Isolation of a recombinant desulfurizing 4,6-diproply dibenzothiophene in n-tetradecane

Rhodococcus erythropolis strain KA2-5-1 is unable to desulfurize 4,6-dipropyl dibenzothiophene (DBT) in the oil phase. The dsz desulfurization gene cluster from R. erythropolis strain KA2-5-1 was transferred into 22 rhodococcal and mycobacterial strains using a transposon-transposase complex. The recombinant strain MR65, from Mycobacterium sp. NCIMB10403, was able to grow on a minimal medium supplemented with 1.0 mM 4,6-dipropyl DBT in n-tetradecane (50%, v v ) as the sole sulfur source. Resting cells of recombinant strain MR65 could desulfurize 68 mg l- of sulfur in light gas oil (LGO) containing 126 mg sulfur l-. Strain MR65 had about 1.5-times the LGO desulfurization activity of R. erythropolis strain KA2-5-1. The application of a recombinant, which is able to utilize 4,6-dipropyl DBT in the oil phase, was effective in enhancing LGO biodesulfurization.     

Analyses of substrate specificity of the desulfurizing bacterium Mycobacterium sp. G3

The substrate specificity of Mycobacterium sp. G3 with desulfurization activity against dibenzothiophene (DBT) was investigated. Desulfurization reactions were carried out using a concentrated cell suspension of G3. The conversion from 4,6-dipropyl DBT, one of the sulfur-containing compounds that is difficult to desulfurize in diesel oil, to 2-hydroxy-3,3'-dipropylbiphenyl as an end -product of the desulfurization reaction was found in the water reaction system and in the oil/water two-phase reaction system. 4,6-Dibutyl DBT and 4,6-dipentyl DBT were metabolized to the hydroxybiphenyl form via the sulfone form in the water reaction system. These results indicate that G3 has high membrane permeability and superior substrate specificity for high molecular weight alkyl DBTs, which are represented by 4,6-dipentyl DBT as C10-DBT. Furthermore, G3 could desulfurize diesel oil, and the sulfur concentration was decreased from 116 mg l(-1) to 48 mg l(-1) within 24 h.     

Identification and functional analysis of genes required for desulfurization of alkyl dibenzothiophenes of Mycobacterium sp. G3

Mycobacterium sp. G3 was reported as a dibenzothiophene (DBT)-degrading microorganism and the first strain to have the ability to degrade high-molecular-weight alkyl DBTs, such as 4,6-dibutyl DBT and 4,6-dipentyl DBT, by the C-S bond cleavage pathway. Three genes (mdsA, mdsB, and mdsC) for desulfurization, which form a cluster, were cloned from Mycobacterium sp. G3. The expression of each gene in Escherichia coli JM109 showed that MdsC oxidized DBT to DBT sulfone, MdsA transformed DBT sulfone into 2-(2'-hydroxyphenyl)benzene sulfinate (HPBS), and MdsB desulfinated HPBS into 2-hydroxybiphenyl (HBP), indicating that the gene products of mdsABC are functional in the recombinant. MdsC oxidized 4,6-dimethyl DBT, 4,6-diethyl DBT, 4,6-dipropyl DBT and 4,6-dibutyl DBT to each sulfone form, suggesting that MdsC covers a broad specificity for alkyl DBTs.     

Biodesulfurization of alkylated forms of dibenzothiophene and benzothiophene by Sphingomonas subarctica T7b

Sphingomonas subarctica T7b was isolated from soil in Toyotomi, Hokkaido, Japan as an organism capable of desulfurizing aromatic hydrocarbons in light gas oil (LGO) through enrichment culture. S. subarctica T7b could grow on mineral salt sulfur-free (MSSF) medium with the n-tetradecane oil phase containing dibenzothiophene (DBT), alkyl dibenzothiophenes (alkyl DBTs) or alkyl benzothiophenes (alkyl BTs) as the sole sulfur source and desulfurize these compounds, but could not utilize the tetradecane as a carbon source. This is the first report of a gram-negative bacterium which can desulfurize 4,6-dibutyl DBT and 4,6-dipentyl DBT. The desulfurized product of DBT produced by this strain was 2-hydroxybiphenyl, as in the case of other DBT-desulfurizing bacteria. S. subarctica T7b could desulfurize LGO and the sulfur content was decreased to 41% within 36 h.     

Biodesulfurization of dibenzothiophene and its derivatives through the selective cleavage of carbon-sulfur bonds by a moderately thermophilic bacterium Bacillus subtilis WU-S2B

Heterocyclic organosulfur compounds such as dibenzothiophene (DBT) in petroleum cannot be completely removed by hydrodesulfurization using chemical catalysts. A moderately thermophilic bacterium Bacillus subtilis WU-S2B, which could desulfurize DBT at 50 degrees C through the selective cleavage of carbon-sulfur (CS) bonds, was newly isolated. At 50 degrees C, growing cells of WU-S2B could degrade 0.54 mM DBT within 120 h to produce 2-hydroxybiphenyl, and the resting cells could also degrade 0.81 mM DBT within 12 h. The DBT-desulfurizing ability of WU-S2B is high over a wide temperature range from 30 to 50 degrees C, and highest at 50 degrees C for both the growing and resting cells, and this is an extremely advantageous property for the practical biodesulfurization. In addition, WU-S2B could also desulfurize DBT derivatives such as 2,8-dimethylDBT, 4,6-dimethylDBT and 3,4-benzoDBT. Therefore, B. subtilis WU-S2B is considered to have more beneficial properties than other desulfurizing bacteria such as Rhodococcus strains previously reported, particularly from the viewpoint of its capacity for thermophilic desulfurization through the CS bond cleavage.     

Effect of sulfur sources on specific desulfurization activity of Rhodococcus erythropolis KA2-5-1 in exponential fed-batch culture

The effects of sulfur sources on the desulfurization activity of Rhodococcus erythropolis KA2-5-1 were investigated by using an exponential fed-batch culture technique. The feed rate of a sulfur source was controlled independently of the feed rate of ethanol, which was used as a carbon and energy source. Among the sulfur sources examined were dibenzothiophene (DBT), ammonium sulfate, L-cysteine, L-methionine, and 2-amino-ethanesulfonic acid. When the fed-medium contained DBT as the sole sulfur source, KA2-5-1 cells showed a maximum desulfurization activity of approximately 130 mmol 2-HBP kg-cell(-1) h(-1). Similar levels of enzyme activity were also achieved with inexpensive ammonium sulfate by using the exponential fed-batch culture technique. In addition, higher levels of desulfurization activity were achieved by increasing the dosage of the DBT desulfurization (dsz) operon and dszD gene in R. erythropolis KA2-5-1. The recombinant strain showed a maximum desulfurization activity of approximately 250 mmol 2-HBP kg-cell(-1) h(-1) in the exponential fed-batch cultures.     

热带假丝酵母ctfat1p基因在吸收脂肪族化合物中的功能研究

以热带假丝酵母(Candida tropicalis)1798中的ctfat1p基因为研究对象,利用重叠PCR将ctfat1p基因内约500 bp片段与G418抗性基因(kanr)相连接,经末端单酶切后电转化至C. tropicalis 1798感受态细胞中,通过一次同源单交换,将抗性基因kanr插入至ctfat1p基因内部,实现目的基因的敲除,并通过发酵实验分析Ctfat1p在热带假丝酵母脂肪酸跨膜转运过程中的功能。结果表明,经过G418抗性筛选和基因组PCR鉴定,成功获得ctfat1p基因缺失菌株C. tropicalis 1798 Δctfat1p;分析发现ctfat1p基因敲除对C. tropicalis 1798以油脂为底物培养12 h后重组菌OD600 nm值仅为野生型菌株的47.9%,表明ctfat1p基因敲除后影响C. tropicalis 1798对油脂吸收利用。通过基因敲除手段构建ctfat1p基因缺失菌株,可以减弱细胞对长链脂肪酸的摄取,验证了ctfat1p基因为热带假丝酵母油脂吸收和利用的关键基因。     

Dibenzothiophene desulfurizing enzymes from moderately thermophilic bacterium Bacillus subtilis WU-S2B: purification, characterization and overexpression

The moderately thermophilic bacterium Bacillus subtilis WU-S2B desulfurized dibenzothiophene (DBT) at 50 degrees C through the selective cleavage of carbon-sulfur bonds. In this study, three enzymes involved in the microbial DBT desulfurization were purified and characterized. The first two enzymes, DBT monooxygenase (BdsC) and DBT sulfone monooxygenase (BdsA), were purified from the wild-type strain, and the last one, 2'-hydroxybiphenyl 2-sulfinic acid desulfinase (BdsB), was purified from the recombinant Escherichia coli overexpressing the gene, bdsB, with chaperonin genes, groEL/ES. The genes of BdsC and BdsA were also overexpressed. The molecular weights of BdsC and BdsA were determined to be 200 and 174 kDa, respectively, by gel filtration chromatography, suggesting that both enzymes had four identical subunits. BdsB had a monomeric structure of 40 kDa. The three enzymes were characterized and compared with the corresponding enzymes (DszC, DszA, and DszB) of mesophilic desulfurization bacteria. The specific activities of BdsC, BdsA, and BdsB were 84.2, 855, and 280 units/mg, respectively, and the latter two activities were higher than those of DszA and DszB. The heat stability and optimum temperature of BdsC, BdsA, and BdsB were higher than those of DszC, DszA, and DszB. Other enzymatic properties were investigated in detail.     

Analysis of dibenzothiophene metabolic pathway in Mycobacterium strain G3

The dibenzothiophene (DBT) metabolic pathway in Mycobacterium strain G3, which is classified as a desulfurizing microorganism with the 4S pathway, was analyzed. 2-Hydroxybiphenyl (HBP), which is an end metabolite in the DBT desulfurization reaction, and 2-methoxybiphenyl (MBP) were found in the reaction mixture, and the methoxylation pathway from HBP to MBP was clarified. Although the substrate in the methoxylation reaction was HBP, there was no relationship between expression of the methoxylation activity and that of the desulfurization activity. Then, 4,6-dimethyl DBT, 4,6-diethyl DBT and benzo[b]naphtho[2,1-d]thiophene were metabolized to their methoxy forms via the desulfurization pathway. We established the methoxylation pathway in Mycobacterium G3.     

Biodegradation of long-chain n-paraffins from waste oil of car engine by Acinetobacter sp

Microorganisms that degrade long-chain n-paraffins from used car engine oil were isolated from soil. For the screening, a fraction of n-paraffin prepared from car engine oil was applied as the sole carbon source. The strain was identified as Acinetobacter sp. The ability of the strain to assimilate long-chain n-paraffins was assessed and characterized. The strain mineralized long-chain n-paraffins (0.1% w/v) in the minimal medium after cultivation for 96 h and also reduced the weight of the waste oil added (1% w/v) by 20% after 72 h without an extracellular biosurfactant. When n-hexadecane was fed as substrate, 1-hexadecanol and 1-hexadecanoic acid were detected as the intermediates by gas chromatography/mass spectrometry. This indicates that the long-chain n-paraffins were metabolized via the terminal oxidation pathway of n-alkane.     

Two acyl-CoA dehydrogenases of Acinetobacter sp. strain M-1 that uses very long-chain n-alkanes

Two genes encoding acyl-CoA dehydrogenases, acdA and acdB, arranged in tandem, were found in the chromosomal DNA of Acinetobacter sp. strain M-1. AcdA was purified from the parental strain and AcdB was purified from an Escherichia coli strain expressing the cloned gene. The substrate specificities of the two enzymes suggest that AcdA is a medium-chain acyl-CoA dehydrogenase and that AcdB is a long-chain acyl-CoA dehydrogenase. Characterization of n-alkane metabolism in Acinetobacter sp. strain M-1 has revealed parallel pathways as well as enzymes with overlapping specificities in a single pathway. The two acyl-CoA dehydrogenases described here provide another example of the physiological complexity underlying n-alkane utilization.     

Biosurfactant-producing bacterium, Pseudomonas aeruginosa MA01 isolated from spoiled apples: physicochemical and structural characteristics of isolated biosurfactant

An extensive investigation was conducted to isolate indigenous bacterial strains with outstanding performance for biosurfactant production from different types of spoiled fruits, food-related products and food processing industries. An isolate was selected from 800 by the highest biosurfactant yield in soybean oil medium and it was identified by 16S rRNA and the two most relevant hypervariable regions of this gene; V3 and V6 as Pseudomonas aeruginosa MA01. The isolate was able to produce 12 g/l of a glycolipid-type biosurfactant and generally less efficient to emulsify vegetable oils compared to hydrocarbons and could emulsify corn and coconut oils more than 50%. However, emulsification index (E(24)) of different hydrocarbons including hexane, toluene, xylene, brake oil, kerosene and hexadecane was between 55.8% and 100%. The surface tension of pure water decreased gradually with increasing biosurfactant concentration to 32.5 mNm(-1) with critical micelle concentration (CMC) value of 10.1mg/l. Among all carbon substrates examined, vegetable oils were the most effective on biosurfactant production. Two glycolipid fractions were purified from the biosurfactant crude extracts, and FTIR and ES-MS were used to determine the structure of these compounds. The analysis indicated the presence of three major monorhamnolipid species: R(1)C(10)C(10), R(1)C(10)C(12:1), and R(1)C(10)C(12); as well as another three major dirhamnolipid species: R(2)C(10)C(10), R(2)C(10)C(12:1), and R(2)C(10)C(12). The strain sweep experiment for measuring the linear viscoelastic of biosurfactant showed that typical behavior characteristics of a weak viscoelastic gel, with storage modulus greater than loss modulus at all frequencies examined, both showing some frequency dependence.     

Cloning and expression of the gene encoding the thermophilic NAD(P)H-FMN oxidoreductase coupling with the desulfurization enzymes from Paenibacillus sp. A11-2

The gene encoding the NAD(P)H-flavin oxidoreductase (flavin reductase) which couples with the thermophilic dibenzothiophene (DBT)-desulfurizing monooxygenases of Paenibacillus sp. A11-2 was cloned in Escherichia coli and designated tdsD. Nucleotide sequence analysis suggested that the gene product consisted of 200 amino acids and showed about 30%, 27% and 26% amino acid sequence similarity to the major flavin reductase of Vibrio fischeri, the NADH dehydrogenase of Thermus thermophilus and several oxygen-insensitive NAD(P)H nitroreductases in the Enterobacteriaceae family, respectively. Both the growing and resting recombinant E. coli, in which tdsD was coexpressed with a set of desulfurizing genes, showed a rate of DBT removal about 5 times higher than the recombinants lacking tdsD. Maximal desulfurization was observed close to 45 degrees C and 55 degrees C in the resting cells and in the cell-free extraction reaction with the tdsD-coexpressing recombinants, respectively. In an organic/aqueous biphasic system, the coexpression of tdsD also markedly enhanced the rate of DBT removal.     

发光细菌PB-P3.9的鉴定与gyrB、luxA基因系统发育分析

平板划线分离法从猪肉上分离得到一株发光细菌PB-P3.9,依据细菌DNA促旋酶B亚基基因(gyrB)、荧光素酶A基因(luxA)序列比对及构建系统发育树鉴定其种属,并探讨其来源。扩增gyrB、luxA基因并测序,两个基因序列比对结果与发光杆菌属的明亮发光杆菌(Photobacterium phosphoreum)分别有99%、98%的序列相似性,结合其生物学特性,确定PB-P3.9为一株明亮发光杆菌。根据GenBank 数据库中明亮发光杆菌标准菌株gyrB、luxA基因序列,采用距离法构建系统发育树,表明PB-P3.9株与标准菌株NCIMB 1279有最近亲缘关系,结合其生长条件,拟确定此发光细菌PB-P3.9来源于海洋深海鱼或中层海水浮游生物。     

Increase in desulfurization activity of Rhodococcus erythropolis KA2-5-1 using ethanol feeding

Rhodococcus erythropolis KA2-5-1 is one of the best strains for the desulfurization of dibenzothiophene (DBT) via a sulfur-specific pathway in which DBT is converted to the end product, 2-hydroxybiphenyl, by releasing sulfite via DBT-sulfone and 2-(2'-hydroxyphenyl) benzene sulphinate. The objective of this research is to develop a culture method in order to attain a high cell density with a high level of specific desulfurization activity. Compared with glucose or glycerol, ethanol was found to be a preferable carbon source for obtaining a high specific activity (SA) of desulfurization. When the amount of DBT fed was restricted by feeding 2.9 mg-DBT/g-ethanol solution, the maximum SA and final cell concentration were 135.5 (mmol-2HBP/kg-dry cell weight-h) and 37 (g-dry cell weight/l), respectively. On the other hand, when glucose or glycerol was used as a carbon source, the SA was lower than 50 (mM-2HBP/kg-dry cell weight-h) and the final cell concentration was also lower than 27 (g-dry cell weight/l). The activities of the desulfurization enzymes in R. erythropolis KA2-5-1 grown on ethanol were remarkably higher than when the strain was grown on glucose or glycerol. It was also suggested that NADH, which is produced by the biochemical reaction of NAD with ethanol catalyzed by alcohol dehydrogenase, might contribute to the conversion of FMN to FMNH2, which is a coenzyme for the activities of desulfurization enzymes.     

Purification and characterization of dibenzothiophene (DBT) sulfone monooxygenase, an enzyme involved in DBT desulfurization, from Rhodococcus erythropolis D-1

Dibenzothiophene (DBT), a model of organic sulfur compound in petroleum, is microbially desulfurized to 2-hydroxybiphenyl by Rhodococcus erythropolis D-1. Three desulfurization (Dsz) enzymes--DszC, A, and B--and flavin reductase are involved in sulfur-specific DBT desulfurization. In this study, DszA was purified, characterized, and crystallized from R. erythropolis D-1. DszA, DBT sulfone monooxygenase, is the second enzyme in microbial DBT desulfurization metabolism and catalyzes the conversion of DBT sulfone to 2'-hydroxybiphenyl 2-sulfinic acid in the presence of flavin reductase with cleavage of the carbon-sulfur bond in the DBT skeleton. Using anion-exchange column chromatography, the four enzyme fractions responsible for DBT desulfurization were separated, and DszA was then purified to homogeneity. Polygonal crystals of DszA were observed within a week. DszA was found to have a molecular mass of 97 kDa and to consist of two subunits with identical masses of 50 kDa. The N-terminal amino acid sequence of the purified DszA completely coincided with the deduced amino acid sequence for dszA of R. erythropolis IGTS8 except for a methionine residue at the latter N-terminal. The optimal temperature and pH for DszA activity were 35 degrees C and about 7.5. The activity of the enzyme was inhibited by Mn2+, Ni2+, 2,2'-bipyridine, and 8-quinolinol, suggesting that a metal might be involved in its activity. DszA acted on not only DBT sulfone but also on dibenz[c,e][1,2]oxathiin 6-oxide and dibenz[c,e][1,2]oxathiin 6,6-dioxide. Dihydroxybiphenyl was formed from the latter two substrates.     

Selective cleavage of the two CS bonds in asymmetrically alkylated dibenzothiophenes by Rhodococcus erythropolis KA2-5-1

The Rhodococcus erythropolis strain KA2-5-1 was characterized by its ability to cleave carbon-sulfur bonds in the dibenzothiophene (DBT) ring by asymmetrically alkyl substitution, such as C2-DBTs (e.g., dimethyl and ethyl DBTs) and C3-DBTs (e.g., trimethyl and propyl DBTs), which are known to remain in hydrodesulfurization-treated diesel fuels. After treatment by solid-phase extraction (SPE) of solvents from microbial reactions of alkylated DBTs (Cx-DBTs), we used gas chromatography (GC), GC-atomic emission detection, GC-mass spectrometry and 1H nuclear magnetic resonance spectroscopy to identify and quantitatively evaluate the Cx-DBT metabolites. Molar ratios of metabolic isomers of the desulfurization products suggested that resting-cell reactions of KA2-5-1 against these Cx-DBTs occurrs through specific carbon-sulfur-bond-targeted cleavages, yielding alkylated hydroxybiphenyls, and that the manner of the attack on the DBT skeleton is affected not only by the position but also by the number and length of the alkyl substituents.     

Cloning and expression of the gene encoding the thermophilic NAD(P)H-FMN oxidoreductase coupling with the desulfurization enzymes from Paenibacillus sp. A11-2

The gene encoding the NAD(P)H-flavin oxidoreductase (flavin reductase) which couples with the thermophilic dibenzothiophene (DBT)-desulfurizing monooxygenases of Paenibacillus sp. A11-2 was cloned in Escherichia coli and designated tdsD. Nucleotide sequence analysis suggested that the gene product consisted of 200 amino acids and showed about 30%, 27% and 26% amino acid sequence similarity to the major flavin reductase of Vibrio fischeri, the NADH dehydrogenase of Thermus thermophilus and several oxygen-insensitive NAD(P)H nitroreductases in the Enterobacteriaceae family, respectively. Both the growing and resting recombinant E. coli, in which tdsD was coexpressed with a set of desulfurizing genes, showed a rate of DBT removal about 5 times higher than the recombinants lacking tdsD. Maximal desulfurization was observed close to 45°C and 55°C in the resting cells and in the cell-free extraction reaction with the tdsD-coexpressing recombinants, respectively. In an organic/aqueous biphasic system, the coexpression of tdsD also markedly enhanced the rate of DBT removal.     

Effect of the synthesized mycolic acid on the biodegradation of diesel oil by Gordonia nitida strain LE31

The dynamics of diesel oil biodegradation were previously investigated at initial substrate concentrations of 1000 to 20,000 ppm using Gordonia nitida isolated from wastewater. Following the gas chromatogram profiles of diesel oil degradation, diesel oil with concentrations of up to 15,000 ppm was efficiently degraded by this strain. At a concentrations of 20,000 ppm, however, the degradation by this strain was not effective. The enhancement of the biodegradation of diesel oi1 (at 15,000 and 20,000 ppm) by a synthetic mycolic acid biosurfactant (at 9, 90 and 900 ppm) was also investigated. In G. nitida inoculated cultures, the degradation of diesel oil was enhanced by the biosurfactant. For comparison, diesel oil degradation in batch incubations was measured after the addition of rhamnolipid and other surfactants. Synthetic mycolic acid enhanced the degradation to a greater extent than any other surfactant tested. Additionally, it was demonstrated that the degradation-enhancing property of synthetic mycolic acid was similar to that of rhamnolipid and Tween 80.