CONSTRUCTION, EXPRESSION, AND CHARACTERIZATION OF RECOMBINANT ?-AMYLASE N205D AND N205A BACILLUS MEGATERIUM NL3

• Main Author: Rofilah, Salsazahra
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/82759
• Institution: Institut Teknologi Bandung

?-Amylase (EC 3.2.1.1) catalyzes the hydrolysis of ?-1,4 glycosidic linkages in starch producing a mixture of oligosaccharides. BmaN2 is an ?-amylase derived from Bacillus megaterium NL3, an isolate from Kakaban Lake, Indonesia. The asparagine amino acid residue at position 205 (N205) in BmaN2 is located in the calcium ion-binding region, serving as a cofactor for BmaN2. The precise role of N205 in BmaN2 remains unclear. This research aims to study the role of N205 in BmaN2 activity. Site directed mutagenesis was used to mutate N205 to alanine (A) and to aspartic acid (D). Escherichia coli TOP10F’ as the cloning host was transformed with pET30a-bmaN2 N205A dan pET30a-bmaN2 N205D. Sequencing was used to confirm the success of BmaN2 N205A and BmaN2 N205D mutation. According to the sequencing results, BmaN2 N205A and BmaN2 N205D mutation was successfully obtained. The codon for asparagine (AAT) was changed into a codon for alanine (GCG) in pET30a-bmaN2 N205A. The pET30a-bmaN2 was also contained a codon for aspartic acid (GAT) in pET30a-bmaN2 N205D. Escherichia coli BL21(DE3) as the expression host was then transformed with pET30a-bmaN2 N205A and pET30a-bmaN2 N205D. Expression of bmaN2, bmaN2 N205A, and bmaN2 N205D was induced with 0.1 mM IPTG at room temperature for 3 hours and 30 minutes. Each of BmaN2 wild type, BmaN2 N205A, and BmaN2 N205D had a size of ± 62 kDa. The activity of BmaN2 wild type, BmaN2 N205A, and BmaN2 N205D was tested using 3,5- dinitrosalisylic acid (DNS) method with 1% soluble starch as substrates. The crude enzyme activities were 11.53 ± 0.06 U/mg, 5.54 ± 0.06 U/mg, and 25.05 ± 0.18 U/mg for BmaN2 wild type, BmaN2 N205A, and BmaN2 N205D, respectively. The BmaN2 wild type, BmaN2 N205A, and BmaN2 N205D proteins were subsequently purified using the Ni- NTA affinity chromatography. Pure BmaN2 wild type, BmaN2 N205A, and BmaN2 N205D proteins were obtained using 100 mM imidazole elution buffer as a single band on the SDS-PAGE. Based on computational analysis, N205 is predicted to be a residue that influences enzyme activity.