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PubMed2003

Macrokinetic and quantitative microbial investigation on a bench-scale biofilter treating styrene-polluted gaseous streams.

We performed a macrokinetic and quantitative microbial investigation of a continuously operating bench-scale biofilter treating styrene-polluted gases. The device was filled with a mixture of peat and glass beads as packing medium and inoculated with the styrene-oxidizing strain, Rhodococcus rhodochrous AL NCIMB 13259. The experimental data of styrene and microbial concentrations, obtained at different biofilter heights, were used to evaluate the pollutant concentration profiles as well as the influence of styrene loading on biomass distribution along the packing medium. Styrene and biomass concentration profiles permitted detection of a linear relationship between the amount of biomass grown in a given section of the biofilter and that of pollutant removed, regardless of the operating conditions tested. Biomass development in the bed appeared to: depend linearly on pollutant concentration at an inlet styrene concentration of <0.10 g m(-3) in the gaseous stream; achieve a maximum value (7. 10(7) colony forming units per gram of packing material) within a wide styrene concentration range (0.10 to 1.0 g m(-3)); and fall sharply beyond this inhibition threshold. The process followed zeroth-order macrokinetics with respect to styrene concentration, which is consistent with zeroth-order microkinetics with either fully active or not fully active biofilm. The maximal volumetric styrene removal rate was found to be 63 g m(packing material) (-3) h(-1) for an influent pollutant concentration of 0.80 g m(-3) and a superficial gas velocity of 245 m h(-1).

Adsorption↗

PubMed2004

Purification and characterization of a chemotolerant alcohol dehydrogenase applicable to coupled redox reactions.

The purification and characterization of an organic solvent tolerant, NADH-dependent medium-chain secondary alcohol dehydrogenase (denoted sec-ADH "A") from Rhodococcus ruber DSM 44541 is reported. The enzyme can withstand elevated concentrations of organic solvents, such as acetone (up to 50% v/v) and 2-propanol (up to 80% v/v). Thus, it is ideally suited for the preparative-scale enantioselective oxidation of sec-alcohol and the asymmetric reduction of ketones, using acetone and 2-propanol, respectively, as cosubstrates for cofactor-regeneration via a coupled-substrate approach. The homodimeric protein was found to bear tightly bound zinc and displayed a molecular mass of 38 kDa per subunit as determined by SDS gel electrophoresis. The optimal temperature ranged from 30-50 degrees C and the half-life at 50 degrees C was 35 h. In addition, excellent storage stability was found. The pH optimum for reduction is pH 6.5; pH 9.0 is preferred for oxidation. The enzyme followed a sequential reaction mechanism. The substrates are medium chain sec-alcohols or (omega-1)-ketones; primary alcohols or aldehydes are not accepted.

Alcohol Dehydrogenase↗

PubMed2004

A novel system for expressing recombinant proteins over a wide temperature range from 4 to 35 degrees C.

Escherichia coli cells are the most commonly used host cells for large-scale production of recombinant proteins, but some proteins are difficult to express in E. coli. Therefore, we tested the nocardioform actinomycete Rhodococcus erythropolis, which grows at temperatures ranging from 4 to 35 degrees C, as an expression host cell. We constructed inducible expression vectors, where the expression of the target genes could be controlled with the antibiotic thiostrepton. Using these expression vectors, several milligrams of reporter proteins could be isolated from 1 liter of culture of R. erythropolis cells grown at a temperature range from 4 to 35 degrees C. Moreover, we successfully purified serum amyloid A1, NADH dehydorogenase 1 alpha subcomplex 4, cytochrome b5-like protein, apolipoprotein A-V, cathepsin D, pancreatic Rnase, and HMG-1 that are all difficult to express in E. coli. In the case of kallikrein 6, mouse deoxyribonuclease I and Kid1, which are also difficult to express in E. coli, the expression level of each protein increased when proteins were expressed at low temperature (4 degrees C). Based on these results, we conclude that a recombinant protein expression system using R. erythropolis as the host cell is superior to respective E. coli systems.

Aminopeptidases↗

PubMed2005

Enhanced biotransformations and product recovery in a membrane bioreactor through application of a direct electric current.

The simultaneous enhancement of biotransformation coupled to product recovery, purification and concentration is presented. The nitrilase of Rhodococcus rhodochrous LL100-21 catalyses the single-step hydrolytic biotransformation of benzonitrile to benzoic acid and ammonia. When a direct electric current is applied across a bioreactor containing the bacterium and benzonitrile, the charged product (benzoic acid) can be removed in situ across an anion exchange membrane and recovered in a separate compartment. Over the course of a 24-hour biotransformation, benzonitrile was converted to benzoic acid which was completely removed from the bioreactor chamber and concentrated 3-fold in a separate chamber. The rate of production of benzoic acid increased by 42% when the current was applied (0.044 mmol/min/g dry cell weight in the presence of current as compared to 0.03 mmol/min/g dry cell weight in its absence). The enhanced reaction rate was achieved irrespective of product separation and therefore appears to be a direct effect upon the bacterial cells. This process has potential for enhanced productivity from biotransformations through a simultaneous increase in metabolic activity and in situ product recovery.

Aminohydrolases↗

PubMed2005

The effect of whole cell immobilisation on the biotransformation of benzonitrile and the use of direct electric current for enhanced product removal.

The nitrilase of Rhodococcus rhodochrous performs a one-step biotransformation of nitriles to their corresponding carboxylic acids. Application of a direct electric current moves the charged carboxylic acid towards an anode, across an anion exchange membrane, into a separate compartment. Cells encapsulated within alginate beads (2.9 mm diameter) for protection against the current biotransformed benzonitrile to benzoic acid with a 26% reduction in the biotransformation rate, from 0.054 mmol/min/g dcw with free cells to 0.040 mmol/min/g dcw with immobilised cells. When the electric current was applied, the biotransformation rate increased to 0.047 mmol/min/g dcw and product recovery increased from 19% to 79%.

Alginates↗

PubMed2006

Overcoming the thermodynamic limitation in asymmetric hydrogen transfer reactions catalyzed by whole cells.

Whole lyophilized cells of an Escherichia coli overexpressing the alcohol dehydrogenase (ADH-'A') from Rhodococcus ruber DSM 44541 were used for the asymmetric reduction of ketones to secondary alcohols. The recycling of the required nicotinamide cofactor (NADH) was achieved in a coupled-substrate process. In the course of the reaction the ketone is reduced to the alcohol and the hydrogen donor 2-propanol is oxidized to acetone by one enzyme. This leads to a thermodynamic equilibrium between all four components determining the maximum achievable conversion. To overcome this limitation an in situ product removal technique (ISPR) for the application with whole cells was developed. In this method the most volatile compound is separated from the reaction vessel by an air flow resulting in a shift of the equilibrium towards the desired secondary alcohol. The so-called stripping process represents a simple and efficient method to overcome the thermodynamic limitation in biocatalytic reactions. Employing this method, the conversion of selected biotransformations was increased up to completeness.

Alcohol Dehydrogenase↗

PubMed2004

Searching for nitrile hydratase using the Consensus-Degenerate Hybrid Oligonucleotide Primers strategy.

Consensus-Degenerate Oligonucleotide Hybrid Primers (CODEHOP) were designed by using the WWW-implemented strategy, based on multiple alignments of nitrile hydratase (NHase) alpha subunit, available from EMBL database. These primers were successfully tested with known NHase-producing bacterial strains such as Agrobacterium tumefaciens DSM 9674, Rhodococcus erythropolis DSM 9675, R. erythropolis 9685 and R. erythropolis 11397 and also allowed amplification from organisms not previously referenced (Variovorax sp. DSM 11402 and Agrobacterium sp. DSM 11401). Hybrid primer utilisation was evaluated by analysing the incorporated sequences in cloned PCR fragments. Several primers from the oligonucleotide pool seemed to participate in the amplification of the correct fragment. Common garden soil was used as a source for both acetonitrile culture enrichment screening and direct DNA extraction. A R. erythropolis strain (CCMI 1005) was isolated by culture enrichment and allowed the PCR amplification of a DNA sequence (AJ548493) identical to the NHase coding sequences commonly described for that species, while the direct use of soil DNA as template led to the CODEHOP detection of two putative NHase coding sequences (AJ548498 and AJ548499), which were significantly different from any other known sequence. The phylogenetic relationship between all sequences obtained and the published NHase coding sequences was assessed by neighbour-joining analysis. The results demonstrate the use of consensus-degenerate primers in NHase detection from organisms known to express it and in the screening for new NHase family members.

Agrobacterium tumefaciens↗

PubMed1990

Steroid-1-dehydrogenases in nocardioform bacteria studied by electrophoresis and immuno blotting techniques.

Fifteen noncardioform bacteria strains, capable of transforming steroid compounds were investigated with regard to their range of inducible steroid-1-dehydrogenase (St1DH)1 activities. The St1DHs of these bacteria were compared due to their immuno reactivity in Western blot experiments with a rabbit antiserum raised against the purified St1DH of Rhodococcus rhodochrous 7030. Four strains exhibited a strong immuno reactivity, irrespective of differences in the electrophoretic mobility of the enzymes. Five strains revealed significantly diminished reactivities, and in five strains with a very low St1DH content, no reactivity was found. One strain, designated as Nocardiaspec. 7151, exhibited a high, inducible St1DH activity, but no immunoreaction was found. The absence of immuno reactivity is discussed in connection with the considerably diminished electrophoretic mobility of this enzyme.

Blotting, Western↗

PubMed1998

Characterization of proteins utilized in the desulfurization of petroleum products by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF/MS) with delayed extraction is utilized in linear, reflected-ion and post-source decay (PSD) modes to directly characterize enzymes being developed for use in a petroleum desulfurization process. The DNA sequence for the genes isolated from Rhodococcus sp. strain IGTS8 that produce three of the four enzymes under study had been previously reported with a discrepancy in residue assignments for one of the enzymes, dsz-C. The use of proteolytic digests followed by MALDI/TOF/MS with delayed extraction in the reflected-ion mode provided sequence-specific information with mass accuracies exceeding 40 ppm over a range of masses and signal-to-noise values. Peptide mapping of >80% of the residues was accomplished for all four proteins. The use of PSD established the true sequence for dsz-C, resolving the discrepancy in the literature. A posttranslational loss of N-terminal methionine was observed for each of the four proteins in linear MALDI/MS and was reconfirmed by peptide mapping for three of the proteins.

Amino Acid Sequence↗

PubMed1998

A novel pathway for the metabolism of caffeine by a mixed culture consortium.

A new oxidative pathway for the degradation of caffeine(1,3,7-Trimethylxanthine, I) by a mixed culture consisting of strains belonging to the genera Klebsiella and Rhodococcus is presented. The mixed culture does not initiate degradation by N-demethylation either complete or partial, but instead carries out oxidation at the C-8 position resulting in the formation of 1,3,7-trimethyluric acid (TMU, II) which further gets degraded to 3,6,8-trimethylallantoin (TMA, III). Both TMU and TMA are hitherto not shown to be formed in the microbial system. Further degradation of TMA (III) by caffeine grown cells yields dimethylurea (VII) as one of the metabolites. Oxygen uptake studies indicated that caffeine(I) grown cells oxidized TMU(II), TMA (III), glyoxalic acid (VI), dimethylurea(VII), and monomethylurea(V), but not monomethyl and dimethyluric acids. The mixed culture does not accept theophylline(1,3-dimethylxanthine), theobromine(3,7-dimethylxanthine), and paraxanthine(1,7-dimethylxanthine) as the carbon source.

Caffeine↗

PubMed1998

Nitrilase catalyzes amide hydrolysis as well as nitrile hydrolysis.

While amides were reported to be completely inert as substrates for all nitrilases reported to date, the nitrilase from Rhodococcus rhodochrous J1, which catalyzes the hydrolytic cleavage of the C-N triple bond in nitrile to form acid and ammonium, was surprisingly found to catalyze hydrolysis of amide to acid and ammonium stoichiometrically. This nitrilase exhibited a Km of 2.94 mM for benzamide, similar to that for benzonitrile as the original substrate (2.10 mM), but the Vmax for benzamide was six orders of magnitude lower than that for benzonitrile. Benzamide inhibited the nitrilase reaction in a reversible, apparently competitive manner. A mutant nitrilase containing alanine or serine instead of Cys165, which is essential for nitrilase catalytic activity, showed no amidase activity. This observation demonstrated that Cys165 plays a crucial role in the hydrolysis of amides as well as nitriles. Together with some reports that certain nitrilases were previously noted to produce low amounts of amide as a by-product from nitrile, the above unexpected findings suggested the existence of a common tetrahedral intermediate in the nitrilase reaction involving nitrile or amide as a substrate.

Aminohydrolases↗

PubMed1999

Hydrazide synthesis: novel substrate specificity of amidase.

The amidase from Rhodococcus rhodochrous J1, which hydrolyses amide to acid and ammonia, was found to catalyze the synthesis of hydrazide using hydrazine as a substrate. This is the first report on the hydrazide synthesis through enzymatic reactions. The enzyme also acted on benzoic acid in the presence of hydrazine, yielding benzoic hydrazide. Together with the finding that benzoic hydrazide was converted into benzoic acid (when it was used as a substrate in the absence of hydrazine), these unique characteristics suggest that the reaction route for the formation of the acid from the hydrazide and that of the hydrazide from the acid are reversible to each other via the acyl-enzyme. Not only aromatic hydrazides but also aliphatic hydrazides were synthesized from the corresponding amides and hydrazine.

Amides↗

PubMed1999

Purification and partial characterization of caffeine oxidase--A novel enzyme from a mixed culture consortium.

Cell-free extract prepared from a mixed culture consisting of strains belonging to the genera Klebsiella and Rhodococcus grown in the presence of caffeine contains a novel enzyme, caffeine (1,3, 7-trimethylxanthine) oxidase which catalyzes the oxidation of caffeine at the C-8 position to produce 1,3,7-trimethyluric acid. The enzyme was purified to homogeneity by a combination of ion-exchange and hydrophobic column chromatographies. Both native and SDS/PAGE of the purified enzyme showed a single protein band and the subunit molecular mass of the protein was determined to be 85 kDa. Dichlorophenol indophenol and cytochrome c served as good electron acceptors but NAD and NADP did not. Caffeine served as the best substrate with an apparent K(m) of 11.4 microM. various analogues of theobromine were also effective substrates for caffeine oxidase. The activity was inhibited by o-phenanthroline, H(2)O(2), and methanol, but salicylate, thiol-group blocking reagents, and sodium arsenite, the known xanthine oxidase inhibitors, did not inhibit the reaction. The spectral characteristics of the purified enzyme suggest that it is a flavoprotein containing non-heme iron.

Caffeine↗

PubMed2000

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.

Amino Acid Sequence↗

PubMed2002

Development of symbionts in triatomine bugs and the effects of infections with trypanosomatids.

In the intestinal tract of fifth instars of the hematophagous reduviid bugs Rhodnius prolixus and Triatoma infestans blood ingestion induced an initial decrease of the concentration of the respective symbiotic bacteria Rhodococcus rhodnii and Nocardia sp. and then within 10 days a 15- or 18-fold increase of the total population/bug to about 0.8 x 10(9) colony-forming units in R. prolixus and 1.8 x 10(9) colony-forming units in T. infestans. About 95-99% of the total populations of both symbionts developed in the anterior midgut regions, i.e., cardia and stomach. The passage from the blood-storing stomach to the digesting small intestine caused a considerable breakdown of symbiont populations, and only about 0.01% of the total population was present in the rectum. These were excreted mainly within 4 h after a blood meal. After infection with three species of trypanosomatids, R. rhodnii, the symbiont of R. prolixus, was affected by Trypanosoma rangeli, but not by Blastocrithidia triatomae or Trypanosoma cruzi. On the other hand, in T. infestans the concentration of Nocardia sp. was reduced after infection with B. triatomae, but not by T. rangeli nor T. cruzi. In long-term B. triatomae-infected T. infestans, this reduction and a reduced diuretic activity after feeding synergistically lowered the symbiont concentration in the singly deposited feces/urine drops drastically compared to uninfected controls. These data strongly support the theory of the mechanisms of pathogenicity of T. rangeli and B. triatomae for R. prolixus and T. infestans, respectively, that the primal point of attack is the host-specific symbiont, R. rhodnii and Nocardia sp., respectively.

Actinomycetales↗

PubMed1992

Isopropylbenzene (cumene)--a new substrate for the isolation of trichloroethene-degrading bacteria.

Various bacterial isolates from enrichments with isopropylbenzene (cumene), toluene or phenol as carbon and energy sources were tested as to their potential to oxidize trichloroethene (TCE). In contrast to toluene and phenol, all isolates enriched on isopropylbenzene were able to oxidize TCE. Two isolates, strain JR1 and strain BD1, were identified as Pseudomonas spec. and as Rhodococcus erythropolis, respectively. TCE oxidation was accompanied by the liberation of stoichiometric amounts of chloride. Initial TCE oxidation rate increased proportional to the substrate concentration from 25 to 200 microM TCE. Maximal initial TCE-degradation rates found here were 4 to 5 nmol.min-1.mg protein-1. The TCE degradation rate decreased with time. The two isolates showed a temperature optimum for TCE degradation between 10 and 20 degrees C. In addition to TCE, R. erythropolis BD1 degraded only cis- and trans-dichloroethene whereas Pseudomonas spec. JR1 was able to oxidize also 1,1-dichloroethene, vinyl chloride, trichloroethane, and 1,2-dichloroethane.

Benzene Derivatives↗

PubMed1990

Selection of trichloroethene (TCE) degrading bacteria that resist inactivation by TCE.

Two isoprene (2-methyl-1,3-butadiene) utilizing bacteria, Alcaligenes denitrificans ssp. xylosoxidans JE 75 and Rhodococcus erythropolis JE 77, were identified as highly efficient cooxidizers of TCE, cis- and trans-dichloroethene, 1,1-dichloroethene and vinyl-chloride. Isoprene grown cells eliminate chloride from TCE in stoichiometric amounts and tolerate high concentrations of TCE.

Alcaligenes↗

PubMed1996

The microbiological fate of polycyclic aromatic hydrocarbons: carbon and oxygen balances for bacterial degradation of model compounds.

A series of pure bacterial strains belonging mainly to the Rhodococcus and Pseudomonas genera were grown on one of the following polycyclic aromatic hydrocarbons (PAH) supplied as sole carbon and energy source; naphthalene, fluorene, phenanthrene, anthracene, fluoranthene and pyrene. In each case, a quantitative evaluation of the carbon repartition of the PAH degraded into CO2, biomass and water-soluble metabolites was carried out. In addition, the kinetics of oxygen consumption and of water-soluble metabolite accumulation during PAH biodegradation was followed with respirometric equipment. Satisfactory carbon balances were obtained and the data correlated well with oxygen consumption values. The results show that growth on PAH presents high mineralization yields (from 56% to 77% of carbon) and sizeable production of biomass (from 16% to 35% of carbon) and limited but significant accumulation of metabolites (from 5% to 23% of carbon). The mineralization yields were higher and biomass yields lower in the case of higher PAH. Some differences between strains were also observed.

Biodegradation, Environmental↗