Laccase-poly(lactic-co-glycolic acid) (PLGA) nanofiber: highly stable, reusable, and efficacious for the transformation of diclofenac

Nanobiocatalysis has received growing attention for use in commercial applications. We investigated the efficiency, stability, and reusability of laccase-poly(lactic-co-glycolic acid) (PLGA) nanofiber for diclofenac transformation. NH stretching vibrations (3400-3500 cm(-1) and 1560 cm(-1)) in FT-IR spectra confirmed immobilization of laccase on PLGA nanofibers. The relative activity of immobilized laccase was 82% that of free laccase. Immobilized laccase had better storage, pH, and thermal stability than free laccase. The immobilized laccase produced complete diclofenac transformation in three reuse cycles, which was extended to 6 cycles in the presence of syringaldehyde. Results suggest that laccase-PLGA nanofiber may be useful for removing diclofenac from aqueous sources and has potential for other commercial applications.     

Recent developments and applications of immobilized laccase

Laccase is a promising biocatalyst with many possible applications, including bioremediation, chemical synthesis, biobleaching of paper pulp, biosensing, textile finishing and wine stabilization. The immobilization of enzymes offers several improvements for enzyme applications because the storage and operational stabilities are frequently enhanced. Moreover, the reusability of immobilized enzymes represents a great advantage compared with free enzymes. In this work, we discuss the different methodologies of enzyme immobilization that have been reported for laccases, such as adsorption, entrapment, encapsulation, covalent binding and self-immobilization. The applications of laccase immobilized by the aforementioned methodologies are presented, paying special attention to recent approaches regarding environmental applications and electrobiochemistry.     

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.     

Laccase immobilization on enzymatically functionalized polyamide 6,6 fibres

Polyamide matrices, such as membranes, gels and non-wovens, have been applied as supports for enzyme immobilization, although in literature the enzyme immobilization on woven nylon matrices is rarely reported. In this work, a protocol for a Trametes hirsuta laccase immobilization using woven polyamide 6,6 (nylon) was developed. A 2⁴ full factorial design was used to study the influence of pH, spacer (1,6-hexanediamine), enzyme and crosslinker concentration on the efficiency of immobilization. The factors enzyme dosage and spacer seem to have played a critical role in the immobilization of laccase onto nylon support. Under optimized working conditions (29 U mL⁻¹ of laccase, 10% of glutaraldehyde, pH = 5.5, with the presence of the spacer), the half-life time attained was about 78 h (18% higher than that of free enzyme), the protein retention was 30% and the immobilization yield was 2%. The immobilized laccase has potential for application in the continuous decolourization of textile effluents, where it can be applied into a membrane reactor.     

Efficient production of Al(OH)3‐immobilized laccase with a Heterobasidion annosum strain selected by microplate screening

Aims: Wild‐type white rot fungi are the most important production organisms for laccase, a promising oxidative biocatalyst with numerous applications. This study aimed at identifying novel highly productive strains, finding optimal cultivation conditions for laccase production and establishing a simple immobilization procedure. Methods and Results: By using a newly developed 96‐well microplate cultivation method, 23 species of white rot fungi, represented by 29 strains, were directly compared with regard to the amount of secreted laccase. Both, with glucose and spruce saw dust as growth substrate a Heterobasidion annosum strain and a Physisporinus vitreus strain were the most productive (730–2200 U l^?1 of secreted laccase). Cultivation conditions for laccase production with H. annosum were optimized in larger‐scale liquid cultures. Aeration with a sparger lead to a 3·8‐fold increase in laccase activity when compared to nonaerated flask cultures. More than 3000 U l^?1 laccase was produced in glucose medium supplemented with yeast extract and the inducer veratryl alcohol. Culture supernatant was incubated with short‐range ordered Al(OH)₃ particles to directly immobilize and concentrate laccase by adsorption. Active laccase was recovered in 40% yield and the Al(OH)₃‐adsorbed laccase was suitable for repeated decolourization of indigo carmine. Conclusions: Microplate cultivation allowed a large‐scale comparison of the capacity of different fungal species for laccase production. Laccase secretion of a highly productive H. annosum strain was found to vary strongly with different cultivation conditions. Adsorption to Al(OH)₃ proved to be suitable as direct immobilization technique. Significance and Impact of the Study: The microplate screening method simplifies strain and medium development for laccase production. Two novel fungal strains suitable for laccase production were identified. Procedures for simple and efficient production of immobilized H. annosum laccase were established.     

金属框架结构材料MOF-199对漆酶的固定化及其性质

以金属框架结构材料MOF-199为载体对漆酶进行固定化,并对固定化酶的性质进行初步研究。首先,以3-氨基丙基三乙氧基硅烷对载体MOF-199进行表面氨基化修饰,再用戊二醛对载体进行活化,最后对漆酶进行固定化。固定化条件优化结果表明:在漆酶质量浓度0.3 g/L,戊二醛用量1%(体积分数),pH 4.8下固定7 h,制得固定化酶活性最高。对固定化酶的研究发现:最适反应温度为40℃,最适pH为5.2,在连续操作7次后,固定化酶的活力仍能保持在51%。固定化漆酶热稳定性,pH耐受性,贮存稳定性均明显高于游离漆酶。     

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.     

Laccase immobilization on enzymatically functionalized polyamide 6,6 fibres

Polyamide matrices, such as membranes, gels and non-wovens, have been applied as supports for enzyme immobilization, although in literature the enzyme immobilization on woven nylon matrices is rarely reported. In this work, a protocol for a Trametes hirsuta laccase immobilization using woven polyamide 6,6 (nylon) was developed. A 2⁴ full factorial design was used to study the influence of pH, spacer (1,6-hexanediamine), enzyme and crosslinker concentration on the efficiency of immobilization. The factors enzyme dosage and spacer seem to have played a critical role in the immobilization of laccase onto nylon support. Under optimized working conditions (29 U mL⁻¹ of laccase, 10% of glutaraldehyde, pH = 5.5, with the presence of the spacer), the half-life time attained was about 78 h (18% higher than that of free enzyme), the protein retention was 30% and the immobilization yield was 2%. The immobilized laccase has potential for application in the continuous decolourization of textile effluents, where it can be applied into a membrane reactor.     

Laccase production using Pleurotus ostreatus 1804 immobilized on PUF cubes in batch and packed bed reactors: influence of culture conditions

The feasibility of laccase production by immobilization of Pleurotus ostreatus 1804 on polyurethane foam (PUF) cubes with respect to media composition was studied in both batch and reactor systems. Enhanced laccase yield was evidenced due to immobilization. A relatively high maximum laccase activity of 312.6 U was observed with immobilized mycelia in shake flasks compared to the maximum laccase activity of free mycelia (272.2 U). It is evident from this study that the culture conditions studied, i.e. biomass level, pH, substrate concentration, yeast extract concentration, Cu2+ concentration, and alcohol nature, showed significant influence on the laccase yield. Gel electrophoretic analysis showed the molecular weight of the laccase produced by immobilized P. ostreatus to be 66 kDa. The laccase yield was significantly higher and more rapid in the packed bed reactor than in the shake flask experiments. A maximum laccase yield of 392.9 U was observed within 144 h of the fermentation period with complete glucose depletion.     

Probing fundamental film parameters of immobilized enzymes--towards enhanced biosensor performance. Part I--QCM-D mass and rheological measurements

Enzyme immobilization is an ever-growing research-area for both analytical and industrial applications. Of critical importance in this area are the effects of immobilization procedures upon the functionality of the immobilized biomolecules. Both beneficial and detrimental effects can be conferred through the selection and tuning of the immobilization procedure. Quartz-crystal microbalance with dissipation (QCM-D) has been previously used to great effect in tracking alterations to thin films of biomolecules immobilized onto quartz transducers.In this study, we investigate the ability of QCM-D to track and monitor film parameters of a monolayer of laccase immobilized on a series of self-assembled monolayers (SAMs), differing in lateral density of binding residues on the SAM and height of the SAM from the quartz surface. Both mass gains and rheological parameters for these varying surfaces were measured and trends later compared to the apparent enzyme kinetics of the immobilized laccase films, assessed electroanalytically (Paper II in this two part study). For covalent attachment of proteins, both shear and viscosity were increased relative to physically adsorbed proteins. An increase in lateral density of protein-binding surface of the SAM components was shown to increase the shear/viscosity of the resultant film while an increase in distance from the electrode (through incorporation of lysine linkers) was shown to decrease the shear/viscosity while simultaneously increasing the wet mass gain of the films. Shear and viscosity may be indicative of both enzyme denaturation and increased lateral protein packing within the film structure hence it is assumed that less distortion occurs with the inclusion of linkers which allow for more optimal protein immobilization.     

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.     

Zinc tetraaminophthalocyanine-Fe3O4 nanoparticle composite for laccase immobilization

Zinc tetraaminophthalocyanine-Fe3O4 nanoparticle composites were prepared by organic-inorganic complex technology and characterized. It has been proved that the ZnTAPc dispersed randomly onto the surface of Fe3O4 nanoparticles to form molecular dispersion layer and there was a relatively strong bond between central zinc cation and oxygen. The nanoparticle composite took the shape of roundish spheres with the mean diameter of about 15 nm. Active amino groups of magnetic carriers could be used to bind laccase via glutaraldehyde. The optimal pH for the activity of the immobilized laccases and free laccase were the same at pH 3.0 and the optimal temperature for laccase immobilization on ZnTAPc-Fe3O4 nanoparticle composite was 45 degrees. The immobilization yields and K(m) value of the laccase immobilized on ZnTAPc-Fe3O4 nanoparticle composite were 25% and 20.1 microM, respectively. This kind of immobilized laccase has good thermal, storage and operation stability, and could be used as the sensing biocomponent for the fiber optic biosensor based on enzyme catalysis.     

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.     

Fungal laccases - occurrence and properties

Laccases of fungi attract considerable attention due to their possible involvement in the transformation of a wide variety of phenolic compounds including the polymeric lignin and humic substances. So far, more than a 100 enzymes have been purified from fungal cultures and characterized in terms of their biochemical and catalytic properties. Most ligninolytic fungal species produce constitutively at least one laccase isoenzyme and laccases are also dominant among ligninolytic enzymes in the soil environment. The fact that they only require molecular oxygen for catalysis makes them suitable for biotechnological applications for the transformation or immobilization of xenobiotic compounds.     

Ceramic honeycomb as support for covalent immobilization of laccase from Trametes versicolor and transformation of nuclear fast red

The covalent immobilization of laccase on an inorganic ceramic support was investigated. The intention was to find a system of enzyme and reactor for a universal immobilization procedure. Laccase from Trametes versicolor as model enzyme was chosen. The special honeycomb structure of the monolith can be applied for intensive mixing of the reaction compounds. An appropriate reactor with ceramic material was constructed allowing different setup for enzyme immobilization and its application. To test the success of the immobilization, 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) was used. The immobilized laccase was found to be stable over a time period of over 3 months. As an example for possible application for treatment of wastewater containing dyes, the conversion of nuclear fast red as model substrate was tested.     

Laccase stabilization by covalent binding immobilization on activated polyvinyl alcohol carrier

AIMS: Attempts were made to immobilize laccase from Panus conchatus. METHODS AND RESULTS: The laccase was immobilized on carboxylated polyvinyl alcohol (PVA) activated by N-hydroxysuccinimide (N-HSI) in aqueous solution at different pHs, temperatures, and with different reaction times. An optimum condition for laccase immobilization is at pH 3.2, 40 degrees C and 12 h, respectively. Immobilization of laccase increased optimal pH for reaction with 2, 2'-azinobis (3-ethylbenzthiazoline-6-solfonate) (ABTS) and pH stability. Immobilized laccase proved to be reacted consecutively 17 times with only a 50% decrease on activity and be used in removal of 2,4,6-trichlorophenol (TCP). CONCLUSIONS: It was possible to immobilize the laccase on carboxylated polyvinyl alcohol by activation with N-hydroxysuccinimide in HAc-NaAc buffer. The immobilized laccase is both stable and reusable. SIGNIFICANCE IMPACT OF THE STUDY: The results indicate that this immobilized laccase can be used in the removal of poisonous effluent from pulp bleaching mills.     

Enhanced removal of crystal violet in water using a facile-fabricated and environmental-friendly laccase immobilized composite membrane

An effort has been made to search a cheaper, easily available and simple alternative for the immobilization of enzymes and practical utilization in dye treatment. In this study, a porous zeolite-like geopolymer membrane (Geo) was used as immobilization support considering environmental friendliness, low cost and chemical/mechanical stability. A facile “cyclic adsorption” method was adopted to prepare the laccase immobilized geopolymer composite membrane (Geo-Lac). The results indicated that the pH-temperature range and stability were improved by adding the Geo support. The feasibility of removing crystal violet (CV) by the Geo-Lac was investigated in a batch mode and a flow-through mode, respectively. More than 99 % of CV (C₀ = 5 mg/L) was removed with the Geo-Lac in a batch mode and the removal efficiency still remained over 93 % within 8 h of high-throughput filtration in a flow-through mode. Moreover, the Geo-Lac was much more durable, and after 4 cycles, it still had a removal efficiency of 90.02 ± 0.33 % for CV within 6 h of filtration. These results indicated that the porous geopolymer membrane is a promising support for both laccase immobilization and further applications in dye removal.     

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.     

漆酶的性质、功能、催化机理和应用

漆酶是一种结合多个铜离子的蛋白,是铜蓝氧化酶蛋白家族的一员。本文叙述漆酶的分子结构、底物特异性及其物理化学特性,并讨论漆酶的酶促反应机理和生物学功能,包括植物漆酶参与细胞壁的形成以及漆酶与病原菌毒力的关系。本文还着重介绍了漆酶在环境生物修复方面的应用。     

漆酶的性质、功能、催化机理和应用

漆酶是一种结合多个铜离子的蛋白,是铜蓝氧化酶蛋白家族的一员。本文叙述漆酶的分子结构、底物特异性及其物理化学特性,并讨论漆酶的酶促反应机理和生物学功能,包括植物漆酶参与细胞壁的形成以及漆酶与病原菌毒力的关系。本文还着重介绍了漆酶在环境生物修复方面的应用。