The white-rot fungi Phlebia radiata and Dichomitus squalens in wood-based cultures: expression of laccases, lignin peroxidases, and oxalate decarboxylase

Basidiomycetous white-rot fungi are the only organisms that can efficiently decompose all the components of wood. Moreover, white-rot fungi possess the ability to mineralize recalcitrant lignin polymer with their extracellular, oxidative lignin-modifying enzymes (LMEs), i.e. laccase, lignin peroxidase (LiP), manganese peroxidase (MnP), and versatile peroxidase (VP). Within one white-rot fungal species LMEs are typically present as several isozymes encoded by multiple genes. This study focused on two effi cient lignin-degrading white-rot fungal species, Phlebia radiata and Dichomitus squalens. Molecular level knowledge of the LMEs of the Finnish isolate P. radiata FBCC43 (79, ATCC 64658) was complemented with cloning and characterization of a new laccase (Pr-lac2), two new LiP-encoding genes (Pr-lip1, Pr-lip4), and Pr-lip3 gene that has been previously described only at cDNAlevel. Also, two laccase-encoding genes (Ds-lac3, Ds-lac4) of D. squalens were cloned and characterized for the first time. Phylogenetic analysis revealed close evolutionary relationships between the P. radiata LiP isozymes. Distinct protein phylogeny for both P. radiata and D. squalens laccases suggested different physiological functions for the corresponding enzymes. Supplementation of P. radiata liquid culture medium with excess Cu2+ notably increased laccase activity and good fungal growth was achieved in complex medium rich with organic nitrogen. Wood is the natural substrate of lignin-degrading white-rot fungi, supporting production of enzymes and metabolites needed for fungal growth and the breakdown of lignocellulose. In this work, emphasis was on solid-state wood or wood-containing cultures that mimic the natural growth conditions of white-rot fungi. Transcript analyses showed that wood promoted expression of all the presently known LME-encoding genes of P. radiata and laccase-encoding genes of D. squalens. Expression of the studied individual LME-encoding genes of P. radiata and D. squalens was unequal in transcript quantities and apparently time-dependent, thus suggesting the importance of several distinct LMEs within one fungal species. In addition to LMEs, white-rot fungi secrete other compounds that are important in decomposition of wood and lignin. One of these compounds is oxalic acid, which is a common metabolite of wood-rotting fungi. Fungi produce also oxalic-acid degrading enzymes of which the most widespread is oxalate decarboxylase (ODC). However, the role of ODC in fungi is still ambiguous with propositions from regulation of intra and extracellular oxalic acid levels to a function in primary growth and concomitant production of ATP. In this study, intracellular ODC activity was detected in four white-rot fungal species, and D. squalens showed the highest ODC activity upon exposure to oxalic acid. Oxalic acid was the most common organic acid secreted by the ODC-positive white-rot fungi and the only organic acid detected in wood cultures. The ODC-encoding gene Ds-odc was cloned from two strains of D. squalens showing the first characterization of an odc-gene from a white-rot polypore species. Biochemical properties of the D. squalens ODC resembled those described for other basidiomycete ODCs. However, the translated amino acid sequence of Ds-odc has a novel N-terminal primary structure with a repetitive Ala-Ser-rich region of ca 60 amino acid residues in length. Expression of the Ds-odc transcripts suggested a constitutive metabolic role for the corresponding ODC enzyme. According to the results, it is proposed that ODC may have an essential implication for the growth and basic metabolism of wood-decaying fungi.

Peroxidases in lignifying xylem of Norway spruce, Scots pine and silver birch

Lignin is a complex plant polymer synthesized through co-operation of multiple intracellular and extracellular enzymes. It is deposited to plant cell walls in cells where additional strength or stiffness are needed, such as in tracheary elements (TEs) in xylem, supporting sclerenchymal tissues and at the sites of wounding. Class III peroxidases (POXs) are secreted plant oxidoreductases with implications in many physiological processes such as the polymerization of lignin and suberin and auxin catabolism. POXs are able to oxidize various substrates in the presence of hydrogen peroxide, including lignin monomers, monolignols, thus enabling the monolignol polymerization to lignin by radical coupling. Trees produce large amounts of lignin in secondary xylem of stems, branches and roots. In this study, POXs of gymnosperm and angiosperm trees were studied in order to find POXs which are able to participate in lignin polymerization in developing secondary xylem i.e. are located at the site of lignin synthesis in tree stems and have the ability to oxidize monolignol substrates. Both in the gymnosperm species, Norway spruce and Scots pine, and in the angiosperm species silver birch the monolignol oxidizing POX activities originating from multiple POX isoforms were present in lignifying secondary xylem in stems during the period of annual growth. Most of the partially purified POXs from Norway spruce and silver birch xylem had highest oxidation rate with coniferyl alcohol, the main monomer in guaiacyl-lignin in conifers. The only exception was the most anionic POX fraction from silver birch, which clearly preferred sinapyl alcohol, the lignin monomer needed in the synthesis of syringyl-guaiacyl lignin in angiosperm trees. Three full-length pox cDNAs px1, px2 and px3 were cloned from the developing xylem of Norway spruce. It was shown that px1 and px2 are expressed in developing tracheids in spruce seedlings, whereas px3 transcripts were not detected suggesting low transcription level in young trees. The amino acid sequences of PX1, PX2 and PX3 were less than 60% identical to each other but showed up to 84% identity to other known POXs. They all begin with predicted N-terminal secretion signal (SS) peptides. PX2 and PX3 contained additional putative vacuolar localization determinants (VSDs) at C-terminus. Transient expression of EGFP-fusions of the SS- and VSD-peptides in tobacco protoplasts showed SS-peptides directed EGFP to secretion in tobacco cells, whereas only the PX2 C-terminal peptide seems to be a functional VSD. According to heterologous expression of px1 in Catharanthus roseus hairy roots, PX1 is a guaicol-oxidizing POX with isoelectric point (pI) approximately 10, similar to monolignol oxidizing POXs in protein extracts from Norway spruce lignifying xylem. Hence, PX1 has characteristics for participation to monolignol dehydrogenation in lignin synthesis, whereas the other two spruce POXs seem to have some other functions. Interesting topics in future include functional characterization of syringyl compound oxidizing POXs and components of POX activity regulation in trees.

Production and Characterization of Lignin Peroxidases from Mangrove Ascomycetes

In this thesis, the production and characterization of ligninolytic enzymes using the fungi isolated from mangrove area are studied. The objective of the present work are isolation and screening of dye decolorizing micro-organisms from mangrove area, screening of the selected microorganisms for the production of lignin degrading enzymes, identification of the potent micro-organisms, characterization of the crude enzyme, lignin peroxidase, of the selected fungi—Aspergillus sp. SIP 11 and Penicillium sp. SIP 10 etc. This included the determination of the optimum pH, temperature, veratryl alcohol and H2O2 concentration. Besides the stability of crude LiP at different pHs and temperatures were studied. The immense applications, particularly in bioremediation, to which the lignin degrading micro-organisms could be used make this study important, the ascomycetes and deuteromycetes fungi, especially form the marine environment were studied with respect to their ligninolytic enzyme system making this study an initial step in unraveling the vast hidden potential of these microbes in bioremediation, the marine microbes are halophilic in nature which make them better suited to cope with the high salinity of industrial effluents thereby giving them added advantage in the filed of bioremediation. The thesis deals with the isolation and screening of lignin degrading enzyme-producing microbes from mangrove area. The identification of the most potent fungal isolates and characterization of LiP from these are also done.

Production and Characterization of Lignin Peroxidases from Mangrove Ascomycetes

The continually growing worldwide hazardous waste problem is receiving much attention lately. The development of cost effective, yet efficient methods of decontamination are vital to our success in solving this problem.Bioremediation using white rot fungi, a group of basidiomycetes characterized by their ability to degrade lignin by producing extracellular LiP, MnP and laccase have come to be recognized globally which is described in detail in Chapter 1.These features provide them with tremendous advantages over other micro-organisms.Chapter 2 deals with the isolation and screening of lignin degrading enzyme producing micoro-organisms from mangrove area. Marine microbes of mangrove area has great capacity to tolerate wide fluctuations of salinitie.Primary and secondary screening for lignin degrading enzyme producing halophilic microbes from mangrove area resulted in the selection of two fungal strains from among 75 bacteria and 26 fungi. The two fungi, SIP 10 and SIP ll, were identified as penicillium sp and Aspergillus sp respectively belonging to the class Ascomycetes .Specific activity of the purified LiP was 7923 U/mg protein. The purification fold was 24.07 while the yield was 18.7%. SDS PAGE of LiP showed that it was a low molecular weight protein of 29 kDa.Zymogram analysis using crystal violet dye as substrate confirmed the peroxidase nature of the purified LiP.The studies on the ability of purified LiP to decolorize different synthetic dyes was done. Among the dyes studied, crystal violet, a triphenyl methane dye was decolorized to the greatest extent.

Heat-inactivated peroxidases and the role of calcium abstraction as a cause of their enhanced lipid oxidation activity: potential effects on the flavour quality of heat-processed vegetables

Cationic swede and anionic turnip peroxidases were partially purified by ion-exchange and gel-filtration chromatography, respectively. Heat treatment of these enzymes and of a commercial high purity horseradish peroxidase (HRP) caused a loss of enzyme activity and a corresponding increase in linoleic acid hydroperoxide formation activity. The hydroperoxide levels in model systems increased only in the early stages of the oxidation reaction and then declined as degradation became more significant. The presence of a dialysed blend of cooked swede markedly lowered the hydroperoxide level formed. Analysis of volatile compounds formed showed that hexanal predominated in a buffer system and in a blend of cooked turnip. In dialysed blends of cooked swede, hexanol was the primary volatile compound generated. After inactivation under mild conditions in the presence of EDTA, the peroxidases showed hydroperoxide formation activity and patterns of volatile compounds from linoleic acid that were similar to those found on heat-inactivation. This suggested that calcium abstraction from the peroxidases was critical for the enhancement of lipid oxidation activity. (C) 2002 Elsevier Science Ltd. All rights reserved.

Amperometric detection of benzoyl peroxide in pharmaceutical preparations using carbon paste electrodes with peroxidases naturally immobilized on coconut fibers

This paper describes the applications of anew carbon paste electrode containing fibers of coconut (Cocus nucifera L) fruit, which are very rich in peroxidase enzymes naturally immobilized on its structure. The new sensor was applied for the amperometric quantification of benzoyl peroxide in facial creams and dermatological shampoos. The amperometric measurements were performed in 0.1 mol L(-1) phosphate buffer (pH 5.2), at 0.0 V (versus Ag/AgCl). On these conditions, benzoyl peroxide was rapidly determined in the 5.0-55 mu mol L(-1), with a detection limit of 2.5 mu mol L(-1) (s/n = 3), response time of 4.1 s (90% of the steady state) and sensitivity limit of 0.33 A mol L(-1) cm(-2). The amperometric results are in good agreement with those obtained by spectrophotometric technique, used as a standard method. (C) 2009 Elsevier B.V. All rights reserved.

Atividade de quitinases e peroxidases em folhas de eucalipto em diferentes estágios de desenvolvimento após tratamento com acibenzolar-S-metil (ASM) e inoculação com Puccinia psidii

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

ATIVIDADE DE PEROXIDASES (EC 1.11.1.7) E TEOR DE PROLINA em FEIJOEIRO Phaseolus vulgaris L. CULTIVADO em CONDIÇÕES DE SALINIDADE

Alterações nos níveis de prolina e na atividade da peroxidase foram estudadas em folhas e raízes de feijoeiro em função da resposta à salinidade do meio. A atividade da peroxidase (E.C. 1.11.1.7) e o teor de prolina das folhas foi maior do que nas raízes. Os resultados mostraram que a atividade da peroxidase diminuiu, enquanto os teores de prolina aumentaram gradualmente nas plantas cultivadas em meio salino. Ocorreu diminuição do teor de potássio e aumento de sódio nos órgãos analisados, com o aumento da concentração salina.

ISOENZYMATIC POLYMORPHISM and ACTIVITY of PEROXIDASES of COMMON BEAN(Phaseolus vulgaris L.) UNDER SALINE STRESS

Uma das utilizações da técnica de cultura de tecidos para o melhoramento vegetal é a identificação de linhas de células que apresentem tolerância ao estresse salino. Para se estudar os mecanismos bioquímicos envolvidos na expressão genética da tolerância a salinidade, calos oriundos de eixos embrionários de quatro cultivares de feijão (Phaseolus vulgaris L.; cultivares IAC - carioca, IAPAR 14, JALO-EEP 558, BAT - 93), foram cultivados em meio sólido Murashige & Skoog (1962), suplementado com NaCl nas concentrações de 0, 20, 40, 60 e 80 mM. Após 14 dias de incubação, os calos foram coletados e analisados quanto aos padrões isoenzimáticos e de atividade das peroxidases. Os cultivares BAT e IAPAR apresentaram duas zonas de atividade em comum na região anódica e apenas uma zona enzimática específica a cada um deles (migração mais rápida).Possivelmente as duas zonas anódicas intermediárias sejam produtos do mesmo loco enzimático, porém com alelos diferentes, consequentemente diferentes mobilidades eletroforéticas. O cv. JALO apresentou duas zonas anódicas de atividade em comum com os cultivares IAC e IAPAR com uma zona anódica exclusiva de migração mais lenta, a qual apresentou atividade mais intensa de todos os cultivares analisados. Este cultivar revelou ainda uma zona catódica provavelmente dimérica e heterozigota nos indivíduos de todos os tratamentos aplicados. Provavelmente, esta é a mesma zona que ocorre em homozigose com fixação do alelo lento para os indivíduos de todos os tratamentos efetuados nos cultivares BAT e IAPAR. O cv. IAC apresentou duas bandas anódicas em comum com os cv. IAPAR e JALO. Apresentou também a banda anódica mais rápida em comum com o cv. IAPAR e uma banda anódica exclusiva de migração mais lenta. Curiosamente, os indivíduos deste cv. mantidos em meio suplementado com 20 mM de NaCl não apresentaram atividade nas três zonas anódicas mais lentas. Ocorreu no cv. IAC uma única zona de atividade catódica, dimérica e heterozigota para os indivíduos provenientes de todos os tratamentos, composta provavelmente de dois alelos diferentes da zona correspondente ao cv. JALO. Amostras provenientes dos tratamentos 40 e 60 mM de NaCl, desta zona catódica, apresentaram maior atividade enzimática. A análise da atividade da peroxidase no extrato bruto, revelou que os cultivares responderam diferentemente ao aumento da concentração salina no meio de cultura, com aumento pronunciado dessa atividade nos cultivares IAC e JALO.

ATIVIDADE DE PEROXIDASES (EC 1.11.1.7) E TEOR DE PROLINA NO EMBRIÃO E COTILÉDONES DE FEIJOEIRO Phaseolus vulgaris L. em CONDIÇÕES DE SALINIDADE

Os efeitos da salinidade sobre a atividade de peroxidases foram acompanhadas no embrião e em cotilédones de feijoeiro Phaseolus vulgaris L. cv. Carioca, observando-se em ambos, aumento na atividade das enzimas. Com relação ao teor de prolina, ocorreu decréscimo constante no embrião de feijoeiro cultivado em condições salinas, entretanto, nos cotilédones, verificou-se aumento progressivo em condições de presença ou ausência de salinidade.

Soluble and bound peroxidases from papaya fruit

Soluble and bound peroxidases were isolated from the pulp of ripening papaya fruit. During papaya ripening, soluble and bound peroxidase activities increased 2.5- and 4.2-fold, respectively. Soluble peroxidase was purified 59-fold by ammonium sulphate precipitation and chromatography on Sephadex G-25, DEAE-cellulose and Sephadex G-100. Bound peroxidase was purified 140-fold by ammonium sulphate precipitation and chromatography on Sephadex G-100 and DEAE-cellulose. Polyacrylamide gel electrophoresis of the purified preparations revealed that both enzymes were highly purified by the procedures adopted. The soluble and bound forms had a Mr of 41 000 and 54 000, respectively. Soluble and bound peroxidases showed optimum activity at pH 6.0 and 5.5, respectively, and were inhibited by p-chloromercuribenzoate, iodoacetamide, N-ethylmaleimide, potassium cyanide and Fe2+. Soluble peroxidase was activated by ammonium sulphate and this activation was prevented by cyanide. © 1990.

Does cotinine act upon reactive oxygen species and peroxidases?

Nicotine, an oxidizing agent, is certainly one of the most widely used alkaloids in the world. It is, together with its main metabolite, cotinine, responsible for tobacco-dependence. The use of tobacco is closely associated with lung disease, morphological leukocyte modification and generation of oxidant species. The aim of this study was to look for a possible relationship between cotinine, oxidant species generation and oxidative processes. After studying the action of cotinine in some chemical oxidation models and on the enzymatic kinetics of peroxidases (myeloperoxidase and horseradish peroxidase), we concluded that cotinine does not act directly upon H 2O 2, HOCl, taurine chloramines, horseradish peroxidase or myeloperoxidase.

Use of hemoglobin as alternative to peroxidases in cholesterol amperometric biosensors

Sophisticated molecular architectures can be produced with the layer-by-layer (LbL) method, which may combine distinct materials on the same film. In this study, we take advantage of this capability to produce cholesterol amperometric biosensors from LbL films containing hemoglobin (Hb) and cholesterol oxidase in addition to the polyelectrolytes poly(allylamine hydrochloride) (PAH) and poly(ethylene imine) (PEI). Following an optimization procedure, we found that an LbL film deposited onto ITO substrates, with the architecture ITO(PEI/Hb)5(PEI/COx)10, yielded a sensitivity of 93.4 μA μmol L-1 cm-2 for cholesterol incorporated into phospholipid liposomes, comparable to state-of-the-art biosensors. Hb acted as efficient electron mediator and did not suffer interference from phospholipids. Significantly, cholesterol could also be detected in real samples from chicken egg yolk, with no effects from potential interferents, including phospholipids. Taken together these results demonstrate the possible fabrication of low cost, easy-to-use cholesterol amperometric biosensors, whose sensitivity can be enhanced by further optimizing the molecular architectures of the LbL films. © 2012 Elsevier B.V.

Imobilização multipontual das peroxidases da casca da soja e chuchu em suportes alternativos de pó de sabugo de milho e celulose bacteriana

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Análise da atividade de peroxidases e polifenoloxidases em diferentes órgãos vegetativos de espécies arbóreas nativas de diferentes grupos sucessionais

As formações vegetais são altamente dinâmicas e dependem principalmente da taxa de crescimento das espécies vegetais, entretanto, esta está intimamente relacionada e suscetível a estresses abióticos, como elevada ou baixa radiação solar, temperaturas extremas, e bióticos, como taque de patógenos e de herbívoros, o que faz com que as plantas necessitem de um sistema de defesa altamente eficiente o qual consiste na atuação de compostos secundários, e de enzimas como por exemplo peroxidases e polifenoloxidases. A atividade das peroxidases constitui a mais importante via do sistema de defesa vegetal a patógenos no que se refere a cicatrização de ferimentos e à herbivoria, propiciando um aumento da lignificação nos tecidos lesados, o que aumenta a resistência dos mesmos, dificultando assim a ocorrência de novas lesões. Quanto às polifenoloxidases, também pertencentes ao grupo das oxidorredutases, são enzimas que também atuam diretamente na via de defesa das plantas ao ataque de patógenos e na cicatrização de ferimentos e processos de senescência. Por meio da extração e análise da atividade de Peroxidases e Polifenoloxidases em porção de raiz, caule, ápice e em folhas de indivíduos de Erythrina speciosa Andrews, Eugenia uniflora L., Hevea brasiliensis M. Arg., Hymenaea courbaril L. var. stilbocarpa (Hayne) Lee et Lang, Joannesia princeps Vell., Licania tomentosa (Benth.) Fritsch, Pachira aquatica Aubl. e Psidium guajava L., obteve-se comportamento pioneiro para as espécies Eugenia uniflora L., Hymenaea courbaril L. var. stilbocarpa (Hayne) Lee et Lang, Joannesia princeps Vell. e Psidium guajava L., e comportamento não-pioneiro para as espécies Erythrina speciosa Andrews, Hevea brasiliensis M. Arg., Licania tomentosa (Benth.) Fritsch e Pachira aquatica Aubl.. Dessa maneira foi possível verificar que a análise da atividade peroxidásica... (Resumo completo, clicar acesso eletrônico abaixo)