Purification and Characterization of Recombinant Alpha Amylase BmaN1dC from Bacillus megaterium NL3 Indri Novia Madhani (a*), Fina Khaerunnisa Frima (a,b), Dessy Natalia (a)
a) Chemistry Department, Institut Teknologi Bandung, Bandung, Indonesia
b) Chemistry Department, Institut Teknologi Sumatera, Lampung Selatan, Indonesia
Abstract
Alpha Amylase (E.C 3.2.1.1) catalyzes the hydrolysis of internal alpha 1,4-glycosidic bond of polysaccharides. Alpha Amylases are used in various industrial purposes such as textiles, detergent, paper, food, and pharmaceutical. A bacterium associated with a sea anemone Bacillus megaterium NL3 from the land-locked marine Kakaban Lake, Derawan Island, East Kalimantan, produces a unique alpha amylase BmaN1. BmaN1 is the first alpha amylase known to have different catalytic residues among members of the GH13 family. BmaN1 was produced in Escherichia coli BL21(DE3) as an inactive inclusion body due to the presence of transmembrane helical region at C-terminal of BmaN1. The aims of this study were to express, purify, characterize of the truncated BmaN1 which has no transmembrane helical region at C-terminal. BmaN1dC was produced in E. coli ArcticExpress (DE3) as a soluble protein with molecular weight of 49 kDa based on SDS-PAGE analysis. BmaN1dC has been purified to homogeneity with 10-fold purification with specific activity of 605 U/mg. The purified of BmaN1dC has exhibits high catalytic efficiency on soluble starch with kcat/KM value of 4.1 mL mg-1s-1. BmaN1dC retained about 35% and 44% on the presence of 10 mM EDTA and SDS, respectively. Despite no additional starch-binding domain, BmaN1dC is able to hydrolyze various raw starches, such as corn and cassava, in which any type of starch granular able to digested by BmaN1dC. In conclusion, these results indicated that BmaN1dC is potential to be used in starch processing industry within the subfamily GH13_45.
