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.

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.

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.

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.