Laccase and tyrosinase activities in lichens

Phenoloxidase activity was found in lichenized ascomycetes belonging to different taxonomic groups. Most of the epigeic and epilithic lichens of the order Peltigerales were found to possess both laccase and tyrosinase activities; the lichens of the order Lecanorales possessed only laccase activity, which was an order of magnitude lower than that of Peltigerales. Water-soluble phenoloxidases were present only in peltigerous lichens: activity that could be washed out from intact thalli comprised 10% of that released from disrupted thalli. The activity of the peltigerous lichens and the release of soluble phenoloxidases into the medium increased when the thalli were rehydrated quickly. In some of the lichens tested, the phenoloxidase activity was stimulated by desiccation-rehydration cycles. The oxidases discovered may play an important role in the phenolic metabolism of lichens and be involved in the biochemical reaction of humus synthesis during primary soil formation, which may be a previously unknown geochemical function of these symbiotic microorganisms.     

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.     

Laccases produced by lichens of the order Peltigerales

'Large' and 'small' fractions of laccase were found in the thalli of lichens Solorina crocea and Peltigera aphthosa. In both lichens, 'large', possibly dimeric, laccases were determined as 175 and 165 kDa (based on the gel filtration data), and 'small' ones were 76 and 97 kDa (according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis data), respectively. By their substrate specificity, pH optima, and thermostability, they were typical laccases. The fractions of 'small' laccases of 45 kDa from S. crocea and 55 kDa from P. aphthosa consisted of two enzymes.     

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.     

出芽短梗霉胞外酸性漆酶

通过愈创木酚法平板检测10株出芽短梗霉,发现5株菌能够分泌胞外多酚氧化酶,反应最适pH在2.0左右,均属于酸性多酚氧化酶。菌株NG的酶活最高,达110 U/mL。添加H2O2、EDTA以及过氧化氢酶不显著影响菌株NG胞外酶活,表明NG分泌的多酚氧化酶中不含有锰过氧化物酶（MnP）和不依赖Mn2＋的过氧化物酶（MiP）,属于漆酶（Lac）。     

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.     

彩绒革盖菌CV—8漆酶和多酚氧化酶活性

彩绒革盖菌（Ｃｏｒｉｌｕｓｖｅｒｓｉｃｏｌｏｒ）是一种具有木质素降解能力的白腐菌,其降解木素的能力主要取决于木质素过氧化物酶、漆酶、多酚氧化酶和愈创木酚氧化酶的活力高低,所以研究其活力和分泌情况对木质素的生物降解、生物制浆和环境保护具有十分重要的意义。国外已有彩绒革盖菌漆酶的报道［１,２］,但主要集中在漆酶对木质素作用后所造成的木质素结构上的变化,有关漆酶和多酚氧化酶的活性分析报道较少［３］;国内有关的研究报道更少［４,５］,至今未见彩绒革盖菌漆酶和多酚氧化酶的研究报道。本文主要测定了彩绒革盖菌…     

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.     

6种植物提取物对酪氨酸酶活性的影响

近年来,中草药美容由于疗效显著、毒副作用小而风靡全球。与此同时,中外众多研究者从中国古代治疗皮肤病的方剂中筛选出多种中药,以现代实验手段探讨它们的作用机理,从而更有效地指导美容用品的研制开发,植物药对酪氨酸酶活性的影响便是其中的研究课题之一。已有证据表明,酪氨酸酶在黑色素生成过程中起着极为关键的作用,用药物调节酪氨酸酶的活性,能医治相关的皮肤病[1]。Ｙｕｋｉｍｉｔｓｕ等[2]选用26种在古方中用于皮肤增白的中药进行了酪氨酸酶活性的抑制性试验;雷铁池等[3]用计算机从古今中医治疗色素增加性皮肤病方剂中精选69首进…     

Aliphatic compounds in some lichens

The presence of free fatty acids in Cetraria nivalis and Cladonia gonecha has been demonstrated. Saturated wax esters occur in Cetraria cucullata and C. nivalis, whilst free alkanols were found in C. cucullata. Triglycerides in C. nivalis were detected by their MS fragmentation patterns. Hexa(α-hydroxyisovalerate) is present in C. nivalis and Cl. gonecha, and the presence of ribitol in C. nivalis was demonstrated by identification of its acetonide in an acetone extract.     

Circular permutation of a bacterial tyrosinase enables efficient polyphenol-specific oxidation and quantitative preparation of orobol

Tyrosinase is a type 3 copper oxygenase that catalyzes a phenol moiety into ortho-diphenol, and subsequently to ortho-quinone. Diverse tyrosinases have been observed across the kingdom including Animalia, Bacteria, Plantae, and Fungi. Among the tyrosinases, bacterial, and mushroom tyrosinases have been extensively exploited to prepare melanin, ortho-hydroxy-polyphenols, or novel plant secondary metabolites during the past decade. And their use as a biocatalyst to prepare various functional biocompounds have drawn great attention worldwide. Herein, we tailored a bacterial tyrosinase from Bacillus megaterium (BmTy) using circular permutation (CP) engineering technique which is a novel enzyme engineering technique to covalently link original N and C termini and create new termini on the middle of its polypeptide. To construct a smart rationally-designed CP library, we introduced 18 new termini at the edge of each nine loops that link α-helical secondary structure in BmTy. Among the small library, seven functional CP variants were successfully identified and they represented dramatic change in their enzyme characteristics including kinetic properties and substrate specificity. Especially, cp48, 102, and 245 showed dramatically decreased tyrosine hydroxylase activity, behaving like a catechol oxidase. Exploiting the dramatic increased polyphenol oxidation activity of cp48, orobol (3′-hydroxy-genistein) was quantitatively synthesized with 1.48 g/L, which was a 6-fold higher yield of truncated wild-type. We examined their kinetic characters through structural speculation, and suggest a strategy to solubilize the insoluble artificial variants effectively.     

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     

A continuous spectrophotometric assay for determination of the aureusidin synthase activity of tyrosinase

Introduction – Aurones (aureusidin glycosides) are plant flavonoids that provide yellow colour to the flowers of some ornamental plants. In this study we analyse the capacity of tyrosinase to catalyse the synthesis of aureusidin by tyrosinase from the chalcone THC (2′,4′,6′,4–tetrahydroxychalcone). Objective – To develop a simple continuous spectrophotometric assay for the analysis of the spectrophotometric and kinetic characteristics of THC oxidation by tyrosinase. Methodology – THC oxidation was routinely assayed by measuring the increase in absorbance at 415 nm vs. reaction time. Results – According to the mechanism proposed for tyrosinase, the enzymatic reaction involves the o‐hydroxylation of the monophenol THC to the o‐diphenol (PHC, 2′,4′,6′,3,4 – pentahydroxychalcone), which is then oxidised to the corresponding o‐quinone in a second enzymatic step. This product is highly unstable and thus undergoes a series of fast chemical reactions to produce aureusidin. In these experimental conditions, the optimum pH for THC oxidation is 4.5. The progress curves obtained for THC oxidation showed the appearance of a lag period. The following kinetic parameters were also determined: K_m = 0.12 mM, V_m = 13 μM/min, V_m/K_m = 0.11/min. Conclusion – This method has made it possible to analyse the spectrophotometric and kinetic characteristics of THC by tyrosinase. This procedure has the advantages of a short analysis time, straightforward measurement techniques and reproducibility. In addition, it also allows the study of tyrosinase inhibitors, such as tropolone. Copyright © 2009 John Wiley & Sons, Ltd.     

Laccase in Anacardiaceae

The presence of a typical laccase is demonstrated in the cavities of the secretory ducts of a number of species of the Anacardiaceae, including Mangifera indica and Schinus molle. In addition mango fruit contains catechol oxidase. The presence of laccase may be of chemotaxonomic value.     

Complete genome sequence of the melanogenic marine bacterium Marinomonas mediterranea type strain (MMB-1(T))

Marinomonas mediterranea MMB-1(T) Solano & Sanchez-Amat 1999 belongs to the family Oceanospirillaceae within the phylum Proteobacteria. This species is of interest because it is the only species described in the genus Marinomonas to date that can synthesize melanin pigments, which is mediated by the activity of a tyrosinase. M. mediterranea expresses other oxidases of biotechnological interest, such as a multicopper oxidase with laccase activity and a novel L-lysine-epsilon-oxidase. The 4,684,316 bp long genome harbors 4,228 protein-coding genes and 98 RNA genes and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Characterization of a new tyrosinase from Pycnoporus species with high potential for food technological applications

AIMS: Tyrosinase production by Pycnoporus cinnabarinus and Pycnoporus sanguineus was screened among 20 strains originating from various geographical areas, particularly from tropical environments. The tyrosinase from the most efficient strain was purified and characterized and tested for food additive applications. METHODS AND RESULTS: Monophenolase and diphenolase activities of tyrosinase were measured from cell lysate from the 20 Pycnoporus strains, for 8-10 days of cultivation. The strain P. sanguineus CBS 614.73 showed the highest productivity (45.4 and 163.6 U g(-1) protein per day for monophenolase and diphenolase respectively). P. sanguineus CBS 614.73 tyrosinase was purified from concentrated cell lysate, anion-exchange, size-exclusion and hydroxyapatite chromatography, with a final yield of 2% and a purification factor of 35-38. The pure enzyme was a monomere with a molecular mass of 45 kDa and it showed four isoforms or isoenzymes with pI between 4.5-5. No N-glycosylation was found. The N-terminal amino acid sequence was IVTGPVGGQTEGAPAPNR. The enzyme was shown to be almost fully active in a pH range of 6-7, in a large temperature range (30-70 degrees C), and was stable below 60 degrees C. The main kinetic constants were determined. The tyrosinase was able to convert p-tyrosol and p-coumaric acid into hydroxytyrosol and caffeic acid, respectively, and it could also catalyse the cross-linking formation of a model protein. CONCLUSIONS: Among the genus Pycnoporus, known for the production of laccase, the strain P. sanguineus CBS 614.73 was shown to produce one other phenoloxidase, a new monomeric tyrosinase with a specific activity of 30 and 84 U mg(-1) protein for monophenolase and diphenolase respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: This study identified P. sanguineus CBS 614.73 as a potential producer of a tyrosinase which demonstrated effectiveness in the synthesis of antioxidant molecules and in protein cross-linking.     

Inhibitory effects of hinokitiol on tyrosinase activity and melanin biosynthesis and its antimicrobial activities

The inhibitory effects of hinokitiol, a constituent of the woody oils isolated from Cupressaceae heartwood, on mushroom tyrosinase and melanin formation in B16 melanoma cells as well as its antimicrobial activity were investigated. Our results showed that hinokitiol could strongly inhibit both monophenolase activity and diphenolase activity of the enzyme and the inhibition was reversible. The IC₅₀ values were estimated as 9.67?μM for monophenolase activity and 0.21?μM for diphenolase activity. The lag time of the monophenolase activity was not obviously lengthened by the compound. Kinetic analyses showed that the inhibition mechanism of hinokitiol was a mixed-type inhibition of the diphenolase activity. Hinokitiol effectively inhibited both cellular tyrosinase activity and melanin biosynthesis in B16 melanoma cells with significant cytotoxicity. Furthermore, it was found that hinokitiol could inhibit the proliferation of Salmonella enteritidis, Escherichia coli, Bacillus subtilis, Staphyloccocus aureus, Klebsiella pneumoniae, and Ralstonia solanacearum to different extents. This research may widen the use of hinokitiol in the fields of food preservation, depigmentation, and insecticide use.     

Abscisic acid in lichens: variation, water relations and metabolism

For the first time the well known drought stress hormone abscisic acid, which is involved in regulating processes increasing desiccation tolerance in many plant systems, was analysed in lichens. ABA was detected in all 26 species investigated. In contrast to higher plants and liverworts, the ABA content increased after hydration of air dry lichen thalli and decreased in desiccating lichen material. Experiments with Baeomyces rufus (Huds.) Rebent indicated that the mycobiont might be the major site of ABA biosynthesis. After incubation of hydrated lichen thalli with radioactive ABA for up to 72 h no metabolism to phaseic acid and dihydrophaseic acid could be detected. Fluctuations of internal ABA might be a result of ABA release to the external medium.     

Upgrading Laccase Production and Biochemical Properties: Strategies and Challenges

Improving laccases continues to be crucial in novel biotechnological developments and industrial applications, where they are concerned. This review breaks down and explores the potential of the strategies (conventional and modern) that can be used for laccase enhancement (increased production and upgraded biochemical properties such as stability and catalytic efficiency). The challenges faced with these approaches are briefly discussed. We also shed light on how these strategies merge and give rise to new options and advances in this field of work. Additionally, this article seeks to serve as a guide for students and academic researchers interested in laccases. This document not only gives basic information on laccases, but also provides updated information on the state of the art of various technologies that are used in this line of investigation. It also gives the readers an idea of the areas extensively studied and the areas where there is still much left to be done. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1015–1034, 2017     

Effect of low water potential on photosynthesis in intact lichens and their liberated algal components

Earlier experiments (T.D. Brock 1975, Planta124, 13–23) addressed the question whether the fungus of the lichen thallus might enable the algal component to function when moisture stress is such that the algal component would be unable to function under free-living conditions. It was concluded that the liberated phycobiont in ground lichen thalli could not photosynthesize at water potentials as low as those at which the same alga could when it was present within the thallus. However, our experience with lichen photosynthesis has not substantiated this finding. Using instrumentation developed since the mid-1970's to measure photosynthesis and control humidity, we repeated Brock's experiments. When applying “matric” water stress (equilibrium with air of constant relative humidity) we were unable to confirm the earlier results for three lichen species including one of the species,Letharia vulpina, had also been used by Brock. We found no difference between the effects of low water potential on intact lichens and their liberated algal components (ground thallus material and isolated algae) and no indication that the fungal component of the lichen symbiosis protects the phycobiont from the adverse effects of desiccation once equilibrium conditions are reached. The photosynthetic apparatus of the phycobiont alone proved to be highly adapted to water stress as it possesses not only the capability of functioning under extremely low degrees of hydration but also of becoming reactivated solely by water vapor uptake.