Evaluation of the Defined Bacterial Consortium Efficacy in the Biodegradation of NSAIDs.

Due to the increasing pollution of wastewater with non-steroidal anti-inflammatory drugs, preparations need to be developed to decompose these drugs. This work aimed to develop a bacterial consortium with a defined composition and boundary conditions for the degradation of paracetamol and selected non-steroidal anti-inflammatory drugs (NSAIDs), including ibuprofen, naproxen, and diclofenac. The defined bacterial consortium consisted of Bacillus thuringiensis B1(2015b) and Pseudomonas moorei KB4 strains in a ratio of 1:2. During the tests, it was shown that the bacterial consortium worked in the pH range from 5.5 to 9 and temperatures of 15-35 °C, and its great advantage was its resistance to toxic compounds present in sewage, such as organic solvents, phenols, and metal ions. The degradation tests showed that, in the presence of the defined bacterial consortium in the sequencing batch reactor (SBR), drug degradation occurred at rates of 4.88, 10, 0.1, and 0.05 mg/day for ibuprofen, paracetamol, naproxen, and diclofenac, respectively. In addition, the presence of the tested strains was demonstrated during the experiment as well as after its completion. Therefore, the advantage of the described bacterial consortium is its resistance to the antagonistic effects of the activated sludge microbiome, which will enable it to be tested in real activated sludge conditions.

Genetic Determinants of Antagonistic Interactions and the Response of New Endophytic Strain Serratia quinivorans KP32 to Fungal Phytopathogens.

Fungal phytopathogens are challenging to control due to their penetration into plant tissues. Therefore, plant-colonizing bacteria could serve as an excellent weapon in fighting fungal infections. In this study, we aim to determine the biocontrol potential of the new endophytic strain Serratia quinivorans KP32, isolated from the roots of Petroselinum crispum L.; identify the related mechanisms; and understand the basis of its antagonistic interaction with taxonomically diverse fungi at the molecular level. The KP32 strain presented biological activity against Rhizoctonia solani, Colletotrichum dematium, Fusarium avenaceum, and Sclerotinia sclerotiorum, and its ability to inhibit the growth of the phytopathogens was found to be mediated by a broad spectrum of biocontrol features, such as the production of a number of lytic enzymes (amylases, chitinases, and proteases), siderophores, volatile organic and inorganic compounds, salicylic acid, and N-acyl-homoserine lactones. The higher expression of chitinase (chiA) and genes involved in the biosynthesis of hydrogen cyanide (hcnC), enterobactin (entB), and acetoin (budA) in bacteria exposed to fungal filtrates confirmed that these factors could act in combination, leading to a synergistic inhibitory effect of the strain against phytopathogens. We also confirm the active movement, self-aggregation, exopolysaccharide production, and biofilm formation abilities of the KP32 strain, which are essential for effective plant colonization. Its biological activity and colonization potential indicate that KP32 holds tremendous potential for use as an active biopesticide and plant growth promoter.

Analysis of the Genome of the Heavy Metal Resistant and Hydrocarbon-Degrading Rhizospheric Pseudomonas qingdaonensis ZCR6 Strain and Assessment of Its Plant-Growth-Promoting Traits.

The Pseudomonas qingdaonensis ZCR6 strain, isolated from the rhizosphere of Zea mays growing in soil co-contaminated with hydrocarbons and heavy metals, was investigated for its plant growth promotion, hydrocarbon degradation, and heavy metal resistance. In vitro bioassays confirmed all of the abovementioned properties. ZCR6 was able to produce indole acetic acid (IAA), siderophores, and ammonia, solubilized Ca3(PO4)2, and showed surface active properties and activity of cellulase and very high activity of 1-aminocyclopropane-1-carboxylic acid deaminase (297 nmol α-ketobutyrate mg-1 h-1). The strain degraded petroleum hydrocarbons (76.52% of the initial hydrocarbon content was degraded) and was resistant to Cd, Zn, and Cu (minimal inhibitory concentrations reached 5, 15, and 10 mM metal, respectively). The genome of the ZCR6 strain consisted of 5,507,067 bp, and a total of 5055 genes were annotated, of which 4943 were protein-coding sequences. Annotation revealed the presence of genes associated with nitrogen fixation, phosphate solubilization, sulfur metabolism, siderophore biosynthesis and uptake, synthesis of IAA, ethylene modulation, heavy metal resistance, exopolysaccharide biosynthesis, and organic compound degradation. Complete characteristics of the ZCR6 strain showed its potential multiway properties for enhancing the phytoremediation of co-contaminated soils. To our knowledge, this is the first analysis of the biotechnological potential of the species P. qingdaonensis.

Genome Mining and Evaluation of the Biocontrol Potential of Pseudomonas fluorescens BRZ63, a New Endophyte of Oilseed Rape (Brassica napus L.) against Fungal Pathogens.

Endophytic bacteria hold tremendous potential for use as biocontrol agents. Our study aimed to investigate the biocontrol activity of Pseudomonas fluorescens BRZ63, a new endophyte of oilseed rape (Brassica napus L.) against Rhizoctonia solani W70, Colletotrichum dematium K, Sclerotinia sclerotiorum K2291, and Fusarium avenaceum. In addition, features crucial for biocontrol, plant growth promotion, and colonization were assessed and linked with the genome sequences. The in vitro tests showed that BRZ63 significantly inhibited the mycelium growth of all tested pathogens and stimulated germination and growth of oilseed rape seedlings treated with fungal pathogens. The BRZ63 strain can benefit plants by producing biosurfactants, siderophores, indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylate (ACC) deaminase, and ammonia as well as phosphate solubilization. The abilities of exopolysaccharide production, autoaggregation, and biofilm formation additionally underline its potential to plant colonization and hence biocontrol. The effective colonization properties of the BRZ63 strain were confirmed by microscopy observations of EGFP-expressing cells colonizing the root surface and epidermal cells of Arabidopsis thaliana Col-0. Genome mining identified many genes related to the biocontrol process, such as transporters, siderophores, and other secondary metabolites. All analyses revealed that the BRZ63 strain is an excellent endophytic candidate for biocontrol of various plant pathogens and plant growth promotion.

Comparative Genomics of Stenotrophomonas maltophilia and Stenotrophomonas rhizophila Revealed Characteristic Features of Both Species.

Although Stenotrophomonas maltophilia strains are efficient biocontrol agents, their field applications have raised concerns due to their possible threat to human health. The non-pathogenic Stenotrophomonas rhizophila species, which is closely related to S. maltophilia, has been proposed as an alternative. However, knowledge regarding the genetics of S. rhizophila is limited. Thus, the aim of the study was to define any genetic differences between the species and to characterise their ability to promote the growth of plant hosts as well as to enhance phytoremediation efficiency. We compared 37 strains that belong to both species using the tools of comparative genomics and identified 96 genetic features that are unique to S. maltophilia (e.g., chitin-binding protein, mechanosensitive channels of small conductance and KGG repeat-containing stress-induced protein) and 59 that are unique to S. rhizophila (e.g., glucosylglycerol-phosphate synthase, cold shock protein with the DUF1294 domain, and pteridine-dependent dioxygenase-like protein). The strains from both species have a high potential for biocontrol, which is mainly related to the production of keratinases (KerSMD and KerSMF), proteinases and chitinases. Plant growth promotion traits are attributed to the biosynthesis of siderophores, spermidine, osmoprotectants such as trehalose and glucosylglycerol, which is unique to S. rhizophila. In eight out of 37 analysed strains, the genes that are required to degrade protocatechuate were present. While our results show genetic differences between the two species, they had a similar growth promotion potential. Considering the information above, S. rhizophila constitutes a promising alternative for S. maltophilia for use in agricultural biotechnology.

Defining the Genetic Basis of Plant⁻Endophytic Bacteria Interactions.

Endophytic bacteria, which interact closely with their host, are an essential part of the plant microbiome. These interactions enhance plant tolerance to environmental changes as well as promote plant growth, thus they have become attractive targets for increasing crop production. Numerous studies have aimed to characterise how endophytic bacteria infect and colonise their hosts as well as conferring important traits to the plant. In this review, we summarise the current knowledge regarding endophytic colonisation and focus on the insights that have been obtained from the mutants of bacteria and plants as well as 'omic analyses. These show how endophytic bacteria produce various molecules and have a range of activities related to chemotaxis, motility, adhesion, bacterial cell wall properties, secretion, regulating transcription and utilising a substrate in order to establish a successful interaction. Colonisation is mediated by plant receptors and is regulated by the signalling that is connected with phytohormones such as auxin and jasmonic (JA) and salicylic acids (SA). We also highlight changes in the expression of small RNAs and modifications of the cell wall properties. Moreover, in order to exploit the beneficial plant-endophytic bacteria interactions in agriculture successfully, we show that the key aspects that govern successful interactions remain to be defined.

A new pathway for naproxen utilisation by Bacillus thuringiensis B1(2015b) and its decomposition in the presence of organic and inorganic contaminants.

Bacillus thuringiensis B1 (2015b) is a bacterial strain that is able to degrade naproxen. However, the potential effect of water co-contaminations on the degradation process and its pathway have not yet been evaluated. The results of our study show that in the presence of aromatic compounds, the B1 (2015b) strain utilised naproxen with an efficiency that was similar to what it was with no aromatic co-contaminations. In the presence of methanol, biodegradation of naproxen was inhibited, while the addition of ethanol increased the decomposition of naproxen. Among the metal ions that were tested, only cobalt (II) and cadmium (II) negatively affected the degradation of the drug. An analysis of the intermediates and enzymes that are engaged in degrading naproxen revealed that the key metabolites are O-desmethylnaproxen, which is the product of tetrahydrofolate-dependent O-demethylase activity, and salicylic acid. Salicylic acid can then be hydroxylated to catechol or gentisic acid or can be cleaved to 2-oxo-3,5-heptadienedioic acid. The high activity level of catechol 1,2-dioxygenase indicated that the main degradative pathway of naproxen in the B1 (2015b) strain is via catechol cleavage.

Naproxen ecotoxicity and biodegradation by Bacillus thuringiensis B1(2015b) strain.

High level of naproxen consumption leads to the appearance of this drug in the environment but its possible effects on non-target organisms together with its biodegradation are not well studied. The aim of this work was to evaluate naproxen ecotoxicity by using the Microbial Assay for Risk Assessment. Moreover, Bacillus thuringiensis B1(2015b) was tested for both ecotoxicity and the ability of this strain to degrade naproxen in cometabolic conditions. The results indicate that the mean value of microbial toxic concentration estimated by MARA test amounts to 1.66 g/L whereas EC50 of naproxen for B1(2015b) strain was 4.69 g/L. At toxic concentration, Bacillus thuringiensis B1(2015b) showed 16:0 iso 3OH fatty acid presence and an increase in the ratio of total saturated to unsaturated fatty acids. High resistance of the examined strain to naproxen correlated with its ability to degrade this drug in cometabolic conditions. The results of bacterial reverse mutation assay (Ames test) revealed that naproxen at concentrations above 1 g/L showed genotoxic effect but the response was not dose-dependent. Maximal specific naproxen removal rate was observed at pH 6.5 and 30 °C, and in the presence of 0.5 g/L glucose as a growth substrate. Kinetic analysis allowed estimation of the half saturation constant (Ks) and the maximum specific naproxen removal rate (qmax) as 6.86 mg/L and 1.26 mg/L day, respectively. These results indicate that Bacillus thuringiensis B1(2015b) has a high ability to degrade naproxen and is a potential tool for bioremediation.

Organic micropollutants paracetamol and ibuprofen-toxicity, biodegradation, and genetic background of their utilization by bacteria.

Currently, analgesics and nonsteroidal anti-inflammatory drugs (NSAIDs) are classified as one of the most emerging group of xenobiotics and have been detected in various natural matrices. Among them, monocyclic paracetamol and ibuprofen, widely used to treat mild and moderate pain are the most popular. Since long-term adverse effects of these xenobiotics and their biological and pharmacokinetic activity especially at environmentally relevant concentrations are better understood, degradation of such contaminants has become a major concern. Moreover, to date, conventional wastewater treatment plants (WWTPs) are not fully adapted to remove that kind of micropollutants. Bioremediation processes, which utilize bacterial strains with increased degradation abilities, seem to be a promising alternative to the chemical methods used so far. Nevertheless, despite the wide prevalence of paracetamol and ibuprofen in the environment, toxicity and mechanism of their microbial degradation as well as genetic background of these processes remain not fully characterized. In this review, we described the current state of knowledge about toxicity and biodegradation mechanisms of paracetamol and ibuprofen and provided bioinformatics analysis concerning the genetic bases of these xenobiotics decomposition.

Paracetamol - toxicity and microbial utilization. Pseudomonas moorei KB4 as a case study for exploring degradation pathway.

Paracetamol, a widely used analgesic and antipyretic drug, is currently one of the most emerging pollutants worldwide. Besides its wide prevalence in the literature only several bacterial strains able to degrade this compound have been described. In this study, we isolated six new bacterial strains able to remove paracetamol. The isolated strains were identified as the members of Pseudomonas, Bacillus, Acinetobacter and Sphingomonas genera and characterized phenotypically and biochemically using standard methods. From the isolated strains, Pseudomonas moorei KB4 was able to utilize 50 mg L-1 of paracetamol. As the main degradation products, p-aminophenol and hydroquinone were identified. Based on the measurements of specific activity of acyl amidohydrolase, deaminase and hydroquinone 1,2-dioxygenase and the results of liquid chromatography analyses, we proposed a mechanism of paracetamol degradation by KB4 strain under co-metabolic conditions with glucose. Additionally, toxicity bioassays and the influence of various environmental factors, including pH, temperature, heavy metals at no-observed-effective-concentrations, and the presence of aromatic compounds on the efficiency and mechanism of paracetamol degradation by KB4 strain were determined. This comprehensive study about paracetamol biodegradation will be helpful in designing a treatment systems of wastewaters contaminated with paracetamol.

Toxicity and biodegradation of ibuprofen by Bacillus thuringiensis B1(2015b).

In recent years, the increased intake of ibuprofen has resulted in the presence of the drug in the environment. This work presents results of a study on degradation of ibuprofen at 25 mg L-1 in the presence of glucose, as an additional carbon source by Bacillus thuringiensis B1(2015b). In the cometabolic system, the maximum specific growth rate of the bacterial strain was 0.07 ± 0.01 mg mL-1 h-1 and K sµ 0.27 ± 0.15 mg L-1. The maximum specific ibuprofen removal rate and the value of the half-saturation constant were q max = 0.24 ± 0.02 mg mL-1 h-1 and K s = 2.12 ± 0.56 mg L-1, respectively. It has been suggested that monooxygenase and catechol 1,2-dioxygenase are involved in ibuprofen degradation by B. thuringiensis B1(2015b). Toxicity studies showed that B. thuringiensis B1(2015b) is more resistant to ibuprofen than other tested organisms. The EC50 of ibuprofen on the B1 strain is 809.3 mg L-1, and it is 1.5 times higher than the value of the microbial toxic concentration (MTCavg). The obtained results indicate that B. thuringiensis B1(2015b) could be a useful tool in biodegradation/bioremediation processes.

Substitutions of Conserved Residues in the C-terminal Region of DnaC Cause Thermolability in Helicase Loading.

The DnaB-DnaC complex binds to the unwound DNA within the Escherichia coli replication origin in the helicase loading process, but the biochemical events that lead to its stable binding are uncertain. This study characterizes the function of specific C-terminal residues of DnaC. Genetic and biochemical characterization of proteins bearing F231S and W233L substitutions of DnaC reveals that their activity is thermolabile. Because the mutants remain able to form a complex with DnaB at 30 and 37 °C, their thermolability is not explained by an impaired interaction with DnaB. Photo-cross-linking experiments and biosensor analysis show an altered affinity of these mutants compared with wild type DnaC for single-stranded DNA, suggesting that the substitutions affect DNA binding. Despite this difference, their activity in DNA binding is not thermolabile. The substitutions also drastically reduce the affinity of DnaC for ATP as measured by the binding of a fluorescent ATP analogue (MANT-ATP) and by UV cross-linking of radiolabeled ATP. Experiments show that an elevated temperature substantially inhibits both mutants in their ability to load the DnaB-DnaC complex at a DnaA box. Because a decreased ATP concentration exacerbates their thermolabile behavior, we suggest that the F231S and W233L substitutions are thermolabile in ATP binding, which correlates with defective helicase loading at an elevated temperature.

Enzymes Involved in Naproxen Degradation by Planococcus sp. S5.

Naproxen is a one of the most popular non-steroidal anti-inflammatory drugs (NSAIDs) entering the environment as a result of high consumption. For this reason, there is an emerging need to recognize mechanisms of its degradation and enzymes engaged in this process. Planococcus sp. S5 is a gram positive strain able to degrade naproxen in monosubstrate culture (27%). However, naproxen is not a suf-ficient growth substrate for this strain. In the presence of benzoate, 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid or vanillic acid as growth substrates, the degradation of 21.5%, 71.71%, 14.75% and 8.16% of naproxen was observed respectively. It was shown that the activity of monooxygenase, hydroxyquinol 1,2-dioxygenase, protocatechuate 3,4-dioxygenase and protocatechuate 4,5-dioxyegnase in strain S5 was induced after growth of the strain with naproxen and 4-hydroxybenzoate. Moreover, in the presence of naproxen activity of gentisate 1,2-dioxygenase, enzyme engaged in 4-hydroxybenzoate metabolism, was completely inhibited. The obtained results suggest that monooxygenase and hydroxyquinol 1,2-dioxygenase are the main enzymes in naproxen degradation by Planococcus sp. S5.

Enzymes Involved in Naproxen Degradation by Planococcus sp. S5.

Naproxen is a one of the most popular non-steroidal anti-inflammatory drugs (NSAIDs) entering the environment as a result of high consumption. For this reason, there is an emerging need to recognize mechanisms of its degradation and enzymes engaged in this process. Planococcus sp. S5 is a gram positive strain able to degrade naproxen in monosubstrate culture (27%). However, naproxen is not a sufficient growth substrate for this strain. In the presence of benzoate, 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid or vanillic acid as growth substrates, the degradation of 21.5%, 71.71%, 14.75% and 8.16% of naproxen was observed respectively. It was shown that the activity of monooxygenase, hydroxyquinol 1,2-dioxygenase, protocatechuate 3,4-dioxygenase and protocatechuate 4,5-dioxyegnase in strain S5 was induced after growth of the strain with naproxen and 4-hydroxybenzoate. Moreover, in the presence of naproxen activity of gentisate 1,2-dioxygenase, enzyme engaged in 4-hydroxybenzoate metabolism, was completely inhibited. The obtained results suggest that monooxygenase and hydroxyquinol 1,2-dioxygenase are the main enzymes in naproxen degradation by Planococcus sp. S5.

Cometabolic Degradation of Naproxen by Planococcus sp. Strain S5.

Naproxen is a non-steroidal anti-inflammatory drug frequently detected in the influent and effluent of sewage treatment plants. The Gram-positive strain Planococcus sp. S5 was able to remove approximately 30 % of naproxen after 35 days of incubation in monosubstrate culture. Under cometabolic conditions, with glucose or phenol as a growth substrate, the degradation efficiency of S5 increased. During 35 days of incubation, 75.14 ± 1.71 % and 86.27 ± 2.09 % of naproxen was degraded in the presence of glucose and phenol, respectively. The highest rate of naproxen degradation observed in the presence of phenol may be connected with the fact that phenol is known to induce enzymes responsible for aromatic ring cleavage. The activity of phenol monooxygenase, naphthalene monooxygenase, and hydroxyquinol 1,2-dioxygenase was indicated in Planococcus sp. S5 culture with glucose or phenol as a growth substrate. It is suggested that these enzymes may be engaged in naproxen degradation.

Altering substrate specificity of catechol 2,3-dioxygenase from Planococcus sp. strain S5 by random mutagenesis.

c23o gene, encoding catechol 2,3-dioxygenase from Planococcus sp. strain S5 was randomly mutagenized to generate variant forms of the enzyme with higher degradation activity. Additionally, the effect of introduced mutations on the enzyme structure was analyzed based on the putative 3D models the wild-type and mutant enzymes. C23OB58 and C23OB81 mutant proteins with amino acid substitutions in close proximity to the enzyme surface or at the interface and in the vicinity of the enzyme active site respectively showed the lowest activity towards all catecholic substrates. The relative activity of C23OC61 mutant towards para-substituted catechols was 20-30% lower of the wild-type enzyme. In this mutant all changes: F191I, C268R, Y272H, V280A and Y293D were located within the conserved regions of C-terminal domain. From these F191I seems to have significant implications for enzyme activity. The highest activity towards different catechols was found for mutant C23OB65. R296Q mutation improved the activity of C23O especially against 4-chlorocatechol. The relative activity of above-mentioned mutant detected against this substrate was almost 6-fold higher than the wild-type enzyme. These results should facilitate future engineering of the enzyme for bioremediation.

Bacterial degradation of naproxen--undisclosed pollutant in the environment.

The presence of non-steroidal anti-inflammatory drugs (NSAIDs) in the environment is an emerging problem due to their potential influence on human health and biocenosis. This is the first report on the biotransformation of naproxen, a polycyclic NSAID, by a bacterial strain. Stenotrophomonas maltophilia KB2 transformed naproxen within 35 days with about 28% degradation efficiency. Under cometabolic conditions with glucose or phenol as a carbon source degradation efficiency was 78% and 40%, respectively. Moreover, in the presence of naproxen phenol monooxygenase, naphthalene dioxygenase, hydroxyquinol 1,2-dioxygenase and gentisate 1,2-dioxygenase were induced. This suggests that degradation of naproxen occurs by its hydroxylation to 5,7,8-trihydroxynaproxen, an intermediate that can be cleaved by hydroxyquinol 1,2-dioxygenase. The cleavage product is probably further oxidatively cleaved by gentisate 1,2-dioxygenase. The obtained results provide the basis for the use of cometabolic systems in the bioremediation of polycyclic NSAID-contaminated environments.

Immobilization as a strategy for improving enzyme properties-application to oxidoreductases.

The main objective of the immobilization of enzymes is to enhance the economics of biocatalytic processes. Immobilization allows one to re-use the enzyme for an extended period of time and enables easier separation of the catalyst from the product. Additionally, immobilization improves many properties of enzymes such as performance in organic solvents, pH tolerance, heat stability or the functional stability. Increasing the structural rigidity of the protein and stabilization of multimeric enzymes which prevents dissociation-related inactivation. In the last decade, several papers about immobilization methods have been published. In our work, we present a relation between the influence of immobilization on the improvement of the properties of selected oxidoreductases and their commercial value. We also present our view on the role that different immobilization methods play in the reduction of enzyme inhibition during biotechnological processes.

Protocatechuate 3,4-dioxygenase: a wide substrate specificity enzyme isolated from Stenotrophomonas maltophilia KB2 as a useful tool in aromatic acid biodegradation.

Protocatechuate 3,4-dioxygenases (P34Os) catalyze the reaction of the ring cleavage of aromatic acid derivatives. It is a key reaction in many xenobiotic metabolic pathways. P34Os characterize narrow substrate specificity. This property is an unfavorable feature in the biodegradation process because one type of pollution is rarely present in the environment. Thus, the following study aimed at the characterization of a P34O from Stenotrophomonas maltophilia KB2, being able to utilize a wide spectrum of aromatic carboxylic acids. A total of 3 mM vanillic acid and 4-hydroxybenzoate were completely degraded during 8 and 4.5 h, respectively. When cells of strain KB2 were grown on 9 mM 4-hydroxybenzoate, P34O was induced. Biochemical analysis revealed that the examined enzyme was similar to other known P34Os, but showed untypical wide substrate specificity. A high activity of P34O against 2,4- and 3,5-dihydroxybenzoate was observed. As these substrates do not possess ortho configuration hydroxyl groups, it is postulated that their cleavage could be connected with their monodentate binding of substrate to the active site. Since this enzyme characterizes untypical wide substrate specificity it makes it a useful tool in applications for environmental clean-up purposes.

Activity of a carboxyl-terminal truncated form of catechol 2,3-dioxygenase from Planococcus sp. S5.

Catechol 2,3-dioxygenases (C23Os, E.C.1.13.12.2) are two domain enzymes that catalyze degradation of monoaromatic hydrocarbons. The catalytically active C-domain of all known C23Os comprises ferrous ion ligands as well as residues forming active site pocket. The aim of this work was to examine and discuss the effect of nonsense mutation at position 289 on the activity of catechol 2,3-dioxygenase from Planococcus strain. Although the mutant C23O showed the same optimal temperature for activity as the wild-type protein (35 °C), it exhibited activity slightly more tolerant to alkaline pH. Mutant enzyme exhibited also higher affinity to catechol as a substrate. Its K(m) (66.17 µM) was approximately 30% lower than that of wild-type enzyme. Interestingly, removal of the C-terminal residues resulted in 1.5- to 1.8-fold (P < 0.05) increase in the activity of C23OB61 against 4-methylcatechol and 4-chlorocatechol, respectively, while towards catechol the activity of the protein dropped to about 80% of that of the wild-type enzyme. The results obtained may facilitate the engineering of the C23O for application in the bioremediation of polluted areas.