Non-phenolic Compounds Research Articles

Synergistic contribution of hydrosulfide and carbonate anions to the β-O-4 bond cleavage of lignin model compounds in a green liquor pretreatment for enzymatic hydrolysis of lignocellulosic materials

To examine why green liquor (GL) pretreatment of lignocellulosic materials effectively facilitates enzymatic saccharification under conditions milder than those of a common alkaline cooking process, dimeric β-O-4 type lignin model compounds with and without free phenolic hydroxyl group were reacted in several alkaline solutions including a model solution of GL, which mainly contains Na2CO3 and Na2S. The β-O-4 bond of the phenolic model compound was cleaved with a sufficient rate in the model solution of GL. The β-O-4 bond cleavage of the non-phenolic model compound was more frequent in the model solution of GL than in other alkaline solutions. These results suggest that β-O-4 bonds present in lignocellulosic materials are effectively cleaved in a GL pretreatment. It was also suggested that HS− and CO3 2− synergistically contribute to the β-O-4 bond cleavage of the non-phenolic model compound under GL pretreatment conditions.

Roles of small laccases from Streptomyces in lignin degradation.

Laccases (EC 1.10.3.2) are multicopper oxidases that can oxidize a range of substrates, including phenols, aromatic amines, and nonphenolic substrates. To investigate the involvement of the small Streptomyces laccases in lignin degradation, we generated acid-precipitable polymeric lignin obtained in the presence of wild-type Streptomyces coelicolor A3(2) (SCWT) and its laccase-less mutant (SCΔLAC) in the presence of Miscanthus x giganteus lignocellulose. The results showed that strain SCΔLAC was inefficient in degrading lignin compared to strain SCWT, thereby supporting the importance of laccase for lignin degradation by S. coelicolor A3(2). We also studied the lignin degradation activity of laccases from S. coelicolor A3(2), Streptomyces lividans TK24, Streptomyces viridosporus T7A, and Amycolatopsis sp. 75iv2 using both lignin model compounds and ethanosolv lignin. All four laccases degraded a phenolic model compound (LM-OH) but were able to oxidize a nonphenolic model compound only in the presence of redox mediators. Their activities are highest at pH 8.0 with a low krel/Kapp for LM-OH, suggesting that the enzymes’ natural substrates must be different in shape or chemical nature. Crystal structures of the laccases from S. viridosporus T7A (SVLAC) and Amycolatopsis sp. 75iv2 were determined both with and without bound substrate. This is the first report of a crystal structure for any laccase bound to a nonphenolic β-O-4 lignin model compound. An additional zinc metal binding site in SVLAC was also identified. The ability to oxidize and/or rearrange ethanosolv lignin provides further evidence of the utility of laccase activity for lignin degradation and/or modification.

The effects of mediator and granular activated carbon addition on degradation of trace organic contaminants by an enzymatic membrane reactor

The removal of four recalcitrant trace organic contaminants (TrOCs), namely carbamazepine, diclofenac, sulfamethoxazole and atrazine by laccase in an enzymatic membrane reactor (EMR) was studied. Laccases are not effective for degrading non-phenolic compounds; nevertheless, 22–55% removal of these four TrOCs was achieved by the laccase EMR. Addition of the redox-mediator syringaldehyde (SA) to the EMR resulted in a notable dose-dependent improvement (15–45%) of TrOC removal affected by inherent TrOC properties and loading rates. However, SA addition resulted in a concomitant increase in the toxicity of the treated effluent. A further 14–25% improvement in aqueous phase removal of the TrOCs was consistently observed following a one-off dosing of 3g/L granular activated carbon (GAC). Mass balance analysis reveals that this improvement was not due solely to adsorption but also enhanced biodegradation. GAC addition also reduced membrane fouling and the SA-induced toxicity of the effluent.

A method for measuring difference in activity of phenolic and non- phenolic groups through 2,2` azino-bis-(3-ethyl-benzothiazoline-6- sulphonate) radical cation.

A 2, 2`azino-bis-(3-ethyl-benzothiazoline-6-sulphonate) radical cation-based method was optimized in 96 well plates to evaluate the comparative scavenging potential of phenolic and non-phenolic group of molecules with respect to control. The interactions of these molecules with ABTS radical cation were quantified on the basis of their relative influence on the bleaching of a bluish-green color complex in a structure- and dose-dependent manner. Experimentally, the developed assay provided evidence that phenolic molecules are more reactive with ABTS radical than non-phenolic compounds because of their resonance and hyper-conjugation effects.

Fungal laccases and their applications in bioremediation.

Laccases are blue multicopper oxidases, which catalyze the monoelectronic oxidation of a broad spectrum of substrates, for example, ortho- and para-diphenols, polyphenols, aminophenols, and aromatic or aliphatic amines, coupled with a full, four-electron reduction of O2 to H2O. Hence, they are capable of degrading lignin and are present abundantly in many white-rot fungi. Laccases decolorize and detoxify the industrial effluents and help in wastewater treatment. They act on both phenolic and nonphenolic lignin-related compounds as well as highly recalcitrant environmental pollutants, and they can be effectively used in paper and pulp industries, textile industries, xenobiotic degradation, and bioremediation and act as biosensors. Recently, laccase has been applied to nanobiotechnology, which is an increasing research field, and catalyzes electron transfer reactions without additional cofactors. Several techniques have been developed for the immobilization of biomolecule such as micropatterning, self-assembled monolayer, and layer-by-layer techniques, which immobilize laccase and preserve their enzymatic activity. In this review, we describe the fungal source of laccases and their application in environment protection.

Olive oil mill wastewaters: Phenolic content characterization during degradation by Coriolopsis gallica

Olive mill wastewaters (OMW) pose a serious environmental concern owing to high polyphenol content. Decolorization and degradation of phenolic compounds (PC) by Coriolopsis gallica was demonstrated in our laboratory as a potential biotreatment of OMW in solid and liquid media. High performance liquid chromatography coupled to electrospray time-of-flight mass spectrometry was used to analyze the evolution of the main phenolic compounds during the C. gallica biodegradation process. Amongst total the compounds characterized in methanolic extracts of OMW, 12 were unknown, 15 were from different polyphenolic families, and 27 were other non-phenolic compounds. The evolution of PC content during the degradation process indicated that, despite the complexity of the OMW phenolic fraction, C. gallica was able to grow on OMW-based media using PC as sources of carbon and energy, particularly acids, alcohols, lignans and flavones. Complete dephenolization of OMW was obtained.

Enhancement of trace organic contaminant degradation by crude enzyme extract from Trametes versicolor culture: Effect of mediator type and concentration

The performance of two redox mediating compounds, namely 1-hydroxybenzotriazole (HBT) and syringaldehyde (SA), was compared in terms of enhancement of enzymatic degradation of a diverse set of 14 phenolic and 16 non-phenolic trace organic contaminants (TrOCs) and the toxicity of the treated media. Extracellular enzyme extract (predominantly containing laccase) from Trametes versicolor culture achieved efficient degradation (70–95%) of nine phenolic and one non-phenolic TrOCs. Mediator dosing extended the spectrum of efficiently degraded TrOCs to 13 phenolic and three non-phenolic compounds, with moderate improvements in removal of a few other non-phenolic compounds. TrOC removal efficiency improved significantly as the HBT dose was increased from 0.1 to 0.5mM, while SA achieved similar removal over dosage range of 0.1–1mM. A particular concern was the toxicity of the treated media (1200–2200 times that of the control) for all SA dosages applied. Overall, HBT at a concentration of 0.5mM achieved the best removal without raising concern regarding toxicity of the treated media. The results are discussed in the light of the redox potential of the enzyme-mediator cocktail, the balance between the stability and reactivity of the radicals generated and their cytotoxic effects.

A Polyvinylpolypyrrolidone (PVPP)-Assisted Folin–Ciocalteu Assay to Assess Total Phenol Content of Commercial Beverages

The use of a Folin–Ciocalteu (FC) assay to assess total phenolics in certain foods may be limited by the inability of this assay to discriminate between phenolics and non-phenolic reducing compounds. In the present study, we have mentioned the usefulness of an insoluble water-synthetic polymer, polyvinylpolypyrrolidone (PVPP), to separate phenolics and non-phenolic reducing compounds (i.e., sugars, ascorbic acid, and sulfite) from their original food matrix. After employing three consecutive cycles adding PVPP, all polyphenols tested (including phenolic and cinnamic acids) showed an adsorption percentage (AP) to PVPP higher than 90 %. When tested in various beverages, the PVPP-based pretreatment affected in different ways the FC index, depending on the food matrix. A low AP was evidenced in the case of orange juice, most probably related to the high content of ascorbic acid in such samples. In contrast, a high adsorption to PVPP was observed in red wine and in powdered fruit samples, showing a higher fraction of polyphenols than non-polyphenolic reducing compounds. We consider that our results strongly support that this PVPP-based procedure allows the evaluation of total phenolic content, by the FC methodology of beverages without the contribution of non-phenolic FC-reducing compounds.

Phenolic Profiles and Antioxidant Activity of Buckwheat (Fagopyrum esculentum Möench and Fagopyrum tartaricum L. Gaerth) Hulls, Brans and Flours

The extracts from hulls, brans and flours of Fagopyrum esculentum Möench (FEM, three varieties) and Fagopyrum tartaricum L. Gaerth (FTG, seven varieties) were screened for free and bound phenolic content or total phenolic content (TPC), as well as 1,1 diphenyl-2-picryl hydrazyl (DPPH) free radical scavenging activity and reducing power. Free phenolics were predominant in buckwheat hulls, brans and flours. FEM hulls extract exhibited the highest reducing power and DPPH free radical scavenging activity with the average EC50 84.54 μg mL−1 and IC50 11.54 μg mL−1 respectively, FTG brans extract had the highest average TPC (24.87 mg GAE g−1 DW), and FEM flours extract showed the lowest TPC, reducing power and radical scavenging activity. Furthermore, the correlations among TPC, DPPH free radical scavenging activity and reducing power of all the samples were investigated. The rank correlation coefficient (rs) between reducing power and DPPH free radical scavenging activity of buckwheat hulls, between TPC and DPPH free radical scavenging activity of buckwheat flours were 0.76 and 0.79, respectively (P<>0.05). However, there is no significant correlation between the remaining indexes of hulls and flours, as well as the ten buckwheat brans. This result indicated that some non-phenolic compounds also contributed to the total antioxidant activity in hulls, brans and flours of buckwheats. This study demonstrated that buckwheat hulls and brans, rather than flours, are good source of antioxidants.

Quantitative Difference in the Rates of the β-O-4 Bond Cleavage between Lignin Model Compounds with and without γ-Hydroxymethyl Groups during the Alkaline Pulping Process

To examine the effect of the presence of γ-hydroxymethyl groups on the rate of the β-O-4 bond cleavage during the alkaline pulping process, the rates of the β-O-4 bond cleavage of non-phenolic lignin model compounds without a γ-hydroxymethyl group, 2-(2-methoxyphenoxy)-1-(3,4-dimethoxyphenyl)ethanol (G’G) and 2-(2,6-dimethoxyphenoxy)-1-(3,4-dimethoxyphenyl)ethanol (G’S), were compared with those of analogous model compounds with a γ-hydroxymethyl group, 2-(2-methoxyphenoxy)-1-(3,4-dimethoxyphenyl)propane-1,3-diol (GG) and 2-(2,6-dimethoxyphenoxy)-1-(3,4-dimethoxyphenyl)propane-1,3-diol (GS), under alkaline pulping conditions. The disappearance of G’G or G’S was accompanied by the quantitative liberation of 2-methoxyphenol or 2,6-dimethoxyphenol, respectively, indicating that the disappearance resulted from the β-O-4 bond cleavage. The disappearance rate of G’G or G’S was in between those of the erythro and threo isomers of GG or GS, respectively. This result seems to be reasonably explained when the steric repulsions of the three staggered conformations are taken into consideration. The disappearance rate of G’G or G’S increased, but the increment became moderate with increasing hydroxide concentration.

Biochemical studies of the multicopper oxidase (small laccase) from Streptomyces coelicolor using bioactive phytochemicals and site‐directed mutagenesis

SummaryMulticopper oxidases can act on a broad spectrum of phenolic and non-phenolic compounds. These enzymes include laccases, which are widely distributed in plants and fungi, and were more recently identified in bacteria. Here, we present the results of biochemical and mutational studies of small laccase (SLAC), a multicopper oxidase from Streptomyces coelicolor (SCO6712). In addition to typical laccase substrates, SLAC was tested using phenolic compounds that exhibit antioxidant activity. SLAC showed oxidase activity against 12 of 23 substrates tested, including caffeic acid, ferulic acid, resveratrol, quercetin, morin, kaempferol and myricetin. The kinetic parameters of SLAC were determined for 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), 2,6-dimethoxyphenol, quercetin, morin and myricetin, and maximum reaction rates were observed with myricetin, where kcat and Km values at 60°C were 8.1 (± 0.8) s−1 and 0.9 (± 0.3) mM respectively. SLAC had a broad pH optimum for activity (between pH 4 and 8) and temperature optimum at 60–70°C. It demonstrated remarkable thermostability with a half-life of over 10 h at 80°C and over 7 h at 90°C. Site-directed mutagenesis revealed 17 amino acid residues important for SLAC activity including the 10 His residues involved in copper coordination. Most notably, the Y229A and Y230A mutant proteins showed over 10-fold increase in activity compared with the wild-type SLAC, which was correlated to higher copper incorporation, while kinetic analyses with S929A predicts localization of this residue near the meta-position of aromatic substrates.Funding Information Funding for this research was provided by the Government of Ontario for the project ‘FFABnet: Functionalized Fibre and Biochemicals’ (ORF-RE-05-005), and the Natural Sciences and Engineering Research Council of Canada.

Quantification of total phenols in bio-oil using the Folin–Ciocalteu method

Bio-oil from fast pyrolysis of biomass contains phenolics derived from the lignin portion of the biomass. Traditional testing for total phenolics in bio-oil is based on either a rough estimate of the weight percent water-insolubles in bio-oil or on tedious liquid–liquid extraction methods. We have evaluated the Folin–Ciocalteu (FC) colorimetry method used for quantifying total phenols in wine to determine total phenols in bio-oil. This method, based on the oxidation of phenolic compounds by the FC reagent, is fast and easy to perform. This study evaluated its accuracy relative to interferents by the use of positive and negative controls. Positive controls included phenol, 4-methylphenol, 3-ethylphenol, guaiacol, 2,6-dimethoxyphenol and eugenol. The negative controls included sugars, furfural, and acids. Potential interferents with the quantification of total phenols by the FC method was calculated for all positive and negative controls by using data obtained when adding the contributor (positive controls) and the interferent (negative controls) into bio-oil using typical concentrations found in bio-oil. The positive and several of the negative controls produced strongly correlated linear relationships between the indicated phenolic content of the bio-oil and the amount of contributor or interferent added. However, the slopes of these relationships for the negative controls were much smaller than those for the positive controls, indicating that the error in the prediction of phenolic content was small even for large concentrations of interferent compounds. For typical concentrations of non-phenolic compounds in bio-oil, the error in predicted phenolic content as a result of their presence was ≤5.8%. Total phenolic content in bio-oil detected by the FC method was comparable to the quantity of total phenolics obtained by liquid–liquid extraction. All results fell within the margin of error and the uncertainty of the measurement by the FC method indicating there was no significant difference in the results between the two methods. The FC method uncertainty of measurement was ±1.1% at the 95% confidence level.

Fractionation of a red grape marc extract by colloidal gas aphrons

The objective of the current study was to investigate the application of colloidal gas aphrons (CGA) as a potential low-cost technology for the fractionation of phenolic extracts obtained from off-skins fermented red grape skins with high sugar content. The trials were aimed to investigate the possibility of using CGA generated from the non-ionic surfactant Tween20 to separate phenolic compounds from non-phenolic compounds (minerals and sugars) and to fractionate different fractions of phenolic compounds (total phenols, cinnamic acids and anthocyanins). Separation tests were carried out in a 0.7 L flotation column investigating the effect of both the volumetric ratio CGA/extract sample and the molar ratio CGA/extract sample on both the recovery yields and the separation factor (the ratio between the concentration of a compound in the recovered aphron phase and in the discharged liquid phase). Results revealed the possibility of reaching high recovery yield even though with a poor selective fractionation of the different compounds present in the extract, with glucose and cinnamic acids showing the highest affinity for the aphron phase.

Comparison of a series of laccase mediators in the electro-oxidation reactions of non-phenolic lignin model compounds

The electro-oxidations of non-phenolic lignin model compounds in an electrolytic mediator system (EMS) have been investigated with several laccase mediators, including N-hydroxyphthalimide (NHPI), 1-hydroxybenzotriazole (HBT), violuric acid (VLA), 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), under the same reaction conditions. In the bulk electrolyses of the monomeric model compound [1-(4′-ethoxy-3′-methoxyphenyl)ethanol (1G)], oxidation with NHPI gave the corresponding CαO product (2G) in high yield, whereas the oxidations with HBT, VLA, and TEMPO afforded 2G in moderate yields. The highest reaction selectivity for the guaiacyl-units was found in the oxidation conducted in the presence of ABTS, although the yield was low. In the bulk electrolyses of the dimeric model compound [4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether (3G)], the oxidation with NHPI gave the corresponding CαO product (4G) in high yield, whereas the oxidations with HBT, VLA, and TEMPO gave 4G in low yields. In contrast, the oxidation with ABTS gave a Cα–Cβ cleavage product (5G) in 5.5% yield. The selectivity of the mediators in the EMS reaction effectively reflected the mechanisms of their reactions, as reported for the laccase mediator system. NHPI was confirmed to be the best mediator in the present system for the selective Cα-carbonylation of the non-phenolic β-O-4 structures in lignin.

Impact of Malolactic Fermentation on the Color and Color Stability of Pinot noir and Merlot Wine

Malolactic fermentation (MLF) is an integral step in red winemaking that results in a decrease in acidity and can also influence additional wine quality parameters. This study examined the impact of MLF on red wine color and the ability of <i>Oenococcus oeni</i> to degrade compounds important to the development of polymeric pigments. Pinot noir and Merlot wines were produced where a portion of the wines underwent a simultaneous alcoholic and malolactic fermentations with <i>O. oeni</i> strain VFO. Post-alcoholic MLFs were conducted using VFO and two additional <i>O. oeni</i> strains in sterile filtered wines. A control wine that did not undergo MLF was pH adjusted to the same pH as wines that had undergone MLF. Wines that underwent MLF (MLF+) contained lower concentrations of acetaldehyde and pyruvic acid than wines that did not undergo MLF (MLF-). Wines that underwent MLF with <i>O. oeni</i> VFO also had higher concentrations of caffeic and <i>trans</i>-<i>p</i>-coumaric acids. MLF+ wines had significantly lower color and polymeric pigment content than MLF-wines and contained significantly higher monomeric anthocyanins. Vitisin B concentrations were also significantly lower in wines that underwent MLF. These differences remained throughout nine months of storage, demonstrating that MLF can affect red wine color independent of pH change. While <i>O. oeni</i> influenced the concentration of phenolic and nonphenolic compounds involved in red wine color development there were no strain specific differences in color and polymeric pigment content.

Cleavage and synthesis function of high and low redox potential laccases towards 4-morpholinoaniline and aminated as well as chlorinated phenols

Laccases are able to mediate both cleavage and synthesis processes. The basis for this dual reaction capability lies in the property of the enzyme laccase to oxidize phenolic, and to some extent non-phenolic substances, to reactive radicals which can undergo on the one hand separations of small substitutents or large molecule parts from the parent compound and on the other hand coupling reactions with other radicals or molecules which are not themselves oxidizable by laccase. The cleavage of the non-phenolic compound 4-morpholinoaniline as well as the deamination of 4-aminophenol and the dechlorination of 4-chlorophenol resulted in the formation of 1,4-hydroquinone which is immediately oxidized by laccase to 1,4-benzoquinone. The formation of the 1,4-hydroquinone/1,4-benzoquinone is the rate limiting step for the synthesis of the heteromolecular dimers and trimers composed of 1,4-benzoquinone and one or two molecules of morpholine. In addition to the synthesis of new compounds from the cleavage products, 4-morpholinoaniline polymerized probably via azo groups and C-N bonds to a homomolecular dimer and trimer. Similarities and differences in cleavage and synthesis reactions catalyzed by the low redox potential laccase of Myceliophthora thermophila (0.46 V) and the high redox potential laccase of Pycnoporus cinnabarinus (0.79 V) were determined. In addition, the dependency of the cleavage and synthesis efficiencies on the (a) structure and redox potential of the laccase, (b) structure and redox potential of the substrate, (c) pH value of the buffer used, (d) incubation temperature, (e) solvent concentration, and (f) laccase activity is discussed in general.

Compound-specific stable carbon isotope ratios of phenols and nitrophenols derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide

We developed an analytical method for measuring compound-specific stable carbon isotope ratios (δ13C) of phenols and nitrophenols in filter samples of particulate organic matter. The method was tested on 13 phenols derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), together with four nonphenolic compounds. The data obtained by our method required two specific corrections for the determination of valid δ13C values: (1) for nitro compounds, the routine correction with use of m/z 46 for the contribution of 12C17O16O molecules) to m/z 45 was modified due to impact of NO2 on the m/z 46 trace, and (2) for the derivatized phenols, measured δ13C values were corrected for the shift in δ13C due to the addition of carbon atoms from the BSTFA moiety. Analysis of standard-spiked filters showed that overall there was a small compound-dependent bias in the δ13C values: the average bias±the standard error of the mean of −0.21±0.1‰ for the standard compounds tested, except 3-methylcatechol, methylhydroquinone, 4-methyl-2-nitrophenol, and 2,6-dimethyl-4-nitrophenol, whereas the average biases±the standard errors of the mean for those were +1.2±0.3‰, +1.2±0.2‰, −1.2±0.2‰, and −1.4±0.5‰, respectively, when the injected mass of a derivatized compound exceeded 15ngC. In situations where such small biases and uncertainties are acceptable, the method described here could be used to obtain valuable information about δ13C values. We also analyzed a real filter sample to demonstrate the practical applicability of the method.

Characterization of a Novel Dye-Decolorizing Peroxidase (DyP)-Type Enzyme from Irpex lacteus and Its Application in Enzymatic Hydrolysis of Wheat Straw

Irpex lacteus is a white rot basidiomycete proposed for a wide spectrum of biotechnological applications which presents an interesting, but still scarcely known, enzymatic oxidative system. Among these enzymes, the production, purification, and identification of a new dye-decolorizing peroxidase (DyP)-type enzyme, as well as its physico-chemical, spectroscopic, and catalytic properties, are described in the current work. According to its N-terminal sequence and peptide mass fingerprinting analyses, I. lacteus DyP showed high homology (>95%) with the hypothetical (not isolated or characterized) protein cpop21 from an unidentified species of the family Polyporaceae. The enzyme had a low optimal pH, was very stable to acid pH and temperature, and showed improved activity and stability at high H2O2 concentrations compared to other peroxidases. Other attractive features of I. lacteus DyP were its high catalytic efficiency oxidizing the recalcitrant anthraquinone and azo dyes assayed (kcat/Km of 1.6 × 10(6) s(-1) M(-1)) and its ability to oxidize nonphenolic aromatic compounds like veratryl alcohol. In addition, the effect of this DyP during the enzymatic hydrolysis of wheat straw was checked. The results suggest that I. lacteus DyP displayed a synergistic action with cellulases during the hydrolysis of wheat straw, increasing significantly the fermentable glucose recoveries from this substrate. These data show a promising biotechnological potential for this enzyme.

Recovery of Natural Antioxidants from Spent Coffee Grounds

Spent coffee grounds (SCG) were extracted with an environmentally friendly procedure and analyzed to evaluate the recovery of relevant natural antioxidants for use as nutritional supplements, foods, or cosmetic additives. SCG were characterized in terms of their total phenolic content by the Folin-Ciocalteu procedure and antioxidant activity by the DPPH scavenging assay. Flavonoid content was also determined by a colorimetric assay. The total phenolic content was strongly correlated with the DPPH scavenging activity, suggesting that phenolic compounds are mainly responsible for the antioxidant activity of SCG. An UHPLC-PDA-TOF-MS system was used to separate, identify, and quantify phenolic and nonphenolic compounds in the SCG extracts. Important amounts of chlorogenic acids (CGA) and related compounds as well as caffeine (CAF) evidenced the high potential of SCG, a waste material that is widely available in the world, as a source of natural phenolic antioxidants.

SUBSTRATE-DEPENDENT EXPRESSION OF LACCASE IN GENETICALLY MODIFIED Escherichia coli: DESIGN AND CONSTRUCTION OF AN INDUCIBLE PHENOL-DEGRADING SYSTEM

Phenolic compounds that are produced by variety of industrial and urban activities pose dangers to live organisms and the environment. Here, an inducible phenol-degrading system was designed and constructed in Escherichia coli as the host. CapR as a transcription activator in Pseudomonas species shows sensitivity towards most common phenolic pollutants. Upon presence of inducible pollutants and conformational changes of CapR, an inducible promoter will trigger the expression of a bacterial laccase gene, which had been isolated previously from a local Bacillus species. Laccase as a multicopper oxidase has the ability to oxidize wide variety of mono and polyphenols. The sensitivity of the inducible system was verified in the presence of phenol with the concentration range of 1 nM–10 mM. A linear correlation was observed between laccase expression and phenol concentration up to 1 mM. Laccase was expressed even in the lowest concentration of phenol (1 nM) after 2 hr of exposure. 2,2-Azinobis (3-ethylbenzthiazoline-6-sulfonate) (ABTS) as a mediator of laccase oxidative reactions could induce laccase expression through conformational changes of CapR. Recognition of ABTS by CapR not only results in expression of the remediating enzyme but also extends its substrate range to nonphenolic compounds.