Document Type : Original Article

Authors

1 Department of Biology, Faculty of Science, Shahid Bahonar University of Kerman, Kerman, Iran

2 Research Dept of Biotechnology, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, IRAN

Abstract

Alcanivorax borkumensis is a marine bacterium that has ability to grow on limited substrates that mainly is alkanes. The ability to use wide range of hydrocarbons is advantage of this bacterium to other marine community bacteria. A. borkumensis have two genetic systems for alkane biodegradation. The First system is alkane hydroxylase (alk-B1and alk-B2) and the second system is  cytochrome P450. Until now there is not any report on cloning of cytochrome P450 gene of A. borkumensis. In this study cytochrome P450 gene from A. borkumensis was cloned. At first P450 gene was cloned in pBluescript plasmid by blunt cloning and insertion was confirmed by colony PCR.  Then P450 gene was cloned in PET-26 expression vector and finally, IPTG induced gene expression in E.coli BL-21 bacterium. It was shown that longer induction time led to more expression level. The recombinant enzyme has difference absorbance in 450 nm that confirmed the activity of this recombinant enzyme.

Keywords

Cappello, S., Denaro, R., Genovese, M., Giuliano, L., Yakimov, M.M. (2006). Predominant growth of Alcanivorax during experiments on oil spill bioremediation in mesocosms. Microbiol Res, 162: 185-190.
 Emtiazi, G., Hassanshahian, M., Golbang, N. (2005). Development of a microtiter plate method for determination of phenol utilization, biofilm formation and respiratory activity by environmental bacterial isolates. Inter Biodeter Biodegrad, 56: 231-235.
Hara, A., Sang-ho, B., Kazuaki, S., Norihiko, M., Smits, T., Vanbeilen, J., Harayama, S. (2004). Cloning and functional analysis of alkB genes in Alcanivorax borkumensis SK2. Environm Microbio, 6 (3): 191–197.
Hasanshahian, M., Emtiazi, G. (2008). Investigation of alkane biodegradation using the microtiter plate method and correlation between biofilm formation, biosurfactant production and crude oil biodegradation. Inter Biodeter Biodegrad, 62: 170-178.
Hassanshahian, M., Emtiazi, G., Cappello, S. (2012).  Isolation and characterization of crude-oil-degrading bacteria from the Persian Gulf and the Caspian Sea. Mar Pollut Bull, 64: 7–12.
Hassanshahian, M., Emtiazi, G., Kermanshahi, R., Cappello, S. (2010). Comparison of oil degrading microbial communities in sediments from the Persian Gulf and Caspian Sea. Soil and Sediment Contamin, 19 (3): 277-291.
Humphreys, M., Clint, C. (2004). Immunodetection and quantification of cytochromees P450 using epitope tagging: immunological, spectroscopic, and kinetic analysisof cinnamate 4-hydroxylase. J Immun Methods, 292: 97–107.
Kim, D., Max, J., Cryle, B., James, J., Voss, P.R., Ortiz, D., Montellano, R. (2007). Functional expression and characterization of cytochromee P450 52A21 from Candida albicans. Arch Biochem Biophysic, 464: 213–220.
Kubota, M., Nodate, M., Yasomoto, H., Taku, U., Osamu, K., Misawa, R. (2005). Isolation and functional analysis of cytochrome P450 genes from various environment. Bio Biotechno Biochem, 69: 2421-2430.
Nodate, M., Mitsutoshi, K., Norihiko, M. (2006). Functional expression system for  cytochromee P450 genes using the reductase domain of self-sufficient P450RhF from Rhodococcus sp. NCIMB 9784. Appl Microbiol Biotechnol, 71: 455–462.
Sambrook, J., Russell, D. (2001). Molecular Cloning III: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 117-187.
Schneiker, S., Martins, D., Yakimov, M.M., Golyshin, P., Timmis, K., Kasir, O. (2006). Genome sequence of the ubiquitous hydrocarbon degrading marine bacterium Alcanivorax borkumensis. Nature Biotechnol, 52: 1-8.
Vanbeilen, J., Martin, N., Smits, T., Christian, R., Stefanie, B., Witholt, B. (2006). Rubredoxins involved in alkane oxidation. J Bacteriol, 184: 1722–1732.
Yakimov, M.M., Golyshin, P.N., Timmis, K.N. )2007(.Obligate oil-degrading marine bacteria. Curr Opin Biotechnol,18 (3):  257-66.
Yakimov, M., Peter, N., Siegmund, L. (1998). Alcanivorax borkumensis gen. nov., sp.  nov., a new, hydrocarbon-degrading and surfactant-producing marine bacterium. Inter J System Bacteriol, 48: 339–348.