Insights into the Binding Interaction of Catechol 1,2-Dioxygenase with Catechol in Achromobacter xylosoxidans DN002.

Microbial remediation has become one of the promising ways to eliminate polycyclic aromatic hydrocarbons (PAHs) pollution due to its efficient enzyme metabolism system. Catechol 1,2-dioxygenase (C12O) is a crucial rate-limiting enzyme in the degradation pathway of PAHs in Achromobacter xylosoxidans DN002 that opens the benzene ring through the ortho-cleavage pathway. However, little attention has been given to explore the interaction mechanism of relevant enzyme-substrate. This study aims to investigate the binding interaction between C12O of strain DN002 and catechol by means of a molecular biological approach combined with homology modeling, molecular docking, and multiple spectroscopies. The removal rate of catechol in the mutant strain of cat A deletion was only 12.03%, compared to the wild-type strain (54.21%). A Ramachandran plot of active site regions of the primary amino acid sequences in the native enzyme showed that 93.5% sequences were in the most favored regions on account of the results of homology modeling, while an additional 6.2% amino acid sequences were found in conditionally allowed regions, and 0.4% in generously allowed regions. The binding pocket of C12O with catechol was analyzed to obtain that the catalytic trimeric group of Tyr164-His224-His226 was proven to be great vital for the ring-opening reaction of catechol by molecular docking. In the native enzyme, binding complexes were spontaneously formed by hydrophobic interactions. Binding constants and thermodynamic potentials from fluorescence spectra indicated that catechol effectively quenched the intrinsic fluorescence of C12O in the C12O/catechol complex via conventional static and dynamic quenching mechanisms of C12O. The results of ultraviolet and visible (UV) spectra, synchronous fluorescence, and circular dichroism (CD) spectra revealed conspicuous changes in the local conformation, and site-directed mutagenesis confirmed the role of predicted key residues during catalysis, wherein His226 had a significant effect on catechol utilization by C12O. This is the first report to reveal interactions of C12O with substrate from the molecular docking results, providing the mechanistic understanding of representative dioxygenases involved in aromatic compound degradation, and a solid foundation for further site modifications as well as strategies for the directed evolution of this enzyme.

Expression and characterization of catechol 1,2-dioxygenase from Oceanimonas marisflavi 102-Na3.

The gene of catechol 1, 2-dioxygenase was identified and cloned from the genome of Oceanimonas marisflavi 102-Na3. The protein was expressed in Escherichia coli BL21 (DE3) and purified to homogeneity of a dimer with molecular mass of 69.2 kDa. The enzyme was highly stable in pH 6.0-9.5 and below 45 °C and exhibited the maximum activity at pH 8.0 and 30 °C. Being the first characterized intradiol dioxygenase from marine bacteria Oceanimonas sp., the enzyme showed catalytic activity for catechol, 3-methylcatechol, 4-methylcatechol, 3-chlorocatechol, 4-chlorocatechol and pyrogallol. For catechol, Km and Vmax were 11.2 µM and 13.4 U/mg of protein, respectively. The enzyme also showed resistance to most of the metal ions, surfactants and organic solvents, being a promising biocatalyst for biodegradation of aromatic compounds in complex environments.

In-situ muconic acid extraction reveals sugar consumption bottleneck in a xylose-utilizing Saccharomyces cerevisiae strain.

BACKGROUND: The current shift from a fossil-resource based economy to a more sustainable, bio-based economy requires development of alternative production routes based on utilization of biomass for the many chemicals that are currently produced from petroleum. Muconic acid is an attractive platform chemical for the bio-based economy because it can be converted in chemicals with wide industrial applicability, such as adipic and terephthalic acid, and because its two double bonds offer great versatility for chemical modification. RESULTS: We have constructed a yeast cell factory converting glucose and xylose into muconic acid without formation of ethanol. We consecutively eliminated feedback inhibition in the shikimate pathway, inserted the heterologous pathway for muconic acid biosynthesis from 3-dehydroshikimate (DHS) by co-expression of DHS dehydratase from P. anserina, protocatechuic acid (PCA) decarboxylase (PCAD) from K. pneumoniae and oxygen-consuming catechol 1,2-dioxygenase (CDO) from C. albicans, eliminated ethanol production by deletion of the three PDC genes and minimized PCA production by enhancing PCAD overexpression and production of its co-factor. The yeast pitching rate was increased to lower high biomass formation caused by the compulsory aerobic conditions. Maximal titers of 4 g/L, 4.5 g/L and 3.8 g/L muconic acid were reached with glucose, xylose, and a mixture, respectively. The use of an elevated initial sugar level, resulting in muconic acid titers above 2.5 g/L, caused stuck fermentations with incomplete utilization of the sugar. Application of polypropylene glycol 4000 (PPG) as solvent for in situ product removal during the fermentation shows that this is not due to toxicity by the muconic acid produced. CONCLUSIONS: This work has developed an industrial yeast strain able to produce muconic acid from glucose and also with great efficiency from xylose, without any ethanol production, minimal production of PCA and reaching the highest titers in batch fermentation reported up to now. Utilization of higher sugar levels remained conspicuously incomplete. Since this was not due to product inhibition by muconic acid or to loss of viability, an unknown, possibly metabolic bottleneck apparently arises during muconic acid fermentation with high sugar levels and blocks further sugar utilization.

Discovery and Functional Analysis of a Salicylic Acid Hydroxylase from Aspergillus niger.

Salicylic acid plays an important role in the plant immune response, and its degradation is therefore important for plant-pathogenic fungi. However, many nonpathogenic microorganisms can also degrade salicylic acid. In the filamentous fungus Aspergillus niger, two salicylic acid metabolic pathways have been suggested. The first pathway converts salicylic acid to catechol by a salicylate hydroxylase (ShyA). In the second pathway, salicylic acid is 3-hydroxylated to 2,3-dihydroxybenzoic acid, followed by decarboxylation to catechol by 2,3-dihydroxybenzoate decarboxylase (DhbA). A. niger cleaves the aromatic ring of catechol catalyzed by catechol 1,2-dioxygenase (CrcA) to form cis,cis-muconic acid. However, the identification and role of the genes and characterization of the enzymes involved in these pathways are lacking. In this study, we used transcriptome data of A. niger grown on salicylic acid to identify genes (shyA and crcA) involved in salicylic acid metabolism. Heterologous production in Escherichia coli followed by biochemical characterization confirmed the function of ShyA and CrcA. The combination of ShyA and CrcA demonstrated that cis,cis-muconic acid can be produced from salicylic acid. In addition, the in vivo roles of shyA, dhbA, and crcA were studied by creating A. niger deletion mutants which revealed the role of these genes in the fungal metabolism of salicylic acid.IMPORTANCE Nonrenewable petroleum sources are being depleted, and therefore, alternative sources are needed. Plant biomass is one of the most abundant renewable sources on Earth and is efficiently degraded by fungi. In order to utilize plant biomass efficiently, knowledge about the fungal metabolic pathways and the genes and enzymes involved is essential to create efficient strategies for producing valuable compounds such as cis,cis-muconic acid. cis,cis-Muconic acid is an important platform chemical that is used to synthesize nylon, polyethylene terephthalate (PET), polyurethane, resins, and lubricants. Currently, cis,cis-muconic acid is mainly produced through chemical synthesis from petroleum-based chemicals. Here, we show that two enzymes from fungi can be used to produce cis,cis-muconic acid from salicylic acid and contributes in creating alternative methods for the production of platform chemicals.

Statistical Optimisation of Phenol Degradation and Pathway Identification through Whole Genome Sequencing of the Cold-Adapted Antarctic Bacterium, Rhodococcus sp. Strain AQ5-07.

Study of the potential of Antarctic microorganisms for use in bioremediation is of increasing interest due to their adaptations to harsh environmental conditions and their metabolic potential in removing a wide variety of organic pollutants at low temperature. In this study, the psychrotolerant bacterium Rhodococcus sp. strain AQ5-07, originally isolated from soil from King George Island (South Shetland Islands, maritime Antarctic), was found to be capable of utilizing phenol as sole carbon and energy source. The bacterium achieved 92.91% degradation of 0.5 g/L phenol under conditions predicted by response surface methodology (RSM) within 84 h at 14.8 °C, pH 7.05, and 0.41 g/L ammonium sulphate. The assembled draft genome sequence (6.75 Mbp) of strain AQ5-07 was obtained through whole genome sequencing (WGS) using the Illumina Hiseq platform. The genome analysis identified a complete gene cluster containing catA, catB, catC, catR, pheR, pheA2, and pheA1. The genome harbours the complete enzyme systems required for phenol and catechol degradation while suggesting phenol degradation occurs via the ß-ketoadipate pathway. Enzymatic assay using cell-free crude extract revealed catechol 1,2-dioxygenase activity while no catechol 2,3-dioxygenase activity was detected, supporting this suggestion. The genomic sequence data provide information on gene candidates responsible for phenol and catechol degradation by indigenous Antarctic bacteria and contribute to knowledge of microbial aromatic metabolism and genetic biodiversity in Antarctica.

Expression and cloning of catA encoding a catechol 1,2-dioxygenase from the 2,4-D-degrading strain Cupriavidus campinensis BJ71.

Catechol 1,2-dioxygenases catalyze catechol ring-opening, a critical step in the degradation of aromatic compounds. Cupriavidus campinensis BJ71, an efficient 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterial strain, was previously isolated from an environment contaminated with 2,4-D. In this study, catA encoding a catechol 1,2-dioxygenase was cloned from the BJ71 strain. The gene was 939 bp long and encoded a polypeptide of 312 amino acids with a molecular weight of 34 kDa. To investigate its enzymatic characteristics, CatA was heterologously expressed in Escherichia coli. Optimal reaction conditions for the pure enzyme were 35 °C and pH 8.0. The enzyme remained stable within a range of 25 °C-45 °C and pH 6.0-9.0, thus indicating that CatA has wide temperature and pH adaptability. After incubation at 45 °C, the enzyme activity of CatA decreased to 37.12%, but its activity was not affected by incubation at pH 9.0. The pure enzyme was able to use catechol, 4-methyl-catechol and 4-chlorocatechol as substrates. Enzyme kinetic parameters Km and Vmax were 39.97 µM and 10.68 U/mg, respectively. This is the first report of the cloning of a gene encoding a catechol 1,2-dioxygenase from a 2,4-D-degrading bacterial strain.

Cloning, expression and characterization of catechol 1,2-dioxygenase from Burkholderia cepacia.

The present study reports on the cloning, expression and characterization of catechol 1,2-dioxygenase (CAT) of bacterial strains isolated from dioxin-contaminated soils in Vietnam. Two isolated bacterial strains DF2 and DF4 were identified as Burkholderia cepacia based on their 16S rRNA sequences. Their genes coding CAT was amplified with a specific pair of primers. Recombinant CAT (rCAT) was expressed in E. coli M15 cells and its activity was confirmed by the detection of cis,cis-muconic acid, a product from catechol, by high-performance liquid chromatography (HPLC) analysis. The rCAT of DF4 had an optimal pH and temperature of 7 and 30°C, respectively. Metal ions, such as Zn2+ and Mn2+, and surfactants, such as SDS, Tween 20 and Triton X100, strongly inhibited enzyme activity, while K+ slightly increased the activity.

Identification and characterization of novel bacterial polyaromatic hydrocarbon-degrading enzymes as potential tools for cleaning up hydrocarbon pollutants from different environmental sources.

Polycyclic aromatic hydrocarbons (PAHs) are recalcitrant hazardous environmental contaminants. Various strategies, including chemical and physical like oxidation, fixation, leaching, and electrokinetic or biological-based techniques are used for remediation of polluted sites. Bioremediation of PAHs, via PAH-degrading endophytic and rhizospheric microbes, represent a time-/cost-effective way for ecorestoration. Four bacterial strains were isolated from contaminated soil on MSM supplemented with anthracene, alpha-naphthalene or catechol as sole carbon sources. These isolates were identified with 16S rRNA as Bacillus anthracis, B. cereus, B. mojavensis and B. subtilis. The degradation efficiency on the selected aromatic compounds was tested by HPLC analysis. B. subtilis showed the highest degradation efficiency of anthracene (99%) after five days of incubation. B. subtilis showed the highest catechol 1, 2 dioxygenase activity in MSM supplemented with anthracene. The enzyme was purified by gel filtration chromatography and characterized (70 kD, Km 2.7 µg and Vmax 178U/mg protein). The catechol 1,2 dioxygenase gene from the identified four bacterial strains were isolated and submitted to GenBank (accession numbers MG255165-MG255168). The gene expression level of catechol 1,2 dioxygenase was upregulated 23.2-fold during the 72 h of incubation period. Furthermore, B. subtilis is a promising strain to be used in bioremediation of aromatic compounds-contaminated environments.

Versatile catechol dioxygenases in Sphingobium scionense WP01T.

The objective was to understand the roles of multiple catechol dioxygenases in the type strain Sphingobium scionense WP01T (Liang and Lloyd-Jones in Int J Syst Evol Microbiol 60:413-416, 2010a) that was isolated from severely contaminated sawmill soil. The dioxygenases were identified by sequencing, examined by determining the substrate specificities of the recombinant enzymes, and by quantifying gene expression following exposure to model priority pollutants. Catechol dioxygenase genes encoding an extradiol xylE and two intradiol dioxygenases catA and clcA that are highly similar to sequences described in other sphingomonads are described in S. scionense WP01T. The distinct substrate specificities determined for the recombinant enzymes confirm the annotated gene functions and suggest different catabolic roles for each enzyme. The role of the three enzymes was evaluated by analysis of enzyme activity in crude cell extracts from cells grown on meta-toluate, benzoate, biphenyl, naphthalene and phenanthrene which revealed the co-induction of each enzyme by different substrates. This was corroborated by quantifying gene expression when cells were induced by biphenyl, naphthalene and pentachlorophenol. It is concluded that the ClcA and XylE enzymes are recruited in pathways that are involved in the degradation of chlorinated aromatic compounds such as pentachlorophenol, the XylE and ClcA enzymes will also play a role in degradation pathways that produce alkylcatechols, while the three enzymes ClcA, XylE and CatA will be simultaneously involved in pathways that generate catechol as a degradation pathway intermediate.

Cloning and characterisation of four catA genes located on the chromosome and large plasmid of Pseudomonas putida ND6

Background: Although the functional redundancy of catechol 1,2-dioxygenase (C12O) genes has been reported in several microorganisms, limited enzymes were characterised, let alone the advantage of the coexistence of the multiple copies of C12O genes. Results: In this study, four novel C12O genes, designated catA, catAI, catAII and catAIII, in the naphthalene-degrading strain Pseudomonas putida ND6, were cloned and characterised. Phylogenetic analysis of their deduced amino acid sequences revealed that the four C12O isozymes each formed independent subtrees, together with homologues from other organisms. All four enzymes exhibited maximum activity at pH 7.4 and higher activity in alkaline than in acidic conditions. Furthermore, CatA, CatAI and CatAIII were maximally active at a temperature of 45°C, whereas a higher optimum temperature was observed for CatAII at a temperature of 50°C. CatAI exhibited superior temperature stability compared with the other three C12O isozymes, and kinetic analysis indicated similar enzyme activities for CatA, CatAI and CatAII, whereas that of CatAIII was lower. Significantly, among metal ions tested, only Cu2+ substantially inhibited the activity of these C12O isozymes, thus indicating that they have potential to facilitate bioremediation in environments polluted with aromatics in the presence of metals. Moreover, gene expression analysis at the mRNA level and determination of enzyme activity clearly indicated that the redundancy of the catA genes has increased the levels of C12O. Conclusion: The results clearly imply that the redundancy of catA genes increases the available amount of C12O in P. putida ND6, which would be beneficial for survival in challenging environments.

Production of muconic acid in plants.

Muconic acid (MA) is a dicarboxylic acid used for the production of industrially relevant chemicals such as adipic acid, terephthalic acid, and caprolactam. Because the synthesis of these polymer precursors generates toxic intermediates by utilizing petroleum-derived chemicals and corrosive catalysts, the development of alternative strategies for the bio-based production of MA has garnered significant interest. Plants produce organic carbon skeletons by harvesting carbon dioxide and energy from the sun, and therefore represent advantageous hosts for engineered metabolic pathways towards the manufacturing of chemicals. In this work, we engineered Arabidopsis to demonstrate that plants can serve as green factories for the bio-manufacturing of MA. In particular, dual expression of plastid-targeted bacterial salicylate hydroxylase (NahG) and catechol 1,2-dioxygenase (CatA) resulted in the conversion of the endogenous salicylic acid (SA) pool into MA via catechol. Sequential increase of SA derived from the shikimate pathway was achieved by expressing plastid-targeted versions of bacterial salicylate synthase (Irp9) and feedback-resistant 3-deoxy-D-arabino-heptulosonate synthase (AroG). Introducing this SA over-producing strategy into engineered plants that co-express NahG and CatA resulted in a 50-fold increase in MA titers. Considering that MA was easily recovered from senesced plant biomass after harvest, we envision the phytoproduction of MA as a beneficial option to add value to bioenergy crops.

Genetic diversity of catechol 1,2-dioxygenase in the fecal microbial metagenome.

Catechol 1,2-dioxygenase is the key enzyme that catalyzes the cleavage of the aromatic ring of catechol. We explored the genetic diversity of catechol 1,2-dioxygenase in the fecal microbial metagenome by PCR with degenerate primers. A total of 35 gene fragments of C12O were retrieved from microbial DNA in the feces of pygmy loris. Based on phylogenetic analysis, most sequences were closely related to C12O sequences from Acinetobacter. A full-length C12O gene was directly cloned, heterologously expressed in Escherichia coli, and biochemically characterized. Purified catPL12 had optimum pH and temperature pH 8.0 and 25 °C and retained 31 and 50% of its maximum activity when assayed at 0 and 35 °C, respectively. The enzyme was stable at 25 and 37 °C, retaining 100% activity after pre-incubation for 1 h. The kinetic parameters of catPL12 were determined. The enzyme had apparent Km of 67 µM, Vmax of 7.3 U/mg, and kcat of 4.2 s-1 for catechol, and the cleavage activities for 3-methylcatechol, 4-methylcatechol, and 4-chlorocatechol were much less than for catechol, and no activity with hydroquinone or protocatechuate was detected. This study is the first to report the molecular and biochemical characterizations of a cold-adapted catechol 1,2-dioxygenase from a fecal microbial metagenome.

The Bark-Beetle-Associated Fungus, Endoconidiophora polonica, Utilizes the Phenolic Defense Compounds of Its Host as a Carbon Source.

Norway spruce (Picea abies) is periodically attacked by the bark beetle Ips typographus and its fungal associate, Endoconidiophora polonica, whose infection is thought to be required for successful beetle attack. Norway spruce produces terpenoid resins and phenolics in response to fungal and bark beetle invasion. However, how the fungal associate copes with these chemical defenses is still unclear. In this study, we investigated changes in the phenolic content of Norway spruce bark upon E. polonica infection and the biochemical factors mediating these changes. Although genes encoding the rate-limiting enzymes in Norway spruce stilbene and flavonoid biosynthesis were actively transcribed during fungal infection, there was a significant time-dependent decline of the corresponding metabolites in fungal lesions. In vitro feeding experiments with pure phenolics revealed that E. polonica transforms both stilbenes and flavonoids to muconoid-type ring-cleavage products, which are likely the first steps in the degradation of spruce defenses to substrates that can enter the tricarboxylic acid cycle. Four genes were identified in E. polonica that encode catechol dioxygenases carrying out these reactions. These enzymes catalyze the cleavage of phenolic rings with a vicinal dihydroxyl group to muconoid products accepting a wide range of Norway spruce-produced phenolics as substrates. The expression of these genes and E. polonica utilization of the most abundant spruce phenolics as carbon sources both correlated positively with fungal virulence in several strains. Thus, the pathways for the degradation of phenolic compounds in E. polonica, initiated by catechol dioxygenase action, are important to the infection, growth, and survival of this bark beetle-vectored fungus and may play a major role in the ability of I. typographus to colonize spruce trees.

Cloning, expression, and characterization of catechol 1,2-dioxygenase from a phenol-degrading Candida tropicalis JH8 strain.

The sequence cato encoding catechol 1,2-dioxygenase from Candida tropicalis JH8 was cloned, sequenced, and expressed in Escherichia coli. The sequence cato contained an ORF of 858 bp encoding a polypeptide of 285 amino acid residues. The recombinant catechol 1,2-dioxygenase exists as a homodimer structure with a subunit molecular mass of 32 KD. Recombinant catechol 1,2-dioxygenase was unstable below pH 5.0 and stable from pH 7.0 to 9.0; its optimum pH was at 7.5. The optimum temperature for the enzyme was 30°C, and it possessed a thermophilic activity within a broad temperature range. Under the optimal conditions with catechol as substrate, the Km and Vmax of recombinant catechol 1,2-dioxygenase were 9.2 µM and 0.987 µM/min, respectively. This is the first article presenting cloning and expressing in E. coli of catechol 1,2-dioxygenase from C. tropicalis and characterization of the recombinant catechol 1,2-dioxygenase.

Purification and Characterization of Catechol 1,2-Dioxygenase from Acinetobacter sp. Y64 Strain and Escherichia coli Transformants.

This study intends to purify and characterize catechol 1,2-dioxygenase (C1,2O) of phenol-degrading Acinetobacter sp. Y64 and of E. coli transformant. Acinetobacter sp. Y64 was capable of degrading 1000 mg/L of phenol within 14 ± 2 h at 30 °C, 160 rpm and pH of 7. One C1,2O of 36 kDa was purified using ammonium sulphate precipitation and Hitrap QFF column chromatograph with 49% recovery and a 10.6-fold increase in purity. Purified Y64 C1,2O had temperature and pH optimum at 37 °C and pH 7.7 respectively with the Michaelis constant of 17.53 µM and the maximal velocity of 1.95 U/mg, respectively. The presence of Fe(3+) or Fe(2+) enhanced the activity of Y64 C1,2O while other compounds such as Ca(2+), and EDTA had an inhibitory effect. 80% of C1,2O activity remained using 4-nitrocatechol as substrate while 2% remained using 3-methylcatechol compared with that using catechol. Y64 catA gene encoding C1,2O was amplified using PCR cloned into pET22b vector and expressed in Escherichia coli BL21 DE3 (pLysS) after transformation. Purified and cloned Y64 C1,2O show no significant differences in the biochemical properties. The phylogenetic tree based on the protein sequences indicates that these C1,2Os possess a common ancestry.

Engineering catechol 1, 2-dioxygenase by design for improving the performance of the cis, cis-muconic acid synthetic pathway in Escherichia coli.

Regulating and ameliorating enzyme expression and activity greatly affects the performance of a given synthetic pathway. In this study, a new synthetic pathway for cis, cis-muconic acid (ccMA) production was reconstructed without exogenous induction by regulating the constitutive expression of the important enzyme catechol 1,2-dioxygenase (CatA). Next, new CatAs with significantly improved activities were developed to enhance ccMA production using structure-assisted protein design. Nine mutations were designed, simulated and constructed based on the analysis of the CatA crystal structure. These results showed that mutations at Gly72, Leu73 and/or Pro76 in CatA could improve enzyme activity, and the activity of the most effective mutant was 10-fold greater than that of the wild-type CatA from Acinetobacter sp. ADP1. The most productive synthetic pathway with a mutated CatA increased the titer of ccMA by more than 25%. Molecular dynamic simulation results showed that enlarging the entrance of the substrate-binding pocket in the mutants contributed to their increased enzyme activities and thus improved the performance of the synthetic pathway.

High-throughput screening for a moderately halophilic phenol-degrading strain and its salt tolerance response.

A high-throughput screening system for moderately halophilic phenol-degrading bacteria from various habitats was developed to replace the conventional strain screening owing to its high efficiency. Bacterial enrichments were cultivated in 48 deep well microplates instead of shake flasks or tubes. Measurement of phenol concentrations was performed in 96-well microplates instead of using the conventional spectrophotometric method or high-performance liquid chromatography (HPLC). The high-throughput screening system was used to cultivate forty-three bacterial enrichments and gained a halophilic bacterial community E3 with the best phenol-degrading capability. Halomonas sp. strain 4-5 was isolated from the E3 community. Strain 4-5 was able to degrade more than 94% of the phenol (500 mg · L(-1) starting concentration) over a range of 3%-10% NaCl. Additionally, the strain accumulated the compatible solute, ectoine, with increasing salt concentrations. PCR detection of the functional genes suggested that the largest subunit of multicomponent phenol hydroxylase (LmPH) and catechol 1,2-dioxygenase (C12O) were active in the phenol degradation process.

Anthranilate degradation by a cold-adapted Pseudomonas sp.

An alpine soil bacterium Pseudomonas sp. strain PAMC 25931 was characterized as eurypsychrophilic (both psychrophilic and mesotolerant) with a broad temperature range of 5-30 °C both for anthranilate (2-aminobenzoate) degradation and concomitant cell growth. Two degradative gene clusters (antABC and catBCA) were detected from a fosmid clone in the PAMC 25931 genomic library; each cluster was confirmed to be specifically induced by anthranilate. When expressed in Escherichia coli, the recombinant AntABC (anthranilate 1,2-dioxygenase, AntDO) converted anthranilate into catechol, exhibiting strict specificity toward anthranilate. Recombinant CatA (catechol 1,2-dioxygenase, C12O) from the organism was active over a broad temperature range (5-37 °C). However, CatA rapidly lost the enzyme activity when incubated at above 25 °C. For example, 1 h-preincubation at 37 °C resulted in 100% loss of enzyme activity, while a counterpart from mesophilic Pseudomonas putida mt-2 did not show any negative effect on the initial enzyme activity. These results suggest that CatA is a new cold-adapted thermolabile enzyme, which might be a product through the adaptation process of PAMC 25931 to naturally cold environments and contribute to its ability to grow on anthranilate there.

Enhanced polyaromatic hydrocarbon degradation by adapted cultures of actinomycete strains.

Fifteen actinomycete strains were evaluated for their potential use in removal of polycyclic aromatic hydrocarbons (PAH). Their capability to degrade of naphthalene, phenanthrene, and pyrene was tested in minimal medium (MM) and MM with glucose as another substrate. Degradation of naphthalene in MM was observed in all isolates at different rates, reaching maximum values near to 76% in some strains of Streptomyces, Rhodococcus sp. 016 and Amycolatopsis tucumanensis DSM 45259. Maximum values of degradation of phenanthrene in MM occurred in cultures of A. tucumanensis DSM 45259 (36.2%) and Streptomyces sp. A12 (20%), while the degradation of pyrene in MM was poor and only significant with Streptomyces sp. A12 (4.3%). Because of the poor performance when growing on phenanthrene and pyrene alone, Rhodococcus sp. 20, Rhodococcus sp. 016, A. tucumanensis DSM 45259, Streptomyces sp. A2, and Streptomyces sp. A12 were challenged to an adaptation schedule of successive cultures on a fresh solid medium supplemented with PAHs, decreasing concentration of glucose in each step. As a result, an enhanced degradation of PAHs by adapted strains was observed in the presence of glucose as co-substrate, without degradation of phenanthrene and pyrene in MM while an increase to up to 50% of degradation was seen with these strains in glucose amended media. An internal fragment of the catA gene, which codes for catechol 1,2-dioxygenase, was amplified from both Rhodococcus strains, showing the potential for degradation of aromatic compounds via salycilate. These results allow us to propose the usefulness of these actinomycete strains for PAH bioremediation in the environment.