PURIFICATION AND CHARACTERIZATION OF Bacillus megaterium NL3 ?-AMYLASE BmaN2 EXPRESSED IN Escherichia coli

• Main Author: Widya, Nurul
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/32381
• Institution: Institut Teknologi Bandung

?-Amylase (EC 3.2.1.1) is an endoenzyme which hydrolizes ?-1,4 glycosidic bonds of oligosaccharides and polysaccharides. ?-Amylases are produced by various organisms, such as bacteria, yeast, fungi, and plant. Starch is a polysaccharide which consists of amylose and amylopectin. Amylose is a linear ?-1,4 glycosidic linked polymer consisting of more than 250 unit of glucoses, while amylopectin is a polymer build by ?-1,4 glycosidic linked glucoses as the main chain and ?-1,6 glycosidic linked braches. Starches from various sources, such as maize, cassava, canna, sago, and rice, have different granule sizes, crystal structures, pores content, percentage of amylose and amylopectin, and other minor characters, hence they might have different degree of hydrolysis. Marine microorganism Bacillus megaterium NL3 produces at least three type of ?-amylases, which are designated as BmaN1, BmaN2 and BmaN3. The aims of this research were to purify and characterize BmaN2 expressed in Escherichia coli. BmaN2 belongs to glycosyl hydrolase GH13 family, sub family 36, has three catalytic residues (Glu297, Asp257, and Asp364), and has no raw starch binding domain. Gene encoding ?-amylase BmaN2 has been cloned in pET30-BmaN2 and has been expressed as in Escherichia coli ArticExpress (DE3) with isopropyl-?-D-thiogalactopyranoside (IPTG) as an inducer. BmaN2 was produced as an intracellular soluble protein with molecular weight of ~61.5 kDa as analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The specific activity of crude protein extract was 0.67 U/mg as determined by 3.5-dinitrosalicyclic acid (DNS) method. One unit was defined as an amount of enzyme required for producing 1 ?mol of reducing sugar per minute under experimental condition. BmaN2 has been purified by Ni-NTA affinity chromatography resulting in an increase of purity of 32 fold with specific activity of 21.7 U/mg and yield of 11%. BmaN2 has an optimum pH and temperature of 8.0 and 40 ?, respectively, and it remains stable in the presence of NaCl up to 500 mM. Specific activities of BmaN2 towards soluble starch, amylopectin, and pullulan, respectively were 12.3 U/mg, 7.32 U/mg, and 0.247 U/mg. Interestingly, despite the absence of starch binding domain, BmaN2 was also capable of hydrolyzing raw starches granules sago, rice, canna, cassava, and maize with total reducing sugars per mg starches of 3.07 mmol, 11.4 mmol, 11.4 mmol, 4.72 mmol, and 8.04 mmol. Taken together, BmaN2 is potential to be used in converting various raw starches into oligosaccharides with higher economic value.