Immobilization of the Tannase From Aspergillus fumigatus CAS21: Screening the Best Derivative for the Treatment of Tannery Effluent Using a Packed Bed Reactor.

Enzyme immobilization is an important alternative to stabilize enzyme properties favoring the efficiency of derivatives (enzyme + support/matrix) for different purposes. According to this, the current study aimed to immobilize the Aspergillus fumigatus CAS21 tannase and the use of the derivatives in the treatment of the effluent produced by the tannery industry. The tannase was immobilized on sodium alginate, DEAE-Sephadex, amberlite, and glass pearls as supports. Calcium alginate was the most adequate support for tannase immobilization with 100% yield and 94.3% for both efficiency and activity. The best tannase activity for the calcium alginate derivative was obtained at 50°C-60°C and pH 5.0. Thermal and pH stabilities evaluated for 24 h at 30°C-60°C and pH 4-7, respectively, were improved if compared to the stability of the free enzyme. Considering the reuse of the calcium alginate derivative, 78% of the initial activity was preserved after 10 catalytic cycles, and after the 9-month storage at 4°C, the activity was maintained in 70%. This derivative was applied in a packed bed reactor (PBR) for the treatment of tannin-rich effluents from the tannery industry. The reduction of the tannin content was effective reaching degradation of 74-78% after 48 h of PBR operation. The concentration of total phenolic compounds was also reduced, and the color and clarity of the effluent improved. In conclusion, the calcium alginate derivative is an attractive alternative as biocatalyst for large-scale treatment of the effluents from the tannery industry.

Expression of F-actin and ß-tubulin genes in free mycelia and robust biofilms of the filamentous fungus Aspergillus niger.

The morphology and growth of the filamentous fungi are influenced by different factors as the culture conditions and the type of fermentative process. The production and secretion of metabolites by these organisms present a direct relationship with their morphology. The organization of the microtubules and actin in the cytoskeleton is determinant for both the fungal growth and morphology. In this context, this study aimed to analyze the expression of the ß-tubulin, F-actin, and glucan synthase in the A. niger mycelia obtained from submerged fermentation and biofilm fermentation through qPCR, as well as the analysis of the nucleus distribution in the hypha. Herein, we showed that ß-tubulin and the F-actin gene were more expressed in the biofilm condition, while the glucan synthase was in the submerged condition. No significant difference was observed in the nucleus distribution between the mycelia obtained from both the fermentative processes. In conclusion, the different morphologies observed for the mycelia from submerged fermentation and biofilm fermentation might be influenced by the differential modulation of genes that codify cytoskeleton proteins, which seems to be potentially regulated by mechanosensing during fungal contact with solid supports.

Stabilization and application of spray-dried tannase from Aspergillus fumigatus CAS21 in the presence of different carriers.

The Aspergillus fumigatus CAS21 tannase was spray dried with ß-cyclodextrin, Capsul® starch, soybean meal, lactose, and maltodextrin as adjuvants. The moisture content and water activity of the products ranged from 5.6 to 11.5% and from 0.249 to 0.448, respectively. The maximal tannase activity was achieved at 40-60 ºC and pH 5.0-6.0 for the powders containing ß-cyclodextrin and Capsul® starch, which was stable at 40 ºC and 40-60 ºC for 120 min, respectively. For all the dried products, tannase retained its activity of over 80% for 120 min at pH 5.0 and 6.0. Salts and solvents influenced the activity of the spray-dried tannase. The activity of the spray-dried tannase was maintained when preserved for 1 year at 4 ºC and 28 ºC. Spray-dried tannase reduced the content of tannins and polyphenolic compounds of leather effluent and sorghum flour and catalyzed the transesterification reaction. The spray drying process stabilized the tannase activity, highlighting the potential of dried products for biotechnological applications.

A novel Trichoderma reesei mutant RP698 with enhanced cellulase production.

A new strain of Trichoderma reesei (teleomorph Hypocrea jecorina) with high cellulase production was obtained by exposing the spores from T. reesei QM9414 to an ultraviolet light followed by selecting fast-growing colonies on plates containing CMC (1% w/v) as the carbon source. The mutant T. reesei RP698 reduced cultivation period to 5 days and increased tolerance to the end-products of enzymatic cellulose digestion. Under submerged fermentation conditions, FPase, CMCase, and Avicelase production increased up to 2-fold as compared to the original QM9414 strain. The highest levels of cellulase activity were obtained at 27 °C after 72 h with Avicel®, cellobiose, and sugarcane bagasse as carbon sources. The temperature and pH activity optima of the FPase, CMCase, and Avicelase were approximately 60 °C and 5.0, respectively. The cellulase activity was unaffected by the addition of 140 mM glucose in the enzyme assay. When T. reesei RP698 crude extract was supplemented by the addition of ß-glucosidase from Scytalidium thermophilum, a 2.3-fold increase in glucose release was observed, confirming the low inhibition by the end-product of cellulose hydrolysis. These features indicate the utility of this mutant strain in the production of enzymatic cocktails for biomass degradation.

Spray Drying of Coloring Extracts Produced by Fungi Isolated from Brazilian Caves

Abstract Pigments produced by submerged fermentation of three filamentous fungi isolated from Brazilian caves, namely Aspergillus keveii, Penicillium flavigenum, and Epicoccum nigrum, were submitted to spray drying in presence of the adjuvants maltodextrin, modified starch or gum arabic. Yellow fine powders with low moisture content and water activity, and high color retention (> 70%) were successfully generated with a high product recovery ratio (> 50%), independently of the adjuvant used. The dried products have enhanced stability and potential to might be used as a natural colorant in food and pharmaceutical applications.

Extracellular Tannase from Aspergillus ochraceus: Influence of the Culture Conditions on Biofilm Formation, Enzyme Production, and Application.

Aspergillus ochraceus biofilm, developed on an inert support, can produce tannase in Khanna medium containing 1.5% (w/v) tannic acid as the carbon source, at an initial pH of 5.0, for 72 h at 28 °C. Addition of 0.1% yeast extract increased enzyme production. The enzyme in the crude filtrate exhibited the highest activity at 30 °C and pH 6.0. At 50 °C, the half-life was 60 min and 260 min at pH 6.0. In general, addition of detergents and surfactants did not affect tannase activity significantly. Tannase has potential applications in various biotechnological processes such as the production of propyl gallate and in the treatment of tannin-rich effluents. The content of tannins and total phenolic compounds in effluents from leather treatment was reduced by 56-83% and 47-64%, respectively, after 2 h of enzyme treatment. The content of tannins and total phenolic compounds in the sorghum flour treated for 120 h with tannase were reduced by 61% and 17%, respectively. Interestingly, the same A. ochraceus biofilm was able to produce tannase for three sequential fermentative process. In conclusion, fungal biofilm is an interesting alternative to produce high levels of tannase with biotechnological potential to be applied in different industrial sectors.

Production of short-chain fructooligosaccharides (scFOS) using extracellular ß-D-fructofuranosidase produced by Aspergillus thermomutatus.

Aspergillus thermomutatus produces an extracellular ß-D-fructofuranosidase when cultured in Khanna medium with sucrose as additional carbon source at 30°C under agitation for 72 hr. Addition of glucose and fructose in the culture medium affected the production of the enzyme negatively. The optimum hydrolytic activity was achieved at 60°C and pH 5.0, with half-life (T50) of 30 hr at 50°C and 62% of its activity maintained at pH 5.0 for 48 hr. The extracellular extract containing ß-D-fructofuranosidase was effective in producing fructooligosaccharides (FOS), mainly 1-kestose. The highest concentration of FOS was obtained at 30°C and 60°C, indicating the existence of at least two enzymes with transfructosylating activity. At 30°C, the maximal FOS concentration was obtained from 48 to 72 hr, while at 60°C, it was achieved only at 72 hr. The best production of FOS (86.7 g/L) was obtained using 500 g/L sucrose as substrate. PRACTICAL APPLICATION: Fructooligosaccharides (FOS) are linear oligomers of fructose units with important applications in the food industry as sweetening agents and biopreservatives. Due to the presence of ß-glycosidic bonds, they cannot be hydrolyzed by human enzymes, allowing the use of FOS-containing products by diabetics. FOS used in the preparation of dairy products imparts humectancy to soft baked products, lowers the freezing point of frozen desserts, provides crispness to low-fat cookies, and provides many other advantages. Diets containing FOS can reduce the levels of triglycerides and cholesterol and improve the absorption of ions, such as Ca2+ and Mg2+ . FOS also exhibit bifidogenic effect on Bifidobacterium and Lactobacillus strains in the colon. Industrially, FOS is produced during the transfructosylation reaction of sucrose catalyzed by ß-D-fructofuranosidase. Identifying new sources of ß-D-fructofuranosidase is an important challenge to meet its industrial demand.

Production and characterization of a thermostable antifungal chitinase secreted by the filamentous fungus Aspergillus niveus under submerged fermentation.

The filamentous fungus Aspergillus niveus produced extracellular antifungal chitinase when cultured under submerged fermentation (SbmF) using crab shells as the carbon source. Maximal chitinase production was achieved at 192 h of cultivation using minimal medium containing 1% chitin. The enzyme was purified 1.97-fold with 40% recovery by ammonium sulfate precipitation and Sephadex G-100 gel filtration. The molecular mass was estimated to be 44 kDa by both 12% SDS-PAGE and Sepharose CL-6B gel filtration. Maximal A. niveus chitinase activity was obtained at 65 °C and pH 5.0. The enzyme was fully stable at 60 °C for up to 120 min and the enzymatic activity was increased by Mn2+. In the presence of reducing and denaturing compounds, the enzyme activity was not drastically affected. The chitinase was able to hydrolyze colloidal chitin, azure chitin, and 4-nitrophenyl N-acetyl-ß-D glucosaminide; for the latter, the K0.5 and maximal velocity (Vmax) were 3.51 mM and 9.68 U/mg of protein, respectively. The A. niveus chitinase presented antifungal activity against Aspergillus niger (MIC = 84 µg/mL), A. fumigatus (MIC = 21 µg/mL), A. flavus (MIC = 24 µg/mL), A. phoenicis (MIC = 24 µg/mL), and Paecilomyces variotii (MIC = 21 µg/mL). The fungus A. niveus was able to produce a thermostable and denaturation-resistant chitinase able to inhibit fungal development, signaling its biotechnological potential.

Characterization of Aspergillus fumigatus CAS-21 tannase with potential for propyl gallate synthesis and treatment of tannery effluent from leather industry.

One of the tannase isoforms produced by the fungus Aspergillus fumigatus CAS-21 under submerged fermentation (SbmF) was purified 4.9-fold with a 10.2% recovery. The glycoprotein (39.1% carbohydrate content) showed an estimated molecular mass of 60 kDa. Optimum temperature and pH for its activity were 30-40 °C and 5.0, respectively. It showed a half-life (t50) of 60 min at 45 and 50 °C, and it was stable at pH 5.0 and 6.0 for 3 h. The tannase activity was insensitive to most salts used, but it reduced in the presence of Fe2(SO4)3 and FeCl3. On contrary, in presence of SDS, Triton-X100, and urea the enzyme activity increased. The Km value indicated high affinity for propyl gallate (3.61 mmol L-1) when compared with tannic acid (6.38 mmol L-1) and methyl gallate (6.28 mmol L-1), but the best Kcat (362.24 s-1) and Kcat/Km (56.78 s-1 mmol-1 L) were obtained for tannic acid. The purified tannase reduced 89 and 25% of tannin content of the leather tannery effluent generated by manual and mechanical processing, respectively, after 2-h treatment. The total phenolic content was also reduced. Additionally, the enzyme produced propyl gallate, indicating its ability to do the transesterification reaction. Thus, A. fumigatus CAS-21 tannase presents interesting properties, especially the ability to degrade tannery effluent, highlighting its potential in biotechnological applications.

Different strategies to kill the host presented by Metarhizium anisopliae and Beauveria bassiana.

Studies conducted over the last decades have shown the potential of entomopathogenic fungi for the biocontrol of some insect pests. Entomopathogenic fungi infect their host through the cuticle, so they do not need to be ingested to be effective. These fungi also secrete secondary metabolites and proteins that are toxic to insect pests. In this context, we analyzed the pathogenicity of Metarhizium anisopliae (Metschn.) strains IBCB 384 and IBCB 425 and Beauveria bassiana (Bals.-Criv.) Vuill. strains E 1764 and E 3158 against Galleria mellonella (Linn.) larvae, during pre-invasion and post-invasion phases. The results showed M. anisopliae, especially strain IBCB 384, was most virulent in the pre-invasion phase against G. mellonella, whereas B. bassiana, especially strain E 1764, was most virulent in the post-invasion phase. During in vivo development and in the production of toxic serum, B. bassiana E 3158 was the most virulent. Different fungal growth (or toxin) strategies were observed for studied strains. Metarhizium anisopliae IBCB 425 prioritizes the growth strategy, whereas strain IBCB 384 and B. bassiana strains E 1764 and E 3158 have a toxic strategy. All strains have pathogenicity against G. mellonella, indicating their possible use for biocontrol.

Characterization of a Thermotolerant Phytase Produced by Rhizopus microsporus var. microsporus Biofilm on an Inert Support Using Sugarcane Bagasse as Carbon Source.

The Rhizopus microsporus var. microsporus biofilm was able to produce increased levels of an extracellular thermotolerant phytase using polyethylene and viscose as an inert support in both modified NBRIP medium and modified Khanna medium containing sugarcane bagasse as the carbon source. The enzyme production was strictly regulated by the phosphorus content with optimal production at 0.5 mM of sodium phytate and KH2PO4. The extracellular phytase, RMPhy1, was purified 4.18-fold with 4.78 % recovery using DEAE-cellulose and CM-cellulose. A single protein band with a molecular mass of 35.4 kDa was obtained when the samples were subjected to 10 % SDS-PAGE. The optimum temperature for activity was 55 °C and the optimum pH was 4.5. R. microsporus var. microsporus phytase exhibited high stability at 30 and 40 °C with a half-life of 115 min at 60 °C. The enzyme activity increased in the presence of Ca (2+) and was inhibited by Zn(2+), arsenate, and sodium phosphate. Phytase demonstrated high substrate specificity for sodium phytate with K m = 0.72 mM and V max = 94.55 U/mg of protein and for p-NPP with K m = 0.04 mM and V max = 106.38 U/mg of protein. The enzyme also hydrolyzed ATP, AMPc, glucose 6-phosphate, glucose 1-phosphate, and UDPG. This is the first report on phytase characterization delivered with biofilm technology. The properties of the enzyme account for its high potential for use in biotechnology and the possibility of application in different industrial sectors as feed in the future.

Secretome Analysis of Metarhizium anisopliae Under Submerged Conditions Using Bombyx mori Chrysalis to Induce Expression of Virulence-Related Proteins.

The entomopathogenic fungus Metarhizium anisopliae is used to control insect pests. This species is specialized for the secretion of an enzymatic complex consisting of proteases, lipases, and chitinases related to pathogenicity and virulence. In this context, the secretomes of strains IBCB 167 and IBCB 384 of M. anisopliae var. anisopliae, grown under submerged fermentation in the presence of chrysalis as an inducer, were analyzed. Analysis of two-dimensional gels showed qualitative and quantitative differences between secreted proteins in both isolates. Around 102 protein spots were analyzed, and 76 % of the corresponding proteins identified by mass spectrometry were grouped into different classes (hydrolases, oxidases, reductases, isomerases, kinases, WSC domains, and hypothetical proteins). Thirty-three per cent of all the proteins analyzed were found to be common in both strains. Several virulence-related proteins were identified as proteases and mannosidases. Endo-N-acetyl-ß-D-glucosaminidase expression was observed to be 10.14-fold higher for strain IBCB 384 than for strain IBCB 167, which may be an important contributor to the high virulence of IBCB 384 in Diatraea ssaccharalis. These results are important for elucidation of the host-pathogen relationship and the differences in virulence observed between the two strains.

Characterization of a multi-tolerant tannin acyl hydrolase II from Aspergillus carbonarius produced under solid-state fermentation

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.

Optimization of culture conditions for tannase production by Aspergillus sp. gm4 in solid state fermentation / Otimização das condições de cultivo para produção de tanase por Aspergillus sp. gm4 em fermentação em estado sólido

Tannase is an industrially important enzyme produced by a large number of microorganisms. This study analyzed the production of tannase by Aspergillus sp. GM4 under solid-state fermentation (SSF) using different vegetable leaves (mango, jamun and coffee) and agricultural residues (coffee husks, rice husks and wheat bran). Among the substrates used jamun leaves yielded high tannase production. The Plackett-Burman design was conducted to evaluate the effects of 12 independent variables on the production of tannase under SSF using jamun leaves as substrate. Among these variables, incubation time, potassium nitrate and tannic acid had significant effects on enzyme production. A lower incubation time was fixed and supplementation with potassium nitrate and tannic acid were optimized using the Central Composite Design. The best conditions for tannase production were: incubation time of 2 days; tannic acid at 1.53% (w w-1) and potassium nitrate at 2.71% (w w- 1). After the optimization process, tannase production increased 4.65-fold, which showed that the statistical experimental design offers a practicable approach to the implementation of optimization of tannase production.

Tanase é uma enzima industrialmente importante produzida por um grande número de microrganismos. Este estudo analisou a produção de tanase por Aspergillus sp. GM4 em fermentação em estado sólido (FES) utilizando diferentes vegetais como folhas de manga, de jambolão, de café e resíduos agrícolas, como a casca de café, casca de arroz e farelo de trigo. Entre os substratos utilizados, as folhas jambolão renderam alta produção de tanase. O planejamento de Plackett-Burman foi conduzido para avaliar os efeitos de 12 variáveis independentes sobre a produção de tanase em FES usando folhas jambolão como substrato. Entre estas variáveis, tiveram efeitos significativos na produção da enzima o tempo de incubação, o nitrato de potássio e o ácido tânico. O menor tempo de incubação foi fixado e a suplementação de nitrato de potássio e ácido tânico foi otimizada utilizando o planejamento composto central rotacional. As melhores condições para a produção de tanase foram o tempo de incubação de dois dias, a concentração de ácido tânico de 1,53% (g g-1) e de nitrato de potássio 2,71% (g gw-1). Após o processo de otimização, a produção tanase aumentou 4,65 vezes, o que mostrou que o delineamento experimental foi um método viável para a otimização da produção de tanase.

Phytase production by Rhizopus microsporus var. microsporus biofilm: characterization of enzymatic activity after spray drying in presence of carbohydrates and nonconventional adjuvants.

Microbial phytases are enzymes with biotechnological interest for the feed industry. In this article, the effect of spray-drying conditions on the stability and activity of extracellular phytase produced by R. microsporus var. microsporus biofilm is described. The phytase was spray-dried in the presence of starch, corn meal (>150 µm), soy bean meal (SB), corn meal (<150 µm) (CM), and maltodextrin as drying adjuvants. The residual enzyme activity after drying ranged from 10.7% to 60.4%, with SB and CM standing out as stabilizing agents. Water concentration and residual enzyme activity were determined in obtained powders as a function of the drying condition. When exposed to different pH values, the SB and CM products were stable, with residual activity above 50% in the pH range from 4.5 to 8.5 for 60 min. The use of CM as drying adjuvant promoted the best retention of enzymatic activity compared with SB. Spray drying of the R. microsporus var. microsporus phytase using different drying adjuvants showed interesting results, being quite feasible with regards their biotechnological applications, especially for poultry diets.

Characterization of the co-purified invertase and ß-glucosidase of a multifunctional extract from Aspergillus terreus.

The filamentous fungus Aspergillus terreus secretes both invertase and ß-glucosidase when grown under submerged fermentation containing rye flour as the carbon source. The aim of this study was to characterize the co-purified fraction, especially the invertase activity. An invertase and a ß-glucosidase were co-purified by two chromatographic steps, and the isolated enzymatic fraction was 139-fold enriched in invertase activity. SDS-PAGE analysis of the co-purified enzymes suggests that the protein fraction with invertase activity was heterodimeric, with subunits of 47 and 27 kDa. Maximal invertase activity, which was determined by response surface methodology, occurred in pH and temperature ranges of 4.0-6.0 and 55-65 °C, respectively. The invertase in co-purified enzymes was stable for 1 h at pH 3.0-10.0 and maintained full activity for up to 1 h at 55 °C when diluted in water. Invertase activity was stimulated by 1 mM concentrations of Mn²âº (161 %), Co²âº (68 %) and Mg²âº (61 %) and was inhibited by Al³âº, Ag⁺, Fe²âº and Fe³âº. In addition to sucrose, the co-purified enzymes hydrolyzed cellobiose, inulin and raffinose, and the apparent affinities for sucrose and cellobiose were quite similar (K(M) = 22 mM). However, in the presence of Mn²âº, the apparent affinity and V(max) for sucrose hydrolysis increased approximately 2- and 2.9-fold, respectively, while for cellobiose, a 2.6-fold increase in V(max) was observed, but the apparent affinity decreased 5.5-fold. Thus, it is possible to propose an application of this multifunctional extract containing both invertase and ß-glucosidase to degrade plant biomass, thus increasing the concentration of monosaccharides obtained from sucrose and cellobiose.

Co-immobilization of fungal endo-xylanase and α-L-arabinofuranosidase in glyoxyl agarose for improved hydrolysis of arabinoxylan.

Plant cell-wall arabinoxylans have a complex structure that requires the action of a pool of debranching (arabinofuranosidases) and depolymerizing enzymes (endo-xylanase). Two Aspergillus nidulans strains over-secreting endo-xylanase and arabinofuranosidase were inoculated in defined 2% maltose-minimum medium resulting in the simultaneously production of these enzymes. To study the synergistic hydrolysis was used arabinoxylan with 41% of arabinose and 59% of xylose residues. Thus, it was adopted different approaches to arabinoxylan hydrolysis using immobilized arabinofuranosidase and endo-xylanase: (i) endo-xylanase immobilized on glyoxyl agarose; (ii) arabinofuranosidase immobilized on glyoxyl agarose; (T1) hydrolysis of arabinoxylan with arabinofuranosidase immobilized on glyoxyl agarose for debranching, followed by a second hydrolysis with endo-xylanase immobilized on glyoxyl agarose; (T2) hydrolysis using (i) and (ii) simultaneously; and (T3) hydrolysis of arabinoxylan with endo-xylanase and arabinofuranosidase co-immobilized on glyoxyl agarose. It was concluded that arabinoxylan hydrolysis using two derivatives simultaneously (T2) showed greater hydrolytic efficiency and consequently a higher products yield. However, the hydrolysis with multi-enzymatic derivative (T3) results in direct release of xylose and arabinose from a complex substrate as arabinoxylan, which is a great advantage as biotechnological application of this derivative, especially regarding the application of biofuels, since these monosaccharides are readily assimilable for fermentation and ethanol production.

Purification and biochemical characterization of glucose-cellobiose-tolerant cellulases from Scytalidium thermophilum.

Two cellulases from Scytalidium thermophilum were purified and characterized, exhibiting tolerance to glucose and cellobiose. Characterization of purified cellulases I and II by mass spectrometry revealed primary structure similarities with an exoglucanase and an endoglucanase, respectively. Molecular masses were 51.2 and 45.6 kDa for cellulases I and II, respectively, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Cellulases I and II exhibited isoelectric points of 6.2 and 6.9 and saccharide contents of 11 and 93 %, respectively. Optima of temperature and pH were 60-65 °C and 4.0 for purified cellulase I and 65 °C and 6.5 for purified cellulase II. Both cellulases maintained total CMCase activity after 60 min at 60 °C. Cysteine, Mn(2+), dithiotreitol and ß-mercaptoethanol-stimulated cellulases I and II. The tolerance to cellulose hydrolysis products and the high thermal stabilities of Scytalidium cellulases suggest good potential for industrial applications.

Extracellular ß-fructofuranosidase from Fusarium graminearum: stability of the spray-dried enzyme in the presence of different carbohydrates.

Microbial enzymes have been used for various biotechnological applications; however, enzyme stabilization remains a challenge for industries and needs to be considered. This study describes the effects of spray-drying conditions on the activity and stability of ß-fructofuranosidase from Fusarium graminearum. The extracellular enzyme ß-fructofuranosidase was spray dried in the presence of stabilizers, including starch (Capsul) (SC), microcrystalline cellulose (MC), trehalose (TR), lactose (LC) and ß-cyclodextrin (CD). In the presence of TR (2% w/v), the enzymatic activity was fully retained. After 1 year of storage, 74% of the enzymatic activity was maintained with the CD stabilizer (10% w/v). The residual activity was maintained as high as 80% for 1 h at 70°C when MC, SC and CD (5% w/v) stabilizers were used. Spray drying with carbohydrates was effective in stabilizing the F. graminearum ß-fructofuranosidase, improved enzymatic properties compared to the soluble enzyme and demonstrated a potential use in future biotechnology applications.

Characterization of a tannase from Emericella nidulans immobilized on ionic and covalent supports for propyl gallate synthesis.

The extracellular tannase from Emericela nidulans was immobilized on different ionic and covalent supports. The derivatives obtained using DEAE-Sepharose and Q-Sepharose were thermally stable from 60 to 75 °C, with a half life (t50) >24 h at 80 °C at pH 5.0. The glyoxyl-agarose and amino-glyoxyl derivatives showed a thermal stability which was lower than that observed for ionic supports. However, when the stability to pH was considered, the derivatives obtained from covalent supports were more stable than those obtained from ionic supports. DEAE-Sepharose and Q-Sepharose derivatives as well as the free enzyme were stable in 30 and 50 % (v/v) 1-propanol. The CNBr-agarose derivative catalyzed complete tannic acid hydrolysis, whereas the Q-Sepharose derivative catalyzed the transesterification reaction to produce propyl gallate (88 % recovery), which is an important antioxidant.