A novel type of aryl sulfotransferase obtained from an anaerobic bacterium of human intestine

A novel type of aryl sulfotransferase is produced by an anaerobic bacterium of human intestine, Eubacterium A-44. Aryl sulfotransferase separated from this bacterium differs from the sulfotransferase which uses 3'-phosphoadenosine 5'-phosphosulfate as a donor. The enzyme catalyzes stoichiometric transfer of a sulfate group from a phenol sulfate ester to other phenolic compounds, with strict specificity. The optimal pH and molecular weight were 8-9 and 315,000, respectively.     

Biodegradation of phenol and its derivatives by engineered bacteria: current knowledge and perspectives

Biodegradation of phenolic compounds is a promising alternative to physical and chemical methods used to remove these toxic pollutants from the environment. The ability of various microorganisms to metabolize phenol and its derivatives (alkylphenols, nitrophenols and halogenated derivatives) has therefore been intensively studied. Knowledge of the enzymes catalyzing the individual reactions, the genes encoding these enzymes and the regulatory mechanisms involved in the expression of the respective genes in bacteria serves as a basis for the development of more efficient degraders of phenols via genetic engineering methods. Engineered bacteria which efficiently degrade phenolic compounds were constructed in laboratories using various approaches such as cloning the catabolic genes in multicopy plasmids, the introduction of heterologous genes or broadening the substrate range of key enzymes by mutagenesis. Efforts to apply the engineered strains in in situ bioremediation are problematic, since engineered strains often do not compete successfully with indigenous microorganisms. New efficient degraders of phenolic compounds may be obtained by complex approaches at the organism level, such as genome shuffling or adaptive evolution. The application of these engineered bacteria for bioremediation will require even more complex analysis of both the biological characteristics of the degraders and the physico-chemical conditions at the polluted sites.     

Recent advances in the development of biosensor for phenol: a review

Phenol and its derivatives are widespread contaminants whose sources are both natural and industrial. Phenol is massively produced and used as a starting material for synthetic polymers and fibers. Although phenolic compounds play important biochemical and physiological roles in living systems, their accumulation in the environment as a result of intensive human activity may result in drastic ecological problem. Various analytical techniques are available for the detection of phenol in environmental samples. But they need complex sample pre-treatment so as are time consuming, costly and use heavy devices. On the other hand a biosensor is a device that gives rapid detection, cost effective and easy. A review study was carried out to accumulate the possible biosensors for the detection of phenolic compounds in environmental samples. A number of biological components including microorganisms, enzymes, antibodies, antigens, nucleic acids etc. can be used for the construction of biosensors that was found to detect phenolic compounds. Of all type of biological components microorganisms and enzymes are mostly used. The microorganisms are Pseudomonas, Moraxella, Arthrobacter, Rhodococcus, and Trichosporon. The most used enzymes are tyrosinase, peroxidase, laccase, glucose dehydrogenase, cellobiose dehydrogenase etc. Antibody sensors can detect a very trace level. The biorecognition of DNA biosensors occur by hybridization of DNA. Biosensors are found to work well when the biological sensing element is immobilized. A variety of immobilization techniques were found to use as adsorption, covalent binding, entrapment, cross-linking etc. For immobilization the matrices used was polyvinyl alcohol, Osmium complex, nafion/sol?Cgel silicate, chitosan, silica gel etc.     

Novel insights into the fungal oxidation of monoaromatic and biarylic environmental pollutants by characterization of two new ring cleavage enzymes

The phenol-degrading yeast Trichosporon mucoides can oxidize and detoxify biarylic environmental pollutants such as dibenzofuran, diphenyl ether and biphenyl by ring cleavage. The degradation pathways are well investigated, but the enzymes involved are not. The high similarity of hydroxylated biphenyl derivatives and phenol raised the question if the enzymes of the phenol degradation are involved in ring cleavage or whether specific enzymes are necessary. Purification of enzymes from T. mucoides with catechol cleavage activity demonstrated the existence of three different enzymes: a classical catechol-1,2-dioxygenase (CDO), not able to cleave the aromatic ring system of 3,4-dihydroxybiphenyl, and two novel enzymes with a high affinity towards 3,4-dihydroxybiphenyl. The comparison of the biochemical characteristics and mass spectrometric sequence data of these three enzymes demonstrated that they have different substrate specificities. CDO catalyzes the ortho-cleavage of dihydroxylated monoaromatic compounds, while the two novel enzymes carry out a similar reaction on biphenyl derivatives. The ring fission of 3,4-dihydroxybiphenyl by the purified enzymes results in the formation of (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid. These results suggest that the ring cleavage enzymes catalyzing phenol degradation are not involved in the ring cleavage of biarylic compounds by this yeast, although some intermediates of the phenol metabolism may function as inducers.     

Isolation and characterization of tyrosinase produced by marine actinobacteria and its application in the removal of phenol from aqueous environment

The present study was focused on screening and characterization of tyrosinase enzyme produced by marine actinobacteria and its application in phenolic compounds removal from aqueous solution. A total of 20 strains were isolated from marine sediment sample and screened for tyrosinase production by using skimmed milk agar medium. Among 20 isolates, two isolates LK-4 and LK-20 showed zone of hydrolysis and these were taken for secondary screening by using tyrosiue agar medium. Based on the result of secondary screening LK-4 was selected for further analysis, such as tyrosinase assay, protein content and specific activity of the enzyme. The tyrosinase enzyme was produced in a SS medium and was partially purified by ammonium sulfate precipitation, dialysis and SDS PAGE. The isolate （LK-4） was identified as Streptomyces espinosus using 16S rRNA gene sequencing and named as ＂Streptomyces espinosus strain LK4 （KF806735）＂. The tyrosinase enzyme was immobilized in sodium alginate which was applied to remove phenolic compounds from water. The enzyme efficiently removed the phenolic compounds from aqueous solution within few hours which indicated that tyrosinasc enzyme produced by Streptomyces espinosus strain LK-4 can be potently used for the removal of phenol and phenolic compounds from wastewater in industries.     

Reaction product affinity regulates activation of human sulfotransferase 1A1 PAP sulfation

Cytosolic sulfotransferase (SULT)-catalyzed sulfation regulates the activity of bio-signaling molecules and aids in metabolizing hydroxyl-containing xenobiotics. The sulfuryl donor for the SULT reaction is adenosine 3′-phosphate 5′-phosphosulfate (PAPS), while products are adenosine 3′,5′-diphosphate (PAP) and a sulfated alcohol. Human phenol sulfotransferase (SULT1A1) is one of the major detoxifying enzymes for phenolic xenobiotics. The mechanism of SULT1A1-catalyzed sulfation of PAP by pNPS was investigated. PAP was sulfated by para-nitrophenyl sulfate (pNPS) in a concentration-dependent manner. 2-Naphthol inhibited sulfation of PAP, competing with pNPS, while phenol activated the sulfation reaction. At saturating PAP, a ping pong kinetic mechanism is observed with pNPS and phenol as substrates, consistent with phenol intercepting the E–PAPS complex prior to dissociation of PAPS. At high concentrations, phenol competes with pNPS, consistent with formation of the E–PAP–phenol dead-end complex. Data are consistent with the previously reported mechanism for sulfation of 2-naphthol by PAPS, and its activation by pNPS [14]. Overall, data are consistent with release of PAP from E–PAP and PAPS from E–PAPS contributing to rate-limitation in both reaction directions.     

Cross-linking and immobilisation of different proteins with recombinant Verrucomicrobium spinosum tyrosinase

This paper reports on the cross-linking and immobilisation of various proteins by the recombinant tyrosinase from Verrucomicrobium spinosum (Vs-tyrosinase). In general it is found that Vs-tyrosinase can readily cross-link proteins with a low degree of complexity, such as casein, but that the enzyme cannot readily cross-link well folded protein substrates such as lysozyme, myoglobin, cytochrome c or Candida antarctica lipase B (CALB). However, the inclusion of phenolic compounds (phenol or caffeic acid) to reaction mixtures of these proteins can greatly enhance the levels of cross-linking. For example it is possible to prepare cross-linked aggregates of industrially applicable enzymes such as CALB by simply incubating it with Vs-tyrosinase and phenol. The resulting aggregates can be collected by centrifugation and retain high levels of activity and may find applications in biocatalysis.     

A novel type of arylsulfotransferase

Arylsulfotransferase catalyzes the transfer of a sulfate group from 3-phosphoadenosine-5-phosphosulfate (PAPS) to a phenolic acceptor substrate. We discovered a novel type of sulfotransferase from an anaerobic bacterium of human intestine, Eubacterium A-44. In the bacterial enzyme PAPS did not serve as a donor and all alcohols did not as acceptors. The new arylsulfotransferase was purified 185-fold from a crude extract of sonicated bacteria to homogeneity. The enzyme (MW 315 kd) was composed of four identical subunits (MW 80 kd) whose N-terminal amino acid was arginine, and its optimal pH and pI were 8–9 and 3.9, respectively. The enzyme catalyzed stoichiometric transfer of a sulfate group from a phenol sulfate ester to other phenols, with strict specificity. With tyramine as an acceptor, p-acetylphenyl sulfate was the best donor, followed by 4-methylumbelliferyl sulfate and p-nitrophenyl sulfate. With p-nitrophenyl sulfate as a donor, naphthol was the best acceptor, followed by estradiol, phenol, tyrosine methylester, tyramine, and epinephrine in decreasing order. Only the 4-position of catecholamines was specifically sulfated. Naturally occurring phenolic compounds, such as flavone, chalcone, and xanthone, were sulfated as well. Tyrosine-containing peptides were enzymatically sulfated: enkephalin, LH-RH, vasopressin, angiotensins, proctorin, CCK-8, and phyllocaerulein were sulfated with high yields. The novel sulfotransferase is expected to be applicable to enzymatic O-sulfation of tyrosine-containing hormones. The ³⁵S-labeled sulfate group from (³⁵S)p-nitrophenyl sulfate was incorporated into a tyrosyl residue at the active site of the enzyme (2 mole ³⁵S/mole of enzyme). The enzyme was inactivated by diethylpyrocarbamate and TLCK, chemical modifying agents for a histidyl residue. The reaction mechanism of arylsulfotransferase was proposed as follows: a donor substrate combines a histidyl residue with concomitant release of a phenolic compound. The sulfate group of the histidyl residue transfers to a tyrosyl residue, and then to an acceptor with the binding of another donor substrate to the histidyl residue.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Phenol and Benzoate Metabolism by Pseudomonas putida: Regulation of Tangential Pathways

Catechol occurs as an intermediate in the metabolism of both benzoate and phenol by strains of Pseudomonas putida. During growth at the expense of benzoate, catechol is cleaved ortho (1,2-oxygenase) and metabolized via the beta-ketoadipate pathway; during growth at the expense of phenol or cresols, the catechol or substituted catechols formed are metabolized by a separate pathway following meta (2,3-oxygenase) cleavage of the aromatic ring of catechol. It is possible to explain the mutually exclusive occurrence of the meta and ortho pathway enzymes in phenol- and benzoate-grown cells of P. putida on the basis of differences in the mode of regulation of these two pathways. By use of both nonmetabolizable inducers and blocked mutants, gratuitous synthesis of some of the meta pathway enzymes was obtained. All four enzymes of the meta pathway are induced by the primary substrate, cresol or phenol, or its analogue. Three enzymes of the ortho pathway that catalyze the conversion of catechol to beta-ketoadipate enol-lactone are induced by cis,cis-muconate, produced from catechol by 1,2-oxygenase-mediated cleavage. Observations on the differences in specificity of induction and function of the two pathways suggest that they are not really either tangential or redundant. The meta pathway serves as a general mechanism for catabolism of various alkyl derivatives of catechol derived from substituted phenolic compounds. The ortho pathway is more specific and serves primarily in the catabolism of precursors of catechol and catechol itself.     

Impact of phenolic compounds on hydrothermal oxidation of cellulose

The effect of phenolic compounds on hydrothermal oxidation of cellulose was studied using a batch reactor at 300 degrees C with H(2)O(2) as oxidant. Intermediate products, as well as the yields of acetic acid produced in the oxidation of cellulose, phenolic compounds, and cellulose-phenolic compound mixtures were examined. Phenolic compounds used were phenol, 1,4-benzenediol, 2-methoxy-4-methylphenol, and 2,6-di-tert-butyl-4-methylphenol. In the case of oxidation of cellulose-phenolic compound mixtures, (1) formic acid, a basic oxidation product from carbohydrates, decreased considerably, (2) 5-hydroxymethyl-2-furaldehyde and 2-furaldehyde, acid-catalyzed dehydration products from carbohydrates, appeared, and (3) the yield of acetic acid increased compared to that in the oxidation of cellulose. From these results, phenolic compounds seem to inhibit the oxidation of cellulose under hydrothermal conditions. The inhibition of the oxidation of cellulose by phenolic compounds seems to be related closer to the stability of phenolic compounds under oxidation conditions rather than the ease to remove phenolic hydrogen on the OH group.     

Glycosylation of Phenolic Compounds by the Site-Mutated β-Galactosidase from Lactobacillus bulgaricus L3

β-Galactosidases can transfer the galactosyl from lactose or galactoside donors to various acceptors and thus are especially useful for the synthesis of important glycosides. However, these enzymes have limitations in the glycosylation of phenolic compounds that have many physiological functions. In this work, the β-galactosidase from Lactobacillus bulgaricus L3 was subjected to site-saturation mutagenesis at the W980 residue. The recombinant pET-21b plasmid carrying the enzyme gene was used as the template for mutation. The mutant plasmids were transformed into Escherichia coli cells for screening. One recombinant mutant, W980F, exhibited increased yield of glycoside when using hydroquinone as the screening acceptor. The enzyme was purified and the effects of the mutation on enzyme properties were determined in detail. It showed improved transglycosylation activity on novel phenolic acceptors besides hydroquinone. The yields of the glycosides produced from phenol, hydroquinone, and catechol were increased by 7.6% to 53.1%. Moreover, it generated 32.3% glycosides from the pyrogallol that could not be glycosylated by the wild-type enzyme. Chemical structures of these glycoside products were further determined by MS and NMR analysis. Thus, a series of novel phenolic galactosides were achieved by β-galactosidase for the first time. This was a breakthrough in the enzymatic galactosylation of the challenging phenolic compounds of great values.     

工业木质素预处理及催化转化苯酚

传统化石能源日益枯竭以及环境污染压力日益加大使得开发以生物质为代表的可替代能源迫在眉睫,木质素作为仅次于纤维素的生物质中第二大主要成分用来制备高附加值化学品是提高生物质资源利用效率的关键。本文将工业木质素进行预处理后采用双液相反应体系将工业木质素转化为苯酚等化合物,使用微波辐照代替传统的加热,考察了预处理方式、温度、时间等条件对产物收率的影响。结果表明,以1-甲基-3-乙基咪唑醋酸盐([EMIM]OAc)处理的工业木质素在微波反应器输出功率为400 W、反应温度为90℃、催化剂为1-甲基-3-胺乙基咪唑四氟硼酸盐([AEMIM]BF4)双液相反应体系中反应60 min苯酚的收率最高可达8.14%。     

Laccase induction in fungi and laccase/N-OH mediator systems applied in paper mill effluent

There has been increasing interest in extracellular enzymes from white rot fungi, such as lignin and manganese peroxidases, and laccases, due to their potential to degrade both highly toxic phenolic compounds and lignin. The optimum cultivation conditions for laccase production in semi-solid and liquid medium by Trametes versicolor, Trametes villosa, Lentinula edodes and Botrytis cinerea and the effects of laccase mediator system in E1 effluent were studied. The higher laccase activity (12756 U) was obtained in a liquid culture of T. versicolor in the presence of 1 mM of 2,5-xylidine and 0.4 mM copper salt as inducers. The effluent biotreatments were not efficient in decolorization with any fungal laccases studied. Maximum phenol reduction was approximately 23% in the absence of mediators from T. versicolor. The presence of 1-hydroxybenzotriazole did not increase phenol reduction. However, acetohydroxamic acid, which was not degraded by laccase, acted very efficiently on E1 effluent, reducing 70% and 73% of the total phenol and total organic carbon, respectively. Therefore, acetohydroxamic acid could be applied as a mediator for laccase bioremediation in E1 effluent.     

Regulation of phenol sulfotransferase expression in cultured bovine bronchial epithelial cells by hydrocortisone

One conjugative pathway for the inactivation of endogenous and exogenous hydroxylated aromatic compounds is catalyzed by phenol (aryl) sulfotransferases (PSTs), which esterify phenolic acceptors with sulfate. The tracheobronchial epithelium is commonly exposed to phenolic drugs and pollutants, and metabolic sulfation and PST activity in this tissue have been previously demonstrated. To determine what factors may control PST expression, extracts of serum-free, growth factor-supplemented cultures of bovine bronchial epithelial cells were assayed for PST activity and PST antigen. The most significant finding was dose-dependent, apparent stimulated expression by hydrocortisone (EC₅₀ = 4 nM, maximal stimulation at 20 nM). Time-course experiments, however, revealed progressive loss of PST in the absence of corticosteroid. After decay of extant PST in steroid-free medium, hydrocortisone reinduced the expression of PST three to fivefold. Western blots using mouse anti-bovine PST revealed corresponding increases in 32 kDa PST protein levels in response to hydrocortisone. Steady state kinetic analyses indicated apparent K_m values of 1—3 μM for 2-naphthol regardless of culture conditions. These results suggest that detoxification of phenolic compounds by sulfation may be regulated by corticosteroids.     

Detoxification of wood hydrolysates with laccase and peroxidase from the white-rot fungus Trametes versicolor

Fermentation of wood hydrolysates to desirable products, such as fuel ethanol, is made difficult by the presence of inhibitory compounds in the hydrolysates. Here we present a novel method to increase the fermentability of lignocellulosic hydrolysates: enzymatic detoxification. Besides the detoxification effect, treatment with purified enzymes provides a new way to identify inhibitors by assaying the effect of enzymatic attack on specific compounds in the hydrolysate. Laccase, a phenol oxidase, and lignin peroxidase purified from the ligninolytic basidiomycete fungus Trametes versicolor were studied using a lignocellulosic hydrolysate from willow pretreated with steam and SO₂. Saccharomyces cerevisiae was employed for ethanolic fermentation of the hydrolysates. The results show more rapid consumption of glucose and increased ethanol productivity for samples treated with laccase. Treatment of the hydrolysate with lignin peroxidase also resulted in improved fermentability. Analyses by GC-MS indicated that the mechanism of laccase detoxification involves removal of monoaromatic phenolic compounds present in the hydrolysate. The results support the suggestion that phenolic compounds are important inhibitors of the fermentation process. Received: 3 November 1997 / Received revision: 4 February 1998 / Accepted: 6 February 1998     

外源酚对凤眼莲体内酚含量和与酚代谢有关酶的影响

凤眼莲能够吸收和在体内聚集外源苯酚,体内的酸含量随着环境中酚浓度的上升而上升。从生长于合酚培养液中的凤眼莲体内能够检测到酚糖苷,说明凤眼莲体内有酚精苷转移酶的存在。浓度小于５０ｍｇ／Ｌ的外源酚能提高凤眼莲体内的多酚氧化酶和过氧化物酶的活性。多酚氯化酶与过氧化物酶在线粒体和微粒体中均有不同程度的分布,而酚糖苷转移酶则不存在于这些细胞器中。     

Sulfate conjugation in drug metabolism: role of inorganic sulfate

Conjugation with sulfate is a major pathway for the biotransformation of phenolic drugs in humans and many animal species. It is a process of limited capacity; the extent of sulfate conjugate formation and the metabolic clearance of drugs subject to conjugation with sulfate depend therefore on the dose, the dosage form, the route of administration, and the rate and duration of administration as well as on the pharmacokinetic parameters of competing processes. The effect of these variables is exemplified by the pharmacokinetics of salicylamide and acetaminophen in humans and rats. In our experience so far, the proximate cause of the nonlinear pharmacokinetics of sulfate conjugation of phenolic drugs is the limited availability and consequent depletion of inorganic sulfate. When this is prevented by direct or indirect (via sulfate donors such as N-acetylcysteine) repletion, the saturability of phenol sulfotransferase (EC 2.8.2.1) activity can become evident. The major mechanism of inorganic sulfate homeostasis is nonlinear renal clearance, which is due largely to saturable renal tubular reabsorption. Systemic depletion of inorganic sulfate secondary to utilization of this anion for the sulfation of drugs affects the availability of sulfate in the central nervous system and may, therefore, modify the disposition of certain neurotransmitters and other endogenous substances that are subject to sulfate conjugation.     

Role of Ca2+ in the metabolism of phenolic compounds in tobacco leaves (Nicotiana tabacum L.)

Given the essential role played by phenol metabolism in many resistance responses to different types of stress, the aim of the present work was to determine how different application rates of calcium may influence this metabolic process. Increased calcium in the nutrient solution in which tobacco plants were grown considerably reduced the foliar concentration of phenolic compounds. Calcium clearly exerted a positive influence on the activities of enzymes (phenylalanine ammonia-lyase, polyphenol oxidase and peroxidase) involved in the metabolism of the phenolics. High dosages of calcium (5 mM) promoted more oxidation than synthesis of these compounds, thus explaining the lower concentration of the phenolics.     

Olfactory responses of tsetse flies to phenols from buffalo urine

Abstract A comparison was made of the EAG responses of males and females of Glossina morsitans morsitans Westwood, G.austeni Newstead and G.tachinoides , Westwood to various doses of compounds known to be components of ox and buffalo urine fractions which are attractive to tsetse in the field (phenol, 3- and 4-methylphenol, 3- and 4-ethylphenol, 4-n-propylphenol, dimethylsulfone). All three species did not respond to dimethylsulfone. The overall responses to the phenolic substances were higher in females than in males in G.m.morsitans and higher in males than in females in G.austeni and G.tachinoides. Response spectra of the species for the phenolic substances suggested that G.m.morsitans and G. austeni were most responsive to 3- and 4-methylphenol and 3-ethylphenol, whereas G. tachinoides was most sensitive to 3-ethylphenol and 3-methylphenol, and only moderately sensitive to 4-methylphenol.
Cross-adaptation experiments, in which l-octen-3-ol, acetone, 4-heptanone and 3-nonanone were also included, revealed that all phenolic compounds stimulated one and the same class of receptors, which differed from the class of receptors activated by l-octen-3-ol. The ketones also had their own receptors. Hence, the flies can obtain information about the presence of attractants by at least three different receptor classes. It was concluded that phenol and any individual alkylphenol found in ox and buffalo urine should be attractive to tsetse flies, provided that stimulus intensity is above threshold and not beyond optimum. One class of receptors may respond more strongly in males than in females, whereas another class is more responsive in females than in males. This may result in a change in sex ratios in catches depending on the odour bait used.     

Biodegradation of phenolic compounds by sulfate-reducing bacteria from contaminated sediments

The biodegradation of phenolic compounds under sulfate-reducing conditions was studied in sediments from northern Indiana. Phenol, p-cresol and 4-chlorophenol were selected as test substrates and added to sediment suspensions from four sites at an initial concentration of 10 mg/liter. Degradative abilities of the sediment microorganisms from the four sites could be related to previous exposure to phenolic pollution. Time to onset of biodegradation of p-cresol and phenol in sediment suspensions from a nonindustrialized site was approximately 70 and 100 days, respectively, in unacclimated cultures. In sediment slurries from three sites with a history of wastewater discharges containing phenolics, time to onset of biodegradation was 50–70 days for p-cresol and 50–70 days for phenol in unacclimated cultures. In acclimated cultures from all four sites, the length of the lag phase was reduced to 14–35 days for p-cresol and 25–60 days for phenol. Length of the biodegradative phase varied from 25 to 40 days for phenol and 10 to 50 days for p-cresol and was not markedly affected by acclimation. Substrate mineralization by sulfate-reducing bacteria was confirmed with radiotracer techniques using an acclimated sediment culture from one site. Addition of molybdate, a specific inhibitor of sulfate reduction, and bacterial cell inactivation inhibited sulfate reduction and substrate utilization. None of the sites exhibited the ability to degrade 4-chlorophenol, nor were acclimated phenol and p-cresol degrading cultures from a particular site able to cometabolize 4-chlorophenol.Correspondence to: D. Dean-Ross