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Biomedical subjects

D B Janssen

Publications and source records attributed to D B Janssen.

At least 91 records · Page 5Linked to original sources

Characterization of the epoxide hydrolase from an epichlorohydrin-degrading Pseudomonas sp.

An epoxide hydrolase was purified to homogeneity from the epichlorohydrin-utilizing bacterium Pseudomonas sp. strain AD1. The enzyme was found to be a monomeric protein with a molecular mass of 35 kDa. With epichlorohydrin as the substrate, the enzyme followed Michaelis-Menten kinetics with a Km value of 0.3 mM and a Vmax of 34 mumol.min-1.mg protein-1. The epoxide hydrolase catalyzed the hydrolysis of several epoxides, including epichlorohydrin, epibromohydrin, epoxyoctane and styrene epoxide. With all chiral compounds tested, both stereoisomers were converted. Amino acid sequencing of cyanogen bromide-generated peptides did not yield sequences with similarities to other known proteins.

Amino Acid Sequence↗

Kinetics of chlorinated hydrocarbon degradation by Methylosinus trichosporium OB3b and toxicity of trichloroethylene.

The kinetics of the degradation of trichloroethylene (TCE) and seven other chlorinated aliphatic hydrocarbons by Methylosinus trichosporium OB3b were studied. All experiments were performed with cells grown under copper stress and thus expressing soluble methane monooxygenase. Compounds that were readily degraded included chloroform, trans-1,2-dichloroethylene, and TCE, with Vmax values of 550, 330, and 290 nmol min-1 mg of cells-1, respectively. 1,1-Dichloroethylene was a very poor substrate. TCE was found to be toxic for the cells, and this phenomenon was studied in detail. Addition of activated carbon decreased the acute toxicity of high levels of TCE by adsorption, and slow desorption enabled the cells to partially degrade TCE. TCE was also toxic by inactivating the cells during its conversion. The degree of inactivation was proportional to the amount of TCE degraded; maximum degradation occurred at a concentration of 2 mumol of TCE mg of cells-1. During conversion of [14C]TCE, various proteins became radiolabeled, including the alpha-subunit of the hydroxylase component of soluble methane monooxygenase. This indicated that TCE-mediated inactivation of cells was caused by nonspecific covalent binding of degradation products to cellular proteins.

Biodegradation, Environmental↗

Purification and characterization of haloalcohol dehalogenase from Arthrobacter sp. strain AD2.

An enzyme capable of dehalogenating vicinal haloalcohols to their corresponding epoxides was purified from the 3-chloro-1,2-propanediol-utilizing bacterium Arthrobacter sp. strain AD2. The inducible haloalcohol dehalogenase converted 1,3-dichloro-2-propanol, 3-chloro-1,2-propanediol, 1-chloro-2-propanol, and their brominated analogs, 2-bromoethanol, as well as chloroacetone and 1,3-dichloroacetone. The enzyme possessed no activity for epichlorohydrin (3-chloro-1,2-epoxypropane) or 2,3-dichloro-1-propanol. The dehalogenase had a broad pH optimum at about 8.5 and a temperature optimum of 50 degrees C. The enzyme followed Michaelis-Menten kinetics, and the Km values for 1,3-dichloro-2-propanol and 3-chloro-1,2-propanediol were 8.5 and 48 mM, respectively. Chloroacetic acid was a competitive inhibitor, with a Ki of 0.50 mM. A subunit molecular mass of 29 kDa was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With gel filtration, a molecular mass of 69 kDa was found, indicating that the native protein is a dimer. The amino acid composition and N-terminal amino acid sequence are given.

Amino Acid Sequence↗

Characterization of the haloacid dehalogenase from Xanthobacter autotrophicus GJ10 and sequencing of the dhlB gene.

The haloacid dehalogenase of the 1,2-dichloroethane-utilizing bacterium Xanthobacter autotrophicus GJ10 was purified from a mutant with an eightfold increase in expression of the enzyme. The mutant was obtained by selecting for enhanced resistance to monobromoacetate. The enzyme was purified through (NH4)2SO4 fractionation, DEAE-cellulose chromatography, and hydroxylapatite chromatography. The molecular mass of the protein was 28 kDa as determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 36 kDa as determined with gel filtration on Superose 12 fast protein liquid chromatography. The enzyme was active with 2-halogenated carboxylic acids and converted only the L-isomer of 2-chloropropionic acid with inversion of configuration to produce D-lactate. The activity of the enzyme was not readily influenced by thiol reagents. The gene encoding the haloacid dehalogenase (dhlB) was cloned and could be allocated to a 6.5-kb EcoRI-BglII fragment. Part of this fragment was sequenced, and the dhlB open reading frame was identified by comparison with the N-terminal amino acid sequence of the protein. The gene was found to encode a protein of 27,433 Da that showed considerable homology (60.5 and 61.0% similarity) with the two other haloacid dehalogenases sequenced to date but not with the haloalkane dehalogenase from X. autotrophicus GJ10.

Amino Acid Sequence↗

Degradation of chlorinated aliphatic hydrocarbons by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase.

Degradation of trichloroethylene (TCE) by the methanotrophic bacterium Methylosinus trichosporium OB3b was studied by using cells grown in continuous culture. TCE degradation was a strictly cometabolic process, requiring the presence of a cosubstrate, preferably formate, and oxygen. M. trichosporium OB3b cells degraded TCE only when grown under copper limitation and when the soluble methane monooxygenase was derepressed. During TCE degradation, nearly total dechlorination occurred, as indicated by the production of inorganic chloride, and only traces of 2,2,2-trichloroethanol and trichloroacetaldehyde were produced. TCE degradation proceeded according to first-order kinetics from 0.1 to 0.0002 mM TCE with a rate constant of 2.14 ml min-1 mg of cells-1. TCE concentrations above 0.2 mM inhibited degradation in cell suspensions of 0.42 mg of cells ml-1. Other chlorinated aliphatics were also degraded by M. trichosporium OB3b. Dichloromethane, chloroform, 1,1-dichloroethane, and 1,2-dichloroethane were completely degraded, with the release of stoichiometric amounts of chloride. trans-1,2-Dichloroethylene, cis-1,2-dichloroethylene, and 1,2-dichloropropane were completely converted, but not all the chloride was released because of the formation of chlorinated intermediates, e.g., trans-2,3-dichlorooxirane, cis-2,3-dichlorooxirane, and 2,3-dichloropropanol, respectively. 1,1,1-Trichloroethane, 1,1-dichloroethylene, and 1,3-dichloropropylene were incompletely converted, and the first compound yielded 2,2,2-trichloroethanol as a chlorinated intermediate. The two perchlorinated compounds tested, carbon tetrachloride and tetrachloroethylene, were not converted.

Biodegradation, Environmental↗

Cloning of 1,2-dichloroethane degradation genes of Xanthobacter autotrophicus GJ10 and expression and sequencing of the dhlA gene.

A gene bank from the chlorinated hydrocarbon-degrading bacterium Xanthobacter autotrophicus GJ10 was prepared in the broad-host-range cosmid vector pLAFR1. By using mutants impaired in dichloroethane utilization and strains lacking dehalogenase activities, several genes involved in 1,2-dichloroethane metabolism were isolated. The haloalkane dehalogenase gene dhlA was subcloned, and it was efficiently expressed from its own constitutive promoter in strains of a Pseudomonas sp., Escherichia coli, and a Xanthobacter sp. at levels up to 30% of the total soluble cellular protein. A 3-kilobase-pair BamHI DNA fragment on which the dhlA gene is localized was sequenced. The haloalkane dehalogenase gene was identified by the known N-terminal amino acid sequence of its product and found to encode a 310-amino-acid protein of molecular weight 35,143. Upstream of the dehalogenase gene, a good ribosome-binding site and two consensus E. coli promoter sequences were present.

Amino Acid Sequence↗

Crystallization of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10.

Haloalkane dehalogenases are enzymes that release chloride or bromide from n-halogenated alkanes. X-ray quality crystals of haloalkane dehalogenase from the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 have been grown at room temperature from 64% saturated ammonium sulfate solutions (pH 6.2 to 6.4). The crystals diffract in the X-ray beam to at least 2.4 A resolution (1 A = 0.1 nm). Their space group is P2(1)2(1)2, with cell dimensions a = 94.1 A, b = 72.8 A, c = 41.4 A and alpha = beta = gamma = 90 degrees. There is one monomer (molecular weight 36,000) per asymmetric unit.

Gram-Negative Aerobic Bacteria↗

Purification and characterization of a bacterial dehalogenase with activity toward halogenated alkanes, alcohols and ethers.

An enzyme that is capable of hydrolytic conversion of halogenated aliphatic hydrocarbons to their corresponding alcohols was purified from a 1,6-dichlorohexane-degrading bacterium. The dehalogenase was found to be a monomeric protein of relative molecular mass 28,000. The affinity for its substrates was relatively low with Km values for short-chain haloalkanes in the range 0.1-0.9 mM. The aliphatic dehalogenase showed a much broader substrate range than has been reported for halidohydrolases so far. Novel classes of substrates include dihalomethanes, C5-C9 1-halo-n-alkanes, secondary alkylhalides, halogenated alcohols and chlorinated ethers. Several of these compounds are important environmental pollutants, e.g. methylbromide, dibromomethane, 1,2-dibromoethane, 1,3-dichloropropene, and bis(2-chloroethyl)ether. The degradation of chiral 2-bromoalkanes appeared to proceed without stereochemical preference. Optically active 2-bromobutane was converted with inversion of configuration at the chiral carbon atom, suggesting that the dehalogenase reaction proceeds by a nucleophilic substitution involving a carboxyl group or base catalysis.

Actinomycetales↗

Degradation of n-haloalkanes and alpha, omega-dihaloalkanes by wild-type and mutants of Acinetobacter sp. strain GJ70.

A 1,6-dichlorohexane-degrading strain of Acinetobacter sp. was isolated from activated sludge. The organism could grow with and quantitatively release halide from 1,6-dichlorohexane, 1,9-dichlorononane, 1-chloropentane, 1-chlorobutane, 1-bromopentane, ethylbromide, and 1-iodopropane. Crude extracts contained an inducible novel dehalogenase that liberated halide from the above compounds and also from 1,3-dichloropropane, 1,2-dibromoethane, and 2-bromoethanol. The latter two compounds were toxic suicide substrates for the organism at concentrations of 10 and 5 microM, respectively. Mutants resistant to 1,2-dibromoethane (3 mM) lacked dehalogenase activity and did not utilize haloalkanes for growth. Mutants resistant to both 1,2-dibromoethane (3 mM) and 2-bromoethanol (30 mM) could no longer oxidize or utilize alcohols and were capable of hydrolytic dehalogenation of 1,2-dibromoethane to ethylene glycol.

Acinetobacter↗

Developments in biotechnology of relevance to drinking water preparation.

This paper discusses strategies to increase the feasibility of microorganisms for the removal of toxic xenobiotics from waste water and drinking water. Based on the principles of adaptational mutations and genetic exchange of catabolic activities, it becomes possible to select and engineer microorganisms that are suitable for the degradation of recalcitrant compounds. The detailed biochemical knowledge that is required for this is now rapidly evolving, and especially for the degradation of chlorinated organics several detoxifying dehalogenation mechanisms have been studied in detail. The feasibility of specialized bacteria for waste and water treatment will be dependent on the possibility to obtain stable performance and maintenance in treatment systems.

Adaptation, Physiological↗

Degradation of halogenated aliphatic compounds by Xanthobacter autotrophicus GJ10.

A bacterium that is able to utilize a number of halogenated short-chain hydrocarbons and halogenated carboxylic acids as sole carbon source for growth was identified as a strain of Xanthobacter autotrophicus. The organism constitutively produces two different dehalogenases. One enzyme is specific for halogenated alkanes, whereas the other, which is more heat stable and has a higher pH optimum, is specific for halogenated carboxylic acids. Haloalkanes were hydrolyzed in cell extracts to produce alcohols and halide ions, and a route for the metabolism of 1,2-dichlorethane is proposed. Both dehalogenases show a broad substrate specificity, allowing the degradation of bromine- and chlorine-substituted organic compounds. The results show that X. autotrophicus may play a role in the degradation of organochlorine compounds and that hydrolytic dehalogenases may be involved in the microbial metabolism of short-chain halogenated hydrocarbons in microorganisms.

Biodegradation, Environmental↗

Purification and characterization of hydrolytic haloalkane dehalogenase from Xanthobacter autotrophicus GJ10.

A new enzyme, haloalkane dehalogenase, was isolated from the 1,2-dichloroethane-utilizing bacterium Xanthobacter autotrophicus GJ10. The purified enzyme catalyzed the hydrolytic dehalogenation of n-halogenated C1 to C4 alkanes, including chlorinated, brominated, and iodinated compounds. The highest activity was found with 1,2-dichloroethane, 1,3-dichloropropane, and 1,2-dibromoethane. The enzyme followed Michaelis-Menten kinetics, and the Km for 1,2-dichloroethane was 1.1 mM. Maximum activity was found at pH 8.2 and 37 degrees C. Thiol reagents such as p-chloromercuribenzoate and iodoacetamide rapidly inhibited the enzyme. The protein consists of a single polypeptide chain of a molecular weight of 36,000, and its amino acid composition and N-terminal sequence are given.

Amino Acid Sequence↗

Catabolite repression and nitrogen control of allantoin-degrading enzymes in Pseudomonas aeruginosa.

The formation of the allantoin-degrading enzymes allantoinase, allantoicase and ureidoglycolase in Pseudomonas aeruginosa was found to be regulated by induction, catabolite repression and nitrogen control. Induction was observed when urate, allantoin or allantoate were included in the growth medium, but not with ureidoglycolate. Tricarboxylic acid cycle intermediates exerted catabolite repression of the synthesis of the three enzymes, while pyruvate and glucose caused less repression. The operation of a nitrogen control mechanism in the regulation of the allantoin-degrading enzymes could be demonstrated with glutamine synthetase-negative mutants, which showed elevated synthesis and escape from catabolite repression when growth was limited for glutamine.

Allantoin↗

Allantoinase from Pseudomonas aeruginosa. Purification, properties and immunochemical characterization of its in vivo inactivation.

The catabolic enzyme allantoinase is rapidly inactivated in cells of Pseudomonas aeruginosa when the stationary phase of growth is reached. This process is irreversible since the protein synthesis inhibitor chloramphenicol completely blocked the reappearance of allantoinase activity that is observed when allantoin is added to stationary cells. Purified alloantoinase appeared to be a protein composed of four identical subunits with a molecular weight of 38,000. With antibodies raised against purified allantoinase it was found that allantoinase inactivation is accompanied by a parallel decrease in immunologically reactive material. This suggests that allantoinase inactivation is caused or followed by rapid proteolysis.

Allantoin↗

Regulation of amidase formation in mutants from Pseudomonas aeruginosa PAO lacking glutamine synthetase activity.

The formation of amidase was studied in mutants from Pseudomonas aeruginosa PAO lacking glutamine synthetase activity. It appeared that catabolite repression of amidase synthesis by succinate was partially relieved when cellular growth was limited by glutamine. Under these conditions, a correlation between amidase and urease formation was observed. The results suggest that amidase formation in strain PAO is subject to nitrogen control and that glutamine or some compound derived from it mediates the nitrogen repression of amidase.

Amidohydrolases↗

Nitrogen control in Pseudomonas aeruginosa: mutants affected in the synthesis of glutamine synthetase, urease, and NADP-dependent glutamate dehydrogenase.

Mutants were isolated from Pseudomonas aeruginosa that were impaired in the utilization of a number of nitrogen sources. In contrast to the wild-type strain, these mutants appeared to be unable to derepress the formation of glutamine synthetase and urease under nitrogen-limited growth conditions, whereas NADP-dependent glutamate dehydrogenase became derepressed. This GlnR- phenotype appeared to be caused by a mutation located in the early region of the P. aeruginosa PAO chromosomal map, close to hisIV59. Partial suppression of the GlnR- phenotype due to a mutation located close to hisII4 was observed. These revertants were different from both the wild-type strain and the GlnR- mutant with respect to the regulation of the synthesis of glutamine synthetase, urease, and NADP-dependent glutamate dehydrogenase (GlnRc phenotype). Also the regulation of glutamine synthetase activity by adenylylation/deadenylylation was altered in the revertants. The results suggest the presence of a regulatory gene that plays a role in the regulation of enzyme formation in response to the availability of ammonia.

Chromosomes, Bacterial↗

Characterization of glutamine-requiring mutants of Pseudomonas aeruginosa.

Revertants were isolated from a glutamine-requiring mutant of Pseudomonas aeruginosa PAO. One strain showed thermosensitive glutamine requirement and formed thermolabile glutamine synthase, suggesting the presence of a mutation in the structural gene for glutamine synthetase. The mutation conferring glutamine auxotrophy was subsequently mapped and found to be located at about 15 min on the chromosomal map, close to and before hisII4. Furthermore, in transduction experiments, it appeared to be very closely linked to gln-2022, a suppressor mutation affecting nitrogen control. With immunological techniques, it could be demonstrated that the glutamine auxotrophs form an inactive glutamine synthetase protein which is regulated by glutamine or a product derived from it in a way similar to other nitrogen-controlled proteins.

Chromosome Mapping↗