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.     

Three Dimensional Carbon Nanosheets as a Novel Catalyst Support for Enzymatic Bioelectrodes

A high performance laccase‐based biofuel cell cathode is developed using carbon nanosheets (CNS) as the catalyst support and buckypaper (BP) as the substrate electrode. Compared to multiwalled carbon nanotube (MWNT)‐based electrodes, CNS‐based electrodes exhibit better electrochemical properties for the oxygen reduction reaction (ORR) under biologically relevant conditions. It is shown that CNSs are conformally coated on the nanotubule bundles within the BP and that laccase is intimately attached to the CNS‐BP. Electrochemical characterization is carried out to derive the kinetic parameters of the ORR at the laccase‐CNS‐BP cathode. The laccase‐CNS‐BP exhibits a steep ORR cathodic wave with a Tafel slope of 19 mV decade^‐1. The onset potential obtained for laccase ORR at CNS‐BP is 20 mV higher than that of the MWNT‐based electrodes, and the laccase‐CNS‐BP cathode has a higher current density than MWNT electrodes.     

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.     

Enhancement of laccase production by Pleurotus ostreatus and its use for the decolorization of anthraquinone dye

The white-rot fungus Pleurotus ostreatus strain 32 is an excellent producer of the industrially important enzyme laccase. Laccase was the only ligninolytic activity detected in the supernatant when the fungus was grown in liquid culture with or without shaking. Growth and laccase production in static cultivation were superior to that in agitated cultivation, and N-limited culture is of benefit to laccase production. When using cellobiose and peptone as carbon and nitrogen source, a higher activity level was obtained. 2,2′-Azino-di-(3-ethylbenzothialozin-6-sulfonic acid) (ABTS) (1 mM) was shown to be the best inducer of laccase production, reaching maximum values of about 400 U/ml. Cu²⁺ (1 mM) also had a positive effect on laccase production, activity being enhanced to 360 U/ml. In addition, anthraquinone dye SN4R can be effectively decolorized by crude laccase (30 U/ml), the rate of which was 66%. The decolorization rate was increased by 90% with ABTS (0.16%) addition as a mediator of laccase.     

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.     

利用根癌农杆菌T-DNA 插入突变寻找参与漆酶葡萄糖阻遏的关键基因

【目的】新型隐球酵母(Cryptococcus neoformans)是人类重要致病真菌,主要毒性因子之一漆酶的表达受葡糖糖阻遏,机制未知。本文拟寻找参与葡萄糖阻遏的关键基因。【方法】建立根癌农杆菌介导的转化方法(Agrobacterium tumefanciens-mediated transformation,ATMT)建立一个容量约200000的随即插入突变文库,在高浓度葡萄糖条件下从中筛选葡萄糖去阻遏的突变株。通过Southern确定突变株中T-DNA的拷贝数,利用反向PCR获得依赖葡萄糖的漆酶阻遏基因序列。【结果】筛选到了30株葡萄糖去阻遏突变株,Southern blot发现83%的葡萄糖去抑制突变株含有单个T-DNA拷贝。初步鉴定了可能参与漆酶阻遏的10个不同生物学功能基因,如参与碳水化合物的代谢,固醇的合成,几丁质的合成,GPI脂锚钩的合成等等。【结论】ATMT突变策略可以找到一些参与漆酶葡萄糖阻遏的关键基因,为理解漆酶在致病过程中的作用机制和工业改进漆酶活性提供参考。     

Co-cultivation of mutant Penicillium oxalicum SAU(E)-3.510 and Pleurotus ostreatus for simultaneous biosynthesis of xylanase and laccase under solid-state fermentation

Co-cultivation of mutant Penicillium oxalicum SAU(E)-3.510 and Pleurotus ostreatus MTCC 1804 was evaluated for the production of xylanase-laccase mixture under solid-state fermentation (SSF) condition. Growth compatibility between mutant P. oxalicum SAU(E)-3.510 and white rot fungi (P. ostreatus MTCC 1804, Trametes hirsuta MTCC 136 and Pycnoporus sp. MTCC 137) was analyzed by growing them on potato dextrose agar plate. Extracellular enzyme activities were determined spectrophotometrically. Under derived conditions, paired culturing of mutant P. oxalicum SAU(E)-3.510 and P. ostreatus MTCC 1804 resulted in 58% and 33% higher levels of xylanase and laccase production, respectively. A combination of sugarcane bagasse and black gram husk in a ratio of 3:1 was found to be the most ideal solid substrate and support for fungal colonization and enzyme production during co-cultivation. Maximum levels of xylanase (8205.31 ± 168.31 IU g(-1)) and laccase (375.53 ± 34.17 IU g(-1)) during SSF were obtained by using 4 g of solid support with 80% of moisture content. Furthermore, expressions of both xylanase and laccase were characterized during mixed culture by zymogram analysis. Improved levels of xylanase and laccase biosynthesis were achieved by co-culturing the mutant P. oxalicum SAU(E)-3.510 and P. ostreatus MTCC 1804. This may be because of efficient substrate utilization as compared to their respective monocultures in the presence of lignin degradation compounds because of synergistic action of xylanase and laccase. Understanding and developing the process of co-cultivation appears productive for the development of mixed enzyme preparation with tremendous potential for biobleaching.     

Production of laccase by membrane-surface liquid culture of Trametes versicolor using a poly(l-lactic acid) membrane

Membrane-surface liquid culture (MSLC) is a promising method for the bioproduction of highly aerobic filamentous fungi [A. Ogawa, A. Yasuhara, T. Tanaka, T. Sakiyama, K. Nakanishi, Production of neutral protease by membrane-surface liquid culture of Aspergillus oryzae IAM2704, J. Ferment. Bioeng. 80 (1995) 35–40]. This paper reports on the production of laccase by Trametes versicolor on a microporous membrane of poly(l-lactic acid) (PLLA), which can be biodegraded via composting after use. The membrane was stable as a support for 24 days at 30 °C. During the first 9 days in MSLC, the fungus produced half as much laccase as it did in liquid-surface culture (LSC); however, the mycelium on the membrane was able to be re-used five times for laccase production. The laccase production was accelerated in the repeated use of the culture while the mycelium in LSC ceased to produce the enzyme. This study shows that compostable PLLA microporous membranes can be used for enzyme production by MSLC of filamentous fungi.     

Improved production of laccase by Daedalea flavida: consideration of evolutionary process optimization and batch-fed culture

The genetic algorithm was used effectively to find the optimal values of eight process variables for the maximum laccase production by Daedalea flavida in a stationary culture. The algorithm was modified suitably to improve laccase production with 18 parallel experiments in 4 generations. A high enzyme titer of 65 % was achieved after the optimization and compared to the titer obtained before optimization. To study the effect of the surface immobilized growth on the enzyme production, the fungus was grown on three solid carriers. When cultured on polymer composite fibers, polyurethane foam, or steel wool, at least 2.5 times more biomass was produced, compared to the biomass produced in support-free growth. On the contrary, the mycelia grown on solid support produced much less laccase than non-adhering mycelia. Four parallel runs of batch-fed cultures were done, using the cell mass of D. flavida to evaluate the influence of four different volumes of medium exchanged on laccase production. For sustainable production of the enzyme, complete exchange of medium was favorable, where the laccase activity increased continuously in six consecutive cycles, though, 50 % exchange of medium produced the maximum laccase in terms of mean enzyme activity obtained in six cycles.     

Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris

Improved expression of recombinant laccase by Pichia pastoris carrying the lcc1 cDNA isolated from Trametes versicolor was achieved by optimization of the cultivation conditions in a fermentor equipped with a methanol sensor system. The results indicated that the activity obtained in fermentor cultivations was at least 7 times higher than in shake-flask cultures. Three different strategies for fermentor cultivations were compared: A (30 degrees C, 1.0% methanol), B (20 degrees C, 1.0% methanol), and C (20 degrees C, 0.5% methanol). The laccase activity, particularly the specific activity, could be improved by decreasing the cultivation temperature. The mechanisms behind the temperature effect on the laccase activity may be ascribed to poor stability, release of more proteases from dead cells, and folding problems at higher temperature. The results showed that the methanol concentration had a marked effect on the production of active heterologous laccase. A fivefold higher volumetric laccase activity was obtained when the methanol concentration was kept at 0.5% instead of 1.0%. The detrimental effect of methanol on the production of recombinant laccase may be attributed to lower laccase stability, a higher proteolytic activity, and folding problems due to higher growth rate at 1.0% methanol.     

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.     

Influence of Several Activators on the Extracellular Laccase Activity and In vivo Decolourization of Poly R‐478 by Semi‐Solid‐State Cultures of Trametes versicolor

The effect of several laccase activity activators,such as ethanol (novel activator), veratryl alcohol, melanin production and aeration level, on the laccase production by Trametes versicolor (CBS100.29) was investigated. The microorganism was cultivated on nylon sponge, functioning as a physical support on which the mycelium was bound. The cultures with veratryl alcohol showed maximum laccase and manganese‐dependent peroxidase (MnP) activities of 238 U/l and 125 U/l, respectively. The laccase activity found is about two times higher than that attained in the control cultures. On the contrary, MnP activity did not appear to be influenced by the addition of this alcohol. Ethanol‐supplemented cultures led to maximum laccase and MnP activity levels of about 102 U/l and 101 U/l, respectively. These activities were approx. 40% lower than those achieved in the reference cultures. The decolourization of the polymeric dye Poly R‐478 by the above‐mentioned cultures was also investigated. A percentage of biological decolourization of around 90% was achieved with control and veratryl alcohol‐supplemented cultures, whereas with ethanol‐supplemented cultures a slightly lower percentage of around 85% was reached after seven days of dye incubation.     

Enhanced formation of laccase activity by the white-rot fungus Trametes pubescens in the presence of copper

The white-rot fungus Trametes pubescens MB 89 has been identified as an outstanding, although not-yet-described, producer of the industrially important enzyme laccase. Extracellular laccase formation could be greatly stimulated by the addition of Cu(II) to a simple, glucose-based culture medium. Using optimum Cu concentrations (1.5-2.0 mM), maximum values for laccase activity of approximately 65 U/ml were obtained. The synthesis of the laccase protein depended on the presence of Cu in the medium as shown by Western blot analysis. Copper had to be supplemented during the exponential phase of growth for its maximal effect; addition during the stationary phase, during which laccase activity is predominantly formed, resulted in markedly reduced laccase productivity. As was shown by X-ray microanalysis of T pubescens mycelia obtained from a laboratory fermentation, Cu was rapidly taken up by the fungal biomass. A possible explanation for this stimulatory effect of Cu on laccase biosynthesis could be a role for this enzyme activity in melanin synthesis. The stimulatory effect of Cu on laccase synthesis was also effective for several other basidiomycetes and hence could be used as a simple method to boost the production of this enzyme.     

粗毛栓菌(Trametes hirsuta) D2固态发酵山核桃蒲壳产漆酶的营养条件研究

【目的】为提高漆酶产量,降低生产成本,以山核桃蒲壳作为基质,对粗毛栓菌D2固态发酵产漆酶的营养条件进行研究。【方法】对不同碳源、氮源、碳氮比、蒲壳含量对漆酶产量的影响进行分析。【结果】山核桃蒲壳是粗毛栓菌生长的良好载体,能够促进漆酶的合成。粗毛栓菌D2漆酶固态发酵培养基干物质组成为:山核桃蒲壳40%(质量比),玉米粉24%(质量比),菜籽饼粉36%(质量比)。发酵6 d时,漆酶活性为126.8 U/g干基。【结论】粗毛栓菌固态发酵山核桃蒲壳产漆酶具有效率高,生产成本低的优点,具有潜在的工业化应用前景。     

Enhancement of copper content and specific activity of CotA laccase from Bacillus licheniformis by coexpression with CopZ copper chaperone in E. coli

Copper depletion of bacterial laccases obtained by heterologous expression in Escherichia coli is a common problem in production of these versatile biocatalysts. We demonstrate that coexpression of small soluble copper chaperones can mitigate this problem. The laccase CotA and the copper chaperone CopZ both from Bacillus licheniformis were used as model system. The use of the E. coli BL21(DE3) strain expressing CopZ and CotA simultaneously from two plasmids resulted in an 20% increase in copper occupancy and in 26% higher specific activity. We conclude that not only intracellular copper ion concentration, but also presence of an appropriate copper chaperone influences copper ion insertion into CotA laccase. Moreover, E. coli BL21(DE3) seems to lack such a copper chaperone which can be partially complemented by heterologous expression thereof. The presented system is simple and can routinely be used for improved heterologous production of bacterial laccase in E. coli.     

Fiber modification of kraft pulp with laccase in presence of methyl syringate

An approach aiming at improving paper properties is to use laccase to copolymerize low-molecular weight phenols with the pulp before papermaking. The addition of methyl syringate (MS) gave a twice wet tensile index of unbleached kraft pulp with laccase treatment but had little effect on the dry tensile index and substantially decreased the brightness of pulp. The radical concentration in laccase-treated fibers increased up to 2.5 times of that in control sample after 60 min. The radical concentration in laccase/MS-treated fibers increased up to 20 times of that in control sample within 2 min, and a highest radical concentration (increased by near 65 times) was obtained after 40 min. A strong agglutination of large-area fibers in handsheet was observed after laccase/MS treatment. The surface lignin coverage of the laccase-treated fibers increased from 54.96% (control) to 59.36%, while that of the laccase/MS-treated fibers increased only up to 55.22%. It is suggested that the graft of MS on fiber and degradation of surface lignin occurred simultaneously. The addition of MS can enhance the activation of fibers and extend the enzymatic oxidation of lignin within the cell wall. An increased bonding area of fibers resulting from interaction of laccase, MS and fibers via radical-coupling reaction maybe account for the significantly improved wet strength of pulp.     

Laccase production and wood degradation by a white-rot fungus Daedalea flavida

A comparative study has been conducted on seven white rot fungi to investigate their abilities to produce laccase and selectively degrade lignin. Laccase was produced constitutively on the different media tested. Of the different lignins, phenolic compounds and sugars involved, the highest laccase yield was obtained on indulin AT. Salicylic acid inhibited enzyme activity. A temperature of 20°C and 0.2% of indulin AT were found to be optimum for enzyme activity. No correlation was found between the amount of enzyme and fungal mass produced. During semisolid degradation of angiospermic wood sawdust, Daedalea flavida caused a total weight loss of 11%, with a lignin loss of 15.77% during two months of decay. Lignin removal was comparatively selective during the first month, during which time laccase production was also higher, indicating its probable role in lignin degradation.     

Molecular cloning and characterization of a novel laccase gene from a white-rot fungus Polyporus grammocephalus TR16 and expression in Pichia pastoris

Aims: To isolate a novel laccase gene from white‐rot fungus Polyporus grammocephalus TR16 and heterologous expression in Pichia pastoris. The characteristics of the heterologously expressed laccase are also studied. Methods and Results: Anchored PCR and 3′ RACE protocol were applied to obtain the full length of the laccase gene, which comprised 12 introns and an opening frame of 1769 bp. The deduced amino acid sequence of the laccase gene had an identity of 45–66% with the laccases reported previously. The cDNA was expressed in Pi. pastoris GS115 with native and α‐factor secretion signal peptides. The laccase activity obtained with the native signal peptide is threefold higher than that obtained with the α‐factor secretion signal peptide. The highest activity of the heterologously expressed laccase reached 893·3 U ml^?1, with its molecular mass estimated to be 65·4 kDa by SDS‐PAGE. The purified heterologously expressed laccase was stable at a pH range of 7·0–10·0. The optimum pH and temperature were 4·5 and 50°C, respectively; the K_m value for ABTS (3‐ethylbenzthiazoline‐6‐sulphonate) was 0·66 mmol l^?1. Conclusion: The novel laccase gene is cloned successfully and heterologously expressed in Pi. pastoris. Significance and Impact of the Study: A novel laccase gene isolated from a tropical fungus serves as a good source for pulp bleaching and wood processing.     

Production of Laccase and Optimization of Its Production by Bacillus sp. Using Distillery Spent Wash as Inducer

A bacillus sp. isolated from the sediments of a distillery mill was used for laccase production under optimized culture conditions. The distillery effluent was used as an inducer for overproduction of laccase by employing the Taguchi approach. Screening of different medium components and their effect on laccase production was studied using an M-16 orthogonal array. The formation of laccase was considerably increased by addition of 1 mM copper sulfate (51.95 U/ml), which was further enhanced by the use of different inducers. The usefulness of the Taguchi method for optimization of culture conditions was investigated with five selected factors at four levels, and it was observed that the optimized medium resulted in a 9-fold increase in extracellular laccase production compared with the control. The optimized medium composition for laccase production was dextrose (1%), tryptone (0.1%), CuSO₄ (1 mM), and an inducer (distillery effluent 10% [v/v]) at pH 7, which altogether resulted in 107.32 U/ml extracellular laccase activity. Hence, the Taguchi approach proved to be a reliable tool in optimizing culture conditions and achieving the best possible combination for enhanced laccase production.     

Effect of different cultivation conditions and inducers on the production of laccase by the litter-dwelling fungal isolate Fusarium incarnatum LD-3 under solid substrate fermentation

The litter-dwelling fungus Fusarium incarnatum LD-3 has been identified as a novel producer of laccase. The present work was oriented towards the optimization of various cultivation conditions for maximizing laccase production under solid substrate fermentation. The process parameters were optimized by the “one factor at a time” approach. Maximum laccsase production was obtained at pH 5.0 and at a temperature of 28 °C with 60 % moisture content using rice bran as a substrate. The laccase production was enhanced in the presence of aromatic inducer, i.e. ortho-dianisidine at a concentration of 0.5 mM. Laccase production was further increased by 52.56 % when the medium was supplemented with 2 % (v/v) alcohol. Among the various amino acids tested as a growth factor and nitrogen source, D-Serine and DL-2 Amino n-butyric acid, DL-Alanine and L-Glycine were found to be the most suitable for laccase production. The highest laccase production (1,352.64 U/g) was achieved under optimized conditions, and was 2.1-fold higher than the unoptimized conditions. Thus, the novel litter-dwelling fungal isolate Fusarium incarnatum LD-3 seems to be an efficient producer of laccase and can be further exploited for biotechnological applications. This is the first report on the optimization of cultivation conditions and inducers for laccase production from Fusarium incarnatum LD-3.