Utilization and polymerization of lignosulfonates by wood-rotting fungi

The susceptibility of lignosulfonates to the action of lignin-degrading wood-rotting fungi was studied by submitting commercial lignosulfonate (Peritan Na) and fractions of calcium lignosulfonate of different molecular weights to the action of selected white rot fungi. As shown by gel filtration chromatography and determinations according to the nitroso method, lignosulfonates, even in conditions which did not support fungal growth, underwent strong polymerization when brought in contact with typical, extracellular polyphenol oxidase-producing white-rot fungi. Owing to the polymerization, nitroso determinations showed a seeming decrease of lignosulfonate. Polyporus dichrous, an “atypical” white-rot fungus which does not produce extracellular polyphenol oxidase and hence does not cause polymerization of lignosulfonates, was found to degrade 11% of the lignosulfonate available in a solid malt extract medium during 19 days. Addition of lignosulfonate to a rich synthetic liquid growth medium increased the mycelial yield of several white-rot fungi. Trametes versicolor was able to grow on a calcium lignosulfonate fraction with molecular weight 1350 which served as sole source of carbon and energy, but not on fractions of higher molecular weight. The utilization/polymerization of lignosulfonates was shown to depend on concentration and on the presence of additional utilizable sources of carbon.     

Sustainable films and coatings from hemicelluloses: a review

This review summarizes the results of past research on films and coatings from hemicelluloses, biopolymers that are as yet relatively unexploited commercially. The targeted uses of hemicelluloses have primarily been packaging films and coatings for foodstuffs as well as biomedical applications. Oxygen permeability of hemicellulose films, an important characteristic for food packaging, was typically comparable to values found for other biopolymer films such as amylose and amylopectin. As expected, the modification of hemicelluloses to create more hydrophobic films reduced the water vapor permeability. However, modified hemicellulose coatings intended for food still exhibited water vapor permeabilities several magnitudes higher than those of other polymers currently used for this purpose. Research on hemicelluloses for biomedical applications has included biocompatible hydrogels and coatings and material surfaces with enhanced cell affinity. Numerous possibilities exist for chemically modifying hemicelluloses, and fundamental studies of films from modified hemicelluloses have identified other potential applications, including selective membranes.     

Siloxane-based biocatalytic films and paints for use as reactive coatings

We have developed a new methodology for preparing films and paints suitable for use as biocatalytic coatings. The hydrolytic enzymes pronase and alpha-chymotrypsin were immobilized by either sol-gel entrapment or by covalent attachment into a polydimethylsiloxane (PDMS) matrix and cast into thin films or incorporated into an oil-based paint formulation. All of the coatings retained enzymatic activity and adhered to several different materials. The enzymatic films and paints also exhibited higher thermostability than enzyme free in solution or covalently attached to the outer surface of PDMS. A porous membrane based on a PDMS-immobilized enzyme was also prepared by an immersion precipitation process. Protein adsorption measurements showed that the enzyme-containing films and paints adsorbed less protein than enzyme-free controls, and that protein adsorption decreased with increasing proteolytic activity of the coating. These coatings thus provide the means to apply a stable enzymatic surface to a wide range of materials, and may be generally useful as biocatalytic paints and films.     

Development of a novel enzyme-redox-mediator system based on a fungal laccase and ruthenium complexes

The laccase produced by the fungus Coriolus hirsutus has been coordinatively modified with ruthenium complexes [Ru(phpy)(phen)(MeCN)2]PF6 and Ru(bpy)2CO3 under aerobic and anaerobic conditions. The amount of the complexes per enzyme molecule does not depend on the oxygen concentration, equaling 5 for [Ru(phpy)(phen)(MeCN)2]PF6 and 3 for Ru(bpy)2CO3. The pH dependence of the enzymatic activity, thermostability, and catalytical and electrocatalytical properties of the modified laccase are reported. It has been shown that, during the modification, at least one molecule of the ruthenium compound was coordinated near the T1 active center of the laccase, being directly involved in the catalysis and enhancing its efficiency.     

Development of a novel enzyme-redox-mediator system based on a fungal laccase and ruthenium complexes

The laccase produced by the fungus Coriolus hirsutus has been coordinatively modified with ruthenium complexes [Ru(phpy)(phen)(MeCN)₂]PF₆ and Ru(bpy)₂CO₃ under aerobic and anaerobic conditions. The amount of the complexes per enzyme molecule does not depend on the oxygen concentration, equaling 5 for [Ru(phpy)(phen)(MeCN)₂]PF₆ and 3 for Ru(bpy)₂CO₃. The pH dependence of the enzymatic activity, thermostability, and catalytical and electrocatalytical properties of the modified laccase are reported. It has been shown that, during the modification, at least one molecule of the ruthenium compound was coordinated near the T1 active center of the laccase, being directly involved in the catalysis and enhancing its efficiency.     

Adsorption characteristics of lignosulfonates in salt-free and salt-added aqueous solutions

Five sodium lignosulfonate (SL) fractions with narrow molecular weight distribution and known salt content were used as the polyanion to build up layer-by-layer self-assembly multilayers with poly(diallyldimethylammonium chloride) (PDAC) as polycation. It is interesting to find that the salt-free SL is hardly adsorbed on the PDAC surface, but the SL in salt-added solutions can be self-assembled well with PDAC to form SL/PDAC multilayers. When the five SL fractions dissolved in saline solutions are adsorbed on the PDAC surface by a self-assembly technique, SL with higher M(w) shows a higher adsorption amount than does SL with lower M(w). The driving forces of self-assembly of SL and PDAC are discussed based on the solution behaviors and adsorption characteristics of SL in salt-free and salt-added aqueous solutions. A possible self-assembled mechanism of SL and PDAC is electrostatic or cation-π interactions, but the SL cannot be adsorbed onto the PDAC surface without a hydrophobic interaction. With the addition of enough salt, the Coulomb interaction of SL becomes negligible, but the adsorption amount increases, indicating that the electrostatic interaction is not the main driving force of SL/PDAC self-assembly. For adsorption of SL in saline solution onto the PDAC surface, the cation-π interaction is the main driving force, and the hydrophobic interaction plays an important role in the adsorbed amount.     

Synergistic effect of laccase mediators on pentachlorophenol removal by Ganoderma lucidum laccase

Laccases have low redox potentials limiting their environmental and industrial applications. The use of laccase mediators has proven to be an effective approach for overcoming the low redox potentials. However, knowledge about the role played by the mediator cocktails in such a laccase-mediator system (LMS) is scarce. Here, we assembled different dual-agent mediator cocktails containing 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), vanillin, and/or acetovanillone, and compared their mediating capabilities with those of each individual mediator alone in oxidation of pentachlorophenol (PCP) by Ganoderma lucidum laccase. Cocktails containing ABTS and either vanillin or acetovanillone strongly promoted PCP removal compared to the use of each mediator alone. The removal enhancement was correlated with mediator molar ratios of the cocktails and incubation times. Analysis of the kinetic constants for each mediator compound showed that G. lucidum laccase was very prone to react with ABTS rather than vanillin and acetovanillone in the cocktails. Moreover, the presence of the ABTS radical (ABTS^+•) and vanillin or acetovanillone significantly enhanced PCP removal concomitant with electron transfer from vanillin or acetovanillone to ABTS^+•. These results strongly suggest that vanillin and acetovanillone mediate the reaction between ABTS and PCP via multiple sequential electron transfers among laccase and its mediators.     

Removable colored coatings based on calcium alginate hydrogels

This article describes the creation of a nontoxic, biodegradable coating using calcium alginate and FD&C approved dyes. The coating is robust but is rapidly removed upon treatment with disodium ethylenediamine tetraacetate (EDTA). Dye leaching from calcium alginate films was studied, and it was determined that the efficiency of dye retention is proportional to the degree of cross-linking. Degradation rates were studied on calcium alginate beads serving as a model for a coating. We determined that degradation rates depend on the gel's cross-linking and on the amount of EDTA used. Bead size also influenced the degradation rates; smaller beads degraded faster than larger beads. We show that the coating can be used as an easily removable and environmentally friendly logotype on an artificial turf surface. Applications of these coatings can be extended to food, cosmetic, medicinal, and textile uses and to wherever nontoxic, easily removable colored coating is desired.     

Hybrid xerogel films as novel coatings for antifouling and fouling release

Hybrid sol-gel-derived xerogel films prepared from 45/55 (mol ratio) n-propyltrimethoxysilane (C3-TMOS)/tetramethylorthosilane (TMOS), 2/98 (mol ratio) bis[3-(trimethoxysilyl)propyl]-ethylenediamine (enTMOS)/tetraethylorthosilane (TEOS), 50/50 (mol ratio) n-octyltriethoxysilane (C8-TEOS)/TMOS, and 50/50 (mol ratio) 3,3,3-trifluoropropyltrimethoxysilane (TFP-TMOS)/TMOS were found to inhibit settlement of zoospores of the marine fouling alga Ulva (syn. Enteromorpha) relative to settlement on acid-washed glass and give greater release of settled zoospores relative to glass upon exposure to pressure from a water jet. The more hydrophobic 50/50 C8-TEOS/TMOS xerogel films had the lowest critical surface tension by comprehensive contact angle analysis and gave significantly greater release of 8-day Ulva sporeling biomass after exposure to turbulent flow generated by a flow channel than the other xerogel surfaces or glass. The 50/50 C8-TEOS/TMOS xerogel was also a fouling release surface for juveniles of the tropical barnacle Balanus amphitrite. X-ray photon electron data indicated that the alkylsilyl residues of the C3-TMOS-, C8-TEOS-, and TFP-TMOS-containing xerogels were located on the surface of the xerogel films (in a vacuum), which contributes to the film hydrophobicity. Similarly, the amine-containing silyl residues of the enTMOS/TEOS films were located at the surface of the xerogel films, which contributes to the more hydrophilic character and increased critical surface tension of these films.     

Effects of laser radiation on rhus vernicifera laccase,type 2 Cu-depleted laccase,and stellacyanin

Laser irradiation in the 450 nm region brings about irreversible changes in the copper sites of Rhus vernicifera laccase and its type 2 Cu-depleted derivative. The absorption band at 614 nm disappears after ~ 2 hr of irradiation with a 200 mW laser beam; the amount of the paramagnetic detectable copper does not decrease, indicating no reduction of these types of copper. No apparent rearrangement of the protein backbone occurs, as ultaviolet dichroic spectra of the enzyme before and after the irradiation do not show appreciable differences. Stellacyanin is insensitive to laser radiation at any wavelength.     

新月旋孢腔菌胞内漆酶的活性测定及漆酶基因ClLac1克隆

漆酶是一种含铜的多酚氧化酶,与植物病原菌致病性、黑色素合成及降解木质素等方面相关。为明确漆酶在新月旋孢腔菌的催化作用及其催化活性,以2,2′-连氮-双(3-乙基苯并噻唑-6-磺酸)（简称ABTS）为底物,利用分光光度计在420nm下测定胞内漆酶活力,结果表明酶活测定最佳反应条件为缓冲液pH2.8、Cu2+浓度500μmol/L和0.6mmol/L ABTS。根据漆酶Cu2+结合保守结构域设计了1条引物,对新月旋孢腔菌漆酶基因进行克隆,并通过RACE技术克隆了其全长cDNA序列。开放阅读框长1,803bp,     

Bioactive coatings of endovascular stents based on polyelectrolyte multilayers

Layer-by-layer self-assembly of two polysaccharides, hyaluronan (HA) and chitosan (CH), was employed to engineer bioactive coatings for endovascular stents. A polyethyleneimine (PEI) primer layer was adsorbed on the metallic surface to initiate the sequential adsorption of the weak polyelectrolytes. The multilayer growth was monitored using a radiolabeled HA and shown to be linear as a function of the number of layers. The chemical structure, interfacial properties, and morphology of the self-assembled multilayer were investigated by time-of-flight secondary ions mass spectrometry (ToF-SIMS), contact angle measurements, and atomic force microscopy (AFM), respectively. Multilayer-coated NiTi disks presented enhanced antifouling properties, compared to unmodified NiTi disks, as demonstrated by a decrease of platelet adhesion in an in vitro assay (38% reduction; p = 0.036). An ex vivo assay on a porcine model indicated that the coating did not prevent fouling by neutrophils. To assess whether the multilayers may be exploited as in situ drug delivery systems, the nitric-oxide-donor sodium nitroprusside (SNP) was incorporated within the multilayer. SNP-doped multilayers were shown to further reduce platelet adhesion, compared to standard multilayers (40% reduction). When NiTi wires coated with a multilayer containing a fluorescently labeled HA were placed in intimate contact with the vascular wall, the polysaccharide translocated on the porcine aortic samples, as shown by confocal microscopy observation of a treated artery. The enhanced thromboresistance of the self-assembled multilayer together with the antiinflammatory and wound healing properties of hyaluronan and chitosan are expected to reduce the neointimal hyperplasia associated with stent implantation.     

Biocomposite films based on alginate and organically modified clay

Sodium alginate/sodium montmorillonite hybrid films were prepared by casting from the suspension of sodium alginate and different clay samples. Clay samples had been modified with a cationic surfactant, a cationic polymer, and a small polar molecule, respectively. Benzethonium chloride, polyethyleneimine and urea were used as clay modifiers. The composite films begin to disintegrate at a higher temperature and with less weight loss than the pure alginate films. This suggests an enhancement of the film thermal stability due to the modification of the alginate with clay samples.     

The synergistic ability of some wood-degrading fungi to transform lignins and lignosulfonates on various media

Summary In order to investigate the ligninolytic activity with mixed cultures of wood-degrading fungi, and the influence of various growth conditions on this activity, 50 wood-degrading fungi were tested for ligninolysis in pure culture and in pair-wise combinations according to a simple plate test recently developed in this laboratory. It was found that a synergistic degradation of lignin and of lignosulfonate was common among fungi inoculated pair-wise on lignin or lignosulfonate media; decomposition was enhanced in the zone where the two mycelia interacted. This synergistic effect was noted with pairs of two different white-rot fungi, with pairs of one white-rot and one brown-rot fungus, and with pairs of one white-rot and one soil Deuteromycete.Lignosulfonate was more susceptible to the synergistic action of pairs of fungi than was lignin. The synergistic attack on lignosulfonate was more pronounced on a meager medium than on a carbohydrate-rich one. On the contrary, the ligninolysis with pure cultures of the fungi was more pronounced on the carbohydrate-rich medium, and lignin was decomposed more easily than was lignosulfonate.     

Studies on the decomposition of lignosulfonates by the fungi Pleurotus ostreatus and Trametes pubescens.

The fungi Pleurotus ostreatus and Trametes pubescens were grown in a mineral medium containing 1% of glucose and 0.9% of lignosulfonates introduced into the culture medium in the form of yeast waste liquor. Chromatography of extracts of the medium and determinations of sulphur and lignosulfonates have revealed that the fungi studied utilized the constituents of the yeast waste liquor (lignosulfonates) as carbon source. This was manifested in an increase of dry mass of the mycelium and protein as compared with the control. The constituents of the yeast waste liquor were also found to have a stimulating effect on the formation of both exo-and endoenzymes, laccase and peroxidase. This may indicate that these oxidases take part in the decomposition of lignosulfonates.     

真菌漆酶的结构与功能

漆酶是一种含铜的多酚氧化酶,能催化氧化酚类和芳香类化合物,同时伴随4个电子的转移,并将分子氧还原成水。漆酶结构的解析是阐明其催化作用机理、了解蛋白质结构与功能关系的基础。综述近年来对真菌漆酶蛋白结构及其功能研究的进展。     

Activity of laccase on unbleached and bleached thermomechanical pulp

The introduction of value-added properties to pulp fibres is an attractive proposition. One interesting option is targeted modification of fibre surfaces by enzymatic activation. In this work, the activity of laccase on pulp and pulp fractions from thermomechanical pulp (TMP) and peroxide bleached TMP was studied on the basis of consumption of the co-substrate oxygen in the reaction and by studying the formation of radicals in the pulp material as analysed by electron paramagnetic resonance spectroscopy (EPR). Laccases obtained from Trametes hirsuta and Myceliophthora thermophila were used in the study. Laccases were found to be active on pulp material of unbleached TMP, whereas only fines from bleached TMP reacted in laccase treatment. Dissolved and colloidal substances (DCS) were assumed to have a mediating role in the laccase-catalysed oxidation of the fibre-bound material.     

Immobilization of commercial laccase on spent grain

The aim of this work was to assess the possibility of using beer spent grain (a byproduct of beer's brewing industry) as a carrier for laccase immobilization. Both adsorption (on spent grain – SG and on digested spent grain – DSG) and covalent binding (using glycidol and glycidol followed by ethylenediamine on DSG) were used. The effect of different immobilization conditions on the immobilization yields and recovered activities such as contact time, enzyme concentration and pH was evaluated. For the best conditions, immobilization yields, recovered activities and thermal, operational and storage stabilities were also evaluated. Finally, the Michaelis–Menten mechanism was applied and the parameter with respect to ABTS oxidation was determined.Enzyme immobilization on DSG led to the best enzyme activities (recovered activities as high as 90%) and to high storage and operational stabilities (10 cycles). Thermal stability was also improved and the half-life of immobilized laccase in SG increased from 0.64 h to 1.1 h at 70 °C.     

基于cDNA克隆的亚热带阔叶林和针叶林生态系统担子菌漆酶基因多样性及其群落结构研究

作为木质素降解的关键酶之一,漆酶在森林生态系统养分循环过程中占有重要的地位。通过构建cDNA克隆文库,从mRNA水平上研究了亚热带喀斯特原生林(常绿落叶阔叶混交林)和红壤马尾松林(针叶林)凋落物层(O层)及表土层(A层,0—20 cm)中担子菌漆酶基因多样性及其群落结构。结果发现,O层担子菌漆酶基因多样性、丰富度和均匀度均以原生林远远高于马尾松林,而A层与O层的趋势相反;同一森林生态系统,原生林土壤O层中担子菌漆酶基因多样性和种群丰富度高于土壤A层,而马尾松林则O层明显低于A层。"目"分类地位上,伞菌目在两森林生态系统中占绝对优势,且在马尾松林O层中比例高达90.2%;原生林O层和马尾松林A层中存在丰富的多孔菌目和钉菇目;红菇目仅在原生林A层中检测到,且比例为12.4%。群落结构上,与原生林O层和马尾松林A层相比,原生林A层和马尾松林O层含漆酶基因的担子菌种群分布极不均匀,分别存在1和2个绝对优势种群(克隆子数占各克隆库20%以上),且分别与伞菌属、小菇属和侧耳属有较高的氨基酸相似性。此外,两森林土壤中漆酶活性、碳、氮、木质素含量均以原生林显著高于马尾松林,且O层显著高于A层。这些结果表明亚热带阔叶林和针叶林不同土壤层位中降解木质素的担子菌漆酶基因多样性和群落结构有着极大的差异,而这种差异与微生物底物的可利用性、多样性以及土壤pH等因素的综合作用密切相关。     

Biosensor for the determination of phenols based on cross-linked enzyme crystals (CLEC) of laccase

Cross-linked enzyme crystals (CLECs) are a versatile form of biocatalyst that can also be used for biosensor application. Laccase from Trametes versicolor (E.C.1.10.3.2) was crystallized, cross-linked and lyophilized with beta-cyclodextrin. The CLEC laccase was found to be highly active towards phenols like 2-amino phenol, guaiacol, catechol, pyrogallol, catechin and ABTS (non-phenolic). The CLEC laccase was embedded in 30% polyvinylpropylidone (PVP) gel and mounted into an electrode to make the sensor. The biosensor was used to detect the phenols in 50-1000 micromol concentration level. Phenols with lower molecular weight such as 2-amino phenol, catechol and pyrogallol gave a short response time where as the higher molecular weight substrates like catechin and ABTS had comparatively a long response time. The optimum pH of the analyte was 5.5-6.0 when catechol was used as substrate. The CLEC laccase retained good activity for over 3 months.