2School of Biosciences and Biotechnology, Faculty Science and Technology, University Kebangsaan Malaysia, Selangor, Malaysia
In this study, benzoate dioxygenase from Rhodococcus ruber UKMP-5M was catalyzed by oxidating the benzene ring to catechol and other derivatives. The benzoate dioxygenase (benA gene) from Rhodococcus ruber UKMP-5M was then expressed, purified, characterized, The benA gene was amplified (642 bp), and the product was cloned into a pGEM-T vector.The recombinant plasmid pGEMT-benA was digested by double restriction enzymes BamHI and HindIII to construct plasmid pET28b-benA and was then ligated into Escherichia coli BL21 (DE3). The recombinant E. coli was induced with 0.5 mM isopropyl β-D-thiogalactoside (IPTG) at 22˚C to produce benzoate dioxygenase. The enzyme was then purified by ion exchange chromatography after 8 purification folds. The resulting product was 25 kDa, determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Benzoate dioxygenase activity was found to be 6.54 U/mL and the optimal pH and temperature were 8.5 and 25°C, respectively. Maximum velocity (Vmax) and Michaelis constant (Km) were 7.36 U/mL and 5.58 µM, respectively. The end metabolite from the benzoate dioxygenase reaction was cyclohexane dione, which was determined by gas chromatography mass spectrometry (GC-MS).
1. Xu Z, Mulchandani A, Chen W. Detection of benzene, toluene, ethyl benzene, and xylenes (BTEX) using toluene dioxygenase-peroxidase coupling reactions. Biotechnol Progr 2003;19:1812-1815.
2. U.S. Environmental Protection Agency. Integrated Risk Information System (IRIS) on Toluene. National Center for Environmental Assessment, Office of Research and Development, Washington, DC. 2005.
3. Zhao K, Guo X, Gong J. A novel benzoate-degrading Rhodococcus strain contains three catA genes with one being transcriptionally active during the growth on benzoate. J Environ Biol 2013;34:40-407.
4. Zeyaullah M, Abdelkafe AS, Zabya WB, Ali A. Biodegradation of catechols by micro-organisms - A short review. Afr J Biotechnol2009;8:2916-2922.
5. Ge Y, Eltis L. Characterization of hybrid toluate and benzoate dioxygenase J Bacteriol 2003;185:5333-5341.
6. Haddad S, Eby DM, Neidle EL. Cloning and expression of the benzoate dioxygenase genes from Rhodococcus sp. strain 19070. Appl Environ Microbiol 2001;67:2507-2514.
7. da Silva MLB, Alvarez PJ. Indole-based assay to assess the effect of ethanol on Pseudomonas putida F1 dioxygenase activity. Biodegradation2010;21:425-430.
8. Larkin MJ, Kulakov LA, Allen CC. Current opinion in biotechnology biodegradation and Rhodococcus- masters of catabolic versatility. Curr Opin Biotechnol2005;16: 282-290.
9. Griffen JA, Kenwright SJ, Abou-Shehada S, Wharry S, Moody TS, Lewis SE. Benzoate dioxygenase from Ralstonia eutropha B9–unusual regiochemistry of dihydroxylation permits rapid access to novel chirons. Org Chem Fron2014;1:79-90.
10. Harayama S, Rekik M, Wubbolts M, Rose K, Leppik RA, Timmis KN. Characterization of five genes in the upper pathway operon of TOL plasmid pWW0 from Pseudomonas putida and ientification of the gene products. J Bacteriol1989; 171:5048-5055.
11. Nadaf NH, Ghosh JS. Purification and characterization of catechol 1, 2 dioxygenase from Rhodococcus sp. NCIM 2891. Res J Environ Earth Sci2011;3:608-613.
12. Kitagawa W, Miyauchi K, Masai E, Fukuda M. Cloning and characterization of benzoate catabolic genes in the gram-positive polychlorinated biphenyl degrader Rhodococcus sp. strain RHA1. J Bacteriol2001;183:6598-6606.
13. Hamzah A, Tavakoli A, Rabu A. Detection of toluene degradation in bacteria isolated from oil contaminated soils. Sains Malays2011;40:1231-1235.
14. Tavakoli A, Hamzah A, Rabu A. Potential characterization of Rhodococcus ruber UKMP-5M for biodegradation of toluene and catechol intermediate compounds. International Congress of malaysian Society for Microbiology 2009; Malaysia.
15. Zamanian M, Mason J R. Benzene dioxygenase in Pseudomonas putida. Subunit composition and immuno cross reactivity with other aromatic dioxygenases. Biochem J 1987;244:611-616.
16. Shaw JP, Harayama S. Purification and characterisation of TOL plasmid-encoded benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase of Pseudomonas putida. Eur J Biochem1990;191:705-714.
17. Retallack DM, Thomas TC, Ying Shao KLH, Resnick SM, Lee VD, Squires CH. Identification of anthranilate and benzoate metabolic operons of Pseudomonas fluorescens and functional characterization of their promoter regions. Microb Cell Fact2006;5:1-13.
18. Williams PA, Murray K. Metabolism of benzoate and the methylbenzoates by Pseudomonas putida (arvilla) mt-2: evidence for the existence of a TOL plasmid. J Bacteriol1974;120:416-423.
19. Suryanto D, Suwanto A. Isolation and characterization of a novel benzoate utilizing Serratia marcescens. Biotropica2003;21:1-10.
20. Yamaguchi M, Fujisawa H. Characterization of NADH-cytochrome c reductase, a component of benzoate 1, 2-dioxygenase system from Pseudomonas arvilla C-1. J Biol Chem1978;253:8848-8853.
21. Wang L, Qiao N, Sun F, Shao Z. Isolation, gene detection and solvent tolerance of benzene, toluene and xylene degrading bacteria from nearshore surface water and Pacific Ocean sediment. Extremophiles 2008;12:335-342.
22. Grund E, Knorr C, Eichenlaub R. Catabolism of benzoate and monohydroxylated benzoates by Amycolatopsis and Streptomyces spp. Appl Environ Microbiol1990; 56:1459-1464.
23. Fahy A, Ball AS, Lethbridge G, Timmis KN, McGenity TJ. Isolation of alkali-tolerant benzene-degrading bacteria from a contaminated aquifer. Lett Appl Microbiol2008;47:60-66.
24. Choi KY, Zylstra GJ, Kim E. Benzoate catabolite repression of the phthalate degradation pathway in Rhodococcus sp. strain DK17. Appl Environ Microbiol2007; 73:1370-1374.
25. Kim JH, Jeong WH, Karegoudar TB, Kim CK. Stable degradation of benzoate by Klebsiella oxytoca C302 immobilized in lginate and polyurethane. Biotechnol Bioprocess Eng2002;7:347-351.
26. Eby DM, Beharry ZM, Coulter ED, Kurtz DM, Neidle EL. Characterization and evolution of anthranilate 1, 2-dioxygenase from Acinetobacter sp. strain ADP1. J Bacteriol2001;183:109-118.
27. Yamaguchi M, Fujisawa H. purification and characterization of an oxygenase component in benzoate 1, 2 dioxygenase system from Pseudomonas arvilla C-1. J Biochem1980;255:5058-5063.
28. Kim D, Kim SW, Choi KY, Lee JS, Kim E. Molecular cloning and functional characterization of the genes encoding benzoate and p-hydroxybenzoate degradation by the halophilic Chromohalobacter sp. strain HS-2. FEMS Microbiol Lett2008; 280:235-241.
29. Goncalves ER, Hara H, Miyazawa D, Davies JE, Elitis LD, Mohn WW. Transcriptomic assessment of lsozymes in the biphenyl pathway of Rhodococcus sp. strain RHA1. Appl Environ Microbiol 2006;72:6183-6193.
30. Aly HA, Huu NB, Wray V, Junca H, Pieper DH. Two angular dioxygenases contribute to the metabolic versatility of dibenzofuran-degrading Rhodococcus sp. strain HA01. Appl Environ Microbiol2008;74:3812-3822.
31. Neidle EL, Hartnett C, Ornston LN, Bairoch A, Rekik M, Harayama S. Nucleotide sequences of the Acinetobacter calcoaceticus benABC genes for benzoate 1, 2-dioxygenase reveal evolutionary relationships among multicomponent oxygenases. J Bacteriol 1991;173:5385-5395.