Perpustakaan Digital ITB

a-Amylase (EC 3.2.1.1) catalyzes the hydrolysis of o-1,4-glycosidic linkages in starch producing linear and branched oligosaccharides. c-Amylase BmaN2, *am Bacillus megaterium NL3 isolated from Kakaban Lake, East Kalimantan, can degrade raw starch. Trp198 is predicted as potential starch binding site (SBS) which helps BmaNZ in degrading raw starch. The aims of this study were to construct bmaN2 mutant, 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. The mutation were confirmed by nucleotide sequencing. BmaN2wild type and mutants (bmaN2 W198F and bmaN2 W198A) were expressed in E. coli ArcticExpress (DE3) using IPTG 0.5 M as an inducer for 24 hours at 15 'C. SDS-PAGE analysis showed that BmaN2 wild type, BmaN2 Wl98F, and BmaN2 W198A have molecular weight of -61.5 kDa. BmN2 activity was determined by DNS method which is based on the amount of reducing sugar at 40'C and pH 8 for 10 minutes. The specific activity of BmaN2 wild type, BmaN2 W198F, and BmaN2 W198A were 72.3 Uimg; 17.0 Ulmg; and 13.3 Ulmg, respectively. The degree of raw starch hydrolysis using rice, wheat, ganyong, corn, potato, cassava and sago in BmaN2 wild type, respectively were 16.53%,25.33ya, l4.4lyo, 13.82o/o, 7 .94oh, 14,.89yo, and l6.7Yr. The degree of hydrolysis of BmaN2 W198F in each raw starch substrates were 14.620/o,25.24yo, 13.48%, 13.33yo,7 .37yo, 12.930 , dan I4.9A%. The degree of hydrolysis of BmaN2 Wl98A in each raw starch substrates were 12.834/o, 21.94/o, ll.99yo, 12.860/o,7 .A3yo, 11 .Ilyo, dan 13.73%