IDENTIFICATION OF A-AMYLASE FROM MARINE BACTERIA BACILLUS SP.

• Main Author: KONO, ALFREDO
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/9881
• Institution: Institut Teknologi Bandung

a-Amylase (EC 3.2.1.1) is an enzyme catalyzing the hydrolysis of a-1,4 glycosidic bonds of starch. This enzyme belongs to family 13 glycosyl hydrolase. The goal of this research is to identify Indonesian marine bacteria producing a-amylase as well as to characterize of its properties. The identification of microbe based on its gene encoding 16S rRNA with PCR technique using reverse primer (UniB1) 5'-GGTTAC(G/C)TTTGTTACGACTT-3' and forward primer (BactF1) 5'-AGAGTTTGATC(A/C)TGGCTCAG-3'. The activity of partial purification of a-amylase was detected by the Fuwa method. The mode of enzyme action on starch degradation was detected using the method of thin layer chromatography. The relative molecular mass was determined using SDS-PAGE, and its activity was determined by zimography. The Indonesian marine bacteria showed a close genetic relationship with Bacillus amyloliquefaciens strain GH58 with sequence homology of 99% on its gene encoding 16S rRNA. Purification of enzyme using anion exchange Resource-Q column resulted in two peaks with amylase activity. The enzyme from first peak had specific activity of 88.46 U/mg, yield of 27.87% and purification fold of 1.4. Meanwhile, the second peak had specific activity of 247.52 U/mg, yield of 22.6% and purification fold of 3.91. Both peak, 1 and 2 showed the similar action on starch degradation. They worked as an endoenzyme containing three protein bands with molecular mass 97 kDa, 55 kDa, and 45 kDa. Analyses of SDS-PAGE as well as native-PAGE indicated that enzymes were multisubunit enzyme.