CLONING, EXPRESSION AND CHARATERIZATION BmaN2 ?-AMYLASE from Bacillus megaterium NL3

• Main Author: Yuliani, Dewi
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/34373
• Institution: Institut Teknologi Bandung

?-Amylase (EC 3.2.1.1) catalyzes the hydrolysis of ?-1,4-glycosidic bonds in starch and has been widely applied in various industries, such as food, beverages, textiles and pharmaceuticals. Starch is conventionally converted into oligosaccharides using thermostable amylase which involves gelatinization process at high temperature. The use of degrading raw starch ?-amylase at below gelatinization temperature will reduce time and cost of production. Researchs on ?-amylases which are capable of raw starch degradation have been carried out, but exploration on ?-amylase from marine microorganisms is still limited. In the previous study, amylases producing Bacillus megaterium NL3 has been isolated from Kakaban lake, East Kalimantan. A gene enconding ?-amylase designated as bmaN2 had been obtained previously. The purposes of this study were to amplify open reading frame (ORF) encoding ?-amylase bmaN2, and to clone the bmaN2 ORF into pGEM-T and pET30a. BmaN2 was then expressed in Escherichia coli ArticExpress (DE3) and the biochemical properties of BmaN2 was characterized. Open reading frame (ORF) of the gene encoding bmaN2 was amplified by PCR using a set of primers. The ~1.5 kb DNA fragment of bmaN2 ORF had been successfully amplified and purified. The bmaN2 DNA fragment was ligated into the cloning vector pGEM-T, and transformed into the host cell E. coli TOP10F'. Transformants were grown and selected by ampicillin resistance and galactosidase activity. The obtained transformants were confirmed by size screening, colony PCR and restriction analysis. Two positive transformant carrying the recombinant plasmid, called as pGEM_bmaN2_B3 pGEM_bmaN2_A21, have been obtained. The resulted recombinant plasmid was then digested with BamHI and EagI and the bmaN2/BamHI-EagI fragment was ligated into the E. coli expression vector pET30a digested with similar enzymes. The pGEM_bmaN2 ligation results were transformed into host cells E. coli TOP10F'. Transformants were selected in selective medium and then analyzed by size screening, colony PCR and restriction analysis. Based on all the results, it was found that the recombinant plasmid pET_bmaN2 has been positively obtained. pET_bmaN2 was used as a template to determine the nucleotide sequence of bmaN2. The single open reading frame for BmaN2 was composed of 1635 nucleotides encoding 545 amino acids, including mature protein and a (His)6 at the N-terminus. Based on the deduced amino acids sequences, BmaN2 belongs to ?-amylase GH13 family 36 sub family and has three catalytic residues that play a role in the hydrolysis reaction. BmaN2 of B. megaterium NL3 has 98% homology with amylase from B. megaterium NCIB with 8 amino acid substitutions. BmaN2 expression in E. coli ArticExpress (DE3) was induced by the addition of 0.5 mM IPTG. SDS PAGE analysis showed that it has molecular wieght of ~61.5 kDa. Characterization of recombinant BmaN2 was done by determining activit y toward various substrates using DNS method. The specific activity of the crude extract of BmaN2 recombinant toward soluble starch, amylopectin, pullulan and ?-cyclodextrin was 0.79 U/mg, 0.90 U/mg, 0.12 U/mg and 0.04 U/mg, respectively. One unit of ?-amylases activity was defined as the amount of enzyme required to produce 1 ?mol of reducing sugar per minute. The degree of hydrolysis of the recombinant BmaN2 toward sago, rice, canna, cassava, and maize at 37 °C was 2.074%, 0.854%, 0.499%, 0.316% and 4.405%, respectively.