MUTANT CONSTRUCTION AND EXPRESSION OF GENE ENCODING ?- AMYLASE BMAN2 W198F AND W198A IN ESCHERICHIA COLI

• Main Author: Elvia Dwifitriayu, Riri
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/32994
• Institution: Institut Teknologi Bandung

?-Amylase (EC 3.2.1.1) catalyzes the hydrolysis of ?-1,4-glycosidic linkages in starch producing linear and branched oligosaccharides. ?-Amylase (BmaN2) of Bacillus megaterium NL3 isolated from Kakaban Lake, East Kalimantan, can degrade raw starch. An amino acid residue Trp198 is predicted as potential starch binding site (SBS) which helps BmaN2 in starch degradation. The aims of this study were to construct two bmaN2 mutants by site directed mutagenesis, and to study the role of Trp198 in starch hydrolysis. A codon TGG at position 592, 593, and 594 encoding Trp198 was changed into TTC and CGG encoding Phe198 (W198F) and Ala198 (W198A), respectively and the resulted mutations were confirmed by nucleotide sequencing. BmaN2 wild type and mutants (bmaN2 W198F and bmaN2 W198A) were expressed in Escherichia coli ArcticExpress (DE3) using 0.5 mM IPTG as an inducer for 24 hours at 15 oC. SDS-PAGE analysis showed that BmaN2 wild type, BmaN2 W198F, and BmaN2 W198A have molecular weight of ~61.5 kDa. BmaN2 activity was assayed by determining the amount of reducing sugar produced from the hydrolysis of soluble and raw starches. The specific activities of BmaN2 wild type, BmaN2 W198F, and BmaN2 W198A were 22.3 U/mg; 17.0 U/mg; and 13.3 U/mg, respectively. The degree of raw starch hydrolysis of BmaN2 towards rice, wheat, ganyong, corn, potato, cassava, and sago, respectively was 16.53%, 25.33%, 14.41%, 13.82%, 7.94%, 14.89%, and 16.7%. Both BmaN2 W198F and BmaN2 W198A have a decrease of raw starch degrading ability compared to BmaN2 wild type in which substitution into alanine resulted in much lower activity