O objetivo deste trabalho foi avaliar se as enzimas secretadas pelo fungo de decomposição parda Gloeophyllum trabeum podem atuar como auxiliares às celulases comerciais na hidrólise de substratos celulósicos oxidados. Para isto foi realizada a oxidação de celulose microcristalina Avicel mediante reação de Fenton (Fe+2, H2O2) e tratamento com CuSO4. O substrato oxidado foi caracterizado e posteriormente foi determinado o efeito desta modificação sobre a eficiência hidrolítica de enzimas comerciais (Celluclast/Novozym) e sobre misturas destas enzimas comerciais suplementadas com extrato enzimático obtido de G. trabeum. As reações realizadas com Avicel indicaram que ambos tratamentos químicos foram efetivos para modificar grupos funcionais presentes na celulose. A caracterização fisicoquímica dos substratos oxidados permitiu concluir que o tratamento Fenton gerou transformações mais intensas na celulose do que o tratamento com CuSO4. O análise dos hidrolisados ácidos das amostras oxidadas por Fenton mediante espectrometria de massas (GC-MS) confirmou que a oxidação ocorre principalmente no C1. As hidrólises enzimáticas utilizando preparações com diferentes cargas das enzimas comerciais apresentaram uma diminuição significativa da conversão de celulose quando as amostras são tratadas com o reagente Fenton. Por outro lado, a celulose oxidada gerada pelo tratamento com CuSO4 ainda é eficientemente hidrolisada pelas enzimas. As duas amostras de celulose Avicel oxidadas também foi hidrolisada utilizando misturas de enzimas comerciais e extratos enzimáticos de G. trabeum. Em todas as condições ensaiadas a suplementação da mistura reacional com extratos enzimáticos do fungo de decomposição parda G. trabeum diminuíram a eficiência de hidrólise enzimática da celulose oxidada. O emprego do dobro de carga enzimática de enzimas comerciais permitiu recuperar os níveis de hidrólise observados na celulose in natura. / The goal of this work was to evaluate whether enzymes produced by the brown-rot fungus Gleophyllum trabeum would be able to act as auxiliary enzymes of commercial cellulases during the enzymatic hydrolysis of oxidized cellulose. To reach this goal an oxidation of microcrystalline-cellulose (Avicel) was performed by two differente reaction systems: Fenton reaction (Fe2+, H2O2) or CuSO4 treatments. The oxidized subtracts were characterized and the effect of cellulose modification on the hydrolytic efficiency of commercial enzymes, supplemented or not with the enzymes recovered from a G. trabeum culture, was determined. The Avicel treatment by the oxidative systems resulted in cellulose structural changes as evidenced by infrared spectroscopy, viscosity measurements and chemical characterization by HPLC and GC/MS chromatography of the monomers recovered after acid hydrolysis. The physicochemical characterization of the oxidized celluloses allowed to conclude that the transformation of the cellulose was more intense when Fenton treatment was used as compared with the CuSO4 treatment. The analysis of the acid hydrolysates by GC-MS indicated that the oxidation by Fenton reaction occurred mainly at the C1 of the cellulose chain, resulting in gluconic acid in the acid hydrolysates. A meaningful decrease of the cellulose conversion was observed when Fetnon-treated samples were submitted to enzymatic hydrolysis using different amounts of commercial enzymes. On the other hand, the CuSO4-oxidized cellulose was still efficiently hydrolyzed by high enzyme dosages. The oxidized Avicel substrates were also hydrolyzed using a mixture of the commercial enzymes with enzymes recovered from G. trabeum cultures. At all tested conditions, the supplementation of the reaction mixture with enzymatic extracts from G. trabeum decreased the enzymatic hydrolysis efficiency. Nevertheless, the normal levels of enzymatic hydrolysis were recovered when the commercial enzymes dosage two-fold increased.

O objetivo deste trabalho foi avaliar se as enzimas secretadas pelo fungo de decomposição parda Gloeophyllum trabeum podem atuar como auxiliares às celulases comerciais na hidrólise de substratos celulósicos oxidados. Para isto foi realizada a oxidação de celulose microcristalina Avicel mediante reação de Fenton (Fe+2, H2O2) e tratamento com CuSO4. O substrato oxidado foi caracterizado e posteriormente foi determinado o efeito desta modificação sobre a eficiência hidrolítica de enzimas comerciais (Celluclast/Novozym) e sobre misturas destas enzimas comerciais suplementadas com extrato enzimático obtido de G. trabeum. As reações realizadas com Avicel indicaram que ambos tratamentos químicos foram efetivos para modificar grupos funcionais presentes na celulose. A caracterização fisicoquímica dos substratos oxidados permitiu concluir que o tratamento Fenton gerou transformações mais intensas na celulose do que o tratamento com CuSO4. O análise dos hidrolisados ácidos das amostras oxidadas por Fenton mediante espectrometria de massas (GC-MS) confirmou que a oxidação ocorre principalmente no C1. As hidrólises enzimáticas utilizando preparações com diferentes cargas das enzimas comerciais apresentaram uma diminuição significativa da conversão de celulose quando as amostras são tratadas com o reagente Fenton. Por outro lado, a celulose oxidada gerada pelo tratamento com CuSO4 ainda é eficientemente hidrolisada pelas enzimas. As duas amostras de celulose Avicel oxidadas também foi hidrolisada utilizando misturas de enzimas comerciais e extratos enzimáticos de G. trabeum. Em todas as condições ensaiadas a suplementação da mistura reacional com extratos enzimáticos do fungo de decomposição parda G. trabeum diminuíram a eficiência de hidrólise enzimática da celulose oxidada. O emprego do dobro de carga enzimática de enzimas comerciais permitiu recuperar os níveis de hidrólise observados na celulose in natura. / The goal of this work was to evaluate whether enzymes produced by the brown-rot fungus Gleophyllum trabeum would be able to act as auxiliary enzymes of commercial cellulases during the enzymatic hydrolysis of oxidized cellulose. To reach this goal an oxidation of microcrystalline-cellulose (Avicel) was performed by two differente reaction systems: Fenton reaction (Fe2+, H2O2) or CuSO4 treatments. The oxidized subtracts were characterized and the effect of cellulose modification on the hydrolytic efficiency of commercial enzymes, supplemented or not with the enzymes recovered from a G. trabeum culture, was determined. The Avicel treatment by the oxidative systems resulted in cellulose structural changes as evidenced by infrared spectroscopy, viscosity measurements and chemical characterization by HPLC and GC/MS chromatography of the monomers recovered after acid hydrolysis. The physicochemical characterization of the oxidized celluloses allowed to conclude that the transformation of the cellulose was more intense when Fenton treatment was used as compared with the CuSO4 treatment. The analysis of the acid hydrolysates by GC-MS indicated that the oxidation by Fenton reaction occurred mainly at the C1 of the cellulose chain, resulting in gluconic acid in the acid hydrolysates. A meaningful decrease of the cellulose conversion was observed when Fetnon-treated samples were submitted to enzymatic hydrolysis using different amounts of commercial enzymes. On the other hand, the CuSO4-oxidized cellulose was still efficiently hydrolyzed by high enzyme dosages. The oxidized Avicel substrates were also hydrolyzed using a mixture of the commercial enzymes with enzymes recovered from G. trabeum cultures. At all tested conditions, the supplementation of the reaction mixture with enzymatic extracts from G. trabeum decreased the enzymatic hydrolysis efficiency. Nevertheless, the normal levels of enzymatic hydrolysis were recovered when the commercial enzymes dosage two-fold increased.

Pentachlorophenol (PCP) is a biocide used by the NZ forestry industry until 1988. Its use was discontinued due to its toxicity to humans and animals. White-rot fungi have been shown to degrade PCP in laboratory and field trials. New Zealand native white-rot fungi were screened to identify organisms suitable for the clean up of PCP contaminated sites. Four criteria were used for the screening: fungal growth at different temperatures, PCP and creosote resistance and PCP degradation in standard liquid medium. Twenty isolates were identified as potentially useful from over 200 that were screened. One unique isolate, Trametes sp. HR577, was chosen for intensive study because it produced the well known laccases previously described from other PCP-degrading white-rot fungi. The white-rot isolate HR577 was assigned to the genus Trametes based on morphological characteristics and gene sequencing studies. The latter showed that the partial laccase gene sequences from Trametes sp. HR 577 had high sequence homology to laccases from other Trametes species, especially T. versicolor and T. villosa. Two laccase isozymes, designated L1c and L2, were purified from Trametes sp. HR577. These isozymes had similar biological properties to other Trametes species laccase isozymes. Both isozymes had a relatively high temperature optima, however, they were not very stable at elevated temperature. The dependence of laccase on dissolved oxygen for catalysis was demonstrated for isozyme L2. Laccase activity was severely inhibited in the absence of dissolved oxygen. This could be restored by reoxygenation into the assay system. Whole cultures of Trametes sp. HR577 grown in liquid culture removed up to 76% of PCP after 72 hours. PCP removal was mostly due to degradation rather than adsorption of PCP to fungal mycelium. Addition of purified and crude laccase isozymes (100 U mL-1) did not enhance PCP degradation. 6-15% of PCP was removed from solutions containing solely purified isozyme L1c or L2 in acetate buffer over 72 hours. Addition of ethanol or the laccase mediator compound 2,2' azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) resulted in increased PCP disappearance from purified laccase cultures. These studies show that the white-rot Trametes sp. HR577 has potential to be used for the clean up of PCP contaminated sites in NZ.

Les substances perturbatrices du système endocrinien sont des substances qui, de par leur capacité à induire des changements hormonaux chez les organismes vivants, génèrent des préoccupations dans le domaine de la qualité des eaux et, par extension, dans le domaine du traitement des effluents aqueux. Particulièrement, ce projet de recherche s’est attardé sur l’élimination des perturbateurs endocriniens phénoliques nonylphénol (NP), bisphénol A (BPA) et triclosan (TCS) en solution aqueuse à l’aide de la laccase (E.C. 1.10.3.2) sécrétée par la souche fongique Coriolopsis polyzona. Cette oxydase est une métalloprotéine pouvant catalyser l’oxydation d’une vaste gamme de substances phénoliques. En premier lieu, l’impact du pH et de la température sur l’élimination de ces composés à l’aide de la laccase libre en utilisant un design factoriel. L’oxydation de ces composés produit des oligomères (dimère à pentamère) via le couplage des radicaux phénoxy produits par l’action de la laccase. Il s’avère que les substances produites suite à l’oxydation du NP et du BPA par la laccase ont perdu leurs similitudes structurales avec l’estrogène. Ainsi, l’élimination de l’activité estrogénique de ces substances est directement liée à la transformation des composés. Finalement, l’utilisation d’ABTS comme médiateur a permis d’augmenter le taux d’oxydation enzymatique de ces composés chimiques. Puis, de façon à augmenter la possibilité d’utilisation de la laccase dans des biotechnologies environnementales, cette enzyme a été immobilisée sur un support siliceux et via la réticulation d’agrégats. L’impact des conditions d’immobilisation sur l’activité enzymatique, la stabilité du catalyseur et les propriété biocatalytiques apparentes a été déterminé pour différentes stratégies d’immobilisation. Globalement, l’immobilisation génère un biocatalyseur stable vis-à-vis les dénaturations chimique, physique et biologique. Particulièrement, l’immobilisation sur un support solide produit un biocatalyseur facile à utiliser ayant une faible activité massique et des propriétés cinétiques moindres que celle de l’enzyme libre. La formation de CLEAs de laccase a permis d’obtenir une activité massique élevée et des propriétés cinétiques supérieures à celle de l’enzyme soluble. Ces biocatalyseurs solides ont étés utilisés pour éliminer en continu le NP, BPA et TCS dans différents types de bioréacteur. Le biocatalyseur sur silice a été utilisé pour éliminer ces substances dans un réacteur garni, tandis que les CLEAs ont été utilisés dans un réacteur à lit fluidisé et un réacteur à perfusion développé au cours de ce projet. Ces différentes configurations de bioréacteur ont permis d’éliminer efficacement ces différents perturbateurs endocriniens. Globalement, les différents résultats obtenus, à l’échelle de laboratoire, au cours de ce projet de recherche démontrent que la laccase et particulièrement les biocatalyseurs formés via les différentes stratégies d’immobilisation testées représentent des approches extrêmement prometteuses pour le développement de biotechnologies environnementales vouées à l’élimination des perturbateurs endocriniens phénoliques.

Os polímeros sintéticos compõem cerca de 20% do lixo urbano no Brasil. Além da não biodegradabilidade, formam uma camada impermeabilizante que impede a passagem de líquidos e gases originados no apodrecimento dos detritos, retardando a estabilização da matéria orgânica. A exploração da madeira produz uma grande quantidade de rejeitos que não são inteiramente aproveitados para gerar energia ou outros produtos, e acarreta sérios problemas ambientais. A finalidade deste trabalho é a produção de compósitos biodegradáveis reunindo rejeitos de madeira e um polímero biodegradável - o poli (álcool vinílico) [PVA]. Para facilitar a degradação do PVA, este foi modificado por anidrido ftálico (AF). A modificação foi estudada através de tempo de gelatinização, calorimetria exploratória diferencial (DSC) e análise termomecânica dinãmica (DMTA). Resíduos da madeira Sugi (Criptomeria japonica) foram refinados até a obtenção de uma farinha com partículas menores que 63 µ m. Esta farinha foi adicionada ao meio de reação AF/PVA. As proporções de AF e PVA, bem como os parâmetros utilizados na prensagem foram determinados segundo um planejamento estatístico fatorial. Os compósitos foram moldados a quente (180 C e 50 MPa). Variando-se a proporção AF/PVA, compósitos com valores de módulo de elasticidade (MOE) de ˜10 GPa e módulo de ruptura (MOR) de ˜90 MPa na flexão foram obtidos. Os valores são inferiores aos apresentados pelo polímero puro, seco e sem plasticizante (acima de 152 MPa), porém superiores às placas de madeira reconstituída de MDF e OSB, disponíveis comercialmente, que apresentam valores de MOR em torno de 49 MPa. A degradação por microorganismos foi avaliada pelo ensaio de soterramento utilizando uma adaptação do método para avaliar a resistência de materiais lignocelulósicos aos fungos da podridão mole (Publicação IPT No. 1157 D5). Os fungos da podridão mole que ocorreram naturalmente nos compósitos foram isolados e identificados segundo a técnica do microcultivo. O gênero mais frequente foi o celulolítico Trichoderma spp. e o mais degradador foi o Chaetomium spp. As mudanças na estrutura e na morfologia dos compósitos foram estudadas através de espectroscopia de infravermelho (IR) e microscopia eletrônica de varredura (MEV). As perdas de massa e das propriedades mecânicas foram monitoradas em intervalos pré-estabelecidos. Compósitos com altas concentrações de AF apresentaram biodegradabilidades superiores à da madeira maciça de Pinus sp. e levemente inferiores à da madeira de Eucalyptus grandis, utilizadas como referências. A biodegradabilidade se deve à facilidade dos grupos ésteres, dos ftalatos de PVA e de celulose, de serem hidrolizados e de regenerar o ácido ftálico. Mesmo sob hidrólise enzimática, a estrutura PVA-AF-celulose, que forma ligações cruzadas entre microfibrilas de celulose, não se desintegra, conservando as propriedades mecânicas por muito tempo. Estas se mantêm superiores, ao término de 180 dias de exposição, às da madeira maciça de Sugi antes de entrar no processo de biodegradação. Uma correlação entre a estrutura, as propriedades físicas e mecânicas, e a taxa de biodegradação dos compósitos de rejeitos de madeira-PVA foi estabelecida. / Synthetic polymers constitute around 20% of urban solid waste in Brazil. Besides being non-biodegradable, they form an impermeable barrier that prevents the liberation of liquids and gas originated in the waste deterioration, retarding organic matter stabilization. The wood industry produces large amounts of waste wood which is not entirely utilized to produce energy or other products, and it brings several environmental problems. The aim of this study is the production of an environmentally friendly wood-based product composed of waste wood and poly (vinyl alcohol) [PVA]. PVA is the most widely produced water soluble and biodegradable synthetic polymer worldwide. However, PVA degradation in aqueous and soil environments has proved to be quite slow under unadapted conditions. To accelerate its degradation, the PVA has been modified by phthalic anhydride (AF). These modifications have been studied by means of gelation time, Differential Scanning Calorimetry (DSC) and (Dynamic Mechanical Thermal Analysis (DMTA). Sugi (Criptomeria japonica) flour (particles size ‹ 63 µm) has been obtained by milling waste samples. Wood flour has been added into AF/PVA reaction medium. AF and PVA ratios and pressing conditions have been set by factorial design. The final pressing temperature and pressure have been set as 180 °C and 50MPa respectively. Varying AF/PVA ratios, composites presenting modulus of elasticity (MOE) values of ˜10GPa and modulus of rupture (MOR) of ˜90 MPa have been obtained. The MOR values are lower than that presented by the pure, dry, no plasticized PVA (higher than 152 MPa), but they are higher than commercial MDF (medium density fiberboard) and OSB (oriented strand board) of around 49 MPa. Degradation by microorganisms has been performed by soil burial test (method adapted from IPT Edition No. 1157 D5). Naturally occurring soft rot fungi have been isolated and identified according to micro cultivation techniques. Most frequent genus has been the cellulolytic Trichoderma spp. and most degrader has been Chaetomium spp. Changes in the composites microstructures and morphology throughout the biodegradation process have been studied by IR and SEM and decreasing in the mechanical properties monitored. The weight Ioss shown by composites with high AF concentration has been higher than the softwood Pinus sp. and comparable to the hardwood Eucalyptus grandis, utilized as witnesses. Even under enzymatic hydrolysis, the PVA-AF-cellulose structure has been only slightly broken, preserving considerable mechanical properties that remain superior to solid Sugi before entering any biodegradation process, even after 180 days of exposure. A correlation has been established between the structure, physical and mechanical properties and biodegradation rate of waste wood-PVA composites

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22854号 / 農博第2437号 / 新制||農||1082(附属図書館) / 学位論文||R2||N5314(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 本田 与一, 教授 田中 千尋, 准教授 坂本 正弘 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

To gain a better understanding of the mechanisms by which the white-rot decay fungus Phanerochaete chrysosporium regulates selective delignification versus simultaneous decay, differential gene expression of its two key enzymes were measured over the course of aspen and birch wood decay tests. The type of decay was determined by differential staining and scanning electron microscopy. Real-time qPCR assessed gene expression of four lignin peroxidase genes and two manganese peroxidase genes at each stage of decay. Differential expression was significant in the mnp2 gene between aspen and birch decay tests during incipient decay; abundant expression of mnp2 in aspen corresponded to early initiation of selective delignification, whereas birch underwent initial simultaneous decay in the absence of abundant mnp2 expression. The lipC gene was the most abundantly expressed lip gene at all time points in both wood species and likely plays an important role in regulating wood decay.

Hydrogen is re-emerging as a serious alternative to fossil fuels. It is a clean gas with high energy density and its combustion only generates water vapour. Nevertheless, the hydrogen industry has a significant carbon footprint since this gas is mostly derived from fossil fuels reforming processes. This project focusses on the development of sustainable alternatives to conventional hydrogen production, in which approaches based on dark fermentation (DF) using an inexpensive residue from the brewery industry as primary feedstock are presented. Firstly, a fungal pre-treatment (FT) was proposed to degrade a high-strength brewery waste slurry (BWS) to obtain an effluent with a lower concentration of chemical oxygen demand (COD) but rich in readily fermentable sugars for the ensuing DF, thus improving hydrogen yields (HY). Secondly, microbial electrolysis and fuel cells (MECs and MFCs) were proposed to assist DF, generating electricity in MFCs while improving HY by MECs. Coupling both microbial electrochemical technologies sequentially after DF did not show any advantage. However, promising results were obtained for electricity and hydrogen production when taking a single-staged approach. Treating BWS directly by MFCs produced 2.0 watts/g COD consumed, while the DF process assisted simultaneously by MECs (DF/MEC) produced 1.6 times more hydrogen than DF alone. An average HY of 2.32 ± 0.06 mol H₂/mol glucose was attained between both DF/MEC and DF after FT, hence approaching the theoretical value of 2.4 mol H₂/mol glucose, representing roughly a 50% improvement compared to DF alone. With an overall COD reduction above 76%, the DF after FT exhibited the highest energy conversion rate per substrate consumed (6.3 kJ/g COD). As valuable by-products obtained, up to 31 g/L of fungal biomass, which is appreciated in many state-of-the-art biomaterials applications, was produced by using BWS. While in the DF/MEC process, 18 g/L of butyric acid were generated, which is three times more than with DF alone. Butyric acid being the precursor to butanol and building block of biodegradable thermoplastics, this result is not without significance. The proposed approaches not only valorize BWS but also validate their economic and environmental attractiveness as promising alternative hydrogen production methods.

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Made available in DSpace on 2014-07-29T16:11:53Z (GMT). No. of bitstreams: 1 DISSERTACAO DEFINITIVA c ficha catalografica.pdf: 2149699 bytes, checksum: ebcbff0f45d07e12d12a9708947a59eb (MD5) Previous issue date: 2009-07-15 / Tyrosinase (E.C.1.14.18.1) is an enzyme of industrial interest that catalyses the ohydroxylation of monophenols (monophenolase activity) and the oxidation of o-diphenols to reactive o-quinones (diphenolase activity), both reactions using molecular oxygen. Pycnoporus sanguineus (L. ex Fries) Murril, is a white rot fungi capable of producing tyrosinase and widely distributed in nature. It is found in regions of mild climate and in tropical forest. The production and characterization of tyrosinase from P. sanguineus were investigated. The selection of inductors, determination of the luminosity influence, biomass and culture media in the production of tyrosinase and the effect of inhibitors on enzyme activity were determined. The fungus produced intracellular tyrosinase and the higher activity was observed using 0.15% L-tyrosine as inducer, in the presence of light, with inoculum of 10 mycelium discs, medium malt extract broth 2%, incubation at 30°C, and constant agitation of 150 rpm, during 2 days. 6 mmol.L-1 salicylhydroxamic acid (SHAM) and 6 mmol.L-1 phenylthiourea (PTU) inhibited 100% of the tyrosinase activity. 0.1 mmol.L-1 sodium azide inhibited 4.15% of tyrosinase activity, while no inhibition was observed after addition of 0.1 mmol.L-1 of phenylmethanesulfonyl fluoride (PMSF). Using L-dopa as substrate, the intracellular crude extract presented optimum pH of 6,6, optimum temperature of 45°C, low stability at 50°C, maintaining about 50% of the activity after 15 min of incubation. The tyrosinase production was confirmed by non-denaturing polyacrylamide gel electrophoresis, using commercial fungal tyrosinase as positive control / A tirosinase (E.C.1.14.18.1), também conhecida como polifenoloxidase ou catecolase, é uma enzima de interesse industrial que catalisa a o-hidroxilação de monofenóis (atividade monofenolase) e a subseqüente oxidação do o-difenol resultante em o-quinonas reativas (atividade difenolase), usando oxigênio molecular. O Pycnoporus sanguineus (L. ex Fries) Murril, um fungo de decomposição branca (white rot) capaz de produzir a tirosinase, é amplamente distribuído na natureza, sendo encontrado em regiões de clima mais ameno e em florestas tropicais. A produção e caracterização da tirosinase produzida por P. sanguineus foi investigada. Foi realizada seleção de indutores, determinação da influência da luminosidade, biomassa e meios de cultura na produção de tirosinase, bem como o efeito de inibidores sobre a atividade enzimática. O extrato bruto enzimático foi caracterizado quanto ao pH ótimo, temperatura ótima e termoestabilidade. Os resultados obtidos demonstraram que o fungo produziu tirosinase intracelular e a mais alta atividade ocorreu com a utilização de 0,15% de L-tirosina como indutor, na presença de luz, com inóculo de 10 discos de micélio fúngico, meio caldo extrato de malte 2%, incubação à 30°C e agitação de 150 rpm, durante 2 dias. Ácido salicilhidroxâmico (SHAM) 6 mmol.L-1 e feniltiouréia (PTU) 6 mmol.L-1 inibiram 100% da atividade de tirosinase. Azida sódica 0,1 mmol.L-1 inibiu 4,15% da atividade de tirosinase, enquanto nenhuma inibição foi observada após adição de fluoreto de fenilmetanosulfonil (PMSF) 0,1 mmol.L-1. Utilizando a L-dopa como substrato, o extrato bruto intracelular apresentou pH ótimo de 6,6, temperatura ótima de 45°C, baixa estabilidade à temperatura de 50°C, mantendo apenas cerca de 50% de atividade após 15 minutos de incubação. A produção da tirosinase foi comprovada através de eletroforese em gel de poliacrilamida não desnaturante, tendo como controle positivo tirosinase fúngica comercial