Correlation between Agar Plate Screening and Solid-State Fermentation for the Prediction of Cellulase Production by Trichoderma Strains

The viability of converting biomass into biofuels and chemicals still requires further development towards the reduction of the enzyme production costs. Thus, there is a growing demand for the development of efficient procedures for selection of cellulaseproducing microorganisms. This work correlates qualitative screening using agar plate assays with quantitative measurements of cellulase production during cultivation under solid-state fermentation (SSF). The initial screening step consisted of observation of the growth of 78 preselected strains of the genus Trichoderma on plates, using microcrystalline cellulose as carbon source. The 49 strains that were able to grow on this substrate were then subjected to a second screening step using the Congo red test. From this test it was possible to select 10 strains that presented the highest enzymatic indices (EI), with values ranging from 1.51 to 1.90. SSF cultivations using sugarcane bagasse and wheat bran as substrates were performed using selected strains. The CG 104NH strain presented the highest EGase activity (25.93 UI·g −1 ). The EI results obtained in the screening procedure using plates were compared with cellulase production under SSF. A correlation coefficient (R 2 ) of 0.977 was obtained between the Congo red test and SSF, demonstrating that the two methodologies were in good agreement

DESAFIOS RELACIONADOS À PRODUÇÃO E APLICAÇÃO DAS ENZIMAS CELULOLÍTICAS NA HIDRÓLISE DA BIOMASSA LIGNOCELULÓSICA

The second generation (2G) ethanol obtained from lignocellulosic biomass has been considered as a potential alternative source of energy for the transportation sector. However, the technologies to convert lignocellulose into fermentable sugars still face several challenges. One of the major challenges of this process is related to the cost of the cellulolytic enzymes. Thus, the development of bioprocesses for enzyme production and strategies to increase the final yield of the enzymatic hydrolysis reactions are required to ensure the economic feasibility of the biomass conversion process. In this review, it is discussed the progress related to fungal enzyme technology over the past few years with major emphasis on the biocatalysts needed for cellulose degradation as well as on cultivation strategies for the production of enzymes on-site. The potential of applying secretome analysis as a tool for the characterization of the enzymatic complexes and some limitations on the use of enzymatic cocktails in the saccharification step, such as the unproductive adsorption of enzymes onto lignin are also discussed in light of the recent literature

Stabilization of glycosylated β-glucosidase by intramolecular crosslinking between oxidized glycosidic chains and lysine residues

Many industrial enzymes can be highly glycosylated, including the β-glucosidase enzymes. Although glycosylation plays an important role in many biological processes, such chains can cause problems in the multipoint immobilization techniques of the enzymes, since the glycosylated chains can cover the reactive groups of the protein (e.g., Lys) and do not allow those groups to react with reactive groups of the support (e.g., aldehyde and epoxy groups). Nevertheless, the activated glycosylated chains can be used as excellent crosslinking agents. The glycosylated chains when oxidized with periodate can generate aldehyde groups capable of reacting with the amino groups of the protein itself. Such intramolecular crosslinks may have significant stabilizing effects. In this study, we investigated if the intramolecular crosslinking occurs in the oxidized β-glucosidase and its effect on the stability of the enzyme. For this, the oxidation of glycosidic chains of β-glucosidase was carried out, allowing to demonstrate the formation of aldehyde groups and subsequent interaction with the amine groups and to verify the stability of the different forms of free enzyme (glycosylated and oxidized). Furthermore, we verified the influence of the glycosidic chains on the immobilization of β-glucosidase from Aspergillus niger and on the consequent stabilization. The results suggest that intramolecular crosslinking occurred and consequently the oxidized enzyme showed a much greater stabilization than the native enzyme (glycosylated). When the multipoint immobilization was performed in amino-epoxy-agarose supports, the stabilization of the oxidized enzyme increases by a 6-fold factor. The overall stabilization strategy was capable to promote an enzyme stabilization of 120-fold regarding to the soluble unmodified enzyme.The authors thank the support from Research Institute of Food Science (CIAL), Embrapa Instrumentation of São Carlos, Foundation for Research Support of Espírito Santo (FAPES), and Federal University of Espírito Santo (UFES).Peer reviewe

The functional properties of a xyloglucanase (gh12) of Aspergillus terreus expressed in Aspergillus nidulans may increase performance of biomass degradation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Filamentous fungi are attractive hosts for heterologous protein expression due to their capacity to secrete large amounts of enzymes into the extracellular medium. Xyloglucanases, which specifically hydrolyze xyloglucan, have been recently applied in lignocellulosic biomass degradation and conversion in many other industrial processes. In this context, this work aimed to clone, express, and determine the functional properties of a recombinant xyloglucanase (AtXEG12) from Aspergillus terreus, and also its solid-state (SSF) and submerged (SmF) fermentation in bioreactors. The purified AtXEG12 showed optimum pH and temperature of 5.5 and 65 A degrees C, respectively, demonstrating to be 90 % stable after 24 h of incubation at 50 A degrees C. AtXEG12 activity increased in the presence of 2-mercaptoethanol (65 %) and Zn+2 (45 %), while Cu+2 and Ag+ ions drastically decreased its activity. A substrate assay showed, for the first time for this enzyme's family, xylanase activity. The enzyme exhibited high specificity for tamarind xyloglucan (K (M) 1.2 mg mL(-1)) and V (max) of 17.4 mu mol min(-1) mg(-1) of protein. The capillary zone electrophoresis analysis revealed that AtXEG12 is an endo-xyloglucanase. The heterologous xyloglucanase secretion was greater than the production by wild-type A. terreus cultivated in SmF. On the other hand, AtXEG12 activity reached by SSF was sevenfold higher than values achieved by SmF, showing that the expression of recombinant enzymes can be significantly improved by cultivation under SSF.Filamentous fungi are attractive hosts for heterologous protein expression due to their capacity to secrete large amounts of enzymes into the extracellular medium. Xyloglucanases, which specifically hydrolyze xyloglucan, have been recently applied in lign1002191339144CNQP - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)563260/2010-62010/52322-32013/16563-42010/10184-32012/20549-42014/18714-2This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho de Desenvolvimento Científico e Tecnológico (CNPq), National System for Research on Biodiversity (Sisbiota-Brazil, CNPq 563260/2010-6/ FAPESP n