Browse > Article
http://dx.doi.org/10.7845/kjm.2013.008

Construction of Overexpression Vectors and Purification of the Oxygenase Component of Alkylphenol Hydroxylase of Pseudomonas alkylphenolia  

Lee, Kyoung (Department of Microbiology and Biomedical Science Institute at CWNU, Changwon National University)
Publication Information
Korean Journal of Microbiology / v.49, no.1, 2013 , pp. 95-98 More about this Journal
Abstract
Following construction of expression vectors in Escherichia coli, a new procedure involving two-step column purifications with a Fast Performance Liquid Chromatography System was developed for purification of the oxygenase component of alkylphenol hydroxylase of Pseudomonas alkylphenolia. From 50 g wet cake of recombinant E. coli BL21(DE3)(pJJPMO2) cells, 110 mg of pure protein in a heterodimeric form containing a stoichiometric amount of iron were obtained and it exhibited a specific activity of 147 nmole/min/mg.
Keywords
seudomonas alkylphenolia; alkylphenol hydroxylase; bacterial multicomponent monooxygenase(BMM); diiron cluster;
Citations & Related Records
Times Cited By KSCI : 1  (Citation Analysis)
연도 인용수 순위
1 Jeong, J.J., Kim, J.H., Kim, C.K., Hwang, I., and Lee, K. 2003. 3- and 4-alkylphenol degradation pathway in Pseudomonas sp. strain KL28: genetic organization of the lap gene cluster and substrate specificities of phenol hydroxylase and catechol 2,3-dioxygenase. Microbiology 149, 3265-3277.   DOI   ScienceOn
2 Lee, K., Kauppi, B., Parales, R.E., Gibson, D.T., and Ramaswamy, S. 1997. Purification and crystallization of the oxygenase component of naphthalene dioxygenase in native and selenomethionine-derivatized forms. Biochem. Biophys. Res. Commun. 241, 553-557.   DOI   ScienceOn
3 Lee, K. and Veeranagouda, Y. 2009. Ultramicrocells form by reductive division in macroscopic Pseudomonas aerial structures. Environ. Microbiol. 11, 1117-1125.   DOI   ScienceOn
4 Notomista, E., Lahm, A., Di Donato, A., and Tramontano, A. 2003. Evolution of bacterial and archaeal multicomponent monooxygenases. J. Mol. Evol. 56, 435-445.   DOI
5 Powlowski, J., Sealy, J., Shingler, V., and Cadieux, E. 1997. On the role of DmpK, an auxiliary protein associated with multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600. J. Biol. Chem. 272, 945-951.   DOI   ScienceOn
6 Sazinsky, M.H., Bard, J., Di Donato, A., and Lippard, S.J. 2004. Crystal structure of the toluene/o-xylene monooxygenase hydroxylase from Pseudomonas stutzeri OX1. Insight into the substrate specificity, substrate channeling, and active site tuning of multicomponent monooxygenases. J. Biol. Chem. 279, 30600-30610.   DOI   ScienceOn
7 Sazinsky, M.H., Dunten, P.W., McCormick, M.S., DiDonato, A., and Lippard, S.J. 2006. X-ray structure of a hydroxylase-regulatory protein complex from a hydrocarbon-oxidizing multicomponent monooxygenase, Pseudomonas sp. OX1 phenol hydroxylase. Biochemistry 45, 15392-15404.   DOI   ScienceOn
8 Sazinsky, M.H. and Lippard, S.J. 2006. Correlating structure with function in bacterial multicomponent monooxygenases and related diiron proteins. Acc. Chem. Res. 39, 558-566.   DOI   ScienceOn
9 Schafer, A., Tauch, A., Jager, W., Kalinowski, J., Thierbach, G., and Puhler, A. 1994. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145, 69-73.   DOI   ScienceOn
10 Shingler, V., Powlowski, J., and Marklund, U. 1992. Nucleotide sequence and functional analysis of the complete phenol/3,4-dimethylphenol catabolic pathway of Pseudomonas sp. strain CF600. J. Bacteriol. 174, 711-724.   DOI
11 Tabor, S. 2001. Expression using the T7 RNA polymerase/promoter system. Curr. Protoc. Mol. Biol. 11, 16.12.11-16.12.11.
12 Amann, E., Ochs, B., and Abel, K.J. 1988. Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene 69, 301-315.   DOI   ScienceOn
13 Cadieux, E. and Powlowski, J. 1999. Characterization of active and inactive forms of the phenol hydroxylase stimulatory protein DmpM. Biochemistry 38, 10714-10722.   DOI   ScienceOn
14 Cadieux, E., Vrajmasu, V., Achim, C., Powlowski, J., and Munck, E. 2002. Biochemical, Mossbauer, and EPR studies of the diiron cluster of phenol hydroxylase from Pseudomonas sp. strain CF 600. Biochemistry 41, 10680-10691.   DOI   ScienceOn
15 Cho, J.H., Jung, D.K., Lee, K., and Rhee, S. 2009. Crystal structure and functional analysis of the extradiol dioxygenase LapB from a long-chain alkylphenol degradation pathway in Pseudomonas. J. Biol. Chem. 284, 34321-34330.   DOI   ScienceOn
16 Cho, A.R., Lim, E.J., Veeranagouda, Y., and Lee, K. 2011. Identification of a p-cresol degradation pathway by a GFP-based transposon in Pseudomonas and its dominant expression in colonies. J. Microbiol. Biotechnol. 21, 1179-1183.   과학기술학회마을   DOI   ScienceOn
17 Haddock, J.D. and Gibson, D.T. 1995. Purification and characterization of the oxygenase component of biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400. J. Bacteriol. 177, 5834-5839.   DOI
18 Divari, S., Valetti, F., Caposio, P., Pessione, E., Cavaletto, M., Griva, E., Gribaudo, G., Gilardi, G., and Giunta, C. 2003. The oxygenase component of phenol hydroxylase from Acinetobacter radioresistens S13. Eur. J. Biochem. 270, 2244-2253.   DOI   ScienceOn