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.We thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, process 2010/52322-3) and Conselho de Desenvolvimento Científico e Tecnológico (CNPq, process 563260-6). This project is also part of National Institute of Science and Technology of the Bioethanol (FAPESP, process 2008/57908-6). Dr. M. L. T. M. Polizeli has a Fellowship of Research Productivity of CNPq. We thank Abilio Borghi for the technical assistance with the English language.info:eu-repo/semantics/publishedVersio

Co-cultivation of Aspergillus nidulans recombinant strains produces an enzymatic cocktail as alternative to alkaline sugarcane bagasse pretreatment

Plant materials represent a strategic energy source because they can give rise to sustainable biofuels through the fermentation of their carbohydrates. A clear example of a plant-derived biofuel resource is the sugar cane bagasse exhibiting 60 % - 80 % of fermentable sugars in its composition. However, the current methods of plant bioconversion employ severe and harmful chemical/physical pretreatments raising biofuel cost production and environmental degradation. Replacing these methods with co-cultivated enzymatic cocktails is an alternative. Here we propose a pretreatment for sugarcane bagasse using a multi-enzymatic cocktail from the co-cultivation of four Aspergillus nidulans recombinant strains. The co-cultivation resulted in the simultaneous production of GH51 arabinofuranosidase (AbfA), GH11 endo-1,4-xylanase (XlnA), GH43 endo-1,5-arabinanase (AbnA) and GH12 xyloglucan specific endo-β-1,4-glucanase (XegA). This core set of recombinant enzymes was more efficient than the alternative alkaline method in maintaining the cellulose integrity and exposing this cellulose to the following saccharification process. Thermogravimetric and differential thermal analysis revealed residual byproducts on the alkali pretreated biomass, which were not found in the enzymatic pretreatment. Therefore, the enzymatic pretreatment was residue-free and seemed to be more efficient than the applied alkaline method, which makes it suitable for bioethanol production

Produção e ação de um pool enzimático de Aspergillus phoenicis com fontes de carbono diferentes

Aspergillus phoenicis is an interesting heat tolerant fungus that can synthesize enzymes with several applications in the food industry due to its great hydrolytic potential. In this work, the fungus produced high enzymatic levels when cultivated on inexpensive culture media consisting of flakes from different origins such as cassava flour, wheat fibre, crushed soybean, agro-industrial wastes, starch, glucose or maltose. Several enzymatic systems were produced from these carbon sources, but amylase was the most evident, followed by pectinase and xylanase. Traces of CMCases, avicelase, lipase, β-xylosidase, β-glucosidase and α-glucosidase activities were also detected. Amylases were produced on rye flakes, starch, oat flakes, corn flakes, cassava flour and wheat fibre. Significant amylolytic levels were produced in the culture medium with glucose or when this sugar was exhausted, suggesting an enzyme in the constitutive form. Cassava flour, rye, oats, barley and corn flakes were also used as substrates in the hydrolytic reactions, aiming to verify the liberation potential of reducing sugars. Corn flakes induced greater liberation of reducing sugars as compared to the others. Thin layer chromatography of the reaction end products showed that the hydrolysis of cassava flour liberated maltooligosaccharides, but cassava flour and corn, rye, oats and barley flakes were hydrolyzed to glucose. These results suggested the presence of glucoamylase and α-amylase as part of the enzymatic pool of A. phoencis

Heterologous production and biochemical characterization of a new highly glucose tolerant GH1 β-glucosidase from Anoxybacillus thermarum

The enzymatic lignocellulosic biomass conversion into value-added products requires the use of enzyme-rich cocktails, including β-glucosidases that hydrolyze cellobiose and cellooligosaccharides to glucose. During hydrolysis occurs accumulation of monomers causing inhibition of some enzymes; thus, glucose/xylose tolerant β-glucosidases could overcome this drawback. The search of new tolerant enzymes showing additional properties,such as high activity, wide-pH range, and thermal stability is very relevant to improve the bioprocess. We describe a novel β-glucosidase GH1 from the thermophilic Anoxybacillus thermarum (BgAt), which stood out by the robustness combination of great glucose/xylose tolerance, thermal stability, and high Vmax. The recombinant his-tagged-BgAt was overexpressed in Escherichia coli, was purified in one step, showed a high glucose/xylose tolerance, and activity stimulation (presence of 0.4M glucose/1.0M xylose). The optimal activity was at 65 °C - pH 7.0. BgAt presented an extraordinary temperature stability (48 h – 50 °C), and pH stability (5.5–8.0). The novel enzyme showed outstanding Vmax values compared to other β-glucosidases. Using p-nitrophenyl-β-D-glucopyranoside as substrate the values were Vmax (7614 U/mg), and KM (0.360 mM). These values suffer a displacement in Vmax to 14,026 U/mg (glucose), 14,886 U/mg (xylose), and KM 0.877mM (glucose), and 1.410mM (xylose).This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, process no 2018/07522-6) and a scholarship to Tassio B. Oliveira (grant 2017/09000-4). MLTMP is a Research Fellow of Conselho de Desenvolvimento Científico e Tecnológico (CNPq, process 301963/2017-7). The project also received grants from National Institute of Science and Technology of Bioethanol,INCT, CNPq 465319/2014-9/FAPESP nº 2014/50884-5). PZA was a fellow from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) funding code 001 and Programa de Doutorado Sanduiche no Exterior, PDSE no 88881.135684/2016-01.Peer reviewe