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Research article
Molecular Biology and Omics
Overexpression and Characterization of Lycopene Cyclase (CrtY) from Marine Bacterium Paracoccus haeundaensis
Department of Microbiology, Pukyong National University, Busan 608-737, Korea
Received: August 27, 2012; Accepted: October 2, 2012
J. Microbiol. Biotechnol. 2013; 23(2): 144-148
Published February 28, 2013 https://doi.org/10.4014/jmb.1208.08068
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Lycopene cyclase converts lycopene to β-carotene by
catalyzing the formation of two beta-rings at each end of
the linear carotene structure. This reaction takes place as
a two-step reaction in which both sides of of the lycopene
molecule are cyclized into β-carotene rings via the
monocyclic γ-carotene as an intermediate. The crtY gene
coding for lycopene cyclase from Paracoccus haeundaensis
consists of 1,158 base pairs encoding 386 amino acids
residues. An expression plasmid containing the crtY gene
(pET44a-CrtY) was constructed and expressed in
Escherichia coli, and produced a recombinant protein of
approximately 43 kDa, corresponding to the molecular
mass of lycopene cyclase. The expressed protein was
purified to homogeneity by His-tag affinity chromatography
and showed enzymatic activity corresponding to lycopene
cyclase. We also determined the lycopene substrate
specificity and NADPH cofactor requirements of the
purified protein. The Km values for lycopene and NADPH
were 3.5 μM and 2 mM, respectively. The results obtained
from this study will provide a wider base of knowledge on
the enzyme characterization of lycopene cyclase at the
molecular level.
Keywords
Carotenoids, Lycopene, Lycopene cyclase, β-carotene, Paracoccus haeundensis
References
- Akyon, Y. 2002. Effect of antioxidants on the immune response of Helicobacter pylori. Clin. Microbiol. Infect. 8: 438-441.
- Amar, Y., V. Kiron, S. Satoh, and T. Watanabe. 2004. Enhancement of innate immunity in rainbow trout (Oncorhynchus mykiss Walbaum) associated with dietary intake of carotenoids from natural products. Fish Shellfish Immunol. 16: 527-537.
- Bertran, J. S. 1999. Carotenoids and gene regulation. Nutr. Rev. 57: 182-191.
- Bhupinder, S. H., A. O. David, B. Peter, K. Hans, and E. H. John. 1993. In vitro expression and activity of lycopene cyclase and β-carotene hydroxylase from Erwinia herbicola. FEBS Lett. 315: 329-334.
- Chemler, J. A., Y. Yan, and M. A. Koffas. 2006. Biosynthesis of isoprenoids, polyunsaturated fatty acids and flavonoids in Saccharomyces cerevisiae. Microb. Cell Fact. 23: 5-20.
- Edge, R., D. J. Garvey, and T. G. Truscott. 1997. The carotenoids as anti-oxidants: A review. J. Photochem. Photobiol. 41: 189-200.
- Francis, X. C., S. Zairen, C. Daniel, H. Joseph, and G. Elisabeth. 1994. Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp Strain PCC7942. Am. Soc. Plant Physiol. 6: 1107-1121.
- Haila, K. M., B. R. Nielsen, M. I. Heininen, and L. H. Skibsted. 1997. Carotenoid reaction with free radicals in acetone and toluene at different oxygen partial pressures. An ESR spintrapping study of structure-activity relationships. Z. Lebensm. Unters. Forsch. 204: 81-87.
- Harker, M., J. Hirschberg, and A. Oren. 1998. Paracoccus marcusii sp. nov., an orange Gram-negative coccus. Int. J. Syst. Bacteriol. 48: 543-548.
- Jorgensen, K. and L. H. Skibsted. 1993. Carotenoid scavenging of radicals. Effect of carotenoid structure and oxygen partial pressure on antioxidative activity. Z. Lebensm. Unters. Forsch. 196: 423-429.
- Jyonouchi, H., L. Ahang, M. Cross, and T. Tomita. 1994. Immunomodulating actions of carotenoids: Enhancement of in vivo and in vitro antibody production to T-dependent antigens. Nutr. Cancer 21: 47-58.
- Lee, J. H., Y. S. Kim, T. J. Choi, W. J. Lee, and Y. T. Kim. 2004. Paracoccus haeundaensis sp. nov., a Gram-negative, halophilic, astaxanthin-producing bacterium. Int. J. Syst. Evol. Microbiol. 54: 1699-1702.
- Lee, J. H. and Y. T. Kim. 2006. Cloning and characterization of the astaxanthin biosynthesis gene cluster from the marine bacterium Paracoccus haeundaensis. Gene 370: 86-95.
- Lee, J. H. and Y. T. Kim. 2006. Functional expression of the astaxanthin biosynthesis genes from a marine bacterium, Paracoccus haeundaensis. Biotechnol. Lett. 28: 1167-1173.
- Yokoyama, A., H. Izumida, and W. Miki. 1994. Production of astaxanthim and 4-ketozeaxanthin by the marine bacterium, Agrobacterium aurantiacum. Biosci. Biotechnol. Biochem. 58:1842-1844.
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Research article
J. Microbiol. Biotechnol. 2013; 23(2): 144-148
Published online February 28, 2013 https://doi.org/10.4014/jmb.1208.08068
Copyright © The Korean Society for Microbiology and Biotechnology.
Overexpression and Characterization of Lycopene Cyclase (CrtY) from Marine Bacterium Paracoccus haeundaensis
Tae Hyug Jeong 1, Keunho Ji 1 and Young Tae Kim 1*
Department of Microbiology, Pukyong National University, Busan 608-737, Korea
Received: August 27, 2012; Accepted: October 2, 2012
Abstract
Lycopene cyclase converts lycopene to β-carotene by
catalyzing the formation of two beta-rings at each end of
the linear carotene structure. This reaction takes place as
a two-step reaction in which both sides of of the lycopene
molecule are cyclized into β-carotene rings via the
monocyclic γ-carotene as an intermediate. The crtY gene
coding for lycopene cyclase from Paracoccus haeundaensis
consists of 1,158 base pairs encoding 386 amino acids
residues. An expression plasmid containing the crtY gene
(pET44a-CrtY) was constructed and expressed in
Escherichia coli, and produced a recombinant protein of
approximately 43 kDa, corresponding to the molecular
mass of lycopene cyclase. The expressed protein was
purified to homogeneity by His-tag affinity chromatography
and showed enzymatic activity corresponding to lycopene
cyclase. We also determined the lycopene substrate
specificity and NADPH cofactor requirements of the
purified protein. The Km values for lycopene and NADPH
were 3.5 μM and 2 mM, respectively. The results obtained
from this study will provide a wider base of knowledge on
the enzyme characterization of lycopene cyclase at the
molecular level.
Keywords: Carotenoids, Lycopene, Lycopene cyclase, β-carotene, Paracoccus haeundensis
References
- Akyon, Y. 2002. Effect of antioxidants on the immune response of Helicobacter pylori. Clin. Microbiol. Infect. 8: 438-441.
- Amar, Y., V. Kiron, S. Satoh, and T. Watanabe. 2004. Enhancement of innate immunity in rainbow trout (Oncorhynchus mykiss Walbaum) associated with dietary intake of carotenoids from natural products. Fish Shellfish Immunol. 16: 527-537.
- Bertran, J. S. 1999. Carotenoids and gene regulation. Nutr. Rev. 57: 182-191.
- Bhupinder, S. H., A. O. David, B. Peter, K. Hans, and E. H. John. 1993. In vitro expression and activity of lycopene cyclase and β-carotene hydroxylase from Erwinia herbicola. FEBS Lett. 315: 329-334.
- Chemler, J. A., Y. Yan, and M. A. Koffas. 2006. Biosynthesis of isoprenoids, polyunsaturated fatty acids and flavonoids in Saccharomyces cerevisiae. Microb. Cell Fact. 23: 5-20.
- Edge, R., D. J. Garvey, and T. G. Truscott. 1997. The carotenoids as anti-oxidants: A review. J. Photochem. Photobiol. 41: 189-200.
- Francis, X. C., S. Zairen, C. Daniel, H. Joseph, and G. Elisabeth. 1994. Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp Strain PCC7942. Am. Soc. Plant Physiol. 6: 1107-1121.
- Haila, K. M., B. R. Nielsen, M. I. Heininen, and L. H. Skibsted. 1997. Carotenoid reaction with free radicals in acetone and toluene at different oxygen partial pressures. An ESR spintrapping study of structure-activity relationships. Z. Lebensm. Unters. Forsch. 204: 81-87.
- Harker, M., J. Hirschberg, and A. Oren. 1998. Paracoccus marcusii sp. nov., an orange Gram-negative coccus. Int. J. Syst. Bacteriol. 48: 543-548.
- Jorgensen, K. and L. H. Skibsted. 1993. Carotenoid scavenging of radicals. Effect of carotenoid structure and oxygen partial pressure on antioxidative activity. Z. Lebensm. Unters. Forsch. 196: 423-429.
- Jyonouchi, H., L. Ahang, M. Cross, and T. Tomita. 1994. Immunomodulating actions of carotenoids: Enhancement of in vivo and in vitro antibody production to T-dependent antigens. Nutr. Cancer 21: 47-58.
- Lee, J. H., Y. S. Kim, T. J. Choi, W. J. Lee, and Y. T. Kim. 2004. Paracoccus haeundaensis sp. nov., a Gram-negative, halophilic, astaxanthin-producing bacterium. Int. J. Syst. Evol. Microbiol. 54: 1699-1702.
- Lee, J. H. and Y. T. Kim. 2006. Cloning and characterization of the astaxanthin biosynthesis gene cluster from the marine bacterium Paracoccus haeundaensis. Gene 370: 86-95.
- Lee, J. H. and Y. T. Kim. 2006. Functional expression of the astaxanthin biosynthesis genes from a marine bacterium, Paracoccus haeundaensis. Biotechnol. Lett. 28: 1167-1173.
- Yokoyama, A., H. Izumida, and W. Miki. 1994. Production of astaxanthim and 4-ketozeaxanthin by the marine bacterium, Agrobacterium aurantiacum. Biosci. Biotechnol. Biochem. 58:1842-1844.
