CLONING AND EXPRESSION ?-Amylase GENE of Bacillus megaterium NL3 (bmaN3) in Hansenula polymorpha

• Main Author: Rahmi Zulkifli, Fifi
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/38236
• Institution: Institut Teknologi Bandung

?-Amylase acts as a catalyst in the hydrolysis of starch and it has been widely used in various industries, such as foods, textiles, health, and energy. The ?-amylase gene of Bacillus megaterium NL3 (bmaN3) obtained from Kakaban Lake, East Kalimantan, has been isolated and characterized. Nucleotide sequence analysis showed that bmaN3 belongs to the Glycoside Hydrolase (GH) family 13, and has 98% identity with putative ?-amylases of B. megaterium DSM319 and ?-amylase of B. megaterium QM B1551. Moreover, it encodes a polypeptide of 484 amino acid residues and has no signal sequence which is indicative of its intracellular localization. The purposes of this research were to clone and to express bmaN3 in yeast Hansenula polymorpha. The bmaN3 gene fragment was first amplified by PCR using a set of primers designed based on the published nucleotide sequence bmaN3 (Accession number 530746390) and a pGEMT-bmaN3F as template. The primers contained the restriction enzyme sites of SalI and HindIII which were later used for the subcloning of the bmaN3 gene fragment into an expression vector. The resulted PCR fragment with size of 1.5 kb was then ligated into the pGEM-T vector to produce a recombinant plasmid pGEM-bmaN3. Subsequently, the bmaN3 DNA fragment which was generated from the SalI and HindIII double digestion of pGEM-bmaN3 was subcloned into a H. polymorpha expression vector pHIPX-4 which had also been cut with similar restriction enzymes. The resulted recombinant plasmid pHIPX4-bmaN3 was integrated into the chromosome of H. polymorpha NCYC495. The integration of plasmid in the chromosome of H. polmorpha NCYC495 transformant was verified by using PCR colony method. The presence of a 1.5 kb PCR fragment indicated that the bmaN3 has been integrated. The bmaN3 expression was regulated by MOX promoter and AOX1 terminator and BmaN3 was localized in the yeast peroxisome. The production of BmaN3 was carried out in mineral medium containing 0.5% methanol as an inducer at 37 °C for 120 h. The crude protein extract of H. polymorpha NCYC495 transformants had a 64.7 U/mg of ?-amylase activity as determined by dinitrosalcylic acid method. In the future, further characterization will be undertaken in order to understand the cellular function as well as the potential application of BmaN3.