mutagenesis using pET30a-baqA containing baqA wildtype as a template and a set of primer with nucleotide changes of TGG→TTT. The presence of mutation in the resulted pET30a-baqA mutant has been confirmed by nucleotide sequence analysis. The BaqA mutant, designated as BaqA01, was expressed in Escherichia coli Arctic and SDS PAGE analysis showed that it has a molecular weight of ~70 kDa. BaqA01 activity towards various types of soluble starches (starch, pulullan, β-cyclodextrin) is lower compared to that of BaqA wildtype. Interestingly, BaqA01 activity on several raw starches (rice starch, sago, and canna) increased up to 20 fold. However, unlike its wildtype counterpart, BaqA01 could not hydrolyze cassava. These results suggested that BaqA interaction with soluble and raw starch might occur through different mechanism.