Immobilization of laccase by encapsulation in a sol-gel matrix and its characterization and use for the removal of estrogens

Laccase from Myceliophthora thermophila was immobilized by encapsulation in a sol-gel matrix based on methyltrimethoxysilane and tetramethoxysilane. The amount of laccase used for the preparation of the hydrogel was in the range 2.2-22 mg of protein/mL sol and the corresponding enzymatic activities were in the range 5.5-17.0 U/g biocatalyst. The kinetic parameters of the encapsulated laccase showed that the immobilized enzyme presented lower affinity for the substrate 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS). However, the stability of laccase was significantly enhanced after immobilization; thus, both pH and thermal stability improved about 10-30% and tolerance to different inactivating agents (NaN(3) , ZnCl(2) , CoCl(2) , CaCl(2) , methanol, and acetone) was 20-40% higher. The reusability of the immobilized laccase was demonstrated in the oxidation of ABTS for several consecutive cycles, preserving 80% of the initial laccase activity after 10 cycles. The feasibility of the immobilized biocatalyst was tested for the continuous elimination of Acid Green 27 dye as a model compound in a packed-bed reactor (PBR). Removals of 70, 58, 57, and 55% were achieved after four consecutive cycles with limited adsorption on the support: only 10-15%. Finally, both batch stirred tank reactor (BSTR) operated in several cycles and PBR, containing the solid biocatalyst were applied for the treatment of a solution containing the endocrine disrupting chemicals (EDCs): estrone (E1), 17β-estradiol (E2), and 17α-ethinylestradiol (EE2). Eliminations of EDCs in the BSTR were higher than 85% and the reusability of the biocatalyst for the degradation of those estrogens was demonstrated. In the continuous operation of the PBR, E1 was degraded by 55% and E2 and EE2 were removed up to 75 and 60%, at steady-state conditions. In addition, a 63% decrease in estrogenic activity was detected.     

Immobilisation of CGTase for continuous production of long-carbohydrate-chain alkyl glycosides: Control of product distribution by flow rate adjustment

Bacillus macerans cyclodextrin glycosyltransferase (CGTase) (EC 2.4.1.19) was covalently immobilised on Eupergit C and used in a packed-bed reactor to investigate the continuous production of long-carbohydrate-chain alkyl glycosides from α-cyclodextrin (α-CD) and n-dodecyl-(1,4)-β-maltopyranoside (C₁₂G₂β). The effects of buffer ion strength and pH, and enzyme loading on the immobilisation yield and the enzyme activity were evaluated. Approximately 98% of the protein and 33% of the total activity were immobilised. At pH 5.15, the enzymatic half-life was 132 min at 60 °C and 18 min at 70 °C. The immobilised enzyme maintained 60% of its initial activity after 28 days storage at 4 °C. The degree of conversion was controlled by simple regulation of the flow rate through the reactor, making it possible to optimise the product distribution. It was possible to achieve a yield of the primary coupling product n-dodecyl-(1,4)-β-maltooctaoside (C₁₂G₈β) of about 50%, with a ratio between the primary and the secondary coupling product of about 10. Thermoanaerobacter sp. CGTase (Toruzyme 3.0 L) immobilised on Eupergit C had good operational stability at 60 and 70 °C thus showing the advantages of using more thermostable enzymes in biocatalysis. However, this enzyme was unsuitable for the production of C₁₂G₈β due to extensive disproportionation reactions, giving a broad product range.     

Immobilization of pycnoporus coccineus laccase on Eupergit C: Stabilization and treatment of olive oil mill wastewaters

The use of olive oil mill wastewaters (OMW) as an organic fertilizer is limited by their phytotoxic effect, due to the high concentration of phenolic compounds. As an alternative to physico-chemical methods for OMW detoxification, the laccase from Pycnoporus coccineus, a white-rot fungus with the ability to decrease the chemical oxygen demand (COD) and color of the industrial effluent, is being studied. In this work, the P. coccineus laccase was immobilized on two acrylic epoxy-activated resins, Eupergit C and Eupergit C 250L. The highest activity was obtained with the macroporous Eupergit C 250L, reaching 110 U g^?1 biocatalyst. A substantial stabilization effect against pH and temperature was obtained upon immobilization. The soluble enzyme maintained ≥80% of its initial activity after 24 h at pH 7.0–10.0, whereas the immobilized laccase kept the activity in the pH range 3.0–10.0. The free enzyme was quickly inactivated at temperatures >50°C, whereas the immobilized enzyme was very stable up to 70°C. Gel filtration profiles of the OMW treated with the immobilized enzyme (for 8 h at room temperature) showed both degradation and polymerization of the phenolic compounds.     

Immobilized laccase for decolourization of Reactive Black 5 dyeing effluent

Reactive Black 5 industrial dyeing effluent was decolourized by free and immobilized laccase. The stability of the enzyme (194 h free and 79 h immobilized) depended on the dyeing liquor composition and the chemical structure of the dye. In the decolourization experiments with immobilized laccase, two phenomenons were observed – decolourization due to adsorption on the support (79%) and dye degradation due to the enzyme action (4%). Dyeing in the enzymatically recycled effluent provided consistency of the colour with both bright and dark dyes.     

Reactive blue 19 decolouration by laccase immobilized on silica beads

Laccase (31.5 U of activity/g or 4.39 μg of protein/m²) from Trametes versicolor was immobilized on controlled-porosity-carrier silica beads and evaluated for the decolouration of Reactive blue 19, an anthraquinone dye. Although there was an initial, rapid adsorption of the dye to the packed bed in a recirculating reactor, about 97.5% of Reactive blue 19 removal was due to enzymatic degradation. The free enzyme lost 52% of its activity in 48 h. However, the activity of the immobilized laccase was unchanged after 4 months of storage in phosphate buffer under ambient conditions followed by three successive decolourations over 120 h. Treating the laccase immobilized beads with ethanolamine reduced dye adsorption by 40%.     

Effect of Different Carbon Sources on Decolourisation of an Industrial Textile Dye Under Alkaline–Saline Conditions

White-rot fungal strains of Trametes versicolor and Phanerochaete chrysosporium were selected to study the decolourisation of the textile dye, Reactive Black 5, under alkaline–saline conditions. Free and immobilised T. versicolor cells showed 100 % decolourisation in the growth medium supplemented with 15 g l^?1 NaCl, pH 9.5 at 30 °C in liquid batch culture. Continuous culture experiments were performed in a fixed-bed reactor using free and immobilised T. versicolor cells and allowed 85–100 % dye decolourisation. The immobilisation conditions for the biomass and the additional supply of carbon sources improved the decolourisation performance during a long-term trial of 40 days. Lignin peroxidase, laccase and glyoxal oxidase activities were detected during the experiments. The laccase activity varied depending on carbon source utilized and glycerol-enhanced laccase activity compared to sucrose during extended growth.     

Characterization and immobilization of the laccase from Pleurotus ostreatus and its use for the continuous elimination of phenolic pollutants

A laccase, the only ligninolytic enzyme produced by the basidiomycete Pleurotus ostreatus strain RK 36 was purified to homogeneity and characterized. The enzyme is a monomeric protein with a molecular weight of 67 000 Da and an isoelectric point of 3.6. Type I and type III Cu(2+) centers were identified by spectrophotometry. With syringaldazine as substrate laccase showed the highest oxidation rates at pH 5.8, 50 degrees C, and in 40 mM phosphate buffer. Among the tested stabilization parameters laccase retained most of its activity in high ionic buffer, pH 10, -20 degrees C, in the presence of 10 mM benzoic acid and with 35% ethylene glycol respectively. Crude laccase was covalently immobilized to Eupergit((R))C. Benzoate was found to stabilize the enzyme during the immobilization process. The activity loss of laccase during 10 days at 25 degrees C storage was 2% on average. Continuous elimination of 2,6-dimethoxyphenol by immobilized laccase was carried out in a packed bed reactor followed by filtration of the formed precipitate. The solubility of the polymerisates of oxidized syringaldazine, o-dianisidine, and 2,6-dimethoxyphenol with respect to temperature, pH-value and organic solvents were examined. The precipitates were found to be insoluble under non-extreme environmental conditions.     

Immobilization and biocatalysis of chlorophyllase in selected organic solvent systems

Chlorophyllase extract from Phaeodactylum tricornutum was immobilized by physical adsorption on DEAE-cellulose and silica gel as well as by covalent binding on Eupergit C, Eupergit C250L, Eupergit C/ethylenediamine (EDA) and Eupergit C250L/EDA. Although the highest immobilization yield (83-93%) and efficiency (51-53%) were obtained when chlorophyllase extract was immobilized on DEAE-cellulose and silica gel, there was no improvement in the thermal stability of chlorophyllase as compared to that of the free one. The immobilization of chlorophyllase extract on Eupergit C250L/EDA resulted by a high recovery of enzymatic activity, with an immobilization efficiency of 44%, and promoted a higher stabilization of chlorophyllase (four times) in the aqueous/miscible organic solvent medium. On the other hand, the inhibitory effect of refined bleached deodorized (RBD) canola oil was reduced by immobilization of chlorophyllase extract onto silica gel as compared to those obtained with other enzyme preparations. However, the re-cycled chlorophyllase extract immobilized on Eupergit C250L/EDA retained more than 75% of its initial enzyme activity after 6 cycles, whereas that immobilized on silica gel was completely inactivated. The highest catalytic efficiency, for both free and immobilized chlorophyllase on Eupergit C250L/EDA, was obtained in the ternary micellar system as compared to the aqueous/miscible organic solvent and biphasic media.     

Application of response surface methodology to study the removal of estrogens in a laccase-mediated continuous membrane reactor

Abstract

A three-level Box–Behnken factorial design combined with response surface methodology (RSM) was applied as a tool to study the laccase-catalyzed removal of three estrogenic compounds: estrone (E1), estradiol (E2), and ethinylestradiol (EE2), in a continuous enzymatic membrane reactor (EMR). Three main factors affecting the treatment efficiency were considered: enzyme activity, hydraulic residence time (HRT) and oxygenation rate. As expected, laccase activity and HRT showed large effects and, interestingly, the relevance of oxygen in improving the oxidation kinetics through raising the dissolved oxygen above saturation levels was demonstrated. When considering elimination rates as the response, optimal conditions were: 1,000 U/L of laccase, 1 h HRT and 60 mgO₂/(L·h) of oxygenation rate, predicting 2.82–3.24 mg eliminated/(L·h), (71–81% of oxidation). These optimum conditions were successfully validated, and 75% of estrogenicity reduction was achieved. On the other hand, only 100 U/L were found as optimal to maximize the efficacy of the enzyme: E1 was oxidized by 0.06 mg/(L·h·U), although the removal of estrogenicity decreased to 60%. The methodology was also applied to maximize the reduction of estrogenic activity: the highest values assayed [1,000 U/L, HRT 4 h and 60 mgO₂/(L·h)] provided 99% detoxification.     

Immobilization of horseradish peroxidase onto kaolin

Kaolin showed as a very perspective carrier for the enzyme immobilization and it was used for the adsorption of horseradish peroxidase (HRP). The effects of the enzyme concentration and pH on the immobilization efficiency were studied in the reaction with pyrogallol and anthraquinone dye C.I. Acid Violet 109 (AV 109). In addition, Fourier transform infrared spectroscopy, scanning electron microscopy and analysis by Brunauer–Emmett–Teller were performed for kaolin, thermally activated kaolin and the immobilized enzyme. It has been shown that 0.1 IU of HRP-kaolin decolorized 87 % of dye solution, under the optimal conditions (pH 5.0, temperature 24 °C, dye concentration 40 mg/L and 0.2 mM of H₂O₂) within 40 min. The immobilized HRP decolorization follows the Ping Pong Bi–Bi mechanism with dead-end inhibition by the dye. The biocatalyst retained 35 ± 0.9 % of the initial activity after seven cycles of reuse in the decolorization reaction of AV 109 under optimal conditions in a batch reactor. The obtained kinetic parameters and reusability study confirmed improvement in performances of k-HRP compared to free, indicating that k-HRP has a great potential for environmental purposes.     

Laccase-mediated oxidation of natural glycosides

Regioselective oxidations of the primary OH's of natural glycosides (thiocolchicoside, colchicoside, amygdalin, asiaticoside, ginsenoside R_E) have been performed on a preparative scale by exploiting the laccase–2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) methodology. The influence of water-miscible organic cosolvents on the stability and activity of a laccase from Trametes pubescens has been investigated. The enzyme has been covalently linked to Eupergit C250L and its performances evaluated. The recovered immobilized enzyme catalyzed several oxidative cycles of thiocolchicoside, without showing significant loss of activity.     

Immobilization of Laccase by Alginate–Chitosan Microcapsules and its Use in Dye Decolorization

Laccase is a ligninolytic enzyme that is widespread in white-rot fungi. Alginate–chitosan microcapsules prepared by an emulsification–internal gelation technique were used to immobilize laccase. Parameters of the immobilization process were optimized. Under the optimal immobilization conditions (2% sodium alginate, 2% CaCl₂, 0.3% chitosan and 1:8 ratio by volume of enzyme to alginate), the loading efficiency and immobilized yield of immobilized laccase were 88.12% and 46.93%, respectively. Laccase stability was increased after immobilization. Both the free and immobilized laccase alone showed a very low decolorization efficiency when Alizarin Red was selected for dye decolorization test. When 0.1 mM 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was added into the decolorization system, the decolorization efficiency increased significantly. Immobilized laccase retained 35.73% activity after three reaction cycles. The result demonstrated that immobilized laccase has potential application in dyestuff treatment.     

Specificities of a chemically modified laccase from Trametes hirsuta on soluble and cellulose-bound substrates

Laccases could prevent fabrics and garments from re-deposition of dyes during washing and finishing processes by degrading the solubilized dye. However, laccase action must be restricted to solubilized dye molecules thereby avoiding decolorization of fabrics. Chemical modification of enzymes can provide a powerful tool to change the adsorption behaviour of enzymes on water insoluble polymers. Polyethylene glycol (PEG) was covalently attached onto a laccase from Trametes hirsuta. Different molecular weights of the synthetic polymer were tested in terms of adsorption behaviour and retained laccase activity. Covalent attachment of PEG onto the laccase resulted in enhanced enzyme stability while with increasing molecular weight of attached PEG the substrate affinity for the laccase conjugate decreased. The activity of the modified laccases on fibre bound dye was drastically reduced decreasing the adsorption of the enzyme on various fabrics. Compared to the 5 kDa PEG laccase conjugate (K/S value 47.60) the K/S value decreased much more (47.96–46.35) after the treatment of dyed cotton fabrics with native laccase.     

Sequential reactors for the removal of endocrine disrupting chemicals by laccase immobilized onto fumed silica microparticles

Abstract

The main objective of this study is the evaluation of the capability of laccase from Myceliophthora thermophila immobilized on fumed silica microparticles (fsMP) for the removal of endocrine disrupting chemicals (EDCs) in two enzymatic reactor configurations. This type of support can also be magnetized to allow the straightforward separation of the biocatalyst under a magnetic field. The support exhibited excellent biocompatibility with the enzyme, superior tolerance to pH and temperature as well as improved stability in comparison with the free enzyme, even in the presence of organic solvents and enzyme inhibitors. The technical feasibility of the removal of EDCs by immobilized laccase was assessed in two types of enzymatic reactors operated in sequential mode: a membrane reactor using fsMP-laccase and a reactor with magnetic separation using magnetized fsMP-laccase. The extent of transformation for the target compounds: bisphenol A (BPA) and 17β-estradiol (E2) was high and comparable to free laccase in both systems (up to 80%). The possibility of reusing the immobilized enzyme, especially for magnetized supports, offers an interesting approach in the development of enzyme based processes for the biotransformation of emerging pollutants.     

Azo dye decolorization by a laccase/mediator system in a membrane reactor: enzyme and mediator reusability

This paper presents the use of a membrane-integrated reactor system with recycling of laccase and mediator for azo dye decolorization. From initial screening of different laccases and mediators, Trametes versicolor laccase and syringaldehyde provided the best system for decolorization. Decolorization yields of 98, 88, 80 and 78% were obtained for Red FN-2BL, Red BWS, Remazol Blue RR and Blue 4BL, respectively. The reaction parameters were optimized and a membrane reactor was set up for dye decolorization in batch mode with reuse of the enzyme. Between 10 and 20 batches could be run with decolorization yields from 95 to 52% depending on the dye type. To study the possibility of reusing both enzyme and mediator, the reactor was run using 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) coupled to polyethylene glycol (PEG). Nine batches were run for the treatment of Remazol Blue RR, providing decolorization yields of 96-78%. Cost analysis of the processes showed that the costs of laccase/syringaldehyde or laccase/TEMPO were almost equal when running 20 batches, but the cost for the PEG-TEMPO was higher. However, the advantages associated with reuse of the mediator should motivate further development of the concept.     

Laccase from Pleurotus sajor-caju on functionalised SBA-15 mesoporous silica: Immobilisation and use for the oxidation of phenolic compounds

Laccase from Pleurotus sajor-caju was immobilised on functionalised SBA-15 mesoporous silica. The immobilisation process reached the equilibrium after about 100 min. In order to study the effect of loading (L) on activity of the immobilised laccase, the adsorption isotherm was built and the activity of the corresponding immobilised biocatalysts was determined. The activity of the immobilised preparations reached a maximum at L = 217 kU g_SBA-15⁻¹, whereas higher loadings gave rise to a less-efficient biocatalyst. The immobilised laccase was used for the oxidation of a mixture of four phenolic compounds (protocatechuic acid, ferulic acid, sinapic acid and caffeic acid) chosen among those present in olive mill wastewaters (OMWs). These compounds determine the phytotoxicity of OMWs. Different reaction rates were observed for the oxidation of the examined phenolic compounds. The biocatalyst was recycled and a conversion of 84 mol% at the 10th reuse and of about 60 mol% after the 14th reuse was obtained. In conclusion, the laccase immobilised on SBA-15 is a potential biocatalyst for bioremediation of OMWs, which is an important environmental problem in the regions around the Mediterranean Sea.     

Immobilisation of Candida rugosa lipase on polyhydroxybutyrate via a combination of adsorption and cross-linking agents to enhance acylglycerol production

In the present study, a combination of immobilisation processes was utilised to prepare robust biocatalysts. First, lipase from Candida rugosa was adsorbed on polyhydroxybutyrate (PHB) particles, followed by cross-linking with glutaraldehyde. Conditions for creating immobilised lipase involved the addition of 0.6 M glutaraldehyde and 45 U mL⁻¹ lipase while mixing at 150 rpm (4 °C) for 30 min. These conditions produced the highest yield of immobilised lipase (92 %) and the highest levels of activity (1.94 mg g⁻¹ support). At 40 °C and pH 9 the immobilised enzyme was optimally active with a K_m and V_maxat 1.2 mM and 2.5 × 10^-3 mmol min⁻¹, respectively. The use of immobilised lipase improved thermal stability, storage stability, and reusability.The immobilised lipase retained 80 % of its activity after incubation at 30–60 °C for 2 h and 4 °C for 30 d in 0.2 M sodium phosphate buffer (pH 7.0). Moreover, the immobilised enzyme retained 50 % of its activity after more than 14 cycles under optimal conditions. The immobilised lipase was used to produce monoacylglycerol MAG. The existence of a carbonyl group at 1,743 and 1,744 cm⁻¹ was identified using attenuated total reflectance (ATR)-Fourier transformed infrared spectroscopy. Results showed that 48 % MAG was produced.     

Nitrate removal in a packed bed reactor using volatile fatty acids from anaerobic acidogenesis of food wastes

A packed bed reactor (PBR) was fed with nitrate containing synthetic wastewater or effluent from a sequencing batch reactor used for nitrification. The C source introduced into the PBR consisted of volatile fatty acids (VFAs) produced from anaerobic acidogenesis of food wastes. When nitrate loading rates ranged from 0.50 to 1.01 kg N/m³·d, the PBR exhibited 100∼98.8% NO₃ ⁻-N removal efficiencies and nitrite concentrations in the effluent ranged from 0 to 0.6 NO₂ ⁻-N mg/L. When the PBR was further investigated to determine nitrate removal activity along the bed height using a nitrate loading rate less than 1.01 kg N/m³·d, 100% nitrate removal efficiency was observed. Approximately 83.2% nitrate removal efficiency was observed in the lower 50% of the packed-bed height. When reactor performance at a C/N ratio of 4 and a C/N ratio of 5 was compared, the PBR showed better removal efficiency (96.5%) of nitrate and less nitrite concentration in the effluent at the C/N ratio of 5. VFAs were found to be a good alternative to methanol as a carbon source for denitrification of a municipal wastewater containing 40 mg-N/L.     

Recombinant sucrose phosphorylase from Leuconostoc mesenteroides: characterization, kinetic studies of transglucosylation, and application of immobilised enzyme for production of alpha-D-glucose 1-phosphate

Sucrose phosphorylase catalyzes the reversible conversion of sucrose (alpha-D-glucopyranosyl-1,2-beta-D-fructofuranoside) and phosphate into D-fructose and alpha-D-glucose 1-phosphate. We report on the molecular cloning and expression of the structural gene encoding sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase) in Escherichia coli DH10B. The recombinant enzyme, containing an 11 amino acid-long N-terminal metal affinity fusion peptide, was overproduced 60-fold in comparison with the natural enzyme. It was purified to apparent homogeneity using copper-loaded Chelating Sepharose and obtained in 20% yield with a specific activity of 190 Umg(-1). LmSPase was covalently attached onto Eupergit C with a binding efficiency of 50% and used for the continuous production of alpha-D-glucose 1-phosphate from sucrose and phosphate (600 mM each) in a packed-bed immobilised enzyme reactor (30 degrees C, pH 7.0). The reactor was operated at a stable conversion of 91% (550 mM product) and productivity of approximately 11 gl(-1)h(-1) for up to 600 h. A kinetic study of transglucosylation by soluble LmSPase was performed using alpha-d-glucose 1-phosphate as the donor substrate and various alcohols as acceptors. D- and L-arabitol were found to be good glucosyl acceptors.     

Kinetics of invertase immobilised on poly(phe-lys) coated polystyrene beads

Summary Surface of polystyrene beads was modified by poly(phe-lys) for invertase immobilisation. The optimum immobilisation conditions of invertase were; 0.01% (w/v) poly(phe-lys), 2% (v/v) glutaraldehyde at 25 °C and pH 4.5. The kinetics of sucrose hydrolysis by free and immobilised invertase in a batch reactor at pH 4.5 and 55 °C gave K_m and V_max values for sucrose with free and immobilised invertase of 81, 114 mM and 10.1, 9.2 mol glucose/min.mg enzyme, respectively. The deactivation rate constants of free and immobilised invertase were 0.0347 and 0.0098 min^–1, respectively.