Simone C. Peixoto-Nogueira

Screening of filamentous fungi for production of enzymes of biotechnological interest

Many enzymes produced by fungi have relevant biotechnological applications in several industrial areas. The purpose of this study was to collect and isolate filamentous fungi from soil and humus, plants and sugar cane bagasse of different regions of the Sao Paulo state. Forty isolates were examined for their ability to produce xylanase, glucose-oxidase, alkaline phosphatase, acid phosphatase, phytase, pectinase and amylase. Among these, twenty three isolates exhibited enzymatic potential. The xylanases produced by two of these isolates (Aspergillus caespitosus and A. phoenicis) showed good potential for pulp bleaching. Among seventeen isolates, at least three produced high levels of glucose-oxidase, being Rhizopus stolonifer and A. versicolor the best producer strains. A. caespitosus, Mucor rouxii, and nine others still not identified were the best producers of phosphatases in submerged fermentation. Pectinase was best produced by IF II and C-8 belong R. stolonifer. Significant levels of amylase were produced by Paecilomyces variotii and A. phoenicis. A remarkable enzyme producer was Rhizopus microsporus var. rhizopodiformis that produced high levels of amylase, alkaline and acid phosphatases, and pectinase. Some morphological structures of this fungus were illustrated using light microscopy (LM) and scanning electron microscopy (SEM). This study contributes to catalogue soil fungi isolated in the state of Sao Paulo, and provides additional information to support future research about the industrial potential of these microorganisms that may produce enzymes and, eventually, also secondary metabolites with anti-microbial or anti-parasitic activities.

Purification and characterization of a thermostable α-amylase produced by the fungus Paecilomyces variotii

An α-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The α-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pI value of 4.5. Temperature and pH optima were 60°C and 4.0, respectively. The enzyme was stable for 1 h at 55°C, showing a t₅₀ of 53 min at 60°C. Starch protected the enzyme against thermal inactivation. The α-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K(m) of α-amylase on Reagen® and Sigma® starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to α-amylases from Bacillus sp. These results confirmed that the studied enzyme was an α-amylase ((1→4)-α-glucan glucanohydrolase).

Regulation of xylanase in Aspergillus phoenicis: a physiological and molecular approach

Microbial xylanolytic enzymes have a promising biotechnological potential, and are extensively applied in industries. In this study, induction of xylanolytic activity was examined in Aspergillus phoenicis. Xylanase activity induced by xylan, xylose or β-methylxyloside was predominantly extracellular (93–97%). Addition of 1% glucose to media supplemented with xylan or xylose repressed xylanase production. Glucose repression was alleviated by addition of cAMP or dibutyryl-cAMP. These physiological observations were supported by a Northern analysis using part of the xylanase gene ApXLN as a probe. Gene transcription was shown to be induced by xylan, xylose, and β-methylxyloside, and was repressed by the addition of 1% glucose. Glucose repression was partially relieved by addition of cAMP or dibutyryl cAMP.

Effects of Aspergillus spp. exogenous fibrolytic enzymes on in vitro fermentation of tropical forages

BACKGROUND Cellulose and hemicellulose are quantitatively the most important structural carbohydrates present in ruminant diets. Rumen micro-organisms produce enzymes that catalyse their hydrolysis, but the complex network formed by structural carbohydrates and lignin reduces their digestibility and restricts efficient utilisation of feeds by ruminants. This study aimed to produce two enzymatic extracts, apply them in ruminant diets to determine the best levels for ruminal digestibility and evaluate their effects on in vitro digestibility. RESULTS In experiment 1 a two-stage in vitro technique was used to examine the effects of different enzymatic levels of Aspergillus japonicus and Aspergillus terricola on tropical forages. Enzyme addition had minor effects on corn silage at the highest enzymatic level. In experiment 2 an in vitro gas production (GP) technique was applied to determine apparent in vitro organic matter digestibility and metabolisable energy. The addition of enzymes in GP showed interesting results. Good data were obtained using sugar cane and Tifton-85 hay supplemented with extracts of A. japonicus and A. terricola respectively. CONCLUSION Overall, the study suggests that addition of crude extracts containing exogenous fibrolytic enzymes to ruminant diets enhances the effective utilisation of ruminant feedstuffs such as forages.

Evidence of high production levels of thermostable dextrinizing and saccharogenic amylases by Aspergillus niveus

The aim of this work was to analyze the effect of several nutritional and environmental parameters on amylase production by a novel, isolated from the thermotolerant filamentous fungus Aspergillus niveus. This strain produced high levels of amylolytic activity in Khanna liquid medium supplemented with commercial starch, initial pH 6.5, under static conditions for 72 h. Among the tested carbon sources, milled corn, oatmeal, soluble potato starch and maisena were the best inducers of enzymatic secretion (220, 180, 170 and 150 U/mL), respectively. The main products of hydrolysis analyzed by thin layer chromatography were glucose, maltose and traces of maltooligosaccharides, suggesting the presence of -amylase and glucoamylase activities in the crude extract. The optimal pH were 4.5 and 5.5 and the optimum temperature was 65°C. The enzymes were fully stable up to 1 h at 55°C. It was possible to verify the presence of three bands with amylolytic activity in non-denaturing polyacrylamide gel electrophoresis (PAGE). These aspects and other properties suggested that the amylases produced by A. niveus might be suitable for biotechnological applications.

Production of cellulase-free xylanase by Aspergillus flavus: Effect of polyols on the thermostability and its application on cellulose pulp biobleaching

The production of xylanase without cellulase is required for prebleaching of pulp in pulp and paper industry. Aspergillus flavus produced high levels of xylanase on agricultural residues with wheat bran and sugarcane bagasse (4.17 U/mg), and wheat bran and corncob (2.97 U/mg). Xylanase was found to be stable at 45°C with 100% of its original activity remaining after 2 h incubation. At 50°C, xylanase was stable for the first twenty minutes, and had half-life of 50 min. The pH stability for the xylanase from A. flavus was most stable in the range of pH 3.0-8.0 retaining more that 100% activity after 1 h. The addition of 5% glycerol, mannitol or xylitol protected the xylanase from thermal inactivation at 50°C. The protective effect by glycerol, xylitol and mannitol resulted in increases of 162, 262.5 and 150% when compared with the control at 120 min, approximately. Increasing the polyols concentration up to 20% (w/v) further improved the thermostability of xylanase after 120 min at 50°C by 300% when compared with the control (no additive). The kappa number reduced 2.56 points, which corresponds to 18.34 kappa efficiency. This xylanase is an attractive enzyme for potential future application in the pulp and paper industries, since industrial application requires a cellulase-free activity, maintenance of high temperature and enzyme stability are desirable. Key words: Aspergillus flavus, polyols, xylanase, biobleaching cellulose pulp.

Gel Electrophoresis for Investigating Enzymes with Biotechnological Application

Maria de Lourdes T. M. Polizeli1*, Simone C. Peixoto-Nogueira1, Tony M. da Silva1, Alexandre Maller2 and Hamilton Cabral3 1Biology Department, Faculty of Philosophy Sciences and Letters of Ribeirao Preto, Sao Paulo University 2Biochemistry and Immunology Department School of Medicine of Ribeirao Preto, Sao Paulo, Sao Paulo University 3Science Pharmaceutical Department School of Pharmaceutical Science of Ribeirao Preto, Sao Paulo University Brazil