Michele Michelin

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).

Effect of phenolic compounds from pretreated sugarcane bagasse on cellulolytic and hemicellulolytic activities.

This work shows both cellulases and hemicellulases are inhibited and deactivated by water-soluble and acetone extracted phenolics from sugarcane bagasse pretreated at 10% (w/v) for 30 min in liquid hot water at 180 or 200°C. The dissolved phenolics in vacuum filtrate increased from 1.4 to 2.4 g/L as temperature increased from 180 to 200°C. The suppression of cellulose and hemicellulose hydrolysis by phenolics is dominated by deactivation of the β-glucosidase or β-xylosidase components of cellulase and hemicellulase enzyme by acetone extract at 0.2-0.65 mg phenolics/mg enzyme protein and deactivation of cellulases and hemicellulases by the water soluble components in vacuum filtrate at 0.05-2mg/mg. Inhibition was a function of the type of enzyme and the manner in which the phenolics were extracted from the bagasse.

Use of cassava peel as carbon source for production of amylolytic enzymes by Aspergillus niveus.

Aspergillus niveus produced high levels of ?-amylase and glucoamylase in submerged fermentation using the agricultural residue cassava peel as a carbon source. In static conditions, the amylase production was substantially greater than in the agitated condition. The optimized culture conditions were initially at pH 5.0, 35°C during 48 hours. Amylolytic activity was still improved (50%) with a mixture of cassava peel and soluble starch in the proportion 1:1 (w/w). The crude extract exhibited temperature and pH optima approximately 70°C and 4.5, respectively. Amylase activity was stable for 1 h at 60°C, and at pH values between 3.0 and 7.0. The enzyme hydrolysed preferentially maltose, starch, penetrose, amylose, isomaltose, maltotriose, glycogen and amylopectin, and not hydrolysed cyclodextrin (? and ß), trehalose and sucrose. In the first hour of reaction on soluble starch, the hydrolysis products were glucose and maltose, but after two hours of hydrolysis, glucose was the unique product formed, confirming the presence in the crude extract of an ?-amylase and a glucoamylase.

Multi-step approach to add value to corncob: production of biomass-degrading enzymes, lignin and fermentable sugars.

This work presents an integrated and multi-step approach for the recovery and/or application of the lignocellulosic fractions from corncob in the production of high value added compounds as xylo-oligosaccharides, enzymes, fermentable sugars, and lignin in terms of biorefinery concept. For that, liquid hot water followed by enzymatic hydrolysis were used. Liquid hot water was performed using different residence times (10-50min) and holding temperature (180-200°C), corresponding to severities (log(R0)) of 3.36-4.64. The most severe conditions showed higher xylo-oligosaccharides extraction (maximum of 93%) into the hydrolysates and higher recovery of cellulose on pretreated solids (maximum of 65%). Subsequently, hydrolysates and solids were used in the production of xylanases and cellulases, respectively, as well as, pretreated solids were also subjected to enzymatic hydrolysis for the recovery of lignin and fermentable sugars from cellulose. Maximum glucose yield (100%) was achieved for solids pretreated at log(R0) of 4.42 and 5% solid loading.

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.

Cellulose nanocrystals from grape pomace: production, properties and cytotoxicity assessment

Cellulose nanocrystals (CNCs) were obtained from grape pomace through chemical and physical pretreatments. Bleached cellulose pulp was subjected to acid hydrolysis (AH) for 30 or 60 min and an ultrasound treatment to obtain CNCs (AH30S and AH60S). Compositional analyses of untreated (UGP) and pretreated (PGP) grape pomace showed the effectiveness of pretreatment in removing non-cellulosic components, recovering 80.1% cellulose in PGP (compared to 19.3% of UGP). Scanning and transmission electron microscopies were used to evaluate the CNCs morphology. AH in combination with ultrasound treatment led to needle-shaped structures and apparently more dispersed suspensions. Crystallinity index and thermal stability were studied by X-ray diffraction and thermogravimetric analysis, respectively. The AH60S sample presented high aspect ratio, crystallinity and thermal stability. CNCs toxicity was evaluated by exposing Caco-2 cells to CNCs suspension and evaluating their viability. Results showed that CNCs are non-toxic, opening the opportunity for their use on food and pharmaceutical applications.

Enzymes Involved in the Biodegradation of Sugarcane Biomass: Challenges and Perspectives

This chapter introduces the role of enzymes in the biomass degradation, namely sugarcane bagasse and straw, leading to the formation of fermentable sugars and second-generation ethanol. The chapter begins with a retrospective of the structuring of the ethanol production chain and then presents current aspects where the deficit of production and its consequences in business can be seen. Subsequently, we list the role of enzymes involved in the biomass hydrolysis, the commercial cocktails, and the proposal of our laboratory in this context. On the other hand, the efficiency of enzymes on the biomass is increased when the bagasse and straw are pretreated. Thus, some technologies that may facilitate the enzymatic hydrolysis and the formation of fermentable sugars are described. Lastly, recent analytical methods that enable a better analysis of the composition and viewing of fiber in the sugarcane biomass are presented.

Use of Lignocellulosic Materials in Bio-based Packaging

This chapter presents the most recent studies on the use of lignocellulosic materials for the development of bio-based packaging materials. It is addressed the incorporation of cellulose and its derivatives, hemicellulose, and lignin in bio-based packaging, and some works where the lignocellulosic materials with few pretreatment are used as filler are also presented. Additionally, it is discussed some bio-based materials extracted from biomass, such as polysaccharides and proteins, but also the chemically synthesized polymers such as polylactic acid (PLA), or obtained through biotechnological routes such as polyhydroxyalkanoates.

Food Applications of Lignocellulosic-Based Packaging Materials

The use of current packaging materials on food products has as main objective to protect the product for the maintenance of food quality and safety during transportation and storage, but also to attract the attention of the consumers for a particular brand while giving information about the food product. The use of cellulose-based materials as secondary and tertiary packaging has been one of the main applications of lignocellulosic materials in food packaging. Nowadays, the use of lignocellulosic materials incorporated in bio-based materials showed to be a good possibility to reduce the use of petroleum-based materials and thus increase the sustainability. During this chapter are given some commercial examples and research studies of the application of lignocellulosic-based materials as food packaging.