Comparative analysis of phenotypic and genotypic characteristics of two desulfurizing bacterial strains, Mycobacterium phlei SM120-1 and Mycobacterium phlei GTIS10

AIM: To compare few phenotypic and genotypic characteristics of two desulfurizing bacterial strains, Mycobacterium phlei SM120-1 and Mycobacterium phlei GTIS10. METHODS AND RESULTS: In the present study, dibenzothiophene (DBT) desulfurizing activity, composition of fatty acids of cell membranes, DBT sulfone monoxygenase gene (bdsA) and the selection pressure applied during the growth and enrichment of the bacterial strains M. phlei SM120-1 and M. phlei GTIS10 were compared in our laboratory. The DBT desulfurization activity of M. phlei SM120-1 was found to be 0.17 +/- 0.02 micromol 2-HBP min(-1) (gram dry cell weight)(-1) and that of the bacterial strain M. phlei GTIS10 was 1.09 +/- 0.05 micromol 2-HBP min(-1) (gram dry cell weight)(-1). Fatty acid methyl ester analysis of cell membranes of these two bacterial strains in the presence of light gas oil showed that both the strains had different fatty acid profiles in their cell membranes. Comparison of the full gene sequences of the desulfurization gene bdsA in the two bacterial strains showed significant difference in the bdsA gene sequences. There was a significant difference observed in the selection pressure applied during the growth and enrichment of the two bacterial strains. CONCLUSIONS: The results of the comparative study of the bacterial strains, M. phlei SM120-1 and M. phlei GTIS10 showed that there were considerable differences in the phenotypic and genotypic characteristics of these two strains. SIGNIFICANCE AND IMPACT OF STUDY: The present study would broaden the understanding of biodesulfurization trait at intra-species level.     

Operon structure and functional analysis of the genes encoding thermophilic desulfurizing enzymes of Paenibacillus sp. A11-2

Paenibacillus A11-2 can efficiently cleave two carbon&bond;sulfur bonds in dibenzothiophene (DBT) and alkyl DBTs, which are refractory by conventional petroleum hydrodesulfurization, to remove sulfur atom at high temperatures. An 8.7-kb DNA fragment containing the genes for the DBT desulfurizing enzymes of A11-2 was cloned in Escherichia coli and characterized. Heterologous expression analysis of the deletion mutants identified three open reading frames that were required for the desulfurization of DBT to 2-hydroxybiphenyl (2-HBP). The three genes were designated tdsA, tdsB, and tdsC (for thermophilic desulfurization). Both the nucleotide sequences and the deduced amino acid sequences show significant homology to dszABC genes of Rhodococcus sp. IGTS8, but there are several local differences between them. Subclone analysis revealed that the product of tdsC oxidizes DBT to DBT-5,5'-dioxide via DBT-5-oxide, the product of tdsA converts DBT-5,5'-dioxide to 2-(2-hydroxyphenyl) benzene sulfinate, and the product of tdsB converts 2-(2-hydroxyphenyl)benzene sulfinate to 2-HBP. Cell-free extracts of a recombinant E. coli harboring all the three desulfurization genes converted DBT to 2-HBP at both 37 and 50 degrees C. In vivo and in vitro exhibition of desulfurization activity of the recombinant genes derived from a Paenibacillus indicates that an E. coli oxidoreductase can be functionally coupled with the monooxygenases of a gram-positive thermophile.     

红球菌SDUZAWQ对有机硫和无机硫的耐受性研究及脱硫基因的克隆

以筛选得到的红球菌SDUZAWQ为对象,研究其在不同浓度的有机硫化合物二苯并噻吩(DBT)存在下的脱硫能力,以及在0.2mmolLDBT和不同浓度Na2SO4同时存在下的脱硫情况。当DBT浓度高达6mmolL时,菌株仍能生长,而且检测出产物2-羟基联苯(2-HBP)的存在,说明该菌株具有耐受较高浓度DBT的能力。当DBT和Na2SO4同时存在时,DBT为菌株SDUZAWQ所利用,并且也检测出2-HBP,并非如文献所报道的红球菌在无机硫存在下不代谢DBT,表明该菌株能够耐受一定浓度的无机硫酸盐。对相关脱硫基因的克隆和测序结果显示,完整脱硫基因dszABC、其上游调控序列和dszD的序列与模式菌株RhodococcuserythropolisIGTS8的同源性分别是99%、100%和100%。     

A novel enzyme, 2'-hydroxybiphenyl-2-sulfinate desulfinase (DszB), from a dibenzothiophene-desulfurizing bacterium Rhodococcus erythropolis KA2-5-1: gene overexpression and enzyme characterization

Dibenzothiophene (DBT), a model of organic sulfur compound in petroleum, is microbially desulfurized to 2-hydroxybiphenyl (2-HBP), and the gene operon dszABC was required for DBT desulfurization. The final step in the microbial DBT desulfurization is the conversion of 2'-hydroxybiphenyl-2-sulfinate (HBPSi) to 2-HBP catalyzed by DszB. In this study, DszB of a DBT-desulfurizing bacterium Rhodococcus erythropolis KA2-5-1 was overproduced in Escherichia coli by coexpression with chaperonin genes, groEL/groES, at 25 degrees C. The recombinant DszB was purified to homogeneity and characterized. The optimal temperature and pH for DszB activity were 35 degrees C and about 7.5, respectively. The K(m) and k(cat) values for HBPSi were 8.2 microM and 0.123.s(-1), respectively. DszB has only one cysteine residue, and the mutant enzyme completely lost the activity when the cysteine residue was changed to a serine residue. This result together with experiments using inhibitors showed that the cysteine residue contributes to the enzyme activity. DszB was also inhibited by a reaction product, 2-HBP (K(i)=0.25 mM), and its derivatives, but not by the other reaction product, sulfite. The enzyme showed a narrow substrate specificity: only 2-phenylbenzene sulfinate except HBPSi served as a substrate among the aromatic and aliphatic sulfinates or sulfonates tested. DszB was thought to be a novel enzyme (HBPSi desulfinase) in that it could specifically cleave the carbon-sulfur bond of HBPSi to give 2-HBP and sulfite ion without the aid of any other proteinic components and coenzymes.     

Sequence and molecular characterization of a DNA region encoding the dibenzothiophene desulfurization operon of Rhodococcus sp. strain IGTS8.

Dibenzothiophene (DBT), a model compound for sulfur-containing organic molecules found in fossil fuels, can be desulfurized to 2-hydroxybiphenyl (2-HBP) by Rhodococcus sp. strain IGTS8. Complementation of a desulfurization (dsz) mutant provided the genes from Rhodococcus sp. strain IGTS8 responsible for desulfurization. A 6.7-kb TaqI fragment cloned in Escherichia coli-Rhodococcus shuttle vector pRR-6 was found to both complement this mutation and confer desulfurization to Rhodococcus fascians, which normally is not able to desulfurize DBT. Expression of this fragment in E. coli also conferred the ability to desulfurize DBT. A molecular analysis of the cloned fragment revealed a single operon containing three open reading frames involved in the conversion of DBT to 2-HBP. The three genes were designated dszA, dszB, and dszC. Neither the nucleotide sequences nor the deduced amino acid sequences of the enzymes exhibited significant similarity to sequences obtained from the GenBank, EMBL, and Swiss-Prot databases, indicating that these enzymes are novel enzymes. Subclone analyses revealed that the gene product of dszC converts DBT directly to DBT-sulfone and that the gene products of dszA and dszB act in concert to convert DBT-sulfone to 2-HBP.     

Biodesulfurization of benzothiophene and dibenzothiophene by a newly isolated Rhodococcus strain

Rhodococcus sp. KT462, which can grow on either benzothiophene (BT) or dibenzothiophene (DBT) as the sole source of sulfur, was newly isolated and characterized. GC and GC-MS analyses revealed that strain KT462 has the same BT desulfurization pathway as that reported for Paenibacillus sp. A11-2 and Sinorhizobium sp. KT55. The desulfurized product of DBT produced by this strain, as well as other DBT-desulfurizing bacteria such as R. erythropolis KA2-5-1 and R. erythropolis IGTS8, was 2-hydroxybiphenyl. A resting cells study indicated that this strain was also able to degrade various alkyl derivatives of BT and DBT.     

Purification, characterization, and overexpression of flavin reductase involved in dibenzothiophene desulfurization by Rhodococcus erythropolis D-1

The dibenzothiophene (DBT)-desulfurizing bacterium, Rhodococcus erythropolis D-1, removes sulfur from DBT to form 2-hydroxybiphenyl using four enzymes, DszC, DszA, DszB, and flavin reductase. In this study, we purified and characterized the flavin reductase from R. erythropolis D-1 grown in a medium containing DBT as the sole source of sulfur. It is conceivable that the enzyme is essential for two monooxygenase (DszC and DszA) reactions in vivo. The purified flavin reductase contains no chromogenic cofactors and was found to have a molecular mass of 86 kDa and four identical 22-kDa subunits. The enzyme catalyzed NADH-dependent reduction of flavin mononucleotide (FMN), and the K(m) values for NADH and FMN were 208 and 10.8 microM, respectively. Flavin adenine dinucleotide was a poor substrate, and NADPH was inert. The enzyme did not catalyze reduction of any nitroaromatic compound. The optimal temperature and optimal pH for enzyme activity were 35 degrees C and 6.0, respectively, and the enzyme retained 30% of its activity after heat treatment at 80 degrees C for 30 min. The N-terminal amino acid sequence of the purified flavin reductase was identical to that of DszD of R. erythropolis IGTS8 (K. A. Gray, O. S. Pogrebinsky, G. T. Mrachko, L. Xi, D. J. Monticello, and C. H. Squires, Nat. Biotechnol. 14:1705-1709, 1996). The flavin reductase gene was amplified with primers designed by using dszD of R. erythropolis IGTS8, and the enzyme was overexpressed in Escherichia coli. The specific activity in crude extracts of the overexpressed strain was about 275-fold that of the wild-type strain.     

Elucidation of 2-hydroxybiphenyl effect on dibenzothiophene desulfurization by Microbacterium sp. strain ZD-M2

The effect of 2-hydroxybiphenyl (2-HBP), the end product of dibenzothiophene (DBT) desulfurization via 4S pathway, on cell growth and desulfurization activity was investigated by Microbacterium sp. The experimental results indicate that 2-HBP would inhibit the desulfurization activity. Providing 2-HBP was added in the reaction media, the DBT degradation rate decreased along with the increase of 2-HBP addition. By contrast, cell growth would be promoted in the addition of 2-HBP at a low concentration (<0.1mM). At high concentration of 2-HBP, the inhibition on the cell growth occurred. Meanwhile, the inhibitory effect of 2-HBP on DBT desulfurization activity was tested both in the oil/aqueous two-phase system and the aqueous system. A mathematical model was developed to explain the product formation kinetics with DBT as the sole sulfur source. The predicted results were close to the experimental data, it elucidated that along with the 2-HBP accumulation, the inhibitory effect of 2-HBP on DBT desulfurization and cell growth was enhanced.     

Biodesulfurization of dibenzothiophene by recombinant Gordonia alkanivorans RIPI90A

The dszABC genes from newly reported dibenzothiophene biodesulfurizing bacterium, Gordonia alkanivorans RIPI90A were cloned and sequenced. The overall nucleotide sequence similarity between the dszABC genes of G. alkanivorans RIPI90A and those of Rhodococcus erythropolis IGTS8 and Gordonia nitida were 83.1% and 83.2%, respectively. A gene transfer system for G. alkanivorans RIPI90A was established employing the Escherichia coli-Rhodococcus shuttle vector pRSG43 as suitable cloning vector, resulting in transformation efficiencies up to 1.6 x 10(5)CFUs microg(-1) plasmid DNA. This stable vector was applied to cloning and efficient expression of the dsz genes under the control of lac promoter. The recombinant strain was able to desulfurize dibenzothiophene in the presence of inorganic sulfate and sulfur-containing amino acids. The maximum desulfurization activity by recombinant resting cells (131.8 microM2-hydroxybiphenylg(dry cell weight)(-1)h(-1)) was increased 2.67-fold in comparison to the highest desulfurization activity of native resting cells.     

Improvement of desulfurization activity in Rhodococcus erythropolis KA2-5-1 by genetic engineering

Rhodococcus erythropolis KA2-5-1 can desulfurize dibenzothiophene (DBT) into 2-hydroxybiphenyl. A cryptic plasmid, pRC4, which was derived from R. rhodochrous IFO3338, was combined with an Escherichia coli vector to construct an E. coli-Rhodococcus shuttle vector. The complete nucleotide sequence of 2582-bp pRC4 was analyzed. Based on the characteristics of its putative replication genes, pRC4 was assigned to the family of pAL5000-related replicons. The desulfurization gene cluster, dszABC, and the related reductase gene, dszD, cloned from KA2-5-1, were reintroduced into KA2-5-1 and efficiently expressed. The DBT desulfurization ability of the transformant carrying two dszABC clusters and one dszD on the vector was about 4-fold higher than that of the parent strain, and the transformant also showed improved desulfurization activity for light gas oil (LGO). Sulfur components in LGO before and after the reaction were analyzed with gas chromatography-atomic emission detection.     

专一性脱硫菌脱硫活性比较与基因保守性研究

对几株能专一性脱除二苯并噻吩(DBT)中硫元素生成2-羟基联苯的细菌,即短芽孢杆菌(Bacillus brevis)R-6、德氏假单孢菌(Pseudomonas delafleldii)R-8、小球诺卡氏菌(Nocardia globerula)R-9、球形芽孢杆菌(Bacillus sphaericus)R-16、红平红球菌(Rhodococcus erythropolis)LSSE8-1和戈登氏菌(Gordonia nitida)LSSEJ-1展开研究。对照研究发现它们对DBT及其衍生物的代谢活性存在着一定的差异。为了从基因水平分析造成这些差别的原因,对这几株菌的脱硫基因展开了研究。根据Rhodococcus erythropolisIGTS8脱硫基因的保守区设计引物,PCR扩增了R-6、R-8的脱硫基因。测序结果表明脱硫基因高度保守,与IGTS8的相关脱硫基因相似性在99%以上。为了进一步验证不同专一性脱硫菌的脱硫基因的保守性,PCR扩增、克隆了LSSEJ-1和R-9的整个脱硫操纵子,结果表明脱硫基因在这两株菌中也是高度保守的。与IGTS8的相关脱硫基因相比较:R-9的dszA与IGTS8的dszA同源性为99.6%,LSSEJ-1的dszA与IGTS8的dszA的同源性为99.9%;R-9和LSSEJ-1的dszB的同源性与IGTS8的dszB都是99.9%;R-9的dszC与IGTS8的dszC同源性是99.9%,LSSEJ-1的dszC与IGTS8的dszC同源性为99.1%。对比研究认为专一性脱硫嗜温菌的脱硫基因的起源可能相同。     

Desulfurization of alkylated forms of both dibenzothiophene and benzothiophene by a single bacterial strain

Thirty-five bacterial strains capable of converting dibenzothiophene into 2-hydroxybiphenyl were isolated. Among them Rhodococcus erythropolis KA2-5-1 was chosen for further characterization because of its ability to retain high desulfurization activity stably. PCR cloning and DNA sequencing of a KA2-5-1 genomic DNA fragment showed that it was practically identical with dszABC genes from Rhodococcus sp. IGTS8, a representative carbon-sulfur-bond-targeted dibenzothiophene-degrading bacterium. KA2-5-1 desulfurized a variety of alkyl dibenzothiophenes through the specific cleavage of their C-S bonds. In addition, unexpectedly, KA2-5-1 also attacked alkyl benzothiophenes in a C-S-bond-targeted fashion. The purified monooxygenase, encoded by dszC of KA2-5-1, converted benzothiophene and dibenzothiophene into benzothiophene sulfone and dibenzothiophene sulfone, respectively, with the aid of an NADH-dependent oxidoreductase. This result raises the possibility that the same enzymatic step may be involved in desulfurization of alkylated forms of both dibenzothiophene and benzothiophene in KA2-5-1 cells.     

脱硫菌Fds-1的分离鉴定及其对柴油脱硫特性的研究

从含硫土壤中分离筛选出一株专一性脱硫菌Fds-1,经生理生化指标和16S rRNA序列分析鉴定其属于枯草芽孢杆菌(Bacillus subtilis)。用Gibb’s试剂显色和气相色谱-质谱联用分析表明,该菌株通过“4S”途径脱除有机硫。实验发现Fds-1的最佳脱硫活性在30℃,在此温度下72h内能脱除约0.5mmol/L DBT中的有机硫。Fds-1菌株对有机硫化合物的利用情况和柴油脱硫前后烃组分比较都进一步证明该菌株适合于柴油生物脱硫。利用休止细胞对不同组分柴油的脱硫研究表明,脱硫菌株Fds-1对精制柴油中的DBT类化合物的降解能力强。因此,该菌株对精制低硫柴油的深度脱硫具有应用意义。     

Thermophilic biodesulfurization of naphthothiophene and 2-ethylnaphthothiophene by a dibenzothiophene-desulfurizing bacterium, Mycobacterium phlei WU-F1

Naphtho[2,1-b]thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and NTH derivatives can be detected in diesel oil following hydrodesulfurization treatment, in addition to DBT derivatives. Mycobacterium phlei WU-F1, which possesses high desulfurizing ability toward DBT and its derivatives over a wide temperature range (20-50 degrees C), could also grow at 50 degrees C in a medium with NTH or 2-ethylNTH, an alkylated derivative, as the sole source of sulfur. At 50 degrees C, the resting cells of WU-Fl degraded 67% and 83% of 0.81 mM NTH and 2-ethylNTH, respectively, within 8 h. By GC-MS analysis, 2-ethylNTH-desulfurized metabolites were identified as 2-ethylNTH sulfoxide, 1-(2'-hydroxynaphthyl)-1-butene and 1-naphthyl-2-hydroxy-1-butene, and it was concluded that WU-F1 desulfurized 2-ethylNTH through a sulfur-specific degradation pathway with the selective cleavage of carbon-sulfur bonds. Therefore, M. phlei WU-Fl can effectively desulfurize asymmetric organosulfur compounds, NTH and 2-ethylNTH, as well as symmetric DBT derivatives under high-temperature conditions, and it may be a useful desulfurizing biocatalyst possessing a broad substrate specificity toward organosulfur compounds.     

Characterization of the desulfurization genes from Rhodococcus sp. strain IGTS8.

Rhodococcus sp. strain IGTS8 possesses an enzymatic pathway that can remove covalently bound sulfur from dibenzothiophene (DBT) without breaking carbon-carbon bonds. The DNA sequence of a 4.0-kb BstBI-BsiWI fragment that carries the genes for this pathway was determined. Frameshift and deletion mutations established that three open reading frames were required for DBT desulfurization, and the genes were designated soxABC (for sulfur oxidation). Each sox gene was subcloned independently and expressed in Escherichia coli MZ1 under control of the inducible lambda pL promoter with a lambda cII ribosomal binding site. SoxC is an approximately 45-kDa protein that oxidizes DBT to DBT-5,5'-dioxide. SoxA is an approximately 50-kDa protein responsible for metabolizing DBT-5,5'-dioxide to an unidentified intermediate. SoxB is an approximately 40-kDa protein that, together with the SoxA protein, completes the desulfurization of DBT-5,5'-dioxide to 2-hydroxybiphenyl. Protein sequence comparisons revealed that the predicted SoxC protein is similar to members of the acyl coenzyme A dehydrogenase family but that the SoxA and SoxB proteins have no significant identities to other known proteins. The sox genes are plasmidborne and appear to be expressed as an operon in Rhodococcus sp. strain IGTS8 and in E. coli.     

Kinetic Analyses of Desulfurization of Dibenzothiophene by Rhodococcus erythropolis in Batch and Fed-Batch Cultures

The DbtS(sup+) phenotype (which confers the ability to oxidize selectively the sulfur atom of dibenzothiophene [DBT] or dibenzothiophene sulfone [DBTO(inf2)]) of Rhodococcus erythropolis N1-36 was quantitatively characterized in batch and fed-batch cultures. In flask cultures, production of the desulfurization product, monohydroxybiphenyl (OH-BP), was maximal at pH 6.0, while specific productivity (OH-BP cell(sup-1)) was maximal at pH 5.5. Quantitative measurements in fermentors (in both batch and fed-batch modes) demonstrated that DBTO(inf2) as the sole sulfur source yielded a greater amount of product than did DBT. Specifically, 100 (mu)M DBT maximally yielded (apprx=)40 (mu)M OH-BP, while 100 (mu)M DBTO(inf2) yielded (apprx=)60 (mu)M OH-BP. Neither maintaining the pH at 6.0 nor adding an additional carbon source increased the yield of OH-BP. The presence of SO(inf4)(sup2-) in growth media repressed expression of desulfurization activity, but SO(inf4)(sup2-) added to suspensions of cells grown in DBT or DBTO(inf2) did not inhibit desulfurization activity.     

Proteomics and Metabolomics Analyses to Elucidate the Desulfurization Pathway of Chelatococcus sp.

Desulfurization of dibenzothiophene (DBT) and alkylated DBT derivatives present in transport fuel through specific cleavage of carbon-sulfur (C-S) bonds by a newly isolated bacterium Chelatococcus sp. is reported for the first time. Gas chromatography-mass spectrometry (GC-MS) analysis of the products of DBT degradation by Chelatococcus sp. showed the transient formation of 2-hydroxybiphenyl (2-HBP) which was subsequently converted to 2-methoxybiphenyl (2-MBP) by methylation at the hydroxyl group of 2-HBP. The relative ratio of 2-HBP and 2-MBP formed after 96 h of bacterial growth was determined at 4:1 suggesting partial conversion of 2-HBP or rapid degradation of 2-MBP. Nevertheless, the enzyme involved in this conversion process remains to be identified. This production of 2-MBP rather than 2-HBP from DBT desulfurization has a significant metabolic advantage for enhancing the growth and sulfur utilization from DBT by Chelatococcus sp. and it also reduces the environmental pollution by 2-HBP. Furthermore, desulfurization of DBT derivatives such as 4-M-DBT and 4, 6-DM-DBT by Chelatococcus sp. resulted in formation of 2-hydroxy-3-methyl-biphenyl and 2-hydroxy –3, 3^/- dimethyl-biphenyl, respectively as end product. The GC and X-ray fluorescence studies revealed that Chelatococcus sp. after 24 h of treatment at 37°C reduced the total sulfur content of diesel fuel by 12% by per gram resting cells, without compromising the quality of fuel. The LC-MS/MS analysis of tryptic digested intracellular proteins of Chelatococcus sp. when grown in DBT demonstrated the biosynthesis of 4S pathway desulfurizing enzymes viz. monoxygenases (DszC, DszA), desulfinase (DszB), and an NADH-dependent flavin reductase (DszD). Besides, several other intracellular proteins of Chelatococcus sp. having diverse biological functions were also identified by LC-MS/MS analysis. Many of these enzymes are directly involved with desulfurization process whereas the other enzymes/proteins support growth of bacteria at an expense of DBT. These combined results suggest that Chelatococcus sp. prefers sulfur-specific extended 4S pathway for deep-desulphurization which may have an advantage for its intended future application as a promising biodesulfurizing agent.     

Description of by-product inhibiton effects on biodesulfurization of dibenzothiophene in biphasic media

As several authors have reported previously, the Biodesulfurization of hydrodesulfurization recalcitrants, such as dibenzothiophene, is not yet commercially viable because mass transfer limitations and feedback inhibition effects are produced during the conversion. This work has been focused to investigate the inhibition process in aqueous and oil-water systems with two different aerobic biocatalysts types, Rhodococcus erythropolis IGTS8 and Pseudomonas putida CECT 5279. The results obtained have proven that global DBT desulfurization process using CECT 5279 was not clearly deactivated due to final product accumulation, under the experimental conditions assayed. Consistently, the desulfurization pattern has been described with the Michaelis-Menten equation, determining the kinetic parameters. On other hand, the assays have shown that important mass transfer limitations produced the decrease of the yields obtained with this Gram(-) strain in biphasic media. With strain IGTS8 it was observed lower mass transfer problems, but contrary the reaction was severely affected by the final product accumulation, in both aqueous and biphasic systems. Therefore it has been proposed an enzymatic kinetic model with competitive inhibition to describe the BDS evolution pattern when this Gram(+) strain was used.     

2-(2′-Hydroxyphenyl)benzene sulfinate desulfinase from the thermophilic desulfurizing bacterium<Emphasis Type="Italic"> Paenibacillus</Emphasis> sp. strain A11-2: purification and characterization

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 degrees 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 Km and kcat 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.     

Comparative studies of phenotypic and genetic characteristics between two desulfurizing isolates of Rhodococcus erythropolis and the well-characterized R. erythropolis strain IGTS8

Two Rhodococcus erythropolis isolates, named A66 and A69, together with the well-characterized R. erythropolis strain IGTS8 were compared biochemically and genetically. Both isolates, like strain IGTS8, desulfurized DBT to 2-hydroxybiphenyl (2-HBP), following the 4S pathway of desulfurization. Strain IGTS8 showed the highest (81.5%) desulfurization activity in a medium containing DBT at 30 °C. Strain A66 showed approximately the same desulfurization activity either when incubated at 30 °C or at 37 °C, while strain A69 showed an increase of desulfurization efficiency (up to 79%) when incubated at 37 °C. Strains A66 and A69 were also able to grow using various organosulfur or organonitrogen-compounds as the sole sulfur or nitrogen sources. The biological responses of A66, A69 and IGTS8 strains to a series of mutagens and environmental agents were evaluated, trying to mimic actual circumstances involved in exposure/handling of microorganisms during petroleum biorefining. The results showed that strains A69 and IGTS8 were much more resistant to UVC treatment than A66. The three desulfurization genes (dszA, dszB and dszC) present in strains A66 and A69 were partially characterized. They seem to be located on a plasmid, not only in the strain IGTS8, but also in A66 and A69. PCR amplification was observed using specific primers for dsz genes in all the strains tested; however, no amplification product was observed using primers for carbazole (car) or quinoline (qor) metabolisms. All this information contributes to broaden our knowledge concerning both the desulfurization of DBT and the degradation of organonitrogen compounds within the R. erythropolis species.