Enzymatic Bioremediation Research Articles

Enzymatic bioremediation of organophosphorus insecticides by recombinant organophosphorous hydrolase

The gene for organophosphorous hydrolase (OPH) (GenBank accession no. M20392) was chemically synthesised with a codon bias toward E. coli, followed by its cloning and heterologous over-expression in E. coli under induction with 0.1 M isopropyl- d-thiogalactopyranoside (IPTG). The protein was localised in the membrane fraction and no amount of the expressed protein was soluble, thus hindering its purification and further downstream utility. The expressed enzyme was solubilised by mild treatment with ionic detergent [sodium dodecyl sulphate (SDS 1.0% (w/v))]. An innovative step of incubation at 4 °C was used to precipitate SDS, resulting in catalytically active OPH in the supernatant and detergent at the bottom. This solubilised, SDS-free, recombinant OPH was able to detoxify parathion and methylparathion ranging between 10–80% and 3.6–45% in enzyme reaction cycles after immobilization on Ca-alginate and agar–agar, respectively.

Cloning and Biochemical Characterization of a Novel Carbendazim (Methyl-1 H -Benzimidazol-2-ylcarbamate)-Hydrolyzing Esterase from the Newly Isolated Nocardioides sp. Strain SG-4G and Its Potential for Use in Enzymatic Bioremediation

A highly efficient carbendazim (methyl-1H-benzimidazol-2-ylcarbamate, or MBC)-mineralizing bacterium was isolated from enrichment cultures originating from MBC-contaminated soil samples. This bacterium, Nocardioides sp. strain SG-4G, hydrolyzed MBC to 2-aminobenzimidazole, which in turn was converted to the previously unknown metabolite 2-hydroxybenzimidazole. The initial steps of this novel metabolic pathway were confirmed by growth and enzyme assays and liquid chromatography-mass spectrometry (LC-MS) studies. The enzyme responsible for carrying out the first step was purified and subjected to N-terminal and internal peptide sequencing. The cognate gene, named mheI (for MBC-hydrolyzing enzyme), was cloned using a reverse genetics approach. The MheI enzyme was found to be a serine hydrolase of 242 amino acid residues. Its nearest known relative is an uncharacterized hypothetical protein with only 40% amino acid identity to it. Codon optimized mheI was heterologously expressed in Escherichia coli, and the His-tagged enzyme was purified and biochemically characterized. The enzyme has a K(m) and k(cat) of 6.1 muM and 170 min(-1), respectively, for MBC. Radiation-killed, freeze-dried SG-4G cells showed strong and stable MBC detoxification activity suitable for use in enzymatic bioremediation applications.

Environmental applications of earthworm esterases in the agroecosystem

Chemical control of pests is still necessary in agriculture, despite the growing efforts to introduce biocontrol-based strategies. Many studies have evidenced the harm of pesticide side-effects on natural populations of pest enemies and other non-target organisms. Moreover, pesticide-contaminated soils can act as a secondary pollution source causing contamination in environmental compartments of critical concern to public health (water resource). These environmental risks need to be assessed and monitored for decision-making related to the post-authorization management of pesticides. Under these considerations, earthworm esterases can be a suitable tool for these regulatory and environmental purposes. Herein, it is suggested the use of earthworm esterases as biomarkers to be included in a field toxicity test. Furthermore, the potential role of gut carboxylesterases (CEs) in the modulation of pesticide toxicity is discussed in view of their contribution to the natural tolerance of earthworms to pesticides, and consequently the appropriate selection of earthworm species for regulatory toxicity testing. Finally, it is postulated that CE secretion into the earthworm gut could be an environmental friendly methodology in the enzymatic bioremediation of pesticide-contaminated soils.

Optimization of Laccase-mediated Benzo[a]pyrene Oxidation and the Bioremedial Application in Aged Polycyclic Aromatic Hydrocarbons-contaminated Soil

Laccase is a polyphenol oxidase with the ability to oxidize a broad range of persistent organic pollutants, including benzo[a]pyrene, the most carcinogenic, mutagenic an dt eratogenic polycyclic aromatic hydrocarbons (PAHs). In this study, the reaction conditions for benzo[a]pyrene oxidation by laccase from Trametes versicolor were optimized in a liquid medium by a series of single factor experiments. The maximal benzo[a]pyrene oxidation rate was observed at 40 ◦ C, pH 4, 10% of acetonitrile and an incubation time of more than 24 hr, and the benzo[a]pyrene oxidation was enhanced significantly by the addition of a mediator, 2,2-azino-bis-(3ethylbenzothiazoline-6-sulfonicacid) (ABTS). Laccase was also applied to aged PAHs polluted soil to examine the efficiency of enzymatic bioremediation. The results showed that the enzyme was still effective in the degradation of anthracene, benzo[a]pyrene and benzo[a]anthracene in soil. Moreover, the degradation rate of most PAHs increased by the addition of ABTS. Our results indicated that the bioremediation of PAHs contaminated soil using laccase is feasible but a suboptimal pH might be a limiting factor in the enzymatic treatment of soil.

Enzymatic bioremediation of cashew nut shell liquid contamination

Cashew nut shell liquid (CNSL), a by-product of the cashew kernel industry, is a caustic, viscous, dark liquid. The process is done manually, which leaves stains on the hands of the workers. The aim was to find the utility of enzymes, oxidoreductases and proteases for the bioremediation of CNSL, which contains phenolics, mainly cardanol (60–65%). The results show that peroxidase reduced the color of the CNSL solution by polymerization and precipitation, where as laccase, papain and fungal and bacterial protease degraded the phenolic constituents. The degradation was mainly at the double bonds of the C15 hydrocarbon chain of the cardanol. To improve the enzyme stability, laccase and papain was separately immobilized in alginate–starch beads. Immobilized laccase can degrade 28.6% CNSL within 2 h, where as papain takes longer duration, and at 73 h, the adsorbed phenols on the alginate (45.86%) also got degraded. MALDI-TOF MS revealed that, immobilized laccase–papain beads combination; 1:1 (w/w) degraded 60% of the cardanol and some phenolic compounds having molecular mass of 374, 390 and 407. These beads are active and stable in aqueous media, can be used to prepare a mild, nontoxic, ecofriendly, cost effective hand wash solution for the removal of phenolic stains.

Enrichment and isolation of endosulfan-degrading microorganism from tropical acid soil

Endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,3,4-benzo-dioxathiepin-3-oxide) is a cyclodiene organochlorine currently used as an insecticide all over the world and its residues are posing a serious environmental threat. This study reports the enrichment and isolation of a microbial culture capable of degrading endosulfan with minimal production of endosulfan sulfate, the toxic metabolite of endosulfan, from tropical acid soil. Enrichment was achieved by using the insecticide as sole sulfur source. The enriched microbial culture, SKL-1, later identified as Pseudomonas aeruginosa, degraded up to 50.25 and 69.77 % of α and β endosulfan, respectively in 20 days. Percentage of bioformation of endosulfan sulfate to total formation was 2.12% by the 20th day of incubation. Degradation of the insecticide was concomitant with bacterial growth reaching up to an optical density of 600 nm (OD600) 2.34 and aryl sulfatase activity of the broth reaching up to 23.93 μg pNP/mL/hr. The results of this study suggest that this novel strain is a valuable source of potent endosulfan–degrading enzymes for use in enzymatic bioremediation. Further, the increase in aryl sulfatase activity of the broth with the increase in degradation of endosulfan suggests the probable involvement of the enzyme in the transformation of endosulfan to its metabolites.

Cloning of Lentinula edodes lemnp2, a manganese peroxidase that is secreted abundantly in sawdust medium

Manganese peroxidase (MnP), which is one of the lignin-degrading enzymes from white-rot fungi, possesses oxidative activity against phenolic compounds, making it a useful enzyme for bioremediation. A novel MnP-encoding gene (lemnp2) was isolated from Lentinula edodes. The deduced amino acid sequence showed approximately 48.8% homology to LeMnP1. The cDNA clone was approximately 1.4 kbp whereas the genomic sequence was 1.9 kbp, and comparison of the two indicated that lemnp2 contains 13 introns. The upstream region of lemnp2 contains putative CAAT, TATA, and metal response elements. Additionally, LeMnP2 contains conserved motifs that are observed in fungal MnPs, including 10 cysteines, a Mn-binding site, and Ca2+-binding sites. The lemnp2 transcript was identified in mycelium cultivated on sawdust medium, and the protein was secreted into the medium. MnP activity was purified from the sawdust medium as one peak during purification. Western blot analysis confirmed that LeMnP2, but not LeMnP1, was secreted into the sawdust medium. These results collectively demonstrate that LeMnP2 is the major MnP secreted into sawdust medium.

Application of immobilized horseradish peroxidase onto modified acrylonitrile copolymer membrane in removing of phenol from water

A non-modified and modified with NaOH and ethylenediamine ultrafiltration membranes prepared from AN copolymer have been used as carriers for the immobilization of horseradish peroxidase (HRP) enzyme. The amount of bound protein onto the membranes and the activity of the immobilized enzyme have been investigated as well as the pH and thermal optimum, and the thermal stability of the free and immobilized HRP. The experiments have proved that the modified membrane is a better support for the immobilization of HRP enzyme. The latter has shown a greater thermal stability than the free enzyme. A possible application has been studied for reducing phenol concentration in water solutions through oxidation of phenol by hydrogen peroxide, in the presence of free and immobilized HRP enzyme on modified AN copolymer membranes. A higher degree of the phenol oxidation has been observed in the presence of the immobilized enzyme. A total removal of phenol has been achieved in the presence of immobilized HRP at concentration of the hydrogen peroxide 0.5 mmol L −1 and concentration of the phenol in the model solutions within the interval 5–40 mg L −1. A high degree of phenol oxidation (95.4%) has been achieved in phenol solution with 100 mg L −1 concentration in the presence of hydrogen peroxide and immobilized HRP, which demonstrates the promising opportunity of using the enzyme for bioremediation of waste waters, containing phenol. The immobilized HRP has shown good operational stability. Deactivation of the immobilized enzyme to 50% of the initial activity has been observed after the 20th day of the enzyme operation.

Enzymatic biotransformation of the azo dye Sudan Orange G with bacterial CotA-laccase

In the present study we show that recombinant bacterial CotA-laccase from Bacillus subtilis is able to decolourise, at alkaline pH and in the absence of redox mediators, a variety of structurally different synthetic dyes. The enzymatic biotransformation of the azo dye Sudan Orange G (SOG) was addressed in more detail following a multidisciplinary approach. Biotransformation proceeds in a broad span of temperatures (30–80 °C) and more than 98% of Sudan Orange G is decolourised within 7 h by using 1 U mL −1 of CotA-laccase at 37 °C. The bell-shape pH profile of the enzyme with an optimum at 8, is in agreement with the pH dependence of the dye oxidation imposed by its acid-basic behavior as measured by potentiometric and electrochemical experiments. Seven biotransformation products were identified using high-performance liquid chromatography and mass spectrometry and a mechanistic pathway for the azo dye conversion by CotA-laccase is proposed. The enzymatic oxidation of the Sudan Orange G results in the production of oligomers and, possibly polymers, through radical coupling reactions. A bioassay based on inhibitory effects over the growth of Saccharomyces cerevisiae shows that the enzymatic bioremediation process reduces 3-fold the toxicity of Sudan Orange G.

Probing the molecular determinants of aniline dioxygenase substrate specificity by saturation mutagenesis

Aniline dioxygenase is a multicomponent Rieske nonheme-iron dioxygenase enzyme isolated from Acinetobacter sp. strain YAA. Saturation mutagenesis of the substrate-binding pocket residues, which were identified using a homology model of the alpha subunit of the terminal dioxygenase (AtdA3), was used to probe the molecular determinants of AtdA substrate specificity. The V205A mutation widened the substrate specificity of aniline dioxygenase to include 2-isopropylaniline, for which the wild-type enzyme has no activity. The V205A mutation also made 2-isopropylaniline a better substrate for the enzyme than 2,4-dimethylaniline, a native substrate of the wild-type enzyme. The I248L mutation improved the activity of aniline dioxygenase against aniline and 2,4-dimethylaniline approximately 1.7-fold and 2.1-fold, respectively. Thus, it is shown that the alpha subunit of the terminal dioxygenase indeed plays a part in the substrate specificity as well as the activity of aniline dioxygenase. Interestingly, the equivalent residues of V205 and I248 have not been previously reported to influence the substrate specificity of other Rieske dioxygenases. These results should facilitate future engineering of the enzyme for bioremediation and industrial applications.

ChrR, a Soluble Quinone Reductase of Pseudomonas putida That Defends against H2O2

Most bacteria contain soluble quinone-reducing flavoenzymes. However, no biological benefit for this activity has previously been demonstrated. ChrR of Pseudomonas putida is one such enzyme that has also been characterized as a chromate reductase; yet we propose that it is the quinone-reducing activity of ChrR that has the greatest biological significance. ChrR reduces quinones by simultaneous two-electron transfer, avoiding formation of highly reactive semiquinone intermediates and producing quinols that promote tolerance of H(2)O(2). Expression of chrR was induced by H(2)O(2), and levels of chrR expression in overexpressing, wild type, and knock-out mutant strains correlated with the H(2)O(2) tolerance and scavenging ability of each strain. The chrR expression level also correlated with intracellular H(2)O(2) levels as measured by protein carbonylation assays and fluorescence-activated cell scanning analysis with the H(2)O(2)-responsive dye H(2)DCFDA. Thus, enhancing the activity of ChrR in a chromate-remediating bacterial strain may not only increase the rate of chromate transformation, it may also augment the capacity of these cells to withstand the unavoidable production of H(2)O(2) that accompanies chromate reduction.

Phylogenetic and biochemical characterisation of a recombinant laccase from Trametes versicolor

Laccases are important enzymes for bioremediation and the best-characterised are from the fungus Trametes versicolor. Here, we describe the cloning and characterisation of a new variant of laccase from T. versicolor and its expression in Saccharomyces cerevisiae. We have performed a sequence-based analysis of Trametes laccases that leads to a proposal for a new nomenclature of this fungus laccases according to their phylogenetic relationships since their nomenclature based on IPs is ambiguous. We also describe the kinetic properties of the recombinant enzyme.

ENZYMATIC BIOREMEDIATION: FROM ENZYME DISCOVERY TO APPLICATIONS

1. Enzymatic bioremediation is potentially a rapid method of removing environmental pesticide residues. Applications include the treatment of residues resulting from agricultural production and processing industries, such as the treatment of irrigation waters, surface-contaminated fruit and vegetables and spent dip liquors. 2. A specific application for some organophosphate-degrading enzymes involves detoxification of nerve agent stockpiles. Effective and affordable remediation requires highly specialized enzymes, so protein engineering techniques are being used to improve properties of various source enzymes to enhance catalytic rates, stability and substrate range. 3. Trials with an optimized organophosphate-degrading enzyme have shown the feasibility of such technology in various applications. 4. The enzymes developed for environmental remediation for specific pesticide classes also have applications as antidotes for high-dose pesticide poisonings and as prophylaxis for people at risk of high pesticide doses.

Enrichment and isolation of endosulfan-degrading microorganisms.

Endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,3,4-benzo-dioxathiepin-3-oxide) is a cyclodiene organochlorine currently used as an insecticide all over the world and its residues are posing a serious environmental threat. This study reports the isolation and identification of enriched microorganisms, capable of degrading endosulfan. Enrichment was achieved by using the insecticide as either the sole source of carbon or sulfur in parallel studies. Two strains each of fungi (F1 and F4) and bacteria (BF2 and B4) were selected using endosulfan as a sole carbon source. A Pandoraea species (Lin-3) previously isolated in our laboratory using lindane (gamma-HCH) as a carbon source was also screened for endosulfan degradation. F1 and F4 (Fusarium ventricosum) degraded alpha-endosulfan by as much as 82.2 and 91.1% and beta-endosulfan by 78.5 and 89.9%, respectively, within 15 d of incubation. Bacterial strains B4 and Lin-3 degraded alpha-endosulfan up to 79.6 and 81.8% and beta-endosulfan up to 83.9 and 86.8%, respectively, in 15 d. Among the bacterial strains isolated by providing endosulfan as a sulfur source, B4s and F4t degraded alpha-endosulfan by as much as 70.4 and 68.5% and beta-endosulfan by 70.4 and 70.8%, respectively, after 15 d. Degradation of the insecticide occurred concomitant with bacterial growth reaching an optical density (OD600) of 0.366 and 0.322 for B4 and Lin-3, respectively. High OD600 was also noted with the other bacterial strains utilizing endosulfan as a sulfur source. Fungal and bacterial strains significantly decreased the pH of the nutrient culture media while growing on endosulfan. The results of this study suggest that these novel strains are a valuable source of potent endosulfan-degrading enzymes for use in enzymatic bioremediation.

Isolation and characterization of a Mycobacterium strain that metabolizes the insecticide endosulfan.

The aim of this study was to isolate and characterize a bacterium capable of metabolizing endosulfan. A endosulfan-degrading bacterium (strain ESD) was isolated from soil inoculum after repeated culture with the insecticide as the sole source of sulfur. Analysis of its 16S rRNA gene sequence, and morphological and physiological characteristics revealed it to be a new fast-growing Mycobacterium, closely related to other Mycobacterium species with xenobiotic-degrading capabilities. Degradation of endosulfan by strain ESD involved both oxidative and sulfur-separation reactions. Strain ESD did not degrade endosulfan when sulfite, sulphate or methionine were present in the medium along with the insecticide. Partial degradation occurred when the culture was grown, with endosulfan, in the presence of MOPS (3-(N-morpholino)propane sulphonic acid), DMSO (dimethyl sulfoxide), cysteine or sulphonane and complete degradation occurred in the presence of gutathione. When both beta-endosulfan and low levels of sulphate were provided as the only sources of sulfur, biphasic exponential growth was observed with endosulfan metabolism being restricted to the latter phase of exponential growth. This study isolated a Mycobacterium strain (strain ESD) capable of metabolizing endosulfan by both oxidative and sulfur-separation reactions. The endosulfan-degrading reactions are a result of the sulfur-starvation response of this bacterium. This describes the isolation of a Mycobacterium strain capable of degrading the insecticide endosulfan. This bacterium is a valuable source of enzymes for use in enzymatic bioremediation of endosulfan residues.

Enrichment of a microbial culture capable of degrading endosulphate, the toxic metabolite of endosulfan.

The aim of this study was to isolate a source of enzymes capable of degrading endosulphate (endosulfan sulphate), the toxic metabolite of the pesticide endosulfan. A microbial broth culture capable of degrading endosulphate was enriched from endosulfan-contaminated soil by providing the metabolite as the sole source of sulphur in broth culture. No microbial growth was observed in the absence of endosulphate. In the presence of endosulphate, growth of the culture occurred with the concomitant formation of three chlorine-containing compounds. Thin layer chromatography and gas chromatography--mass spectral analysis identified these metabolites as endosulfan monoaldehyde, 1,2,3,4,7,7-hexachloro-5,6-bis(methylene)bicyclo[2.2.1]-2-heptene and 1,2,3,4,7,7-hexachloro-5-hydroxymethylene-6-methylenebicyclo[2.2.1]-2-heptene. The second and third compounds have not been reported in previous metabolic studies. The enriched culture was also able to utilize alpha- and beta-endosulfan as sulphur sources, each producing the hydrolysis product endosulfan monoaldehyde as the sole chlorine-containing metabolite. Alpha-endosulfan was more readily hydrolysed than the beta-isomer. This study isolated a mixed microbial culture capable of degrading endosulphate. The products of degradation were characterized as novel endosulfan metabolites. This study describes the isolation of a mixed microbial culture that is potentially a valuable source of hydrolysing enzymes for use in enzymatic bioremediation, particularly of endosulphate and alpha-endosulfan residues.

Enrichment of an endosulfan-degrading mixed bacterial culture.

An endosulfan-degrading mixed bacterial culture was enriched from soil with a history of endosulfan exposure. Enrichment was obtained by using the insecticide as the sole source of sulfur. Chemical hydrolysis was minimized by using strongly buffered culture medium (pH 6.6), and the detergent Tween 80 was included to emulsify the insecticide, thereby increasing the amount of endosulfan in contact with the bacteria. No growth occurred in control cultures in the absence of endosulfan. Degradation of the insecticide occurred concomitant with bacterial growth. The compound was both oxidized and hydrolyzed. The oxidation reaction favored the alpha isomer and produced endosulfate, a terminal pathway product. Hydrolysis involved a novel intermediate, tentatively identified as endosulfan monoaldehyde on the basis of gas chromatography-mass spectrometry and chemical derivatization results. The accumulation and decline of metabolites suggest that the parent compound was hydrolyzed to the putative monoaldehyde, thereby releasing the sulfite moiety required for growth. The monoaldehyde was then oxidized to endosulfan hydroxyether and further metabolized to (a) polar product(s). The cytochrome P450 inhibitor, piperonyl butoxide, did not prevent endosulfan oxidation or the formation of other metabolites. These results suggest that this mixed culture is worth investigating as a source of endosulfan-hydrolyzing enzymes for use in enzymatic bioremediation of endosulfan residues.

Insect enzymes for bioremediation

Professor Devonshire from Rothamsted (UK) has joined CSIRO Entomology (Australia) for 12 months to work on a special case of organophosphate pesticide bioremediation, using insect enzymes. ‘Our work has shown that resistant insects have developed the ability to break down the harmful chemicals in insecticide sprays. If we can produce larger quantities of these enzymes in a form which can be sprayed or applied in the field, residues of the harmful chemicals can also be broken down.’ he says. Pesticide biochemistry is an area of broad and ongoing mutual interest both with CSIRO and with other Australian agricultural research institutions.

Microbial genomes.

Microbial genome sequencing is driven by the need to understand and control pathogens and to exploit extremophiles and their enzymes in bioremediation and industry. It is hard for the traditional bacteriologist to grasp the scale and pace of the venture. Around two dozen microbial genomes have now been completed and, within a decade, genomes from every significant species of bacterial pathogen of humans, animals and plants will have been sequenced. Indeed, we will often have more than one sequence from a species or genus--for example, we already have sequences from two strains of Helicobacter pylori, from two strains of Mycobacterium tuberculosis and from three species of Pyrococcus. However, genome sequencing risks becoming expensive molecular stamp-collecting without the tools to mine the data and fuel hypothesis-driven laboratory-based research. Bioinformatics, twinned with the new experimental approaches forming functional genomics', provides some of the needed tools. Nonetheless, there will be an increasing need for us to explore the detailed implications of genomic findings. Microbial genome sequencing thus represents not a threat, but an exciting opportunity for molecular microbiologists.

Engineering of improved microbes and enzymes for bioremediation

Bioremediation with microorganisms is an attractive alternative to conventional techniques, such as incineration and chemical treatment, for disposing of pollutants. Recent progress in molecular biology, microbiology, and genetics is providing the driving force towards engineering improved microbes and enzymes for bioremediation. A number of genetic engineering approaches have been developed in the past several years that have proven useful in introducing/evolving the desired properties for different biodegradative pathways or enzymes. The initial excitement generated in this area should continue to pave the way for rational or irrational design of microbes or enzymes with novel remedial properties.