Water purification through bioconversion of phenol compounds by tyrosinase and chemical adsorption by chitosan beads

Enzymatic removal of various phenol compounds from artificial wastewater was undertaken by the combined use of mushroom tyrosinase (EC 1.14.18.1) and chitosan beads as function of pH value, temperature, tyrosinase dose, and hydrogen peroxide-to-substrate ratio. Chitosan film incubated in a p-crersol+tyrosinase mixture had the main peaks at 400-470 nm assigned to chemically adsorbed quinone derivatives, which increased over the immersion time. These results indicate that removal of phenol compounds is caused by their tyrosinase-catalyzed oxidation to the corresponding quinone derivatives and the subsequent chemical adsorption on the chitosan film. The optimum conditions for quinone adsorption were determined to be pH 7 and 45 degrees C for p-cresol. Some alkyl-substituted phenol compounds were removed by adsorption of quinone derivatives enzymatically generated on the chitosan beads, and the % removal for p-cresol, 4-ethylphenol, 4-n-propylphenol, 4-n-butylphenol, and p-chlorophenol went up to 93%. In addition, 4-tert-butylphenol underwent tyrosinase-catalyzed oxidation in the presence of hydrogen peroxide. This procedure was applicable to removal of chlorophenols and alkyl-substituted phenols.     

Color and toxicity removal following tyrosinase-catalyzed oxidation of phenols

The products of phenol oxidation catalyzed by mushroom tyrosinase (polyphenol oxidase, EC 1.14.18.1) were assessed in terms of their residual color and toxicity. The addition of aluminum sulfate had little effect on the removal of colored products from phenol solutions treated with tyrosinase. Although chitosan was used successfully to remove the color when added before the reaction initiation or after the reaction completion, the required dose of chitosan was lower when it was added after the reaction. In this case, the minimum doses of chitosan required to achieve 90% color removal were proportional to the logarithm of the initial concentration of phenol. The color removal induced by chitosan addition appeared to be the result of chemical interaction followed by a coagulation mechanism. All treated solutions of phenol and chlorophenols, except 2,4-dichlorophenol, had substantially lower toxicities than their corresponding initial toxicities, as measured using the Microtox assay. Chitosan addition significantly enhanced the reduction in toxicity. The toxicities of the phenol solutions treated with tyrosinase were markedly lower than previously reported toxicities of solutions treated with peroxidase enzymes.     

Preparation and characterization of epoxy-functionalized magnetic chitosan beads: laccase immobilized for degradation of reactive dyes

Cross-linked magnetic chitosan beads were prepared by phase-inversion technique in the presence of epichlorohydrin under alkaline condition, and used for covalent immobilization of laccase. The activity of the immobilized laccase on the magnetic chitosan was about 260 U (g/dry beads) with an enzyme loading of about 16.33 ± 0.39 mg [(g/dry beads) mg/g]. Kinetic parameters, V _max and K _m values were determined as 21.7 U/mg protein and 9.4 μM for free enzyme, and 15.6 U/mg protein and 19.7 μM for the immobilized laccase, respectively. The operational and thermal stabilities of the immobilized laccase were improved compared to free counterpart. The immobilized laccase was operated in a batch reactor for the decolorization of reactive dyes from aqueous solution. The laccase immobilized on magnetic chitosan beads was very effective for removal of textile dyes from aqueous solution which creates an important environmental problem in the discharged textile dying solutions.     

Mass spectrometric profiling of glucosamine, glucosamine polymers and their catecholamine adducts. Model reactions and cuticular hydrolysates of Toxorhynchites amboinensis (Culicidae) pupae.

Glucosamine (Gln), glucosamine polymers, and their catecholamine adducts were characterized using positive ion electrospray mass spectrometry (ESMS) and tandem mass spectrometry (ESMS-MS). N-acetyldopamine (NADA), a catecholamine found in many insect cuticles, was oxidized using mushroom tyrosinase, and the resulting quinone derivatives were reacted with Gln, (Gln)3, and polymeric glucosamine (chitosan). Adducts of glucosamine and its trisaccharide with NADA were readily identified as [M + H]+ ions in ESMS spectra, and ESMS-MS of selected ions confirmed the condensation of 1-3 NADA residues with Gln. In addition to Gln modification by the quinone derivatives of NADA, other spectra were consistent with the formation of adducts with N-acetylnoradrenaline and moieties formed by intramolecular cyclization following oxidation. The primary amine of glucosamine was involved in initial adduct formation, but the sites for subsequent additions of oxidized NADA to glucosamine, presumably via hydroxyl groups, could not be identified by ESMS alone. The ESMS spectra of chitosan films infused into the spectrometer following solubilization in acidic methanol/water produced spectra similar to that of (Gln)3 up to m/z 502. Ions of gradually decreasing intensity consistent with (Gln)x, where x = 4-8, were observed. Modification of chitosan films following incubation with NADA plus tyrosinase rendered the films insoluble in dilute acid, simulating the cross-linking process proposed to occur during insect cuticle sclerotization. Acid hydrolysates of the pupal stage of the mosquito Toxorhynchites amboinensis, using only two pupal exuviae for the hydrolyses, were infused into the mass spectrometer without preliminary chromatography. Eight amino acids, glucosamine, N-acetylglucosamine, catecholamines, and a variety of polymers incorporating these compound classes were identified.     

Removal of phenols from wastewater by soluble and immobilized tyrosinase

An enzymatic method for removal of phenols from industrial wastewater was investigated. Phenols in an aqueous solution were removed after treatment with mushroom tyrosinase. The reduction order of substituted phenols is catechol > p-cresol > p-chlorophenol > phenol > p-methoxyphenol. In the treatment of tyrosinase alone, no precipitate was formed but a color change from colorless to dark-brown was observed. The colored products were removed by chitin and chitosan which are available abundantly as shellfish waste. In addition, the reduction rate of phenols was observed to be accelerated in the presence of chitosan. Tyrosinase, immobilized by using amino groups in the enzyme on cation exchange resins, can be used repeatedly. By treatment with immobilized tyrosinase, 100% of phenol was removed after 2 h, and the activity was reduced very little even after 10 repeat treatments. (c) 1993 John Wiley & Sons, Inc.     

Model sclerotization studies. 4. Generation of N-acetylmethionyl catechol adducts during tyrosinase-catalyzed oxidation of catechols in the presence of N-acetylmethionine

Incubation of catechol with mushroom tyrosinase in the presence of N-acetylmethionine resulted in the generation of an adduct. This product was identified to be N-acetylmethionyl catechol, on the basis of spectral characteristics and well-characterized chemical reaction of o-benzoquinone with N-acetylmethionine. Enzyme-catalyzed oxidation of catechol and the subsequent nonenzymatic addition of the resultant quinone to N-acetylmethionine accounted for the observed reaction. That the reaction is not confined to catechol alone, but is of general occurrence, can be demonstrated by the facile generation of similar adducts in incubation mixtures containing N-acetylmethionine, tyrosinase, and different N-acetylmethionines, such as 4-methylcatechol and N-acetyldopamine. Attempts to duplicate the reaction with insect cuticular phenoloxidases were not successful, as the excess N-acetylmethionine used in the reaction inhibited their activity. Nevertheless, occurrence of this nonenzymatic reaction between N-acetylmethionine and mushroom tyrosinase-generated quinones indicates that a similar reaction between enzymatically generated quinones in the cuticle with protein-bound methionine moiety is likely to occur during in vivo quinone tanning as well. Arch. Insect Biochem. Physiol. 38:44–52, 1998. © 1998 Wiley-Liss, Inc.     

Immobilization of tyrosinase on chitosan-clay composite beads

Tyrosinase was immobilized on glutaraldehyde crosslinked chitosan-clay composite beads and used for phenol removal. Immobilization yield, loading efficiency and activity of tyrosinase immobilized beads were found as 67%, 25% and 1400 U/g beads respectively. Optimum pH of the free and immobilized enzyme was found as pH 7.0. Optimum temperature of the free and immobilized enzyme was determined as 25-30 °C and 25 °C respectively. The kinetic parameters of free and immobilized tyrosinase were calculated using l-catechol as a substrate and K(m) value for free and immobilized tyrosinase were found as 0.93 mM and 1.7 mM respectively. After seven times of repeated tests, each over 150 min, the efficiency of phenol removal using same immobilized tyrosinase beads were decreased to 43%.     

Trapping of transiently formed quinone methide during enzymatic conversion of N-acetyldopamine to N-acetylnorepinephrine

We have demonstrated that quinone methide formation is an important aspect of insect physiology and proposed that enzymatically generated quinone methides react nonenzymatically with water or other nucleophiles to form Michael-1,6-addition products [(1988) Adv. Insect Physiol. 21, 179-231; (1989) J. Cell. Biochem. suppl. 13C, 58]. Using a purified o-quinone isomerase from the larval cuticle of Sacrophaga bullata and mushroom tyrosinase, we now demonstrate that transiently formed N-acetyldopamine quinone methide from N-acetyldopamine can be trapped by methanol to produce beta-methoxy N-acetyldopamine. The methanol adduct thus formed was found to be a racemic mixture and can be resolved into the optical isomers on cyclodextrin chiral column. These results confirm our contention that enzymatically generated quinone methides are nonenzymatically and nonstereoselectively transformed to Michael-1,6-adducts by reaction with water or other nucleophiles.     

Removal of aqueous phenol using immobilized enzymes in a bench scale and pilot scale three-phase fluidized bed reactor

The main objective of this work was to investigate the removal of aqueous phenol using immobilized enzymes in both bench scale and pilot scale three-phase fluidized bed reactors. The enzyme used in this application was a fungal tyrosinase [E.C. 1.14.18.1] immobilized in a system of chitosan and alginate. The immobilization matrix consisted of a chitosan matrix cross-linked with glutaraldehyde with an aliginate-filled pore space. This support matrix showed superior mechanical properties along with retaining the unique adsorptive characteristics of the chitosan. Adsorption of the o-quinone product by the chitosan reduced tyrosinase inactivation that is normally observed for this enzyme under these conditions. This approach allowed reuse of the enzyme in repeated batch applications. For the bench scale reactor (1.2-l capacity) more than 92% of the phenol could be removed from the feed water using an immobilized enzyme volume of 18.5% and a residence time of the liquid phase of 150 min. Removal rates decreased with subsequent batch runs. For the pilot scale fluidized bed (60 l), 60% phenol removal was observed with an immobilized enzyme volume of 5% and a residence time of the liquid phase of 7 h. Removal decreased to 45% with a repeat batch run with the same immobilized enzyme.     

Studies on chitosan: 2. Solution stability and reactivity of partially N-acetylated chitosan derivatives in aqueous media

The stability of the solutions of partially N-acetylated chitosans was studied by two methods: (1) 1% solutions of the chitosan derivatives in 0.1 M aqueous acetic acid were added dropwise to buffer solutions with pH from 8.6 to 12 and to a 0.1 M NaOH solution; (2) to each 0.5% solution of the derivatives in 0.1 M acetic acid was added the desired amount of a 1 M NaOH solution. The stability data obtained were summarized with respect to the degree of N-acetylation. It was found that the solutions of the derivatives with more than 50% acetyl content were stable even in alkaline conditions and the gelation and precipitation of the solutions did not occur. The reactivity of the derivatives with the degree of N-acetylation of more than 50% was studied using methyl 4-azidobenzoimidate (MABI) and ethylene glycol diglycidyl ether in homogeneous states. It was found that MABI reacted with amino groups of the chitosans only at neutral pH and glycidyl groups reacted at neutral and alkaline pH. It seems that these unique properties of chitosans with a degree of N-acetylation of more than 50% will enable us to prepare new chitosan derivatives.     

In vitro protein-polysaccharide conjugation: tyrosinase-catalyzed conjugation of gelatin and chitosan

The enzyme tyrosinase was used for the in vitro conjugation of the protein gelatin to the polysaccharide chitosan. Tyrosinases are oxidative enzymes that convert accessible tyrosine residues of proteins into reactive o-quinone moieties. Spectrophotometric and dissolved oxygen studies indicate that tyrosinase can oxidize gelatin and we estimate that 1 in 5 gelatin chains undergo reaction. Oxidized tyrosyl residues (i.e., quinone residues) can undergo nonenzymatic reactions with available nucleophiles such as the nucleophilic amino groups of chitosan. Ultraviolet/visible, (1)H-NMR, and ir provided chemical evidence for the conjugation of oxidized gelatin with chitosan. Physical evidence for conjugation was provided by dynamic viscometry, which indicated that tyrosinase catalyzes the sol-to-gel conversion of gelatin/chitosan mixtures. The gels formed from tyrosinase-catalyzed reactions were observed to differ from gels formed by cooling gelatin. In contrast to gelatin gels, tyrosinase-generated gels had different thermal behavior and were broken by the chitosan-hydrolyzing enzyme chitosanase. These results demonstrate that tyrosinase can be exploited for the in vitro formation of protein-polysaccharide conjugates that offer interesting mechanical properties.     

A mediator-free phenol biosensor based on immobilizing tyrosinase to ZnO nanoparticles

A mediator-free phenol biosensor was developed. The low-isoelectric point tyrosinase was adsorbed on the surface of high-isoelectric point ZnO nanoparticles (nano-ZnO) facilitated by the electrostatic interactions and then immobilized on the glassy carbon electrode via the film forming by chitosan. It was found that the nano-ZnO matrix provided an advantageous microenvironment in terms of its favorable isoelectric point for tyrosinase loading and the immobilized tyrosinase retaining its activity to a large extent. Moreover, there is no need to use any other electron mediators. Phenolic compounds were determined by the direct reduction of biocatalytically generated quinone species at -200mV (vs. saturated calomel electrode). The parameters of the fabrication process and the various experimental variables for the enzyme electrode were optimized. The resulting biosensor can reach 95% of steady-state current within 10s, and the sensitivity was as high as 182microAmmol(-1)L. The linear range for phenol determination was from 1.5x10(-7) to 6.5x10(-5)molL(-1) with a detection limit of 5.0x 10(-8)molL(-1) obtained at a signal/noise ratio of 3. In addition, the apparent Michaelis-Menten constant (K(m)(app)) and the stability of the enzyme electrode were estimated. The performance of the developed biosensor was compared with that of biosensors based on other immobilization matrices.     

Effects of the immobilization supports on the catalytic properties of immobilized mushroom tyrosinase: a comparative study using several substrates

Mushroom tyrosinase was immobilized from an extract onto glass beads covered with one of the following compounds: the crosslinked totally cinnamoylated derivatives of glycerine, D-sorbitol, D-manitol, 1,2-O-isopropylidene-alpha-D-glucofuranose, D-glucuronic acid, D-gulonic acid, sucrose, D-glucosone, D-arabinose, D-fructose, D-glucose, ethyl-D-glucopyranoside, inuline, dextrine, dextrane or starch, or the partially cinnamoylated derivative 3,5,6-tricinnamoyl-D-glucofuranose which was obtained by the acid hydrolysis of 1,2-O-isopropylidene-alpha-d-glucofuranose. The enzyme was immobilized by direct adsorption onto the support and the quantity of tyrosinase immobilized was found to increase with the hydrophobicity of the supports. The kinetic constants of immobilized tyrosinase acting on the substrates, 4-tert-butylcatechol, dopamine and DL-dopa, were studied. When immobilized tyrosinase acted on 4-tert-butylcatechol, the values of K(m)(app) were lower than these obtained for tyrosinase in solution while, when dopamine and DL-dopa were used, the K(m)(app) were higher. The order of the substrates as regards their ionizable groups, DL-dopa (two ionizable groups)>dopamine (one ionizable group)>4-tert-butylcatechol (no ionizable group) coincided with the order of the K(m)(app) values shown by tyrosinase immobilized on the hydrophobic supports, and was the inverse of that observed for tyrosinase in solution. The K(m)(app) values of immobilized tyrosinase were in all cases higher than those of soluble tyrosinase and depended on the nature of the support and the hydrophobicity of the substrate, meaning that it is possible to design supports with different degrees of selectivity towards a mixture of enzyme substrates in the reaction medium.     

Enzymatic grafting of carboxyl groups on to chitosan--to confer on chitosan the property of a cationic dye adsorbent

Chitosan (CTS) is a good adsorbent for dyes but lacks the ability to adsorb cationic dyes. In this study, chitosan was modified to possess the ability to adsorb cationic dyes from water. Four kinds of phenol derivatives: 4-hydroxybenzoic acid (BA), 3,4-dihydroxybenzoic acid (DBA), 3,4-dihydroxyphenyl-acetic acid (PA), hydrocaffeic acid (CA) were used individually as substrates of tyrosinase to graft onto chitosan. FTIR analysis provided supporting evidence of phenol derivatives being grafted. The grafting amounts of these phenol derivatives onto chitosan were examined by the adsorption of an anionic dye (amaranth) and reached a plateau value. The final contents of carboxyl groups in chitosan (mmol carboxyl groups per kg chitosan) were measured as 46.36 for BA, 70.32 for DBA, 106.44 for PA, and 113.15 for CA. These modified chitosans were used in experiments on uptake of the cationic dyes crystal violet (CV) and bismarck brown Y (BB) by a batch adsorption technique at pH 7 for CV and at pH 9 for BB and 30 degrees C. Langmuir type adsorption was found, and the maximum adsorption capacities for both dyes were increased with the following order CTS-CA>CTS-PA>CTS-DBA>CTS-BA.     

Tyrosinase Reaction and Subsequent Chitosan Adsorption for Selective Removal of a Contaminant from a Fermentation Recycle Stream

In the industrial production of penicillin V, the phenoxyacetate precursor is added to the fermentor to direct biosynthesis. When used for producing semisynthetic penicillins, the penicillin V is often hydrolyzed to 6-aminopenicillanic acid with the regeneration of the phenoxyacetate precursor. To reduce raw-material as well as waste-disposal costs, it is desirable to recycle the phenoxyacetate precursor. Unfortunately, the recycle stream is generally contaminated by the p-hydroxylated derivative of this precursor. We examined a two-step approach to eliminate this contaminant. In the first step the tyrosinase enzyme was used to selectively convert the p-hydroxyphenoxyacetate contaminant to a reactive intermediate—presumably its quinone. In the second step, the tyrosinase-generated reactive intermediate was allowed to react with and strongly bind to chitosan. In contrast, the phenoxyacetate precursor was neither oxidized by tyrosinase nor bound to chitosan. When concentrated phenoxyacetate solutions were tested, the combination of tyrosinase and chitosan effectively converted low levels of the p-hydroxyphenoxyacetate contaminant and removed its products from solution, while the concentration of the phenoxyacetate precursor was unaffected.     

Correlation of chitosan's rheological properties and its ability to electrospin

Chitosan-based, defect-free nanofibers with average diameters ranging from 62 +/- 9 nm to 129 +/- 16 nm were fabricated via electrospinning blended solutions of chitosan and polyethylene oxide (PEO). Several solution parameters such as acetic acid concentration, polymer concentration, and polymer molecular weight were investigated to optimize fiber consistency and diameter. These parameters were evaluated using the rheological properties of the solutions as well as images produced by scanning electron microscopy (SEM) of the electrospun nanofibers. Generally, SEM imaging demonstrated that as total polymer concentration (chitosan + PEO) increased, the number of beads decreased, and as chitosan concentration increased, fiber diameter decreased. Chitosan-PEO solutions phase separate over time; as a result, blended solutions were able to be electrospun with the weakest electric field and the least amount of complications when solutions were electrospun within 24 h of initially being blended. The addition of NaCl stabilized these solutions and increased the time the blended solutions could be stored before electrospinning. Pure chitosan nanofibers with high degrees of deacetylation (about 80%) were unable to be produced. When attempting to electrospin highly deacetylated chitosan from aqueous acetic acid at concentrations above the entanglement concentration, the electric field was insufficient to overcome the combined effect of the surface tension and viscosity of the solution. Therefore, the degree of deacetylation is an extremely important parameter to consider when attempting to electrospin chitosan.     

Adsorptive removal of copper and nickel ions from water using chitosan coated PVC beads

A new biosorbent was developed by coating chitosan, a naturally and abundantly available biopolymer, on to polyvinyl chloride (PVC) beads. The biosorbent was characterized by FTIR spectra, porosity and surface area analyses. Equilibrium and column flow adsorption characteristics of copper(II) and nickel(II) ions on the biosorbent were studied. The effect of pH, agitation time, concentration of adsorbate and amount of adsorbent on the extent of adsorption was investigated. The experimental data were fitted to Langmuir and Freundlich adsorption isotherms. The data were analyzed on the basis of Lagergren pseudo first order, pseudo-second order and Weber-Morris intraparticle diffusion models. The maximum monolayer adsorption capacity of chitosan coated PVC sorbent as obtained from Langmuir adsorption isotherm was found to be 87.9 mg g(-1) for Cu(II) and 120.5 mg g(-1) for Ni(II) ions, respectively. In addition, breakthrough curves were obtained from column flow experiments. The experimental results demonstrated that chitosan coated PVC beads could be used for the removal of Cu(II) and Ni(II) ions from aqueous medium through adsorption.     

Equilibrium and kinetics studies of adsorption of copper (II) on chitosan and chitosan/PVA beads

The adsorption of Cu(II) ions from aqueous solution by chitosan and chitosan/PVA beads was studied in a batch adsorption system. Chitosan solution was blended with poly(vinyl alcohol) (PVA) in order to obtain sorbents that are insoluble in aqueous acidic and basic solution. The adsorption capacities and rates of Cu(II) ions onto chitosan and chitosan/PVA beads were evaluated. The Langmuir, Freundlich and BET adsorption models were applied to describe the isotherms and isotherm constants. Adsorption isothermal data could be well interpreted by the Langmuir model. The kinetic experimental data properly correlated with the second-order kinetic model, which indicates that the chemical sorption is the rate-limiting step. The Cu(II) ions can be removed from the chitosan and chitosan/PVA beads rapidly by treatment with an aqueous EDTA solution. Results also showed that chitosan and chitosan/PVA beads are favourable adsorbers.     

Tyrosinase‐catalyzed oxidation of rhododendrol produces 2‐methylchromane‐6,7‐dione,the putative ultimate toxic metabolite: implications for melanocyte toxicity

RS‐4‐(4‐Hydroxyphenyl)‐2‐butanol (rhododendrol, RD) was used as a skin‐whitening agent until it was reported to induce leukoderma in July 2013. To explore the mechanism underlying its melanocyte toxicity, we characterized the tyrosinase‐catalyzed oxidation of RD using spectrophotometry and HPLC. Oxidation of RD with mushroom tyrosinase rapidly produced RD‐quinone, which was quickly converted to 2‐methylchromane‐6,7‐dione (RD‐cyclic quinone) and RD‐hydroxy‐p‐quinone through cyclization and addition of water molecule, respectively. RD‐quinone and RD‐cyclic quinone were identified as RD‐catechol and RD‐cyclic catechol after NaBH₄ reduction. Autoxidation of RD‐cyclic catechol produced superoxide radical. RD‐quinone and RD‐cyclic quinone quantitatively bound to thiols such as cysteine and GSH. These results suggest that the melanocyte toxicity of RD is caused by its tyrosinase‐catalyzed oxidation through production of RD‐cyclic quinone which depletes cytosolic GSH and then binds to essential cellular proteins through their sulfhydryl groups. The production of ROS through autoxidation of RD‐cyclic catechol may augment the toxicity.     

Tyrosinase-containing chitosan gels: A combined catalyst and sorbent for selective phenol removal

There are a series of examples in which phenols appear as contaminants in process streams and their selective removal is required for waste minimization. For the selective removal of a phenol from a mixture, we are exploiting the substrate specificity of the enzyme tyrosinase to convert phenols into reactive o-quinones which are then adsorbed onto the amine-containing polymer chitosan. To effectively package the enzyme and sorbent, tyrosinase was immobilized between two chitosan gel films. The entrapment of tyrosinase between the films led to little loss of activity during immobilization, while tyrosinase leakage during incubation was limited. The chitosan gels rapidly adsorb the tyrosinase-generated product(s) of phenol oxidation while the capacity of the gels is substantially greater than the capacity of chitosan flakes. The performance of tyrosinase-containing chitosan gels significantly depends on the ratio of tyrosinase-to-chitosan. High tyrosinase-to-chitosan ratios result in less efficient use of tyrosinase, presumably due to suicide inactivation. However, the efficiency of chitosan use increases with increased tyrosinase-to-chitosan ratios. (c) 1996 John Wiley & Sons, Inc.