Cloning and expression of a haloacid dehalogenase fromPseudomonas sp. strain 19 S

A dehalogenase gene specifying the utilization of a variety of haloacids byPseudomonas sp. Strain 19S has been cloned and expressed inE. coli. Our cloning strategy employed specific amplification of a fragment homologous toPseudomonas dehalogenase gene by Polymerase Chain Reaction (PCR). The PCR amplicon successfully acted as a probe to detect the dehalogenase gene in the Southern Blot of the digestedPseudomonas total DNA. Corresponding fragments were cloned into pUC 18 vector and amplified inE. coli MV 1190. One clone with a substantial dehalogenation activity carried a recombinant plasmid containing a 5.5 kb insert.Abbreviations 2-CPA 2-chloropropionate - MCA monochloro acetate - IPTG isopropyl-1-thio--D-galactoside - NBT nitroblue tetrazolium salt - PCR polymerase chain reaction - X-gal 5-bromo-4-chloro-3-indolyl--D-galactoside - X-phosphate 5-bromo-4-chloro-3-indolyl phosphate     

Overexpression and feasible purification of thermostable L-2-halo acid dehalogenase ofPseudomonas sp. YL

The gene encoding thermostable L-2-halo acid dehalogenase ofPseudomonas sp. YL was isolated, and its over-expression system was constructed. Gene library was prepared fromSau3AI fragments of total DNA fromPs. sp. YL, pUC118 as a vector andEscherichia coli JM109 as a host. The recombinant cells resistant to bromoacetate, a germicide, were isolated and shown to produce L-2-halo acid dehalogenase. Subsequently, subcloning was carried out with pKK223-3 as a vector, and the length of DNA inserted was reduced to 1.1 kbp. One of the subclones showed very high activity, and the amount of the dehalogenase produced corresponded to about 30% of the soluble protein. From 5 g (wet weight) of cells, 105 mg of dehalogenase was efficiently purified by heat treatment and DEAE-Toyopearl chromatography. This overexpression system provides a large amount of the thermostable enzyme to enable us to study the properties, structure and application of the enzyme.Abbreviations IPTG isopropyl -D-thiogalactopyranoside - KPB potassium phosphate buffer - SDS sodium dodecyl sulfate - X-gal 5-bromo-4-chloro-3-indolyl--D-galactoside     

Purification, characterization, and gene cloning of a novel fluoroacetate dehalogenase from Burkholderia sp. FA1

Fluoroacetate dehalogenase catalyzes the hydrolytic defluorination of fluoroacetate to produce glycolate. The enzyme is unique in that it catalyzes the cleavage of the highly stable carbon–fluorine bond in an aliphatic compound. The bacterial isolate FA1, which was identified as Burkholderia, grew on fluoroacetate as the sole carbon source to produce fluoroacetate dehalogenase (FAc-DEX FA1). The enzyme was purified to homogeneity and characterized. The molecular weights were estimated to be 79,000 and 34,000 by gel filtration and SDS-polyacrylamide gel electrophoresis (PAGE), respectively, suggesting that the enzyme is a dimer. The purified enzyme was specific to haloacetates, and fluoroacetate was the best substrate. The activities toward chloroacetate and bromoacetate were less than 5% of the activity toward fluoroacetate. The K_m and V_max values for the hydrolysis of fluoroacetate were 5.1 mM and 11 μmol per minute milligram, respectively. The gene coding for the enzyme was isolated, and the nucleotide sequence was determined. The open reading frame consisted of 912 nucleotides, corresponding to 304 amino acid residues. Although FAc-DEX FA1 showed high sequence similarity to fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX H1) (61% identity), the substrate specificity of FAc-DEX FA1 was significantly different from that of FAc-DEX H1: FAc-DEX FA1 was more specific to fluoroacetate than FAc-DEX H1.     

Identification of 4-chlorobenzoyl-coenzyme A as intermediate in the dehalogenation catalysed by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3

The intermediate in the reaction catalysed by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 was identified as 4-chlorobenzoyl-CoA. One component of 4-chlorobenzoate debalogenase worked as a a 4-chlorobenzoyl-CoA ligase catalysing the formation of 4-chlorobenzoyl-CoA from 4-chlorobenzoate, coenzyme A and ATP. This intermediate was detected spectrophotometrically and by HPLC. 4-chlorobenzoyl-CoA was the substrate for the dehalogenase component, which catalysed the conversion to 4-hydroxybenzoate with concomitant release or coenzyme A.     

The purification and characterisation of 4-chlorobenzoate:CoA ligase and 4-chlorobenzoyl CoA dehalogenase from Arthrobacter sp. strain TM-1

4-Chlorobenzoate:CoA ligase, the first enzyme in the pathway for 4-chlorobenzoate dissimilation, has been partially purified from Arthrobacter sp. strain TM-1, by sequential ammonium sulphate precipitation and chromatography on DEAE-Sepharose and Sephacryl S-200. The enzyme, a homodimer of subunit molecular mass approximately 56 kD, is dependent on Mg2+-ATP and coenzyme A, and produces 4-chlorobenzoyl CoA and AMP. Besides Mg2+, Mn2+, Co2+, Fe2+ and Zn2+ are also stimulatory, but not Ca2+. Maximal activity is exhibited at pH 7.0 and 25 degrees C. The ligase demonstrates broad specificity towards other halobenzoates, with 4-chlorobenzoate as best substrate. The apparent Michaelis constants (Km) of the enzyme for 4-chlorobenzoate, CoA and ATP were determined as 3.5, 30 and 238 microM respectively. 4-Chlorobenzoyl CoA dehalogenase, the second enzyme, has been purified to homogeneity by sequential column chromatography on hydroxyapatite, DEAE-Sepharose and Sephacryl S-200. It is a homotetramer of 33 kD subunits with an isoelectric point of 6.4. At pH 7.5 and 30 degrees C, Km and kcat for 4-CBCoA are 9 microM and 1 s(-1) respectively. The optimum pH is 7.5, and maximal enzymic activity occurs at 45 degrees C. The properties of this enzyme are compared with those of the 4-chlorobenzoyl CoA dehalogenases from Arthrobacter sp. strain 4-CB1 and Pseudomonas sp. strain CBS-3, which differ variously in their N-terminal amino acid sequences, optimal pH values, pI values and/or temperatures of maximal activity.     

R-2-氯丙酸脱卤酶的定向进化

通过易错PCR手段将R-2-氯丙酸脱卤酶定向进化,并使用基于Cl-浓度显色反应的高通量筛选得到有效突变子库,发现突变子DehDIV-G2和DehDIV-E7的酶比活力分别提高25.2%和38.7%。通过SYBYL对酶与底物进行分子对接显示,DehDIV-G2的活化能下降0.961 4 kJ/mol,DehDIV-E7的活化能下降2.549 8 kJ/mol。由于酶和底物R-2-氯丙酸的活化能下降,亲和能力提高,从而提高酶的比活力。     

Biochemical and molecular characterisation of the 2,3-dichloro-1-propanol dehalogenase and stereospecific haloalkanoic dehalogenases from a versatile <Emphasis Type="Italic">Agrobacterium</Emphasis> sp.

We previously reported the presence of both haloalcohol and haloalkanoate dehalogenase activity in the Agrobacterium sp. strain NHG3. The versatile nature of the organism led us to further characterise the genetic basis of these dehalogenation activities. Cloning and sequencing of the haloalcohol dehalogenase and subsequent analysis suggested that it was part of a highly conserved catabolic gene cluster. Characterisation of the haloalkanoate dehalogenase enzyme revealed the presence of two stereospecific enzymes with a narrow substrate range which acted on d -2-chloropropionic and I-2-chloropropionoic acid, respectively. Cloning and sequencing indicated that the two genes were separated by 87 bp of non-coding DNA and were preceded by a putative transporter gene 66 bp upstream of the d-specific enzyme.     

Biochemical and structural studies of a l-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii

Haloacid dehalogenases have potential applications in the pharmaceutical and fine chemical industry as well as in the remediation of contaminated land. The l-2-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii has been cloned and over-expressed in Escherichia coli and successfully purified to homogeneity. Here we report the structure of the recombinant dehalogenase solved by molecular replacement in two different crystal forms. The enzyme is a homodimer with each monomer being composed of a core-domain of a β-sheet bundle surrounded by α-helices and an α-helical sub-domain. This fold is similar to previously solved mesophilic l-haloacid dehalogenase structures. The monoclinic crystal form contains a putative inhibitor l-lactate in the active site. The enzyme displays haloacid dehalogenase activity towards carboxylic acids with the halide attached at the C2 position with the highest activity towards chloropropionic acid. The enzyme is thermostable with maximum activity at 60°C and a half-life of over 1 h at 70°C. The enzyme is relatively stable to solvents with 25% activity lost when incubated for 1 h in 20% v/v DMSO.     

X-ray structure and mechanism of ZgHAD,a l-2-haloacid dehalogenase from the marine Flavobacterium Zobellia galactanivorans

Haloacid dehalogenases are potentially involved in bioremediation of contaminated environments and few have been biochemically characterized from marine organisms. The l -2-haloacid dehalogenase (l -2-HAD) from the marine Bacteroidetes Zobellia galactanivorans Dsij^T (ZgHAD) has been shown to catalyze the dehalogenation of C2 and C3 short-chain l -2-haloalkanoic acids. To better understand its catalytic properties, its enzymatic stability, active site, and 3D structure were analyzed. ZgHAD demonstrates high stability to solvents and a conserved catalytic activity when heated up to 60°C, its melting temperature being at 65°C. The X-ray structure of the recombinant enzyme was solved by molecular replacement. The enzyme folds as a homodimer and its active site is very similar to DehRhb, the other known l -2-HAD from a marine Rhodobacteraceae. Marked differences are present in the putative substrate entrance sites of the two enzymes. The H179 amino acid potentially involved in the activation of a catalytic water molecule was confirmed as catalytic amino acid through the production of two inactive site-directed mutants. The crystal packing of 13 dimers in the asymmetric unit of an active-site mutant, ZgHAD-H179N, reveals domain movements of the monomeric subunits relative to each other. The involvement of a catalytic His/Glu dyad and substrate binding amino acids was further confirmed by computational docking. All together our results give new insights into the catalytic mechanism of the group of marine l -2-HAD.     

一株兼性氧化亚氮还原菌的还原N2O能力

【目的】从水稻土中分离筛选出一株兼性氧化亚氮还原菌,并探索其在不同条件下还原N_2O的能力,为减少温室气体N_2O的排放提供重要依据。【方法】通过微生物富集培养分离技术从水稻土中分离得到纯菌;利用nosZ基因和16S rRNA的测序分析鉴定菌株;通过测定菌株在不同条件下N_2O的还原量,分析该菌株还原N_2O的能力及调控因子。【结果】经鉴定,该菌株含有nos Z基因,属于假单胞菌属,在温度30°C、厌氧条件下还原N_2O速率高达0.0219μmol/min以上,改变不同温度和氧气浓度后其能力相对减弱,但仍具备较强的还原N_2O作用。【结论】从水稻土中分离筛选得到的兼性氧化亚氮还原菌为假单胞菌,它在不同环境条件下都具备较强的还原N_2O能力,该菌株可能为减少土壤N_2O排放提供新途径,对保障生态环境安全具有重要的应用价值。     

Construction of a Tn5 derivative encoding bioluminescence and its introduction in Pseudomonas, Agrobacterium and Rhizobium

Summary A simple method based upon the use of a Tn5 derivative, Tn5-Lux, has been devised for the introduction and stable expression of the character of bioluminescence in a variety of gram-negative bacteria. In Tn5-Lux, the luxAB genes of Vibrio harveyi encoding luciferase are inserted on a SalI-BglII fragment between the kanamycin resistance (Km^r) gene and the right insertion sequence. The transposon derivative was placed on a transposition suicide vehicle by in situ recombination with the Tn5 suicide vector pGS9, to yield pDB30. Mating between Escherichia coli WA803 (pDB30) and a strain from our laboratory, Pseudomonas sp. RB100C, gave a Km^r transfer frequency of 10^-6 per recipient, a value 10 times lower than that obtained with the original suicide vehicle pGS9. Tn5-Lux was also introduced by insertion mutagenesis in other strains of gram-negative soil bacteria. The bioluminescence marker was expressed in the presence of n-decanal, and was monitored as chemiluminescence in a liquid scintillation counter. The recorded light intensities were fairly comparable among the strains, and ranged between 0.2 to 1.8x10⁶ cpm for a cell density of 10³ colony forming units/ml. Nodules initiated by bioluminescent strains of Rhizobium leguminosarum on two different hosts were compared for intensity of the bioluminescence they produced.     

Studies on the anaerobic degradation of benzoic acid and 2-aminobenzoic acid by a denitrifying Pseudomonas strain

The growth of a denitrifying Pseudomonas strain on benzoic acid and 2-aminobenzoic acid (anthranilic acid) has been studied. The organism grew aerobically on benzoate, 2-aminobenzoate, and gentisate, but not on catechol or protocatechuic acid. These and other findings suggest that aerobic degradation of benzoic acid was via gentisic acid. Under completely anaerobic conditions in the presence of nitrate, benzoate and 2-aminobenzoate (5 mM each) were oxidized to CO₂ with the concurrent reduction of NO ₃ ^- to NO ₂ ^- . Only after complete NO ₃ ^- consumption was NO ₂ ^- reduced to N₂. Cells contained a NADP-specific 2-oxoglutaate dehydrogenase, in contrast to a NAD-specific pyruvate dehydrogenase. During anaerobic metabolism of [carboxyl-¹⁴C]benzoic acid, 16% of the label of metabolized benzoic acid was incorporated into cell material; this excludes intermediary decarboxylation during anaerobic metabolism. Extracts catalysed the activation of benzoic acid and a variety of its derivatives to the respective aryl-coenzyme A thioesters, ATP being cleaved to AMP and PP_i; two synthetase activites were present. Extracts from 2-aminobenzoate-grown cells catalyzed a NADH-dependent reduction of 2-aminobenzoyl-CoA (100 nmol·min^-1·mg^-1 cell protein) to an unidentified CoA thioester, with a stoichiometric release of NH₃ and a stoichiometry of 3 mol NADH oxidized per mol 2-aminobenzyol-CoA reduced when tested under aerobic conditions. The 2-aminobenzoyl-CoA reductase activity was lacking in anaerobic benzoate-grown cells and in aerobic cells. This is taken as evidence that 2-aminobenzoyl-CoA reductase is a key enzyme in a novel reductive pathway of anaerobic 2-aminobenzoic acid metabolism.Dedicated to Prof. Charles W. Evans     

A novel biotransformation of 2-formyl-6-naphthoic acid to 2,6-naphthalene dicarboxylic acid by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. for the purification of crude 2,6-naphthalene dicarboxylic acid

Crude 2,6-naphthalene dicarboxylic acid was purified by Pseudomonas sp. HN-72 which biotransformed the major impurity of 2-formyl-6-naphthoic acid into 2,6-naphthalene dicarboxylic acid. The biotransformation yield reached 100% when the reaction was performed at 40°C for 1 h, in 200 ml KH₂PO₄/KOH buffer (50 mM, pH 8.0), with 0.2% (w/v) crude 2,6-naphthalene dicarboxylic acid and 2.5 mg dry cell wt/ml.     

Properties of tetrachloroethene and trichloroethene dehalogenase of Dehalospirillum multivorans

Some properties of tetrachloroethene and trichloroethene dehalogenase of the recently isolated, tetrachloroethene-utilizing anaerobe, Dehalospirillum multivorans, were studied with extracts of cells grown on pyruvate plus fumarate. The dehalogenase catalyzed the oxidation of reduced methyl viologen with tetrachloroethene (PCE) or trichloroethene (TCE) as electron acceptor. All other artificial or physiological electron donors tested were ineffective. The PCE and TCE dehalogenase activity was insensitive towards oxygen in crude extracts. When extracts were incubated under anoxic conditions in the presence of titanium citrate as reducing agent, the dehalogenase was rapidly inactivated by propyl iodide (50 M). Inactivation did not occur in the absence of titanium citrate. The activity of propyl-iodide-treated extracts was restored almost immediately by illumination. The dehalogenase was inhibited by cyanide. The inhibition profile was almost the same under oxic and anoxic conditions independent of the presence or absence of titanium citrate. In addition, N₂O, nitrite, and ethylene diamine tetra-acetate (EDTA) were inhibitors of PCE and TCE dehalogenase. Carbon monoxide and azide had no influence on the dehalogenase activity. Trans-1,2-dichloroethene or 1,1-dichloroethene, both of which are isomers of the dechlorination product cis-1,2-dichloroethene, neither inhibited nor inactivated the dehalogenase. PCE and TCE dechlorination appeared to be mediated by the same enzyme since the inhibitors tested had nearly the same effects on the PCE and TCE dehalogenating activity. The data indicated the involvement of a corrinoid and possibly of an additional transition metal in reductive PCE and TCE dechlorination.Abbreviations PCE Tetrachloroethene - TCE Trichloroethene - DCE Dichloroethene - EDTA Ethylene diamine tetra-acetate - MV Methyl viologen - BV Benzyl viologen - PI Propyl iodide, 1-iodopropane - TC Titanium(III) citrate     

7,10-Dihydroxy-8(E)-octadecenoic acid produced by Pseudomonas 42A2: evaluation of different cultural parameters of the fermentation

A fermentation process for the microbial production of a new lipid surface-active compound, 7,10-dihydroxy-8 (E)-octadecenoic acid (OCD), has been established using a vegetable oil as carbon source in a coordinated carbon/nitrogen feed strategy. The surfactant was produced during the logarithmic growth phase. Aeration was the most critical parameter for product formation. Up to 7 g product/l was produced.The authors are with the Laboratorio de Microbiologia, Facultad de Farmacia, Avenida Diagonal 643, Universidad de Barcelona, 08028 Barcelona, Spain     

Biodegradation and detoxification of nicotine in tobacco solid waste by a Pseudomonas sp.

A Pseudomonas sp. grew with nicotine optimally 3 g l^–1 and at 30 °C and pH 7. Nicotine was fully degraded within 10 h. The resting cells degraded nicotine in tobacco solid waste completely within 6 h in 0.02 m sodium phosphate buffer (pH 7) at maximally 56 mg nicotine h^–1 g dry cell^–1.     

Biodegradation of haloalkanes

Halogenated alkanes constitute a significant group among the organic pollutants of environmental concern. Their industrial and agricultural uses are extensive, but until 1978 they were considered to be non-biodegradable. In recent years, microorganisms were described that could degrade, partially or fully, singly or in consortia, many of the compounds tested. The first step in haloalkane degradation appears to be universal: removal of the halogen atom(s). This is mediated by a group of enzymes, generally known as dehalogenases, acting in most cases either as halidohydrolases or oxygenases. Nevertheless, information is still severely lacking regarding the biochemical pathways involved in these processes, as well as their genetic control.A recently isolated Pseudomonas strain, named ES-2, was shown to possess a very wide degradative spectrum, and to contain at least one hydrolytic dehalogenase. The utilization by this organism of water-insoluble haloalkanes, such as 1-bromooctane, appears to consist of three phases: extracellular emulsification by a constitutively excreted surface active agent, periplasmic dehalogenation by an inducible dehalogenase, and intracellular degradation of the residual carbon skeleton.     

A new -2-haloacid dehalogenase acting on 2-haloacid amides: purification, characterization, and mechanism

-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of - and -2-haloalkanoic acids to produce the corresponding - and -2-hydroxyalkanoic acids, respectively. We have constructed an overproduction system for -2-haloacid dehalogenase from Pseudomonas putida PP3 ( -DEX 312) and purified the enzyme to analyze the reaction mechanism. When a single turnover reaction of -DEX 312 was carried out in H₂¹⁸O by use of a large excess of the enzyme with - or -2-chloropropionate as a substrate, the lactate produced was labeled with ¹⁸O. This indicates that the solvent water molecule directly attacked the substrate and that its oxygen atom was incorporated into the product. This reaction mechanism contrasts with that of -2-haloacid dehalogenase, which has an active-site carboxylate group that attacks the substrate to displace the halogen atom. -DEX 312 resembles -2-haloacid dehalogenase from Pseudomonas sp. 113 ( -DEX 113) in that the reaction proceeds with a direct attack of a water molecule on the substrate. However, -DEX 312 is markedly different from -DEX 113 in its substrate specificity. We found that -DEX 312 catalyzes the hydrolytic dehalogenation of 2-chloropropionamide and 2-bromopropionamide, which do not serve as substrates for -DEX 113. -DEX 312 is the first enzyme that catalyzes the dehalogenation of 2-haloacid amides.     

Biotransformation of oleic acid into 10-hydroxy-8E-octadecenoic acid by Pseudomonas sp. 42A2

Pseudomonas sp. 42A2 when incubated for 36 h with oleic acid (20 g l^–1) in a stirred bioreactor, accumulated 10-hydroxy-8E-octadecenoic acid. Production in a 2 l bioreactor with 1.4 l of working volume, was increased from 0.65 g l^–1 to 7.4 g l^–1 with K _L a values ranging between 15 and 200 h^–1. A linear relationship was found between volumetric productivity and oxygen transfer rates and an exponential relation between the specific rate of product formation and specific growth rate.     

Degradation of carbazole and its derivatives by a Pseudomonas sp.

Carbazole, carbazoles with monomethyl or dimethyls substituted on different positions (C₁-carbazoles or C₂-carbazoles), and benzocarbazoles, as toxic and mutagenic components of petroleum and creosote contamination, were biodegradable by an isolated bacterial strain Pseudomonas sp. XLDN4-9. C₁-carbazoles were degraded in preference to carbazole and C₂-carbazoles. The biodegradation of C₁-carbazoles or C₂-carbazoles was influenced by the positions of methyl substitutions. Among C₁-carbazole isomers, 1-methyl carbazole was the most susceptible. C₂-carbazole isomers with substitutions on the same benzo-nucleus were more susceptible at a concentration of less than 3.4 μg g⁻¹ petroleum, especially when harboring one substitution on position 1. In particular, 1,5-dimethyl carbazole was the most recalcitrant dimethyl isomer.