2-(2′-Hydroxyphenyl)benzene sulfinate desulfinase from the thermophilic desulfurizing bacterium Paenibacillus sp. strain A11-2: purification and characterization
Tóm tắt
2-(2′-Hydroxyphenyl)benzene sulfinate (HPBSi) desulfinase (TdsB), which catalyzes the final step of desulfurization of dibenzothiophene (DBT), was purified from a thermophilic DBT- and benzothiophene (BT)-desulfurizing bacterium: Paenibacillus sp. strain A11-2. The molecular mass of the purified enzyme was 31 kDa and 39 kDa by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis, respectively, suggesting a monomeric structure. The optimal temperature and pH for the reaction involving TdsB was 55°C and the enzyme was more resistant to heat treatment than DszB, a counterpart purified from Rhodococcus erythropolis. The optimum pH for TdsB activity was pH 8. TdsB converted HPBSi to 2-hydroxybiphenyl (2-HBP) and sulfite stoichiometrically. The K
m and k
cat values for HPBSi were 0.33 mM and 0.32 s−1, respectively. TdsB was inactivated by SH reagents such as p-chloromercuribenzoic acid and 5,5′-dithio-bis-2-nitrobenzoic acid, but was not inhibited by chelating reagents such as EDTA and o-phenanthroline. TdsB was also inhibited by o-hydroxystyrene, the final desulfurized product of BT. However, 2-HBP and its derivatives showed only a weak inhibitory effect. TdsB desulfurized 2-(2′-hydroxyphenyl)ethen-1-sulfinate to yield o-hydroxystyrene, but DszB could not. A site-directed mutagenesis study revealed the cysteine residue at position 17 to be essential to the catalytic activity of TdsB.
Tài liệu tham khảo
Denome SA, Olson ES, Young KD (1993) Identification and cloning of genes involved in specific desulfurization of dibenzothiophene by Rhodococcus sp. strain IGTS8. Appl Environ Microbiol 59:2837–2843
Gallagher JR, Olson ES, Stanley DC (1993) Microbial desulfurization of dibenzothiophene: a sulfur-specific pathway. FEMS Microbiol Lett 107:31–36
Gray KA, Pogrebinsky OS, Mrachko GT, Xi L, Monticello DJ, Squires CH (1996) Molecular mechanisms of biocatalytic desulfurization of fossil fuels. Nat Biotechnol 14:1705–1709
Hanson G, Kemp DS (1981) Convenient routes to 4,4″ functionalised o-terphenyls and 2,2′functionalised biphenyls. Org Chem 46:5441–5443
Ishii Y, Konishi J, Okada H, Hirasawa K, Onaka T, Suzuki M (2000) Operon structure and functional analysis of the genes encoding thermophilic desulfurizing enzymes of Paenibacillus sp. A11–2. Biophys Biochem Res Commun 270:81–88
Izumi Y, Ohshiro T, Ogino H, Hine Y, Shimao M (1994) Selective desulfurization of dibenzothiophene by Rhodococcus erythropolis D-1. Appl Environ Microbiol 60:223–226
Kilbane JJ (1989) Desulfurization of coals: the microbial solution. Trends Biotechnol 7:97–101
Konishi J, Ishii Y, Onaka T, Okumura K, Suzuki M (1997) Thermophilic carbon-sulfur-bond-targeted biodesulfurization. Appl Environ Microbiol 63:3164–3169
Konishi J, Ishii Y, Onaka T, Ohta Y, Suzuki M, Maruhashi K (2000) Purification and characterization of dibenzothiophene sulfone monooxygenase and FMN-dependent NADH oxidoreductase from the thermophilic bacterium Paenibacillus sp. strain A11–2. J Biosci Bioeng 90:607–613
Konishi J, Ishii Y, Onaka T, Maruhashi K (2002) Purification and characterization of the monooxygenase catalyzing sulfur-atom specific oxidation of dibenzothiophene and benzothiophene from the thermophilic bacterium Paenibacillus sp. strain A11-2. Appl Microbiol Biotechnol 60:128–133
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
McFarland BL (1999) Biodesulfurization. Curr Opin Microbiol 2:257–264
Nakayama N, Matsubara T, Ohshiro T, Moroto Y, Kawata Y, Koizumi K, Hirakawa Y, Suzuki M, Maruhashi K, Izumi Y, Kurane R (2002) A novel enzyme, 2′-hydroxybiphenyl-2- sulfinate desulfinase (DszB), from a dibenzothiophene-desulfurizing bacterium Rhodococcus erythropolis KA2–5-1: gene overexpression and enzyme characterization. Biochim Biophys Acta 1598:122–130
Ohshiro T, Izumi Y (1999) Microbial desulfurization of organic sulfur compounds in petroleum. Biosci Biotechnol Biochem 63:1–9
Ohshiro T, Suzuki K, Izumi Y (1996) Regulation of dibenzothiophene degrading enzyme activity of R. erythropolis D-1. J Ferment Bioeng 81:121–124
Omori T, Monna L, Saiki Y, Kodama T (1992) Desulfurization of dibenzothiophene by Corynebacterium sp. strain SY1. Appl Environ Microbiol 58:911–915
Piddington CS, Kovachvich BR, Rambosek J (1995) Sequence and molecular characterization of a DNA region encoding the dibenzothiophene desulfurization operon of Rhodococcus sp. strain IGTS8. Appl Environ Microbiol 64:2327–2331
Wang P, Kraviec S (1994) Desulfurization of dibenzothiophene to 2-hydroxybiphenyl by some newly isolated bacterial strains. Arch Microbiol 161:266–271
Webb R (1963) Enzyme and metabolic inhibitors, vol 1. Academic Press, New York, p 78