Overexpression and Purification of Monooxygenases Cloned from Arthrobacter chlorophenolicus A6 for Enzymatic Decomposition of 4-Chlorophenol

doi:10.7857/JSGE.2014.19.3.047

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2014 Vol.19, Issue 3 Preview Page Next Page

Overexpression and Purification of Monooxygenases Cloned from Arthrobacter chlorophenolicus A6 for Enzymatic Decomposition of 4-Chlorophenol 4-Chlorophenol 분해박테리아 Arthrobacter chlorophenolicus A6로부터의 monooxygenase의 복제 및 대량발현과 정제 그리고 기질분해활성도 분석: 류송정;이소라;김한승;
Ryu, Song-Jung;Kang, Christina S.;Kim, Han S.;

30 June 2014. pp. 47 ~ 55

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Abstract

Arthrobacter chlorophenolicus A6 possesses several monooxygenases (CphC-I, CphC-II, and CphB) that can catalyze the transformation of 4-chlorophenol (4-CP) to hydroxylated intermediates in the initial steps of substrate metabolism. The corresponding genes of the monooxygenases were cloned, and the competent cells were transformed with these recombinant plasmids. Although CphC-II and CphB were expressed as insoluble forms, CphC-I was successfully expressed as a soluble form and isolated by purification. The specific activity of the purified CphC-I was analyzed by using 4-CP, 4-chlorocatechol (4-CC), and catechol (CAT) as substrates. The specific activities for 4-CP, 4-CC, and CAT were determined to be 0.312 U/mg, 0.462 U/mg, 0.246 U/mg, respectively. The results of this study indicated that CphC-I is able to catalyze the degradation of 4-CC and CAT in addition to 4-CP, which is a primary substrate. This research is expected to provide the fundamental information for the development of an eco-friendly biochemical degradation of aromatic hydrocarbons.

Keywords

4-Chlorophenol;Arthrobacter chlorophenolicus A6;Monooxygenase;Enzyme cloning and expression;

References

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Information

Publisher :The Korean Society of Soil and Groundwater Environment
Publisher(Ko) :한국지하수토양환경학회
Journal Title :Journal of Soil and Groundwater Environment
Journal Title(Ko) :지하수토양환경
Volume : 19
No :3
Pages :47 ~ 55
DOI :https://doi.org/10.7857/JSGE.2014.19.3.047

[1] Chen, W., Bruhlmann, F., Richins, R.D., and Mulchandani, A., 1999, Engineering of improved microbes and enzymes for bioremediation, Curr. Opin. Biotechnol., 10, 137-141.10.1016/S0958-1669(99)80023-8

[2] Czaplicka, M., 2004, Sources and transformations of chlorophenols in the natural environment, Sci. Total Environ., 322, 21-39.10.1016/j.scitotenv.2003.09.015

[3] Dumon-Seignovert, L., Cariot, G., and Vuillard, L., 2004, The toxicity of recombinant proteins in Escherichia coli: a comparison of overexpression in BL21(DE3), C41(DE3), and C43(DE3), Protein Expr. Purif., 37, 203-206.10.1016/j.pep.2004.04.025

[4] El-Sayed, W.S., Ismaeil, M., and El-Beih, F., 2009, Isolation of 4-chlorophenol-degrading bacteria, Bacillus subtilis OS1 and Alcaligenes sp. OS2 from petroleum oil-contamination soil and characterization of its catabolic pathway, Aust. J. Basic Appl. Sci., 3(2), 776-783.

[5] Ferraroni, M., Kolomytseva, M.P., Solyanikova, I.P., Scozzafava, A., Golovleva, L.A., and Briganti, F., 2006, Crystal structure of 3-chlorocatechol 1,2-dioxygenase key enzyme of a new modified ortho-pathway from the gram-positive Rhodococcus opacus 1CP grown on 2-chlorophenol, J. Mol. Biol., 360, 788-799.10.1016/j.jmb.2006.05.046

[6] Gisi, M.R. and Xun, L., 2003, Characterization of chlorophenol 4-monooxynenase (TftD) and NADH:Flavin adenine dinucleotide oxidoreductase (TtfC) of Burkholderia cepacia AC1100, J. Bacteriol., 185, 2786-2792.10.1128/JB.185.9.2786-2792.2003

[7] Hernandez, S.R., Kergaravat, S.V., and Pividori, M.I., 2013, Enzymatic electrochemical detection coupled to multivariate calibration for the determination of phenolic compounds in environmental samples, Talanta., 106, 399-407.10.1016/j.talanta.2013.01.024

[8] Hollender, J., Hopp, J., and Dott, W., 1997, Degradation of 4- chlorophenol via the meta cleavage pathway by Comamonas testosteroni JH5, Appl. Environ. Microbiol., 63, 4567-4572.

[9] Jegannathan, K.R. and Nielsen, P.H., 2013, Environmental assessment of enzyme use in industrial production- a literature review, J. Clean. Prod., 42, 228-240.10.1016/j.jclepro.2012.11.005

[10] Khan, K., Madhavan, T.P.V., and Muniyappa, K., 2010, Cloning, overexpression and purification of functionally active Saccharomyces cerevisiae Hop1 protein from Escherichia coli, Protein Expr. Purif., 72, 42-47.10.1016/j.pep.2010.03.016

[11] Konovalova, E.I., Solyanikova, I.P., and Golovleva, L.A., 2009, Degradation of 4-chlorophenol by the strain Rhodococcus opacus 6a, Microbiology., 78, 805-807.10.1134/S0026261709060204

[12] Lee, S.H., Lee, S.H., Ryu, S.J., Kang, C.S., Suma, Y., and Kim, H.S., 2013, Effective biochemical decomposition of chlorinated aromatic hydrocarbons with a biocatalyst immobilized on natural enzyme support, Bioresour. Technol., 141, 89-96.10.1016/j.biortech.2013.01.159

[13] Li, J., Cai, W., and Zhu, L., 2011, The characteristics and enzyme activities of 4-chlorophenol biodegradation by Fusarium sp., Bioresour. Technol., 102, 2985-2989.10.1016/j.biortech.2010.10.006

[14] Nordin, K., Unell, M., and Jansson, J.K., 2005, Novel 4-chlorophenol degradation gene cluster and degradation route via hydroxyquinol in Arthrobacter chlorophenolicus A6, Appl. Environ. Microbiol., 71, 6538-6544.10.1128/AEM.71.11.6538-6544.2005

[15] Olaniran, A.O. and Igbinosa, E.O., 2011, Chlorophenols and other related derivatives of environmental concern: properties, distribution and microbial degradation processes, Chemosphere., 83, 1297-1306.10.1016/j.chemosphere.2011.04.009

[16] Papaneophytou, C.P., Rinotas, V., Douni, E., and Kontopidis, G., 2013, A statistical approach for optimization of RANKL overexpression in Escherichia coli: purification and characterization of the protein, Protein Expr. Purif., 90, 9-19.10.1016/j.pep.2013.04.005

[17] Perry, L.L. and Zylstra, G.J., 2007, Cloning of a gene cluster involved in the catabolism of p-nitrophenol by Arthrobacter sp. strain JS443 and characterization of the p-nitrophenol monoxygenase, J. Bacteriol., 189, 7563-7572.10.1128/JB.01849-06

[18] Pieper, D.H. and Reineke, W., 2000, Engineering bacteria for bioremediation, Curr. Opin. Biotechnol., 11, 262-270.10.1016/S0958-1669(00)00094-X

[19] Sahoo, N.K., Pakshirajan, K., and Ghosh, P.K., 2010, Enhancing the biodegradation of 4-chlorophenol by Arthrobacter chlorophenolicus A6 via medium development, Int. Biodeter. Biodegr., 64, 474-480.10.1016/j.ibiod.2010.05.008

[20] Sanakis, Y., Mamma, D., Christakopoulos, P., and Stamatis, H., 2003, Catechol 1,2-dioxygenase from Pseudomonas putida in organic media-an electron paramagnetic resonance study, Int. J. Biol. Macromol., 33, 101-106.10.1016/S0141-8130(03)00073-4

[21] Shah, B. and Chen, A., 2012, Novel electrochemical approach for the monitoring of biodegradation of phenolic pollutants and determination of enzyme activity, Electrochem. Commun., 25, 79-82.10.1016/j.elecom.2012.09.009

[22] Shumkova, E.S., Solyanikova, I.P., Plotnikova, E.G., and Golovleva, L.A., 2009, Phenol degradation by Rhodococcus opacus strain 1G, Appl. Biochem. Microbiol., 45, 51-57.

[23] Sorensen, H.P. and Mortensen, K.K., 2005, Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli, Microb. Cell. Fact., 4, 1-8.10.1186/1475-2859-4-1

[24] Tobajas, M., Monsalvo, V.M., Mohedano, A.F., and Rodriguez, J.J., 2012, Enhancement of cometabolic biodegradation of 4- chlophenol induced with phenol and glucose as carbon sources by Comamonas testosteroni, J. Environ. Manage., 95, S116-S121.10.1016/j.jenvman.2010.09.030

[25] van de Pas, B.A., Smidt, H., Hagen, W.R., van der Oost, J., Schraa, G., Stams, A.J.M., and de Vos, W.M., 1999, Purification and molecular characterization of ortho-chlorophenol reductive dehalogenase, a key enzyme of halorespiration in Desulfitobacterium dehalogenans, J. Biol. Chem., 274, 20287-20292.10.1074/jbc.274.29.20287

[26] Wang, J., Ma, X., Liu, S., Sun, P., Fan, P., and Xia, C., 2012, Biodegradation of phenol and 4-chlorophenol by Candida tropicalis W1, Procedia Environ. Sci., 16, 299-303.10.1016/j.proenv.2012.10.042

[27] Wojcieszy ska, D., Gre , I., Hupert-Kocurek, K., and Guzik, U., 2011, Modulation of FAD-dependent monooxygenase activity from aromatic compounds-degrading Stenotrophomonas maltophilia strain KB2, Acta. Biochim. Pol., 58, 421-426.

Journal of Soil and Groundwater Environment ISSN:1598-6438(Print) 2287-8831(Online) 지하수토양환경

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