Leonora Rios de Souza Moreira

Biomass-Derived Inhibitors of Holocellulases

Enzymes constitute a major monetary cost in the bioconversion of holocellulose to ethanol. Identifying enzyme inhibitors and moderating their effects is one approach that may help to overcome this issue. Most inhibitors that reduce the hydrolysis activity of holocellulases are released as the holocellulosic biomass is broken down in the pretreatment and hydrolysis steps. Recent reports in the literature have shown that the major inhibitors or deactivators of cellulases are phenols and xylooligosaccharides. The bioconversion of hemicelluloses by hemicellulases also has important practical applications in various agro-industrial processes in addition to the conversion of hemicellulosic biomass to fuels and chemicals. Hemicellulases, such as β-xylosidases, may also help alleviate the inhibitory effect of xylooligosaccharides to cellulases. However, compared to cellulases, less is known about the inhibition or deactivation of hemicellulases and pectinases, especially for inhibitors that are generated during pre-treatment and the hydrolysis of lignocellulosic substrates. Considering the importance of such enzymes for the complete degradation of lignocellulosic substrates, this review provides a broad view of the effect of inhibitors of holocellulases (cellulases, hemicellulases, and pectinases).

Two β-xylanases from Aspergillus terreus: Characterization and influence of phenolic compounds on xylanase activity

Sugarcane bagasse was used as an inexpensive alternative carbon source for production of β-xylanases from Aspergillus terreus. The induction profile showed that the xylanase activity was detected from the 6th day of cultivation period. Two low molecular weight enzymes, named Xyl T1 and Xyl T2 were purified to apparent homogeneity by ultrafiltration, gel filtration and ion exchange chromatographies and presented molecular masses of 24.3and 23.60 kDa, as determined by SDS-PAGE, respectively. Xyl T1 showed highest activity at 50 °C and pH 6.0, while Xyl T2 was most active at 45 °C and pH 5.0. Mass spectrometry analysis of trypsin digested Xyl T1 and Xyl T2 showed two different fingerprinting spectra, indicating that they are distinct enzymes. Both enzymes were specific for xylan as substrate. Xyl T1 was inhibited in greater or lesser degree by phenolic compounds, while Xyl T2 was very resistant to the inhibitory effect of all phenolic compounds tested. The apparent km values of Xyl T2, using birchwood xylan as substrate, decreased in the presence of six phenolic compounds. Both enzymes were inhibited by N-bromosuccinimide and Hg(2+) and activated by Mn(2+). Incubation of Xyl T1 and Xyl T2 with L-cysteine increased their half-lives up to 14 and 24 h at 50 °C, respectively. Atomic force microscopy showed a bimodal size distribution of globular particles for both enzymes, indicating that Xyl T1 is larger than Xyl T2.

Production and characterization of an enzyme complex from a new strain of Clostridium thermocellum with emphasis on its xylanase activity

A new bacterial strain (ISO II) was isolated from manure cow and identified as phylogenetically close to the thermophilic cellulolytic bacterium Clostridium thermocellum. The new strain produced extracellular xylanase, pectinase, mannanase and cellulase activities when grown in liquid culture medium containing banana stem as carbon source. The enzyme production profile after growth on banana stem showed that xylanase and cellulase activities were detected in different incubation periods. An enzyme complex containing xylanase, cellulase and mannanase activities was isolated from culture supernatant samples of strainISO II. The complex was partially purified by ultrafiltration and gel filtration chromatography on Sephacryl S-300. Zymogram analysis after SDS-PAGE presented at least 05 subunits with xylanase activity. The enzyme showed single protein and xylanase activity bands after electrophoresis under non-denaturing conditions. The hydrolysis of xylan was optimal at temperature range of 55-75oC and pH 6.0. Xylanase activity was quite stable at 65oC, retaining 80% of its original activity after 12 h incubation. The apparent Km values, using insoluble and soluble arabinoxylans as substrates, were 1.54 and 11.53 mg/mL, respectively. Xylanase was activated by dithiothreitol, L-tryptophan and L-cysteine and strongly inhibited by N-bromosuccinimide and CoCl2. The characterization of mannanase showed Km and temperature optimum of 0.846 mg/mL and 65oC, respectively and pH 8.0. By contrast to xylanase, it was less stable at 65oC with half-life of 2.5 h and inhibited by dithiothreitol and Ca2+.

Enzymology of Plant Cell Wall Breakdown: An Update

Vast quantities of lignocellulosic material are available for exploitation as potential source of food and biofuel. Lignocellulose structure is degraded by an arsenal of enzyme systems that works synergically. Basically, two enzyme types are responsible for the efficient degradation of lignocelluloses: hydrolytic enzyme system, which degrades the holocellulose structure and oxidative enzyme system, which acts on lignin and open phenyl rings. For a variety of reasons, enzymatic conversion of lignocellulose is preferred over chemical conversion procedures. This chapter shows a comprehensive picture of the main enzymes involved in lignocellulose breaking down.

The hydrolysis of agro-industrial residues by holocellulose-degrading enzymes

Holocellulose structures from agro-industrial residues rely on main and side chain attacking enzymes with different specificities for complete hydrolysis. Combinations of crude enzymatic extracts from different fungal species, including Aspergillus terreus, Aspergillus oryzae, Aspergillus niger and Trichoderma longibrachiatum, were applied to sugar cane bagasse, banana stem and dirty cotton residue to investigate the hydrolysis of holocellulose structures. A. terreus and A. oryzae were the best producers of FPase and xylanase activities. A combination of A. terreus and A. oryzae extracts in a 50% proportion provided optimal hydrolysis of dirty cotton residue and banana stem. For the hydrolysis of sugar cane bagasse, the best results were obtained with samples only containing A. terreus crude extract.

Exploring Trichoderma and Aspergillus secretomes: Proteomics approaches for the identification of enzymes of biotechnological interest

Filamentous fungal secretomes comprise highly dynamic sets of proteins, including multiple carbohydrate active enzymes (CAZymes) which are able to hydrolyze plant biomass polysaccharides into products of biotechnological interest such as fermentable sugars. In recent years, proteomics has been used to identify and quantify enzymatic and non-enzymatic polypeptides present in secretomes of several fungi species. The resulting data have widened the scientific understanding of the way filamentous fungi perform biomass degradation and offered novel perspectives for biotechnological applications. The present review discusses proteomics approaches that have been applied to the study of fungal secretomes, focusing on two of the most studied filamentous fungi genera: Trichoderma and Aspergillus.

Xylan-degrading enzymes from Aspergillus terreus: Physicochemical features and functional studies on hydrolysis of cellulose pulp

Two endo-β-1,4-xylanases named XylT1 and XylT2, previously purified from Aspergillus terreus, were structurally investigated by fluorescence quenching and characterized with respect to their binding properties with phenolic compounds. Neutral and charged quenchers had access to both enzymes in neutral and alkaline pHs. The greatest access was noted for the negative quencher, possibly due to positive amino acid residues in the vicinity of tryptophan. These tryptophan environments may partially explain the conformational differences and lower binding constants of phenolic compounds for XylT2 than XylT1Phenolic compounds had lower binding constants for XylT2 than XylT1. These results show that xylanases present structural and functional differences, despite belonging to similar families. XylT1 and XylT2 were also evaluated for their ability to hydrolyze cellulose pulp in different stages of bleaching. Both enzymes promoted hydrolysis of cellulose pulps, which was confirmed by the release of total reducing sugars, pentoses and chromophoric material. Analysis of released xylooligosaccharides demonstrated a preferential release of xylobiose. None of xylanases released glucose, showing that they do not hydrolyze the cellulose present in the pulp, making both enzymes excellent choices for bio-bleaching applications.

An Overview of Cellulose-Degrading Enzymes and Their Applications in Textile Industry

Abstract This chapter overviews the cellulose-degrading enzymes produced by microorganisms and their utility in the textile industry. The cellulose-degrading enzyme system is composed of cellobiohydrolases I and II (EC 3.2.1.76 and EC 3.2.1.91), endo-1,4-β-glucanase (EC 3.2.1.4), and β-glucosidase (EC 3.2.1.21), acting in synergy for the conversion of cellulose into glucose. In addition, recently discovered enzymes, such as CBM33 and GH61, were found to increase the efficiency of cellulose breakdown by acting on its surface. The use of cellulases in the textile industry has proven to be an important tool to make textile processing environmentally friendly and to reduce energy demand and water consumption. Their applications in the textile industry include bioscouring, biopolishing, and biostonewashing.

Hydrolysis of sugarcane bagasse with enzyme preparations from Acrophialophora nainiana grown on different carbon sources

Abstract The filamentous fungus Acrophialophora nainiana, isolated from a hot spring in Brazil, was grown in liquid culture on different cellulosic and lignocellulosic carbon sources for seven days and enzyme extracts were characterised with respect to their carbohydrase activity profile. The enzyme extracts obtained from growing A. nainiana on cellulose, dirty-cotton residue, sugarcane bagasse and banana stem were used in the hydrolysis of sugarcane bagasse untreated (UT), pre-treated by steam explosion (SET) and pre-treated by acid-catalysed steam explosion (SAT). The carbohydrase activity profile of the enzyme preparations varied significantly with the used carbon source. The highest enzyme activities, especially total cellulase (0.0132 IU) and xylanase (0.0774 IU) activities, were obtained with banana stem as the carbon source. Pectinase activity was produced on all carbon sources at comparable levels. On sugarcane bagasse, total cellulase activity on filter paper and pectinase activities were predominant, but a very low amount of xylanase and CMCase activity, 0.0011 IU and 0.0019 IU, respectively, was found. The exocellulase/endocellulase activity ratio (FPAsol/FPAinsol) of the cellulases produced varied between 1 and 4 depending on the substrate. The highest endocellulase activity (FPAinsol) content was obtained when grown on sugarcane bagasse. Conversions to reducing sugars of the differently pre-treated sugarcane bagasse substrates with enzyme preparations from A. nainiana were in general low. The highest conversion to reducing sugars (˜18%) was obtained for the SET bagasse by the banana stem enzyme preparation, while conversions with the other enzyme preparations were below 5%. In most cases a very low conversion (below 1%) was obtained for the SAT bagasse, but better conversions were achieved for the UT. These results are mainly attributable to the hydrolysis of the hemicellulose fraction and the low cellulase and β-glucosidase activities in the enzyme preparations. Hydrolysis data were also analysed and successfully fitted with a fractal kinetics model, and model parameters are discussed with respect to the carbon source used for A. nainiana enzyme production and substrate pre-treatment.

Enzymes and Food Industry: A Consolidated Marriage

Abstract The development of food processing techniques is intertwined with the history of mankind. Some processes, such as brewing, are dated from the dawn of civilization. In this context, the use of biological agents has been of utmost importance for both the improvement of nutritional and sensorial characteristics of foods or development of new products. In the last century, the development of enzyme-based technologies greatly impacted the food industry, since these biomolecules catalyze biochemical reactions with high specificity and mild physicochemical conditions. Moreover, they are safe tools for use in food processing. Enzymatic modification of foods was crucial for the development and expansion of some segments of food industry. The main classes of enzymes of considerable importance for the food industry include carbohydrate-acting enzymes, lipases, and proteases, which are used in food processing, such as baking, cereal processing, juice production, meat processing, and dairy production. This chapter aims at providing an overview of enzymatic applications in food industry, as well as the newly progresses in this area.