Immobilization of laccase from Streptomyces psammoticus and its application in phenol removal using packed bed reactor

Laccase was produced from Streptomyces psammoticus under solid-state fermentation. The enzyme was partially purified by ammonium sulphate precipitation and was immobilized in alginate beads by entrapment method. Calcium alginate beads retained 42.5% laccase activity, while copper alginate beads proved a better support for laccase immobilization by retaining 61% of the activity. Phenol and colour removal from a phenol model solution was carried out using immobilized laccase. Batch experiments were performed using packed bed bioreactor, containing immobilized beads. Reusability of the immobilized matrix was studied for up to 8 successive runs, each run with duration of 6 h. The system removed 72% of the colour and 69.9% of total phenolics from the phenol model solution after the initial run. The immobilized system maintained 50% of its efficiency after eight successive runs. The degradation of phenolic compounds by immobilized laccase was evaluated and confirmed by Thin layer chromatography and nuclear magnetic resonance spectroscopy.     

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.     

A comparison of entrapped and covalently bonded laccase: Study of its leakage,reusability, and the catalytic efficiency in TEMPO-mediated glycerol oxidation

This article presents the comparison for reusability and leakage between entrapped and covalently bonded laccase and their performances towards the selective oxidation of glycerol. The reusability of immobilized laccase enzyme was studied by reacting a batch of immobilized laccase with ABTS for 15 cycles. The investigation of the leakage of immobilized laccase was carried out by storing the immobilized laccase in acetate buffer solution for 32 days. The data show that the retained enzyme activities of entrapped and covalently bonded enzyme after being reused for eight cycles were well above 60% and the leakages after storing for a month in the acetate buffer at 4?°C were well below 15%. The entrapped laccase coupled with TEMPO was found to perform better and gave a two-fold higher yield of glyceraldehyde and glyceric acid in the selective oxidation of glycerol compared to covalently bonded laccase. Hence, physical entrapment of laccase would be a suitable immobilization method in the laccase-mediated selective oxidation of glycerol.     

Specific immobilization of laccase onp-benzoquinone-activated supports

Summary A specific immobilization of laccase (EC 1.10.3.2) onto a ready-to-usep-benzoquinone-activated agarose support is described. The single-step procedure leads to a laccase protein coupling of I8% and an enzyme activity immobilization yield of 27%, while the retained specific activity of the immobilized enzyme was 150% of the specific activity of the free laccase. This peculiar result is thought to be related to the fact that during the process of support activation byp-benzoquinone, a significant amount of the hydroquinone by-product of the activation process is coupled to the support. These coupled derivatives constitute substrate (hydroquinone) analogues for which laccase exhibits a high affinity. Therefore, simultaneous affinity retention on the hydroquinone groups and covalent coupling on the p-benzoquinone groups allow the binding of the enzyme in an advantageous conformation which can generate this increase specific activity by immobilization. The entire process can be considered as an affinity immobilization. The immobilized enzyme is much more stable to the inhibitory action of chloride and azide ions, with a recovery of 100% of the activity, than the free laccase, with a recovery of 67% and 32%, respectively, after removal of the inhibitors by dialysis. The stability was 95% after storage for 14 months at 4° C.Abbreviations HQ hydroquinone - p-BQ p-benzoquinone - U enzyme units Part of the work was presented at the Satellite FEBS 1989 Symposium onBiochemical and biophysical approaches to the study of copper proteins, Camerino, Italy.     

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.     

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.     

Study on Remediation-improvement of 2,4-Dichlorophenol Contaminated Soil by Organic Fertilizer Immobilized Laccase

ABSTRACT

In this paper, laccase is immobilized by the cross-linking method, using organic fertilizer as a carrier and glutaraldehyde as a cross-linking agent. Here, the optimal conditions of laccase immobilization were explored and the optimal operating conditions and stabilities of free laccase and immobilized laccase were also studied. Then, free laccase and immobilized laccase were applied to the soil remediation. Meanwhile, the effect of soil improvement treated with immobilized laccase was studied through ecological evaluation. The results showed that the optimal conditions for laccase immobilization were: the volume fraction of glutaraldehyde was 5%, the amount of enzyme added was 15 mL, and the immobilization time was 6 h. Under the same conditions, thermal stability and acid-base stability of immobilized laccase were better than free laccase. Under the optimal conditions, using laccase to treat 2,4-dichlorophenol in the soil, it was found that the free laccase group degraded 44.4% within 5 days, while the immobilized laccase group degraded 58.6%. Although both the degradation trends and route are the same, the degradation effect of the latter is obviously better. Ecological evaluation showed that organic fertilizer carrier had an impact on soil physical and chemical properties and soil enzymes, playing a positive role in soil ecological security and improving the soil.     

Stability and kinetic behavior of immobilized laccase from Myceliophthora thermophila in the presence of the ionic liquid 1‐ethyl‐3‐methylimidazolium ethylsulfate

The use of ionic liquids (ILs) as reaction media for enzymatic reactions has increased their potential because they can improve enzyme activity and stability. Kinetic and stability properties of immobilized commercial laccase from Myceliophthora thermophila in the water‐soluble IL 1‐ethyl‐3‐methylimidazolium ethylsulfate ([emim][EtSO₄]) have been studied and compared with free laccase. Laccase immobilization was carried out by covalent binding on glyoxyl–agarose beads. The immobilization yield was 100%, and the activity was totally recovered. The Michaelis‐Menten model fitted well to the kinetic data of enzymatic oxidation of a model substrate in the presence of the IL [emim][EtSO₄]. When concentration of the IL was augmented, the values of V_max for free and immobilized laccases showed an increase and slight decrease, respectively. The laccase–glyoxyl–agarose derivative improved the laccase stability in comparison with the free laccase regarding the enzymatic inactivation in [emim][EtSO₄]. The stability of both free and immobilized laccase was slightly affected by small amounts of IL (<50%). A high concentration of the IL (75%) produced a large inactivation of free laccase. However, immobilization prevented deactivation beyond 50%. Free and immobilized laccase showed a first‐order thermal inactivation profile between 55 and 70°C in the presence of the IL [emim][EtSO₄]. Finally, thermal stability was scarcely affected by the presence of the IL. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:790–796, 2014     

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.     

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%.     

Immobilized laccase of Cerrena unicolor for elimination of endocrine disruptor micropollutants

The white-rot fungus Cerrena unicolor C-139 produced 450?000 U l⁻¹ of laccase when cultivated in submerged (50 ml) fermentation of wheat bran. Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2.), from C. unicolor C-139 was immobilized covalently on control porosity carrier silica beads. The activity of the immobilized laccase was approximately 15.8 units per gram of silica beads. The pH optimum was between 2.5 and 3.0 for free and immobilized laccase. The immobilization of enzyme appeared to be the main factor for retention of laccase activity at high temperature of 80 °C. The apparent K_m value (100 μmol) of immobilized laccase from C. unicolor C-139 was 6.7 times higher than free laccase (15 μmol) using 2,2-azino-bis-[3-ethylthiazoline-6-sulfonate] (ABTS) as the substrate. Immobilized laccase was able to eliminate 80 % of Bisphenol A, 40 % of Nonylphenol, and 60 % of Triclosan from solutions containing 50 μmol of each micropollutant separately. The experiments were run three times consecutively with the same immobilized laccase without loss of enzyme activity.     

Oxidation of aromatic compounds in organic solvents with laccase from Trametes versicolor

Summary Laccase purified from Trametes versicolor oxidizes 2,6-dimethoxyphenol (2,6-DMP) and syringaldazine in hydrophobic solvents presaturated with water, and in hydrophilic organic solvents provided that a sufficient amount of water is added. Ease of performance of the laccase test in organic solvents is improved after immobilization of the enzyme by entrapping in Sepharose CL-6B during enzyme filtration through the gel beads. The gel-enzyme association has been shown to be stable in water-presaturated solvents. Efficiency of the immobilized laccase in organic solvents containing 7% water was 10%–20% of that in potassium-citrate buffer. Immobilized laccase in organic solvents showed good stability and high tolerance to elevated temperatures.     

Stability of immobilized laccase on Luffa Cylindrica fibers and assessment of synthetic hormone degradation

In this work were studied the pH, thermal, and storage stability of free and immobilized laccases. Enzymes were produced by Pleurotus ostreatus on potato dextrose (PD) broth and potato dextrose modified (PDM) broth, and immobilized using Luffa cylindrica fibers as support. Both free and immobilized enzymes were assessed on their respective enzymatic activities and for 17α-ethinylestradiol (EE2) degradation. The optimum pH conditions concerning laccase activity ranged from 3.6 to 4.6, while temperature ranged between 30?°C and 50?°C for both free and immobilized enzyme. Laccase produced using PD broth presented greater storage stability and thermal stability than that of PDM. Best EE2 removals were of 79.22% and 75.00% for the free and immobilized enzymes, respectively. Removal rates were assessed during 8?h at pH 5. The removal of 17α-ethinylestradiol was stabilized in the fourth cycle of use. Results imply that immobilization promoted stability towards pH and temperature variations, although media played a decisive role in the enzymatic activity. Both free and immobilized laccases of P. ostreatus were able to degrade EE2, whereas immobilized laccase in PDM medium presented possible reuse applicability, albeit removal was not optimal when compared to other reports.     

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.     

Decolorization and detoxification of textile dyes with a laccase from Trametes hirsuta

Trametes hirsuta and a purified laccase from this organism were able to degrade triarylmethane, indigoid, azo, and anthraquinonic dyes. Initial decolorization velocities depended on the substituents on the phenolic rings of the dyes. Immobilization of the T. hirsuta laccase on alumina enhanced the thermal stabilities of the enzyme and its tolerance against some enzyme inhibitors, such as halides, copper chelators, and dyeing additives. The laccase lost 50% of its activity at 50 mM NaCl while the 50% inhibitory concentration (IC(50)) of the immobilized enzyme was 85 mM. Treatment of dyes with the immobilized laccase reduced their toxicities (based on the oxygen consumption rate of Pseudomonas putida) by up to 80% (anthraquinonic dyes). Textile effluents decolorized with T. hirsuta or the laccase were used for dyeing. Metabolites and/or enzyme protein strongly interacted with the dyeing process indicated by lower staining levels (K/S) values than obtained with a blank using water. However, when the effluents were decolorized with immobilized laccase, they could be used for dyeing and acceptable color differences (DeltaE*) below 1.1 were measured for most dyes.     

White‐rot fungi combined with lignite granules and lignitic xylite to decolorize textile industry wastewater

The feasibility of using immobilized fungi to decolorize textile industry wastewater containing dyes was examined in experiments with: two species of white‐rot fungi (a Marasmius species from Indonesia, which produces copious biomass, and Trametes hirsuta, which produces high levels of laccase); two types of lignite products as adsorbents and solid substrates (lignitic xylite and lignite granules); and four simulated wastewaters, each containing a different kinds of reactive textile azo dye. The growth, extracellular enzyme production, dye degradation and dye absorption parameters afforded by each permutation of fungus, substrate and dye were then measured. Both fungal species grew poorly on xylite, but much better on lignite granules. Marasmius sp. produced up to 67 U/L laccase on lignite granules, but just 10 U/L on xylite, and no other detectable extracellular enzymes. T. hirsuta produced 1343 U/L laccase and up to 12 U/L unspecific peroxidase when immobilized on lignite granules, and 898 U/L laccase with 14 U/L unspecific peroxidase when immobilized on xylite. The amount of color lost from the dye solutions depended on both the type of dye and the enzyme levels in the fermenter.     

Immobilization of wood-rotting fungi laccases on modified cellulose and acrylic carriers

Extracellular laccases produced by three different wood-rotting fungi, Cerrena unicolor, Heterobasidion annosum and Trametes versicolor, were immobilized via covalent bonds formation on DEAE-Granocel 500, CM-Granocel 500, and acrylic carriers. Out of the tested carriers, only the DEAE-Granocel 500, which was activated by divinyl sulphone appeared to be a suitable matrix for the expression of enzymic activity. Only one laccase of all the tested enzymes produced by C. unicolor showed the best binding to the carrier and a satisfactory enzymic activity. The immobilized laccase exhibited the highest enzymic activity at pH 5.2 and it was more resistant to thermal denaturation than the native enzyme. At 90 °C, it retained 75% activity compared to the free enzyme. It was also more stable during storage at 4 °C: after 4 months the immobilized laccase retained 98% of initial activity. Immobilized C. unicolor laccase was active in 10–60% concentration of methanol, acetone, isopropanol or acetonitrile. The best enzymic activity was observed in 20% solution of acetonitrile in buffer.     

桦褶孔菌漆酶固定化及其对染料的降解

采用壳聚糖交联法和海藻酸钠-壳聚糖包埋交联法固定化桦褶孔菌产生的漆酶,探讨最佳固定化条件,固定化漆酶的温度,pH稳定性及操作稳定性,并以两种固定化酶分别对4种染料进行了降解.结果表明:(1)壳聚糖交联法固定化漆酶的最佳条件为:壳聚糖2.5%,戊二醛7%,交联时间2h,固定化时间5h,给酶量1g壳聚糖小球:1mL酶液(1U/mL),固定化效率56%;(2)海藻酸钠-壳聚糖包埋交联法固定化漆酶的最佳条件为:海藻酸钠浓度4%,壳聚糖浓度0.7%,氯化钙浓度5%,戊二醛浓度0.6%,给酶量4mL 4%海藻酸钠:1mL酶液(1U/mL),固定化效率高达86%;(3)固定化的漆酶相比游离漆酶有更好的温度和pH稳定性;(4)比较两种固定化漆酶,海藻酸钠-壳聚糖包埋交联法固定化酶的温度及酸度稳定性要优于壳聚糖固定化酶,但可重复操作性要弱于后者,两者重复使用8次后的剩余酶活比率分别为71%及64%;(5)两种固定化酶对所选的4种不同结构的合成染料均有较好的降解效果,其中壳聚糖固定化酶对茜素红的降解效果及重复使用性极佳,重复降解40mg/L的茜素红10次,降解率仍保持在100%.     

Transformation of 2,4,6-trichlorophenol by free and immobilized fungal laccase

Laccase from the white rot fungus Coriolus versicolor was immobilized on Celite R-637 by covalent binding with glutaraldehyde. After a sharp primary decline in activity (up to 50%), the retained enzyme activity was stable over a storage period of 33 days at 4 degrees C. A comparative study of soluble and immobilized laccases revealed the increased resistance of immobilized enzyme to the unfavourable effects of alkaline pH, high temperature and the action of inhibitors. A combination of these properties of immobilized laccase resulted in the ability to oxidize 2,4,6-trichlorophenol (2,4,6-TCP) at 50 degrees C at pH 7.0. The reactions of soluble and immobilized laccase with 2,4,6-TCP were examined in the presence and absence of redox mediators. 3,5-Dichlorocatechol, 2,6-dichloro-1,4-benzoquinone and 2,6-dichloro-1,4-hydroquinone were found to be the primary products of 2,4,6-TCP oxidation by laccase; oligo- and polymeric compounds were also found.     

Bacterial cellulose as carrier for immobilization of laccase: Optimization and characterization

Bacterial cellulose (BC) has attracted attention as a new functional material due to its excellent mechanical strength, tridimensional nanostructure, high purity, and increased water absorption, compared to plant cellulose. In this work, commercial laccase was immobilized on BC and the influence of enzyme concentration, contact time, and pH was optimized toward the recovery activity of immobilized laccase. This optimization was carried out using a 3³ experimental design and response surface methodology. Enzyme concentration played a critical role in laccase immobilization. Under optimized conditions (0.15 μL L^?1 of enzyme concentration, 4.8 h of contact time, pH 5.4), the predicted and experimental response were equal to 47.88 and 49.30%, respectively. The thermal stability of the immobilized laccase was found to increase notably at 60 and 70°C presenting stabilization factor equal to 1.79 and 2.11, respectively. The immobilized laccase showed high operational stability, since it retained 86% of its initial activity after seven consecutive biocatalytic cycles of reaction with 2,2′‐azinobis‐(3‐ethylbenzothiazoline‐6‐sulfonic acid). Kinetic studies showed that the values of Michaelis–Menten constant and maximum reaction rate decreased upon immobilization (9.9‐ and 1.6‐fold, respectively). Globally, the use of immobilized laccase on BC offers an interesting tool for industrial biocatalytic applications.