Enzymatic cross-linking of gelatine with laccase and tyrosinase

Conventional cross-linking of proteins involves the use of toxic chemicals. Here, cross-linking of gelatine and gelatine hydrolysates with tyrosinases from Botryosphaeria obtusa (BoT1 and BoT2), Agaricus bisporus (AbT) and from Verrucomicrobium spinosum (VsT) and with laccases from Trametes hirsuta (ThL) and T. versicolor (TvL) was demonstrated. Enzymatic oxidation of tyrosine residues was indicated by UV/VIS and fluorescence spectroscopy and further confirmed by oxygen consumption measurements. Using a model substrate (Tyr-Ala) dimerization was demonstrated by using RP-HPLC and LC-MS. Enzymatic cross-linking significantly increased the molecular weight of the soluble material up to the point of precipitation as demonstrated by both SDS-PAGE and size exclusion chromatography. The effect of cross-linking was further enhanced in the presence of phenolic molecules such as catechin.     

Characterization of tyrosinase and accompanying laccase from Amorphophallus campanulatus

Tyrosinase and laccase activities were detected in the corm of Amorphophallus campanulatus after extraction with ethanol followed by ammonium sulphate precipitation (20-60%) and dialysis against 10 mM Na2HPO4 buffer at pH 7.0. Tyrosinase was found to be the predominant enzyme exhibiting mono- and di-phenolase activities, specificity for L-DOPA as substrate, optimum pH being 6.0, optimum temperature at 40 degrees C and Km at 1.05 mM. Laccase showed substrate specificity for p-phenylenediamine (p-PD), Km at 2.7 mM, optimum pH being 5.0 and was inactivated above 40 degrees C. Three isoforms of tyrosinase were detected on SDS-PAGE with apparent molecular mass approximately 127, 31 and 27 kDa respectively. On staining sections of A. campanulatus with L-DOPA as substrate and 3-methyl benzothiazolinone hydrazone (MBTH) for colour development, tyrosinase was detected in the intercellular spaces of the plant tissue. The cytosolic region did not show any colour indicating the absence of the enzyme.     

Assays for mammalian tyrosinase: a comparative study

This work describes a comparative study of the tyrosinase activity determined using three methods which are the most extensively employed; two radiometric assays using L-tyrosine as substrate (tyrosine hydroxylase and melanin formation activities) and one spectrophotometric assay using L-dopa (dopa oxidase activity). The three methods were simultaneously employed to measure the activities of the soluble, melanosomal, and microsomal tyrosinase isozymes from Harding-Passey mouse melanoma through their purification processes. The aim of this study was to find any correlation among the tyrosinase activities measured by the three different assays and to determine whether that correlation varied with the isozyme and its degree of purification. The results show that mammalian tyrosinase has a greater turnover number for L-dopa than for L-tyrosine. Thus, enzyme activity, expressed as mumol of substrate transformed per min, is higher in assays using L-dopa as substrate than those using L-tyrosine. Moreover, the percentage of hydroxylated L-tyrosine that is converted into melanin is low and is affected by several factors, apparently decreasing the tyrosinase activity measured by the melanin formation assay. Bearing these considerations in mind, average interassay factors are proposed. Their values are 10 to transform melanin formation into tyrosine hydroxylase activity, 100 to transform tyrosine hydroxylase into dopa oxidase activity, and 1,000 to transform melanin formation into dopa oxidase activity. Variations in these values due to the presence in the tyrosinase preparations of either inhibitors or regulatory factors in melanogenesis independent of tyrosinase are also discussed.     

Recent developments in the use of tyrosinase and laccase in environmental applications

Our current global environmental challenges include the reduction of harmful chemicals and their derivatives. Bioremediation has been a key strategy to control the massive presence of chemicals in the environment. Enzymes including the phenoloxidases, laccases and tyrosinases, are increasingly being investigated as “green products” in the removal of many chemical contaminants in waters and soils. Both phenoloxidases are widespread in nature and attractive biocatalysts due to their ability to use readily available molecular oxygen as sole cofactor for their catalytic elimination of a large number of chemicals. Taking advantage of their catalytic potentials, remarkable advances have been made in the engineering of laccases to produce suitable biocatalysts in environmental applications. Studies about novel strategies of laccase immobilization and insolubilization for the treatment of chemical contaminants were provided. Likewise, tyrosinases are gaining increasing interest in environmental applications due to their catalytic similarities with laccases although they remain far less investigated to date. This disparity was addressed in this review along with the molecular features and catalytic mechanism of tyrosinases relevant in environmental applications. A perspective on the future use of laccases and tyrosinases in bioremediation was discussed.     

Optimization of simultaneous production of tyrosinase and laccase by Neurospora crassa

Increasing demand for efficient and environmentally benign oxidation technologies has resulted in a focus on the use of oxidoreductases. Laccases and tyrosinases, which utilize molecular oxygen and produce water as by-product, are particularly attractive. Simultaneous production of laccase and tyrosinase was studied in Neurospora crassa FGSC #321 as the fungal strain which has the ability to produce tyrosinase intracellularly while producing laccase extracellularly. Using one-variable-at-a-time experiments and a Taguchi orthogonal L9 array demonstrated that a Vogel minimal medium containing 2.5% sucrose at pH 6.5 and 25?°C with no agitation or oxygen purging were the optimum conditions for N. crassa FGSC #321 growth. Conditions were adjusted to obtain the highest laccase and tyrosinase production. Results indicate that the control mechanisms for the production of both enzymes in N. crassa FGSC #321 are similar but not necessarily identical. Results revealed that transferring the harvested cells from the growth medium into the phosphate buffer (pH 6.8, 0.1M) containing cycloheximide (2?μM) and fluorouracil (2?mM) and increasing the temperature to 30?°C were the best conditions for simultaneous production of laccase and tyrosinase (1278 and 410?U/g of biomass, respectively). Nonetheless, starvation at 35?°C is proposed as the most cost-effective means for inducing laccase. The N. crassa laccase was characterized by using its molecular weight, pI value, optimal pH and temperature and stability.     

Co-occurrence of the multicopper oxidases tyrosinase and laccase in lichens in sub-order peltigerineae

BACKGROUND AND AIMS: Following previous findings of high extracellular redox activity in lichens and the presence of laccases in lichen cell walls, the work presented here additionally demonstrates the presence of tyrosinases. Tests were made for the presence of tyrosinases in 40 species of lichens, and from selected species their cellular location and molecular weights were determined. The effects of stress and inhibitors on enzyme activity were also studied. METHODS: Tyrosinase and laccase activities were assayed spectrophotometrically using a variety of substrates. The molecular mass of the enzymes was estimated using polyacrylamide gel electrophoresis. KEY RESULTS: Extracellular tyrosinase and laccase activity was measured in 40 species of lichens from different taxonomic groupings and contrasting habitats. Out of 20 species tested from the sub-order Peltigerineae, all displayed significant tyrosinase and laccase activity, while activity was low or absent in other species tested. Representatives from both groups of lichens displayed low peroxidase activities. Identification of the enzymes as tyrosinases was confirmed by the ability of lichen thalli or leachates derived by shaking lichens in distilled water to metabolize substrates such as L-dihydroxyphenylalanine (DOPA), tyrosine and epinephrine readily in the absence of hydrogen peroxide, the sensitivity of the enzymes to the inhibitors cyanide, azide and hexylresorcinol, activation by SDS and having typical tyrosinase molecular masses of approx. 60 kDa. Comparing different species within the Peltigerineae showed that the activities of tyrosinases and laccase were correlated to each other. Desiccation and wounding stimulated laccase activity, while only wounding stimulated tyrosinase activity. CONCLUSIONS: Cell walls of lichens in sub-order Peltigerineae have much higher activities and a greater diversity of cell wall redox enzymes compared with other lichens. Possible roles of tyrosinases include melanization, removal of toxic phenols or quinones, and production of herbivore deterrents.     

白腐真菌漆酶的纯化及性质

液体发酵培养白腐真菌F9,粗酶液经盐析、透析浓缩、葡聚糖G-100柱层析、DEAE-纤维素离子交换层析四步分离纯化,得电泳纯漆酶。经SDS-PAGE法测定酶的相对分子质量约为6×104,酶活回收率达46.47%,纯度提高了18.86倍。F9漆酶最适反应温度为40℃,最适反应pH为4.8,在35℃以下、pH 4.8～5.4的范围内稳定性较强。其催化愈创木酚的Km为4.61 mmol/L,vm为6.27 mmol/(L.min)。K+对其有激活作用,而Fe2+、Fe3+对其有明显抑制作用。     

Role of laccase in lignin degradation by white-rot fungi

Abstract Laccase is commonly found in white-rot fungi and catalyses the abstraction of one electron from the phenolic hydroxyl group to polymerize or depolymerize lignin model compounds. Laccase degrades both β-1 and β-O-4 dimers via C _α - C _β cleavage, C _α oxidation and alkyl-aryl cleavage. Also, aromatic ring cleavage may be detected following the action of laccase. Laccase can also oxidize non-phenolic compounds when primary mediators, such as 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate), are co-present. Laccase produces Mn(III) chelates which allow wood-decaying enzymes to penetrate wood cell walls. Laccase is considered to be capable of degrading lignin together with lignin peroxidase and manganese peroxidase.     

Enzymatic polymerization of phenolic compounds using laccase and tyrosinase from Ustilago maydis

Flavonoids are a big group of polyphenols of low molecular weight with in vitro antioxidant properties. In this study, the laccase and tyrosinase from Ustilago maydis were partially characterized and their effect on the antioxidant activity of some phenolic compounds was investigated. Since enzymatic polymerization of the phenolic compounds was detected, the size of the aggregates was determined and related to their antioxidant activity. Morphology of the polymers was analyzed by atomic force microscopy. The results showed that the laccase- and tyrosinase-catalyzed polymerization of quercetin produced aggregates with relatively low molecular weight and higher antioxidant activity than the monomeric quercetin. In the case of kaempferol, the aggregates reached higher sizes in the first 2 h of reaction and their antioxidant activity was increased. In the last case, the aggregates adopted fractal-ordered shapes similar to coral in the case of the kaempferol-laccase system and to fern in the case of the kaempferol-tyrosinase system. The kaempferol and quercetin polymers at low concentration had strong scavenging effect on Reactive oxygen species (ROS) and inhibition of lipoperoxidation in human hepatic cell line WRL-68.     

Occurrence of fusicoccanes in plants and fungi

The literature on substances structurally related to fusicoccin shows that its molecular design is not unique. Compounds of this type with a dicyclopenta[a,d]cyclooctane skeleton (the 585 ring system) are often encountered in nature. The compounds, systematically reviewed, comprise a single class of fusicoccane-type terpenoids characterized by the presence of the dicyclopenta[a,d]cyclooctane system in the structure of these molecules. The review is arranged according to the taxonomy of the organisms producing these terpenoids; it is shown that compounds of this type, including physiologically active ones, are found in the major systematic divisions: fungi, lower and higher plants, and animals (insects). Chemical, analytical, and biogenetic data are given for each compound, as well as data on their physiological activity and the mechanism of their action on plants. Nomenclature is proposed for the fusicoccane-type compounds.     

Oxidation of milled wood lignin with laccase, tyrosinase and horseradish peroxidase

In this paper the oxidation of milled wood lignin (MWL), catalysed by three enzymes, i.e. laccase, tyrosinase and horseradish peroxidase (HRP) was studied. The oxidation was followed by measuring the consumption of O₂ during laccase and tyrosinase treatment and of H₂O₂ during HRP treatment. Both laccase and HRP were found to oxidise lignin effectively, whereas the effect of tyrosinase was negligible. The changes in MWL molecular-weight distributions caused in the reactions were analysed by gel permeation chromatography. Both laccase and HRP treatments were found to polymerise MWL. Peroxidase treatment was found to decrease the amount of phenolic hydroxyls in MWL, whereas no such effect could be detected in the laccase-treated sample. Both laccase and HRP treatments were, however, found to increase the amount of conjugated structures in MWL. The formation of phenoxy radicals during the treatments was studied by electron paramagnetic resonance spectroscopy. Phenoxy radicals were detected in both laccase and HRP-treated samples. The amount of the formed phenoxy radicals was found to be essentially constant during the detected time (i.e. 20–120 min after the addition of enzyme).     

影响真菌漆酶表达及其活性的因素

丝状真菌同一菌株中存在动力学、理化特性各异的多个漆酶同工酶。金属离子,碳、氮等培养基成分,培养条件,以及异生物质、热休克处理和共培养的菌株等多种生物和非生物的因素对真菌漆酶同工酶表现出选择性诱导效应。影响真菌漆酶表达及其调控因素的研究能指导漆酶的选择性合成,以满足不同的应用需求。主要阐述影响真菌漆酶表达及其活性的因素的相关研究进展。     

Isolation and biochemical characterization of laccase and tyrosinase activities in a novel melanogenic soil bacterium

Bacterial polyphenol oxidases (PPOs) with a high oxidizing capacity for a wide range of substrates could make their applications in phenolic biotransformation, food processing, cosmetics, and textile industry. We have isolated a melanogenic soil bacterium by differential screening of a number of strains which were isolated from the Iranian microflora. The taxonomic characterization of this strain indicates that it belongs to the genus Bacillus (HR03), and has the ability to produce all types of PPOs; cresolase (EC 1.14.18.1), cathecolase (EC.1.10.3.1), and laccase (EC 1.10.3.2). We studied the tyrosinase activity using l-tyrosine and l-dopa as substrates and the laccase activity with specific substrates such as syringaldazine and 2,6-dimethoxyphenol. The optimum pH and temperature, obtained for all types of polyphenol oxidases, were at about pH 5.5 and 55 °C, respectively. Tyrosinase-like enzyme of this strain shows a lag period in its tyrosine hydroxylase activity that could be avoided by the addition of small amounts of l-dopa and sodium dodecyl sulfate (SDS). In addition, tyrosinase and laccase were activated by SDS below the critical micelle concentration and were inhibited by 1 mM EDTA. We tested the resistance of melanized-cells against UVA, UVC and H₂O₂. Results show that melanin protects strain HR03 against UV lights and the oxidant.     

Removal of phenols from mixtures by co-immobilized laccase/tyrosinase and Polyclar adsorption

An enzymatic method for removal of phenols from their mixtures was investigated. Phenols in an aqueous solution were removed after a two-step treatment with co-immobilized laccase and tyrosinase and Polyclar (polyvinylpolypyrrolidone). A laccase from Pyricularia oryzae and mushroom tyrosinase were co-immobilized on Mikroperl in a fixed-bed tubular bioreactor by a rapid and simple method. The support immobilized 95% of the total laccase units and 35% of the total tyrosinase units. Different mixtures of phenols were passed through the column with co-immobilized laccase and tyrosinase. This method removed 42–90% of different phenolic substances by a single passage through the bioreactor. The second step employed Polyclar for additional removal of phenolic substances from mixtures. The degree of removal depends on the nature of the phenols. Complete removal was achieved for a-naphthol, 2,4-dichlorophenol, 4-methoxyphenol, b-naphthol, 4-chloro-3-methylphenol and catehin. The operational stability of the immobilized system was 10–90 h depending on the substrate. The biocatalyst was capable of continuous transformation of different phenols in mixtures. Journal of Industrial Microbiology & Biotechnology (2000) 24, 383–388. Received 12 August 1999/ Accepted in revised form 18 February 2000     

Occurrence of cyclosporins and cyclosporin-like peptolides in fungi

Apart from 17 previously listed fungal taxa producing cyclosporin A and its natural congeners B to Z, several additional and taxonomically diverse strains producing the single novel component [Thr², Leu⁵, Leu¹⁰]cyclosporin or cyclosporin-like peptolides (eg SDZ 214-103=[Thr², Leu⁵, D-Hiv⁸, Leu¹⁰]cyclosporin) have recently been described. We report here the isolation of two further and novel cyclosporins, [Thr², Leu⁵, Ala¹⁰]cyclosporin (2) and [Thr², Ile⁵] cyclosporin (3), from strains classified asAcremonium luzulae (Fuckel) W Gams andLeptostroma anamorph ofHypoderma eucalyptii Cooke & Harkn, respectively. In both new strains the usual pattern of cyclosporins A to Z is not found. The structure elucidations of2 and3 are based on NMR spectroscopy, and biological data (immunosuppressive activity, cyclophilin-binding affinity and antifungal effects) are presented.     

Heterogeneous production of laccase by mycelium of white-rot fungi

Mycelium of white-rot fungi secretes laccase into the medium. It was found by cultivation on malt-agar plates that the mycelium does not produce laccase equally in all its parts. The youngest hyphae at the margins of the colony represent usually the maximum producers, whereas older hyphae produce less or none at all. An exception here isCollybia velutipes which is the weakest producer of laccase of all the fungi studied and where only the older hyphae begin to secrete it. Manometric estimation of laccase showed that maximum specific activity of laccase is achieved at the boundary between the phases of initial and linear growth and i₁₁ some cases during the first half of linear growth. Ageing of the mycelium characterized by certain changes in its metabolism is reflected in changes of enzyme production by fungal hypha of different age.     

Determination of polyphenolic complex in wines by electrochemical methods and using the enzymes tyrosinase and laccase

Several red wines were studied to find a correlation between physicochemical parameters characterizing the antioxidant status of wine and total content of phenols in samples. The content of dissolved oxygen (its value varied from 0.75 to 3.28 mg/ml), pH (3.10-3.63), redox potential (-186 to -106 mV), mass concentration of free and total sulfur dioxide (10-30 and 36-200 mg/dm3, respectively), absorption spectra, and total phenol content were determined. The wines fell into two main groups-with a relatively low (1850-2050 mg/dm3) and high (2300-2900 mg/dm3) contents of polyphenols. It was demonstrated that physicochemical parameters (except for the content of sulfur dioxide) correlate with the total phenol content in the wines studied.     

红树植物对根域真菌生态的影响

植物根系具有丰富的微生物种类和生物量,为了掌握红树植物根系真菌的生态状况,在广东湛江红树林国家级自然保护区内6种红树植物群落中采集样品分离根际与非根际真菌,分析红树植物对根域真菌生态的影响以及根域真菌与环境因子的关系。从分离培养的6种红树植物的根域样本中,鉴定出5属11种真菌。桐花树、木榄、无瓣海桑和秋茄根际真菌的丰度与种类均高于非根际真菌,说明这4种红树植物对根际真菌生长有促进作用,而红海榄与白骨壤则相反,说明其对根际真菌有抑制作用。总体上,红树林林内真菌种类数远高于林外,表明红树植物覆盖对促进真菌物种多样性有积极作用。根际真菌丰度与总有机碳(TOC)呈显著负性相关关系,非根际真菌种类数与硝态氮(NO3-N)呈显著正相关关关系,说明根际TOC中可能含有抑制真菌生长作用的物质,而在非根际,红树植物根系分泌物影响较小。红树植物根系分泌物对根际微生物数量和种类数有一定的影响。     

木腐菌氧化酶系检定及漆酶产生的研究

采用丁香醛连氮、苯胺蓝平板脱色等4种方法,定性检测了27种真菌的木素氧化酶系组成。结果表明5个菌种同时具有漆酶(Lac)、木素过氧化物酶(Lip)和锰过氧化物酶(Mnp)3种酶的活性,5个菌种同时具有Lac和Lip2种酶的活性,8个菌种具有1种酶的活性;12个菌种具有Lac活性。选择其中生长速度快、Lac活性高的5个菌种进行Lac产生的研究,发现木蹄层孔菌Fomes fomentarius诱导、静止培养条件下产生的Lac酶活峰值高达9496U/mL,远远高于其它已报道过的菌种;静止培养条件下贝形刺革菌Hymenochaete badio-ferruginea和乳白耙菌Irpexlacteus产Lac,其峰值也分别达到了652和292U/mL,均明显高于其它培养方式,说明木蹄层孔菌等3种真菌静止培养可代替振荡培养进行Lac产生、制备等相关的后续研究。木蹄层孔菌等5种真菌对刚果红等染料脱色作用研究表明,各菌种对4类染料均具有高效广谱的脱色作用。     

Biosensors based on acrylic microgels: a comparative study of immobilized glucose oxidase and tyrosinase

Acrylic microgels are proposed as enzyme immobilizing support in amperometric biosensors. Two enzymes, glucose oxidase and tyrosinase, were entrapped in this matrix and their behaviour is compared. The optimum cross-linking of the polymeric matrix required to retain the enzyme, and to allow the diffusion of the substrate is different for each enzyme, 3.2% for glucose oxidase and 4.5% for tyrosinase. The effect of pH and temperature on the biosensor responses has been studied by experimental design methodology and predictions have been compared with independently performed experimental measurements. A quadratic effect of the variables studied (pH and T) on the biosensor response and the small or null interaction between them was confirmed. The pH results obtained with both methods are coincident revealing an reversible effect on the enzyme. However, the temperature optimum value obtained by experimental design was 10 degrees C lower as a result of an activity decay due to irreversible thermal denaturation of both enzymes.