Resumo Poligalacturonases são enzimas com potencial biotecnológico para aplicação na clarificação de sucos de frutas e para aumento da eficiência da filtragem. O objetivo deste trabalho foi avaliar a produção de poligalacturonase pelo fungo Neosartorya glabra, por fermentação submersa e em estado sólido, usando resíduos de casca de frutas como fonte de carbono, além de avaliar também a aplicação desta enzima na clarificação e na diminuição de viscosidade de sucos de maracujá e maçã. A maior produção de poligalacturonase (4,52 U/g/h) foi obtida por fermentação submersa em meio de Vogel (1964) contendo cascas de laranja (Citrus sinensis), variedade Bahia, na concentração 1,5% (m/v, massa seca), a 30-35°C por 72 h. A poligalacturonase do extrato bruto apresentou atividade ótima a 50-60°C e pH 5,5. A enzima reteve cerca de 90% da atividade inicial após 180 minutos, a 40°C, e 50% da atividade inicial após 150 minutos, a 50°C. A enzima mostrou-se estável em pH ácido (3,0 a 6,5) após 120 minutos, a 25°C. Todas estas propriedades enzimáticas fazem desta poligalacturonase uma alternativa atrativa para a aplicação na indústria de sucos de frutas ricas em pectina, uma vez que a aplicação do extrato enzimático bruto no processamento de suco de polpa de maracujá (Passiflora edulis) causou uma redução da viscosidade de 80% e uma diminuição da absorbância da luz de 75%. No processamento de suco de polpa de maçã (Malus domestica), houve uma redução da viscosidade de 50% e uma diminuição da absorbância da luz de 78%.

Summary Polygalacturonases are enzymes with the biotechnological potential for use in fruit juice clarification and for the enhancement of filtration efficiency. The aim of this work was to assess the production of polygalacturonase by the fungus Neosartorya glabra by means of solid-state and submerged fermentation using fruit peel residues as the carbon source, and also apply the enzyme in the clarification and decrease in viscosity of passion fruit and apple juices. The highest polygalacturonase (4.52 U/g/h) production was obtained by means of submerged fermentation in Vogel´s medium (1964) containing orange peel – Bahia variety (Citrus sinensis), at a concentration of 1.5% (w/v, dried mass) at 30-35°C for 72 h. The polygalacturonase of the crude extract presented optimal activity at 60°C and pH 5.5. The enzyme retained around 90% of the initial activity after 180 minutes at 40°C, and 50% of the initial activity after 150 minutes at 50°C. The enzyme was shown to be stable at acid pH values (3.0-6.5) after 120 minutes at 25oC. All these favourable enzymatic properties make the polygalacturonase attractive for potential uses in the industry of pectin-rich fruit juices, since the application of the crude extract to passion fruit (Passiflora edulis) juice caused an 80% reduction in viscosity and 75% decrease in light absorbance. In the processing of apple pulp juice (Malus domestica), there was a 50% reduction in viscosity and 78% decrease in light absorbance.

Background Tannases are enzymes with biotechnological potential produced mainly by microorganisms as filamentous fungi. In this context, the production and characterization of a multi-tolerant tannase from Aspergillus carbonarius is described. Results The filamentous fungus A. carbonarius produced high levels of tannase when cultivated under solid-state fermentation using green tea leaves as substrate/carbon source and tap water at a 1:1 ratio as the moisture agent for 72 h at 30°C. Two tannase activity peaks were obtained during the purification step using DEAE-Cellulose. The second peak (peak II) was purified 11-fold with 14% recovery from a Sepharose CL-6B chromatographic column. The tannase from peak II (tannase II) was characterized as a heterodimeric glycoprotein of 134.89 kDa, estimated through gel filtration, with subunits of 65 kDa and 100 kDa, estimated through SDS-PAGE, and 48% carbohydrate content. The optimal temperature and pH for tannase II activity was 60°C and 5.0, respectively. The enzyme was fully stable at temperatures ranging from 20-60°C for 120 min, and the half-life (T1/2) at 75°C was 62 min. The activation energy was 28.93 kJ/mol. After incubation at pH 5.0 for 60 min, 75% of the enzyme activity was maintained. However, enzyme activity was increased in the presence of AgNO3 and it was tolerant to solvents and detergents. Tannase II exhibited a better affinity for methyl gallate (Km = 1.42 mM) rather than for tannic acid (Km = 2.2 mM). Conclusion A. carbonarius tannase presented interesting properties as, for example, multi-tolerance, which highlight its potential for future application.

Plant cell wall is mainly composed by cellulose, hemicellulose and lignin. The heterogeneous structure and composition of the hemicellulose are key impediments to its depolymerization and subsequent use in fermentation processes. Thus, this study aimed to perform a screening of thermophilic and thermotolerant filamentous fungi collected from different regions of the São Paulo state, and analyze the production of β-xylosidase and arabinanase at different temperatures. These enzymes are important to cell wall degradation and synthesis of end products as xylose and arabinose, respectively, which are significant sugars to fermentation and ethanol production. A total of 12 fungal species were analyzed and 9 of them grew at 45 ºC, suggesting a thermophilic or thermotolerant character. Additionally Aspergillus thermomutatus anamorph of Neosartorya and A. parasiticus grew at 50 ºC. Aspergillus niger and Aspergillus thermomutatus were the filamentous fungi with the most expressive production of β-xylosidase and arabinanase, respectively. In general for most of the tested microorganisms, β-xylosidase and arabinanase activities from mycelial extract (intracellular form) were higher in cultures grown at high temperatures (35-40 ºC), while the correspondent extracellular activities were favorably secreted from cultures at 30 ºC. This study contributes to catalogue isolated fungi of the state of São Paulo, and these findings could be promising sources for thermophilic and thermotolerant microorganisms, which are industrially important due to their enzymes.

Background: Tannases are enzymes that may be used in different industrial sectors as, for example, food and pharmaceutical. They are obtained mainly from microorganisms, as filamentous fungi. However, the diversity of fungi stays poorly explored for tannase production. In this article, Aspergillus ochraceus is presented as a new source of tannase with interesting features for biotechnological applications. Results: Extracellular tannase production was induced when the fungus was cultured in Khanna medium with tannic acid as carbon source. The extracellular tannase was purified 9-fold with 2% recovery and a single band corresponding to 85 kDa was observed in SDS-PAGE. The native apparent molecular mass was estimated as 112 kDa. Optima of temperature and pH were 40ºC and 5.0, respectively. The enzyme was fully stable from 40ºC to 60ºC during 1 hr. The activity was enhanced by Mn2+ (33-39%) and NH4+ (15%). The purified tannase hydrolyzed tannic acid and methyl gallate with Km of 0.76 mM and 0.72 mM, respectively, and Vmax of 0.92 U/mg protein and 0.68 U/mg protein, respectively. The analysis of a partial sequence of the tannase encoding gene showed an open read frame of 567 bp and a sequence of 199 amino acids were predicted. TLC analysis revealed the presence of gallic acid as a tannic acid hydrolysis product. Conclusion: The extracellular tannase produced by A. ochraceus showed distinctive characteristics such as monomeric structure and activation by Mn2+, suggesting a new kind of fungal tannases with biotechnological potential. Further, it was the first time that a partial gene sequence for A. ochraceus tannase was described.

Aspergillus phoenicis é um fungo termotolerante interessante, uma vez que pode sintetizar enzimas com diversas aplicações em indústrias alimentícias em função de seu grande potencial de hidrólise. Neste trabalho, verificou-se que esse fungo produziu níveis enzimáticos elevados, quando o mesmo foi cultivado em meio de cultura de baixo custo, constituído de flocos de diferentes origens, como farinha de mandioca, fibra de trigo, soja triturada, resíduos agroindustriais, amido e glicose ou maltose. Diversos sistemas enzimáticos foram produzidos a partir dessas fontes de carbono, mas a amilase foi a mais evidente, seguida de pectinase e xilanase. Traços de atividades de CMcases, avicelase, lipase, β-xilosidase, β-glicosidase e α-glicosidase também foram detectados. Amilases foram produzidas em flocos de centeio, amido, flocos de aveia, flocos de milho, farinha de mandioca e fibra de trigo. Níveis amilolíticos significantes foram produzidos em meio de cultura contendo glicose ou quando esse açúcar não estava mais presente, sugerindo uma forma constitutiva dessa enzima. Farinha de mandioca, flocos de centeio, aveia, cevada e milho também foram utilizados como substratos nas reações de hidrólise, com o objetivo de verificar a potencial liberação de açúcares redutores. Flocos de milho induziram maior formação de açúcares redutores, quando comparados com os outros substratos. A análise por cromatografia em camada delgada dos produtos finais da reação revelou que a hidrólise da farinha de mandioca liberou malto-oligossacarídeos, mas farinha de mandioca e flocos de milho, centeio, aveia e cevada foram hidrolisados até glicose. Esses resultados sugeriram a presença de glucoamilase e α-amilase como parte da mistura enzimática de A. phoenicis.

Aspergillus phoenicis is an interesting heat tolerant fungus that can synthesize enzymes with several applications in the food industry due to its great hydrolytic potential. In this work, the fungus produced high enzymatic levels when cultivated on inexpensive culture media consisting of flakes from different origins such as cassava flour, wheat fibre, crushed soybean, agro-industrial wastes, starch, glucose or maltose. Several enzymatic systems were produced from these carbon sources, but amylase was the most evident, followed by pectinase and xylanase. Traces of CMCases, avicelase, lipase, β-xylosidase, β-glucosidase and α-glucosidase activities were also detected. Amylases were produced on rye flakes, starch, oat flakes, corn flakes, cassava flour and wheat fibre. Significant amylolytic levels were produced in the culture medium with glucose or when this sugar was exhausted, suggesting an enzyme in the constitutive form. Cassava flour, rye, oats, barley and corn flakes were also used as substrates in the hydrolytic reactions, aiming to verify the liberation potential of reducing sugars. Corn flakes induced greater liberation of reducing sugars as compared to the others. Thin layer chromatography of the reaction end products showed that the hydrolysis of cassava flour liberated maltooligosaccharides, but cassava flour and corn, rye, oats and barley flakes were hydrolyzed to glucose. These results suggested the presence of glucoamylase and α-amylase as part of the enzymatic pool of A. phoencis.

The effect of several nutritional and environmental parameters on Penicillium purpurogenum growth and sacharogenic amylase production was analyzed. High enzyme levels (68.2 U mg-1) were obtained with Khanna medium at initial pH 6.0, incubated at 30ºC for 144 hours. The optimum pH and temperature activities were 5.0 and 65ºC, respectively. The enzyme presented a half-life (t50) of 60 min, at 65ºC. Only glucose was detected after 24 hours of reaction using soluble starch as substrate.

The filamentous fungus A. phoenicis produced high levels of ?-D-fructofuranosidase (FFase) when grown for 72 hrs under Solid-State Fermentation (SSF), using soy bran moistened with tap water (1:0.5 w/v) as substrate/carbon source. Two isoforms (I and II) were obtained, and FFase II was purified 18-fold to apparent homogeneity with 14% recovery. The native molecular mass of the glycoprotein (12% of carbohydrate content) was 158.5 kDa with two subunits of 85 kDa estimated by SDS-PAGE. Optima of temperature and pH were 55�C and 4.5. The enzyme was stable for more than 1 hr at 50�C and was also stable in a pH range from 7.0 to 8.0. FFase II retained 80% of activity after storage at 4�C by 200 hrs. Dichroism analysis showed the presence of random and ?-sheet structure. A. phoenicis FFase II was activated by Mn2+, Mg2+ and Co2+, and inhibited by Cu2+, Hg2+ and EDTA. The enzyme hydrolyzed sucrose, inulin and raffinose. Kd and Vmax values were 18 mM and 189 U/mg protein using sucrose as substrate.

Fungi collected from Brazilian soil and decomposing plants were screened for pectinase production. R. microsporus var. rhizopodiformis was the best producer and was selected to evaluate the pectic enzyme production under several nutritional and environmental conditions. The pectinase production was studied at 40ºC, under 28 carbon sources-supplemented medium. The inducer effect of several agro-industrial residues such as sugar cane bagasse, wheat flour and corncob on polygalacturonase (PG) activity was 4-, 3- and 2-fold higher than the control (pectin). In glucose-medium, a constitutive pectin lyase (PL) activity was detected. The results demonstrated that R. microsporus produced high levels of PG (57.7 U/mg) and PL (88.6 U/mg) in lemon peel-medium. PG had optimum temperature at 65 ºC and was totally stable at 55 ºC for 90 min. Half-life at 70 ºC was 68 min. These results suggested that the versatility of waste carbon sources utilization by R. microsporus, produce pectic enzymes, which could be useful to reduce production costs and environmental impacts related to the waste disposal.

O fungo filamentoso Aspergillus caespitosus foi um bom produtor de invertases intracelular e extracelular em fermentação submersa (FSbm) ou em estado sólido (FES), usando resíduos agroindustriais como fonte de carbono, sendo que para ambas as condições de cultivo, a maior produtividade foi obtida empregandose farelo de trigo. A produção da forma extracelular em FES mantido a 30ºC, por 72 horas, foi aumentada usandose solução de sais SR (1:1, m/v) para umidificar o substrato, sendo aproximadamente 5,5 vezes maior se comparada a FSbm (Meio Khanna) com a mesma fonte de carbono. Entretanto, a mistura de farelo de trigo e farinha de aveia em FES levou a um aumento de 2,2 vezes na produção enzimática se comparada ao uso isolado do farelo de trigo. A produção enzimática, em ambas as condições de cultivo, foi afetada pela adição suplementar de fontes de nitrogênio e fosfato. A adição de glicose em FSbm e em FES promoveu a diminuição da enzima extracelular, mas favoreceu um acúmulo intracelular de 35 vezes maior. A temperatura ótima de atividade para as invertases produzidas em FES e em FSbm foi de 50ºC e 60ºC, respectivamente, sendo estáveis a 50ºC por mais de 60 minutos. Todas as formas enzimáticas apresentaram atividade máxima em uma faixa de pH de 4.0-6.0.

The filamentous fungus Aspergillus caespitosus was a good producer of intracellular and extracellular invertases under submerged (SbmF) or solid-state fermentation (SSF), using agroindustrial residues, such as wheat bran, as carbon source. The production of extracellular enzyme under SSF at 30ºC, for 72h, was enhanced using SR salt solution (1:1, w/v) to humidify the substrate. The extracellular activity under SSF using wheat bran was around 5.5-fold higher than that obtained in SbmF (Khanna medium) with the same carbon source. However, the production of enzyme with wheat bran plus oat meal was 2.2-fold higher than wheat bran isolated. The enzymatic production was affected by supplementation with nitrogen and phosphate sources. The addition of glucose in SbmF and SSF promoted the decreasing of extracellular activity, but the intracellular form obtained in SbmF was enhanced 3-5-fold. The invertase produced in SSF exhibited optimum temperature at 50ºC while the extraand intracellular enzymes produced in SbmF exhibited maximal activities at 60ºC. All enzymatic forms exhibited maximal activities at pH 4.0-6.0 and were stable up to 1 hour at 50ºC.