A celulose é a mais abundante fonte de carbono presente na madeira e nos resíduos agrícolas, e a sua hidrólise completa é realizada pela ação sinergística de diferentes enzimas, como: as endo-1,4-ß-D-glucanase, exo-1,4-ß-glucanase e ß-glucosidase ou celobiase. O presente trabalho descreve algumas propriedades fisiológicas e bioquímicas do sistema ß-glucosidásico do fungo termofílico Scytalidium thermophilum. Tal fungo foi isolado originalmente do solo da Índia e gentilmente cedido pelo Dr. G. Straastma (Holanda). O meio M8 favoreceu a produção das ß-glucosidases. Entre os açúcares testados como fonte de carbono, avicel e celobiose foram os melhores indutores das ß-glucosidases extracelular e micelial. Quando o fungo foi crescido em dois estágios, observou-se inicialmente a repressão da síntese por glicose e a indução por avicel ou celobiose. Utilizando-se ciclo-heximida, observou-se a síntese \"de novo\" das proteínas. A ß-glucosidase extracelular foi purificada utilizando-se um fracionamento protéico e uma coluna de troca-iônica DEAE-celulose, de onde foram obtidos duas atividades enzimáticas denominadas ß-glucosidases I e II. A ß-glucosidase I foi aplicada em coluna de troca iônica CM-celulose, enquanto que a ß-glucosidase II foi aplicada em Sephadex G-100. A ß-glucosidase I foi purificada 2 vezes com 4.0% de recuperação, ao passo que a ß-glucosidase II foi purificada 2,4 vezes com 2.0% de recuperação. A ß-glucosidase micelial foi purificada utilizando-se um choque térmico, fracionamento protéico, coluna de filtração Sephadex G-100 e uma coluna troca-iônica DEAE-celulose. Foi purificada 23 vezes com recuperação de 25%. A ß-glucosidases extracelular I e micelial apresentaram um temperatura ótima aparente de 70 e 60°C e um pH de 5.5 e 6.0, respectivamente. Ambas enzimas foram inibidas por Ag+2 e Hg+2. A ß-glucosidases extracelular I e micelial possuem um peso molecular de 40.7 kDa e 39kda (SDS-Page) e 57 kDa e 33,8 kda (Sephadex G-100), respectivamente. A ß-glucosidase extracelular I foi capaz de hidrolisar PNP-glu, PNP-xil, celobiose, xilana e CMC, enquanto que a ß-glucosidase micelial hidrolisou PNP-glu, PNP-fuc, PNP-xil, PNPgal, ONPG e lactose. Ambas enzimas foram ativadas por glicerol a 1M. A ß-glucosidase extracelular I foi ativada por xilose, frutose e lactose, e se mostrou resistente a glicose 50mM, enquanto que a ß-glucosidase micelial foi ativada por glicose e xilose. ß-glucosidases extracelular I e micelial apresentaram um PI de 4.0 e 6.5, respectivamente. Os parâmetros cinéticos estimados para a ß-glucosidase extracelular I foram de Km 4,33 e 0,342mM e Vmáx de 5,37 e 2,0µmoles/min/mg prot. para celobiose e PNP-glu, respectivamente. O valor de Ki (Constante de Inibição) foi de 71mM para glicose. Para a ß-glucosidase micelial, os valores de Km e Vmáx foram de 0,29mM e 13,27µmoles/min/mg prot; 0,5 mM e 7,25µmoles/min/mg prot e 1,61 mM e 4,12µmoles/min/mg prot para os substratos PNP-glu, PNP-fuc e celobiose, respectivamente. Na presença de glicose e xilose os valores de Km e Vmáx foram de 1,26mM e 40,04 µmoles/min/mg.prot, e 1,33mM, e 30,49 µmoles/min/mg prot, respectivamente para o PNP-glu. O valor de Ki (Constante de Inibição) foi de 1,32mM para celobiose. A análise dos produtos de hidrólise das ß-glucosidases extracelular I e micelial foram anlisadas em TLC, e revelaram que ambas enzimas realizam hidrólise quando celobiose foi utilizada a 10mM, e transglicosilação quando celobiose foi utilizada a 250mM. Os resultados aqui apresentados demonstram importante papel importante do Scytalidium como produtor de ß-glucosidase com potencial na sacarificação enzimática da celulose. / Cellulose is the most abundant carbon source found in woods and waste residues. In nature the complete hidrolysis of cellulose occurs by the sinergistic action of several enzymes such endo-1,4-ß-D-glucanase, exo-1,4-ß-glucanase e ß-glucosidase or cellobiase. The present work describe some physiological and biochemical properties of ß-glucosidase system from thermophilic fungus Scytalidium thermophilum. The fungus was gift to Dr. Straastma (Mushroom Experimental Station, The Netherlands). The culture medium M8 enhance the production of ß-glucosidase. Among carbohydrates tested as carbon source, avicel and cellobiose were the best inducers of ß-glucosidase extracellular and mycelial. When the fungus was grown in two stages, observed the repression by glucose, and induction by avicel or cellobiose. The presence of cycloheximide inhibited the syntesis of ß-glucosidase, suggesting that the enzyme produced in the presence of indutors required \"de novo\" synthesis. Extracellular ß-glucosidase was purified using the precipitation with 75% amonium sulfate, ion exchange cromatography column DEAE-cellulose, and were obtained two activities: ß-glucosidase extracellular I and II. The ß-glucosidase I was applied to a CM-cellulose colunm, while ß-glucosidase II was applied to a Sephadex G-100 colunm. The ß-glucosidase II was purified two times and 4% yield, and the ß-glucosidase II was purified 2,4 times and 2% yield. The mycelial ß-glucosidase was purified using the termic treatment, a precipitation with 75% amonium sulfate followed by Sephadex G-100 and DEAEcellulose. The enzyme was purified 23 time with 23% yield. The ß-glucosidase extracellular II and mycelial shown optima of temperature and pH of 60°C and 70°C, 4.4 and 6.0, respectively. Hg+2 and Ag+2 ions were strong inhibitors of ß-glucosidase extracellular I and mycelial. The molecular weight of ß-glucosidase extracellular I and mycelial was stimated as 40.7 KDa and 39KDa (SDS-PAGE) and 57kDa and 33.8 kDa (Sephadex G-100). The ß-glucosidase extracellular I hydrolyzed PNP-glu, PNP-xyl, cellobiose,xylan and CMC, while ß-glucosidase mycelial hydrolyzed PNP-fuc, PNP-xyl, PNP-gal, ONPG and lactose. Both enzymes were activeted by glycerol 1M. The ß-glucosidase extracellular I was activeted by xylose, fructose and lactose, and show strong at glucose 50mM. The ß-glucosidase mycelial was activeted by glucose and xylose. ß-glucosidase extracellular I and mycelial shows PI 4.0 and 6.5, respectively. The kinects studies reveled for ß-glucosidase extracellular I a Km of 4,33 and 0,342mM and Vmáx of 5,37 and 2,0µmoles/min/mg prot for cellobiose and PNP-glu, respectively. The Ki values obtained from Dixon plots was 71mM for glucose. To ß-glucosidase mycelial the Km and and Vmáx were 0,29mM e 13,27µmoles/min/mg prot; 0,5 mM e 7,25µmoles/min/mg prot and 1,61 mM and 4,12µmoles/min/mg prot for PNP-glu, PNPfuc and cellobiose, respectively. Using xylose or glucose the Km and Vmáx was 1,26mM e 40,04 µmoles/min/mg.prot, and 1,33mM, e 30,49 µmoles/min/mg prot, respectively for PNP-glu. The Ki values obtained from Dixon plots was 1,32mM using cellobiose. The products of hydrolisis of cellobiose by the action of purified enzymes glucosidase extracellular I and mycelial were analised in thin-layer-cromatography, and show hydrolisis of cellobiose at 10mM,and transglycosilation reaction when cellobiose was using at 250mM. The intrinsic biochemical and regulatory properties the ß-glucosidase system of Scytalidium support the idea that organism may be useful for biotechnological applications.

A celulose é a mais abundante fonte de carbono presente na madeira e nos resíduos agrícolas, e a sua hidrólise completa é realizada pela ação sinergística de diferentes enzimas, como: as endo-1,4-ß-D-glucanase, exo-1,4-ß-glucanase e ß-glucosidase ou celobiase. O presente trabalho descreve algumas propriedades fisiológicas e bioquímicas do sistema ß-glucosidásico do fungo termofílico Scytalidium thermophilum. Tal fungo foi isolado originalmente do solo da Índia e gentilmente cedido pelo Dr. G. Straastma (Holanda). O meio M8 favoreceu a produção das ß-glucosidases. Entre os açúcares testados como fonte de carbono, avicel e celobiose foram os melhores indutores das ß-glucosidases extracelular e micelial. Quando o fungo foi crescido em dois estágios, observou-se inicialmente a repressão da síntese por glicose e a indução por avicel ou celobiose. Utilizando-se ciclo-heximida, observou-se a síntese \"de novo\" das proteínas. A ß-glucosidase extracelular foi purificada utilizando-se um fracionamento protéico e uma coluna de troca-iônica DEAE-celulose, de onde foram obtidos duas atividades enzimáticas denominadas ß-glucosidases I e II. A ß-glucosidase I foi aplicada em coluna de troca iônica CM-celulose, enquanto que a ß-glucosidase II foi aplicada em Sephadex G-100. A ß-glucosidase I foi purificada 2 vezes com 4.0% de recuperação, ao passo que a ß-glucosidase II foi purificada 2,4 vezes com 2.0% de recuperação. A ß-glucosidase micelial foi purificada utilizando-se um choque térmico, fracionamento protéico, coluna de filtração Sephadex G-100 e uma coluna troca-iônica DEAE-celulose. Foi purificada 23 vezes com recuperação de 25%. A ß-glucosidases extracelular I e micelial apresentaram um temperatura ótima aparente de 70 e 60°C e um pH de 5.5 e 6.0, respectivamente. Ambas enzimas foram inibidas por Ag+2 e Hg+2. A ß-glucosidases extracelular I e micelial possuem um peso molecular de 40.7 kDa e 39kda (SDS-Page) e 57 kDa e 33,8 kda (Sephadex G-100), respectivamente. A ß-glucosidase extracelular I foi capaz de hidrolisar PNP-glu, PNP-xil, celobiose, xilana e CMC, enquanto que a ß-glucosidase micelial hidrolisou PNP-glu, PNP-fuc, PNP-xil, PNPgal, ONPG e lactose. Ambas enzimas foram ativadas por glicerol a 1M. A ß-glucosidase extracelular I foi ativada por xilose, frutose e lactose, e se mostrou resistente a glicose 50mM, enquanto que a ß-glucosidase micelial foi ativada por glicose e xilose. ß-glucosidases extracelular I e micelial apresentaram um PI de 4.0 e 6.5, respectivamente. Os parâmetros cinéticos estimados para a ß-glucosidase extracelular I foram de Km 4,33 e 0,342mM e Vmáx de 5,37 e 2,0µmoles/min/mg prot. para celobiose e PNP-glu, respectivamente. O valor de Ki (Constante de Inibição) foi de 71mM para glicose. Para a ß-glucosidase micelial, os valores de Km e Vmáx foram de 0,29mM e 13,27µmoles/min/mg prot; 0,5 mM e 7,25µmoles/min/mg prot e 1,61 mM e 4,12µmoles/min/mg prot para os substratos PNP-glu, PNP-fuc e celobiose, respectivamente. Na presença de glicose e xilose os valores de Km e Vmáx foram de 1,26mM e 40,04 µmoles/min/mg.prot, e 1,33mM, e 30,49 µmoles/min/mg prot, respectivamente para o PNP-glu. O valor de Ki (Constante de Inibição) foi de 1,32mM para celobiose. A análise dos produtos de hidrólise das ß-glucosidases extracelular I e micelial foram anlisadas em TLC, e revelaram que ambas enzimas realizam hidrólise quando celobiose foi utilizada a 10mM, e transglicosilação quando celobiose foi utilizada a 250mM. Os resultados aqui apresentados demonstram importante papel importante do Scytalidium como produtor de ß-glucosidase com potencial na sacarificação enzimática da celulose. / Cellulose is the most abundant carbon source found in woods and waste residues. In nature the complete hidrolysis of cellulose occurs by the sinergistic action of several enzymes such endo-1,4-ß-D-glucanase, exo-1,4-ß-glucanase e ß-glucosidase or cellobiase. The present work describe some physiological and biochemical properties of ß-glucosidase system from thermophilic fungus Scytalidium thermophilum. The fungus was gift to Dr. Straastma (Mushroom Experimental Station, The Netherlands). The culture medium M8 enhance the production of ß-glucosidase. Among carbohydrates tested as carbon source, avicel and cellobiose were the best inducers of ß-glucosidase extracellular and mycelial. When the fungus was grown in two stages, observed the repression by glucose, and induction by avicel or cellobiose. The presence of cycloheximide inhibited the syntesis of ß-glucosidase, suggesting that the enzyme produced in the presence of indutors required \"de novo\" synthesis. Extracellular ß-glucosidase was purified using the precipitation with 75% amonium sulfate, ion exchange cromatography column DEAE-cellulose, and were obtained two activities: ß-glucosidase extracellular I and II. The ß-glucosidase I was applied to a CM-cellulose colunm, while ß-glucosidase II was applied to a Sephadex G-100 colunm. The ß-glucosidase II was purified two times and 4% yield, and the ß-glucosidase II was purified 2,4 times and 2% yield. The mycelial ß-glucosidase was purified using the termic treatment, a precipitation with 75% amonium sulfate followed by Sephadex G-100 and DEAEcellulose. The enzyme was purified 23 time with 23% yield. The ß-glucosidase extracellular II and mycelial shown optima of temperature and pH of 60°C and 70°C, 4.4 and 6.0, respectively. Hg+2 and Ag+2 ions were strong inhibitors of ß-glucosidase extracellular I and mycelial. The molecular weight of ß-glucosidase extracellular I and mycelial was stimated as 40.7 KDa and 39KDa (SDS-PAGE) and 57kDa and 33.8 kDa (Sephadex G-100). The ß-glucosidase extracellular I hydrolyzed PNP-glu, PNP-xyl, cellobiose,xylan and CMC, while ß-glucosidase mycelial hydrolyzed PNP-fuc, PNP-xyl, PNP-gal, ONPG and lactose. Both enzymes were activeted by glycerol 1M. The ß-glucosidase extracellular I was activeted by xylose, fructose and lactose, and show strong at glucose 50mM. The ß-glucosidase mycelial was activeted by glucose and xylose. ß-glucosidase extracellular I and mycelial shows PI 4.0 and 6.5, respectively. The kinects studies reveled for ß-glucosidase extracellular I a Km of 4,33 and 0,342mM and Vmáx of 5,37 and 2,0µmoles/min/mg prot for cellobiose and PNP-glu, respectively. The Ki values obtained from Dixon plots was 71mM for glucose. To ß-glucosidase mycelial the Km and and Vmáx were 0,29mM e 13,27µmoles/min/mg prot; 0,5 mM e 7,25µmoles/min/mg prot and 1,61 mM and 4,12µmoles/min/mg prot for PNP-glu, PNPfuc and cellobiose, respectively. Using xylose or glucose the Km and Vmáx was 1,26mM e 40,04 µmoles/min/mg.prot, and 1,33mM, e 30,49 µmoles/min/mg prot, respectively for PNP-glu. The Ki values obtained from Dixon plots was 1,32mM using cellobiose. The products of hydrolisis of cellobiose by the action of purified enzymes glucosidase extracellular I and mycelial were analised in thin-layer-cromatography, and show hydrolisis of cellobiose at 10mM,and transglycosilation reaction when cellobiose was using at 250mM. The intrinsic biochemical and regulatory properties the ß-glucosidase system of Scytalidium support the idea that organism may be useful for biotechnological applications.

In this study, Scytalidium thermophilum laccase gene was first cloned into E. coli and then heterologously expressed in A. sojae. S. thermophilum is a thermophilic fungus with an important role in determining selectivity of compost produced for growing Agaricus bisporus. S. thermophilum laccase gene was first cloned by Novo Nordisk Bio Tech, Inc. in 1998. This laccase gene (lccS) has an open reading frame of 2092bp. It is composed of five exons punctuated by four small introns. The coding region, excluding intervening sequences is very GC-rich (60.8% G+C) and encodes a preproenzyme of 616 amino acids: a 21 amino acid signal peptide and a 24 amino acid predicted propeptide. lccS gene was amplified using specific primers to exclude the signal and pro-peptide coding regions and ligated to expression vector pAN52-4. The recombinant plasmid was used to transform Aspergillus sojae ATCC11906 (pyrG-). Heterologuos expression was observed in glucose-containing media, under the control of the glyceraldehydes 3-phosphate dehydnogenese promoter and the secretion signal of glucoamylase gene. Laccase gene is an important step towards the high level expression of this enzyme in a GRAS eucaryotic host and for further biotransformation and enzyme engineering studies. In this study also bioinformatic analysis of N-terminal and C-terminal propeptide cleavage sites of fungal proteins including laccases were studied.

Scytalidium thermophilum is a termophilic fungus that effectively produces the extracellular enzyme catalase phenol oxidase (CATPO). The enzyme is distinct among catalases with its bifunctionality of oxidising phenolic compounds in the absence of H2O2. CATPO is capable of oxidizing catechol, chlorogenic acid, caffeic acid and catechin which are ortho &ndash / diphenolic compounds. Diphenolic compounds are known as strong antioxidants. Catalase is one of the important antioxidant enzymes. Therefore, in this thesis the effect of CATPO on the final antioxidant capacity of the oxidized products was analysed. Antioxidant capacity measurements of oxidized and unreacted phenolic compounds were done using the two widely used methods TEAC and FRAP. CATPO oxidized catechol showed 2.4 fold increase when compared to its nonoxidized form, which was highest among others. Catechol was followed by caffeic acid, chlorogenic acid, and catechin. This finding is new to the literature and may be of importance to the antioxidant mechanism of organisms. Results have also shown that the most well known phenol oxidases, laccase and tyrosinase, do not result in such high increases in antioxidant capacity upon oxidation of the substrates tested. Due to this finding, as a possible means of applying CATPO to increase the antioxidant capacity of products daily consumed, tea was selected. Tea is the second most consumed beverage after water and it is known to possess high amounts of flavanols. Green tea is rich in catechins whereas black tea is a rich source of theaflavins and thearubigins. Fermentation is a critical process for production of good quality tea and is the key step differing between green and black tea production. During this process phenol oxidases catalyze the oxidation of polyphenolic compounds present in tea leaves to their corresponding o-quinones. Utilization of CATPO in tea samples resulted in an increase in antioxidant capacity and its effect was enhanced by an increase in brewing time. Interestingly, the addition of sugar decreased antioxidant capacity. Laccase and tyrosinase were ineffective in increasing the antioxidant capacity of tea samples.

Endo-b-1,4 xylanases (EC. 3.2.1.8) are typically produced as a mixture of different hydrolytic enzymes such as b-1,4-xylosidase (EC. 3.2.1.37) , a-Larabinofuranosidases (EC. 3.2.1.55), and feruloyl esterase (EC 3.1.1.73) that hydrolyze xylan molecule, which constitutes 20-30% of the weight of wood and agricultural wastes. Thus, xylan, a renewable biomass, can be utilized as a substrate for the preparation of many products such as fuels, solvents and pharmaceuticals. Besides, xylanolytic enzymes themselves are also used in food,feed, textile industries and pre-bleaching of kraft. In the first part of the study, xylanolytic systems of a soil isolate Bacillus pumilus SB-M13 and a thermophilic fungus Scytalidium thermophilum were investigated. Production rate and type of xylanolytic changed depending on the carbon source and the microorganism. However, xylanolytic enzyme production was found to be sequential, in synergy and under the control of carbon catabolite repression for both microorganisms. In the second part, B. pumilus SB-M13 b-1,4 xylanase was purified and biochemically characterized. The enzyme was stable at alkaline pHs and highest activity was observed at 60&deg / C and pH 7.5. Enzyme Km and kcat values were determined as 1.87 mg/ml and 43,000 U/mg, respectively. B. pumilus SB-M13 and S .thermophilum a-L-arabinofuranosidases were also purified and biochemically characterized. Although produced from a mesophilic microorganism, B. pumilus SB-M13 arabinofuranosidase was quite thermostable. Moreover, unlike other fungi, S. thermophilum produced alkaline stable arabinofuranosidases. Both enzymes were multimeric, alkaline stable and most active at 70&deg / C and pH 7.0. However, when compared to S. thermophilum, catalytic power of B. pumilus SB-M13 arabinofuranosidase was higher.

In this study, the aim was identification and classification of the enzyme having phenol oxidase activity produced by a thermophilic fungus, Scytalidium thermophilum. For this purpose, enzyme production, purification, biochemical characterization and structural analysis by X-ray crystallography studies have been performed. At the beginning of the research, this enzyme was considered as a phenol oxidase and analyzed accordingly. However, during purification, amino acid sequencing and structural studies, the enzyme was shown to be a catalase, with an additional catechol oxidase activity. This novel bifunctional catalase-catechol oxidase (CCO) was purified 10 fold with 45 % yield by anion exchange and gel filtration chromatographies. CCO was determined as a tetrameric protein having total and subunit molecular weights of 320 and 80 kDa, respectively. Isoelectric point of CCO was verified as 5.0. CCO catalase and catechol oxidase activities were characterized in terms of their kinetic behavior at different pH and temperatures. Depending on the substrate specificity and inhibitor studies of CCO, the phenol oxidase activity was determined as catechol oxidase but not tyrosinase or laccase. The best crystallization condition for CCO was determined and X-ray diffraction data was collected at the Daresbury Synchrotron Radiation Source (United Kingdom) at 2.7 &Aring / resolution. The preliminary structure was solved by molecular replacement method using Penicilium vitale catalase structure. CCO was verified to have a tetrameric structure with two homodimers and a metal center in each polypeptide chain.

Scytalidium thermophilum is a thermophilic fungus with an important role in the composting process of mushroom cultivation. An extracellular phenol oxidase of Scytalidium thermophilum (STEP) with novel features was previously studied in our laboratory. This enzyme later turned out to be a catalase having phenol oxidase activity. The aim of this study was to clone Scytalidium thermophilum bifunctional catalase/phenol oxidase encoding gene and express the gene in Aspergillus sojae for future site directed mutagenesis studies. Scytalidium thermophilum catalase gene was first cloned into E. coli XL1 Blue MRF&rsquo / and then heterologously expressed in Aspergillus sojae ATCC11906. For that aim, the catalase gene was amplified using specific primers, excluding the signal and pro-peptide coding regions and amplified fragment was then cloned into E.coli XL1 Blue MRF&rsquo / and sequenced. It was observed that the cloned gene, named as catpo, was 10 amino acids different from the amino acid sequence of the S.thermophilum catalase gene formerly cloned by Novo Nordisk. The catpo gene encoding a mature protein of 681 amino acids was then ligated onto expression vector pAN52-4 and the recombinant plasmid was transformed into Aspergillus sojae ATCC11906. Heterologous expression was observed under the control of the glyceraldehydes 3-phosphate dehydrogenese promoter of Aspergillus nidulans and the secretion signal of the glucoamylase gene of Aspergillus niger. Catalase activity of the transformants reached at a level of 13206 U/g at the end of the fourth day of cultivation. However, this is still lower than the catalase activity of the gene donor strain of Scytalidium thermophilum.

Scytalidium thermophilum produces a novel phenol oxidase, which has turned out to be a bifunctional catalase-phenol oxidase (CATPO) during the course of this work, by other researchers of our group. Therefore, in the beginning of the studies, substrate specificity and inhibitor assays were conducted on the crude enzyme, followed by production, purification, cloning, expression, and mutagenesis and crystallography studies for further functional and structural analysis of CATPO. Accordingly, substrate specificity and inhibitory tests applied for crude enzyme characterisation presented the similarity of the phenol oxidase nature of CATPO essentially to catechol oxidase. Production studies were performed to investigate the effects of different factors including induction time, growth temperature, exogenous iron and hydrogen peroxide addition. In view of that, CATPO is constitutively produced in a growth associated manner. However, some phenolic compounds enhance its production. In this study, 15 phenolic compounds were tested for their ability to affect CATPO production. Of the phenolic compounds tested, catechol, resorcinol and vanillic acid caused repression of CATPO production. On the other hand, caffeic acid, myricetin and resveratrol enhanced CATPO production. As a biocatalyst, the efficiency of CATPO was examined and found to be a good candidate for getting pharmaceutically important drug intermediates. Its dual mechanism was analysed through side-directed mutagenesis. Two conserved residues (His101 and Val142) were mutated to discriminate catalase and phenol oxidase activities. Spectroscopic and mutagenesis studies exhibited the presence of heme d centre. Lastly, its structure was analysed by X-ray crystallography and found to have a tetrameric structure.

This thesis was aimed to analyze phenol oxidation by the bifunctional catalase/phenol oxidase of the thermophilic fungus Scytalidium thermophilum. Several reactive oxygen species (ROS) are continuously produced in fungi under oxidative stress. Depending on the nature of the ROS species, some are highly toxic and are rapidly detoxified by various cellular enzymatic mechanisms, including the production of catalase. S. thermophilum produces a novel bifunctional catalase-phenol oxidase (CATPO) which is capable of oxidizing phenolics in the absence of hydrogen peroxide. Phenol oxidases convert phenolic compounds to quinones, which are then polymerized mainly by free- radical mediated reactions. In this study, 14 phenolic compounds were selected according to their different chemical structures and functional properties and were analyzed as substrates of CATPO. Among 14 phenolic compounds, only in catechol, chlorogenic acid, catechin and caffeic acid distinct oxidation products were observed by HPLC. The oxidation products of catechol, caffeic acid, chlorogenic acid and catechin were characterized by LC-ESI-MS. Dimer, trimer, tetramer and oligomer formations were detected. While the maximum conversion efficiency, at 1 hour of reaction, was observed with catechin, minimum conversion efficiency was attained by caffeic acid, under the specified conditions. The oxidation products observed after oxidation of catechol, chlorogenic acid, catechin and caffeic acid by CATPO was compared with the same phenolic compounds oxidation products oxidized by laccase and tyrosinase. CATPO was incapable of oxidizing tyrosinase and laccase-specific substrates tyrosine and ABTS respectively. However, the oxidizing spectrum of substrates indicates that the nature of phenol oxidation by CATPO appears to resemble mainly those of laccase.

ABSTRACT UTILIZATION OF SCYTALIDIUM THERMOPHILUM PHENOL OXIDASE IN BIOORGANIC SYNTHESES Kaptan, Yelda M.S., Department of Biotechnology Supervisor: Prof. Dr. Z&uuml / mr&uuml / t B. &Ouml / gel Co-supervisor: Prof Dr. Ufuk Bakir September 2004, 90 pages In this study, the ultimate aim was to utilize phenol oxidases of Scytalidium thermophilum in bioorganic syntheses. For this purpose, studies were conducted towards enhancing the production of phenol oxidases by Scytalidium thermophilum, developing a suitable method for laccase activity assays, analyzing the effects of organic solvents on phenol oxidase activity and analysis of the biotransformation of a number of organic substrates by phenol oxidases of Scytalidium thermophilum. In order to enhance the production of phenol oxidases, induction experiments were carried out with gallic acid, syringaldazine and chlorogenic acid. Gallic acid was found as the most effective inducer for phenol oxidase production. Inductive effect of edible mushroom Agaricus bisporus was also assayed, however, the phenolic compounds released by mushroom did not represent any induction for phenol oxidase activity of Scytalidium thermophilum. Different substrates were tested and catechol was determined as the most suitable substrate rather than syringaldazine and ABTS. Molar extinction coefficient (e) of catechol was calculated as 3450 M-1 cm-1 and 3700 M-1 cm-1 by using &ldquo / substrate blank&rdquo / and &ldquo / enzyme blank&rdquo / respectively at 420 nm. Kinetic parameters, Km and Vmax for the enzymatic reactions in which catechol was used as substrate were calculated as 52.03 mM and 0.253 U/ml respectively from Lineweaver-Burk plot and as 41.25 mM and 0.2055 U/ml from Hanes-Woolf plot. Effect of some organic solvents on phenol oxidases of Scytalidium thermophilum was assayed and DMSO was found as an appropriate solvent for the organic substrates. Phenol oxidase containing culture supernatant could oxidize benzoin, hydrobenzoin and benzoyl benzoin.