Abstract
L-cysteine desulfhydrase (CD) plays an important role in L-cysteine decomposition. To identify the CD gene in Pseudomonas sp. TS1138 and investigate its effect on the L-cysteine biosynthetic pathway, the CD gene was cloned from Pseudomonas sp. TS1138 by polymerase chain reaction (PCR) method. The nucleotide sequence of CD gene was determined to be 1,215 bp, and its homology with other sequences encoding CD was analyzed. Then the CD gene was subcloned into pET-21a(+) vector and expressed in Escherichia coli (E. coli) by isopropyl-β-D-thiogalactopyranoside (IPTG) inducement. The recombinant CD was purified by Ni-NTA His-Bind resin, and its activity was identified by the CD activity staining. The enzymatic properties of the recombinant CD were characterized and its critical role involved in the L-cysteine biosynthetic pathway was also discussed.
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Awano N, Wada M, Kohdoh A, Oikawa T, Takagi H, Nakamori S (2003). Effect of cysteine desulfhydrase gene disruption on L-cysteine overproduction in Escherichia coli. Appl Microbiol Biotechnol, 62: 239–243
Awano N, Wada M, Mori H, Nakamori S, Takagi H (2005). Identification and functional analysis of Escherichia coli cysteine desulfhydrases. Appl Environ Microb, 71: 4149–4152
Candura F (1959). Determination of transaminases with photometric methods using 2, 4-dinitrophenyl-hydrazine. Boll Soc Ital Biol Sper, 35: 697–700
Fukamachi H, Nakano Y, Yoshimura M, Koga T (2002). Cloning and characterization of the L-cysteine desulfhydrase gene of Fusobacterium nucleatum. FEMS Microbiol Lett, 215(1): 75–80
Gaitonde M K (1967). A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acid. Biochem J, 104: 627–633
Jin Y J, Yang W B, Liu Z, Bai G, Sun D (2004). Purification and the properties of L-cysteine desulfhydrase of Pseudomonas sp. TS1138. Acta Scientiarum Naturalium Universitatis Nankaiensis, 37(4): 100–104 (in Chinese)
Kurzban G P, Chu L, Ebersole J L, Holt S C (1999). Sulfhemoglobin formation in human erythrocytes by cystalysin, an L-cysteine desulfhydrase from Treponema denticola. Oral Microbiol Immunol, 14(3):153–164
Liu Z, Yang W B, Bai G, Tian W, Jin Y J (2003). Microbial enzyme conversion of L-cysteine and L-cystine. Microbiology, 30(6): 16–21 (in Chinese)
Nelson K E, Weinel C, Paulsen I T, Dodson R J, Hilbert H, Martins dos Santos V A P, Fouts D E, Gill S R, Pop M, Holmes M, Brinkac L, Beanan M, DeBoy R T, Daugherty S, Kolonay J, Madupu R, Nelson W, White O, Peterson J, Khouri H, Hance I, Chris Lee P, Holtzapple E, Scanlan D, Tran K, Moazzez A, Utterback T, Rizzo M, Lee K, Kosack D, Moestl D, Wedler H, Lauber J, Stjepandic D, Hoheisel J, Straetz M, Heim S, Kiewitz C, Eisen J, Timmis K N, Düsterhöft A, Tümmler B, Fraser C M (2002). Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol, 4: 799–808
Ohmachi T, Nishino M, Kawata M, Edo N, Funaki H, Narita M, Mori K, Tamura Y, Asada Y (2002). Identification, cloning, and sequencing of the genes involved in the conversion of DL-2-amino-Δ2-thiazoline-4-carboxylic acid to L-cysteine in Pseudomonas sp. Strain ON-4a. Biosci Biotechnol Biochem, 66: 1097–1104
Pae K M, Ryu O H, Yoon H S (1992). Kinetic properties of a L-cysteine desulfhydrase-deficient mutant in the enzymatic formation of L-cysteine from DL-ATC. Biotechnol Lett, 4: 1143–1148
Ryu O H, Ju J Y, Shin C S (1997). Continuous L-cysteine production using immobilized cell reactors and product extractors. Process Biochem, 32: 201–209
Ryu O H, Shin C S (1991). Analysis of the reaction steps in the bioconversion of DL-ATC to L-cysteine. J Microbiol Chem, 1: 50–53
Sambrook J, Fritsch E F, Maniatis T (2001). Molecular Cloning: A Laboratory Manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press
Sano K, Mitsugi K (1978). Enzyme production of L-Cysteine from DL-2-amino-Δ2-thiazoline-4-carboxylic acid by Pseudomonas thiazolinophilum: Optimal condition for the enzyme formation and enzymatic reaction. Agric Biol Chem, 42: 2315–2321
Sano K, Yokozeki K, Tamura F (1977). Microbial conversion of DL-ATC to L-cysteine and L-cystine: Screening of microorganisms and identification of products. Appl Environ Microbio, 34: 806–810
Shiba T, Takeda K, Yajima M, Tadano M (2002). Genes from Pseudomonas sp. strain BS involved in the conversion of L-2-amino-Δ2-thiazoline-4-carboxylic acid to L-cysteine. Appl Environ Microbiol, 68: 2,179–2,187
Tamura Y, Nishino M, Ohmachi T, Asada Y (1998). N-carbamyl-Lcysteine as an intermediate in the bioconversion from DL-2-amino-Δ2-thiazoline-4-carboxylic acid to L-cysteine by Pseudomonas sp. On-4a. Biosci Biotechnol Biochem, 62: 2226–2229
Wada M, Awano N, Haisa K, Takagi H, Nakamori S (2002). Purification, characterization and identification of cysteine desulfhydrase of Corynebacterium glutamicum, and its relationship to cysteine production. FEMS Microbiol Lett, 217(1): 103–107
Zdych E, Peist R, Reidl J, Boos W (1995). MalY of Escherichia coli is an enzyme with the activity of a β C-S lyase (cystathionase). J Bacteriol, 177: 5,035–5,039
Zheng L, White R H, Cash V L, Jack R F, Dean D R (1993). Cysteine desulfurase activity indicates a role for NIFS in metallocluster biosynthesis. Proc Natl Acad Sci, 90: 2754–2758
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Translated from Microbiology, 2006, 33(4): 21–26 [译自: 微生物学通报]
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Yu, Y., Bai, G., Liu, C. et al. Cloning, expression and characterization of L-cysteine desulfhydrase gene from Pseudomonas sp. TS1138. Front. Biol. China 2, 391–396 (2007). https://doi.org/10.1007/s11515-007-0059-6
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DOI: https://doi.org/10.1007/s11515-007-0059-6