ORIGINAL ARTICLES   

Minerva Biotecnologica 2016 June;28(2):81-5

Copyright © 2016 EDIZIONI MINERVA MEDICA

lingua: Inglese

Cloning, expression and characterization of CYP109B1 gene from Bacillus subtilis

Younes GHASEMI ^{1, 2}, Niloofar AFRASIABI ³, Narjes EBRAHIMI ¹, Mohammad B. GHOSHOON ¹, Navid NEZAFAT ¹, Mozhdeh ZAMANI ¹, Mohammad J. RAEE ¹

¹ Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran; ² Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran; ³ International Branch Student Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran

BACKGROUND: CYP450 enzymes comprise a superfamily of heme B containing monooxygenases. They play an important role in drug metabolism and biosynthesis. CYP450 enzymes are of considerable interest because of their applications in bioremediation, biosensors, and biotransformation and synthesis of fine chemicals. CYP109B1, has a key role in the production of secondary metabolites such as pravastatin, nootkatol and (+)-nootkatone (commercial flavorings). Due to its application in various fields, in this study we cloned the yjiB gene from Bacillus subtilis in Escherichia coli BL21 (DE3) for the study of its structure and application in desired enzymatic reactions.
METHODS: yjiB gene from Bacillus subtilis, PTCC 1023, was amplified using specific primers and cloned into pET-28a vector. It was transformed and expressed in Escherichia coli BL21 (DE3). The expression of recombinant protein was analyzed by SDS-PEAGE after IPTG induction. The enzyme was purified by Ni-NTA spin columns. The 3D structure of the enzyme was modeled and its binding sites were predicted.
RESULTS: According to our results, yjiB gene coding for CYP109B1 was actively expressed in E. coli. The DNA sequence alignments resulting from the BLAST search of yjiB showed 99% sequence identity with the other strains of Bacillus subtilis. The amino acid sequence differed in one position comparing to all recorded data in NCBI.
CONCLUSIONS: By expressing the yjiB gene coding for CYP109B1 in E. coli, we can have a great supply of this recombinant protein for secondary metabolite production and further studies on structure, activity or protein engineering. The modeled 3D structure and predicted binding sites of this enzyme can help us in understanding and improving enzyme activity.