Thesis (MSc (Microbiology))--Stellenbosch University, 2008. / Complete degradation of cellulose produces mainly glucose, which can be fermented to ethanol. Therefore cellulose presents an abundant renewable energy resource for the production of an alternative, environmentally friendly, transportation fuel. Enzymatic degradation of cellulose is achieved by the synergistic action of three cellulase enzyme groups: endoglucanases, exoglucanases and -glucosidases. However, cellulolytic organisms do not produce significant amounts of ethanol. Therefore, a need has arisen to develop a recombinant microorganism with the ability to produce cellulolytic enzymes, hydrolyze cellulose and ferment the resulting sugars to ethanol in a single process step, referred to as “Consolidated Bioprocessing” (CBP). This would provide a cost-effective, economically feasible strategy for the production of bioethanol. The naturally fermentative yeast, Saccharomyces cerevisiae, is often used as host for the expression of recombinant proteins due to several characteristics, including its robustness in industrial processes, the well developed genetic tools available for manipulation and its proven safety status. A number of cellulase genes have previously been successfully expressed by recombinant S. cerevisiae strains. In this study, all three components of the cellulase system were co-expressed in S. cerevisiae to test the ability of the yeast to effectively produce the heterologous proteins, and consequently produce enough glucose for growth on an amorphous cellulosic substrate. The Trichoderma reesei endoglucanase gene egII (Cel5A) was successfully expressed by a S. cerevisiae Y294 strain. Recombinant EGII displayed activities of 19.6 nkat.ml-1 and 22.3 nkat.ml-1 towards CMC and barley -glucan, respectively. The major endoglucanase gene, egI (Cel7B) from T. reesei was subjected to random mutagenesis by propagating the egI-containing plasmid in an E. coli mismatch repair deficient strain. Screening of S. cerevisiae transformants revealed a strain, S. cerevisiae Y294[pLEM1], with improved levels of endoglucanase activity (21.8 nkat.ml-1), compared to S. cerevisiae Y294[pAZ40], expressing the wild type gene (10.3 nkat.ml-1). Through subcloning of the mutated ENO1 promoter region and the mutated egI gene fragment, it was established that the mutations located in both the promoter- and gene sequences were responsible for the improved levels of activity displayed by S. cerevisiae Y294[pLEM1]. The egII gene and the altered egI gene were co-expressed with a codon optimised T. reesei cellobiohydrolase (sCBHI) and a -glucosidase from Saccharomycopsis fibuligera. This resulted in a reduction in endoglucanase levels, possibly due to the metabolic burden placed on the yeast by co-expressing the different cellulases. The hydrolysis products produced by cellulase co-expressing strains were cellotriose, cellobiose and glucose, although the glucose yield was insufficient to enable growth on cellulose as sole carbon source. As the major hydrolysis product was cellobiose, it is likely that a bottleneck exists at its conversion to glucose, suggesting inadequate -glucosidase activity. This study has provided insight into co-expression of cellulase enzymes by the yeast S. cerevisiae. The knowledge obtained could be applied in optimizing cellulase cocktails for efficient cellulose degradation and eventual production of ethanol by recombinant yeast. It has also demonstrated the applicability of random mutagenesis for improving the activity of cellulases.

This project aimed investigated cellulase in-situ production for large-scale on-farm production of lignocellulosic biofuel. Cellulase activity and glucose released by T. reesei with corn stover and wheat bran as co-substrates for solid state cultivation (SSC) were examined. Co-cultivation has previously increased T. reesei cellulase, but corn stover and wheat bran have not been co-cultivated (Dhillon, Oberoi et al. 2011). This work compared cellulase activity and glucose concentration of corn stover co-cultivated with 0-40% wheat bran in high solids. Samples with at least 20% wheat bran exhibited increased cellulase activity. However, the average glucose concentration without wheat bran was 3.29 g/L compared to 16.7 g/L with wheat bran. Glucose released by T. reesei on pretreated corn stover with 0-40% wheat bran was compared at the optimal temperatures for fungal growth and for cellulase activity after SSC. Previous research has rarely used cellulase from SSC to hydrolyze lignocellulose. Following SSC of T. reesei at 30°C for seven days, samples were warmed to 50°C for five days. Glucose concentration increased to 12.1 and 32.7 g/L for samples with and without wheat bran. This strategy could reduce lignocellulosic fuel production costs by eliminating need for commercial cellulase and is promising for efficient cellulose hydrolysis.

Extensive research into enzyme-induced bio-conversion of lignocellulose to soluble sugars has been conducted and research continues in this area. Several approaches have been taken to attempt to alleviate the economic problems associated with utilisation of lignocellulose in fuel ethanol production. By expressing cellulase genes in planta, it is hoped that the cost of enzyme-mediated hydrolysis of cellulose to its soluble sugar monomers, will be reduced. Some accomplishments have been made in this area using nuclear genetic transformation (Abdeev et al., 2003; Abdeev et al., 2004; Austin-Phillips et al., 1999; Biswas et al., 2006; Dai et al., 2000a,b; Dai et al., 2005; Jin et al., 2003; Kawazu et al., 1999; Sakka et al., 2000; Ziegelhoffer et al., 1999; Ziegelhoffer et al., 2001; Ziegler et al., 2000), but more research is required to bring the levels of cellulase enzyme expression in plants to levels that will make the process economically competitive. Chloroplasts of N. tabacum were selected as a target for transformation for high level expression due to their extremely high rates of transcription and translation. These were transformed with two genes, the e1 gene from A. cellulolyticus, and the cbh1 gene from T. reesei. Further aims included the investigation of the effects of using different promoters, and the novel use of both nuclear and chloroplast-based expression in a single plant, on the level of protein production in the heterologous host. Heterologous expression of the cbh1 gene was not successful. This is thought to be due to toxicity of the protein in a prokaryotic environment. Future studies should focus on trying to avoid this toxicity by targeting of the chloroplast-expressed enzyme to specific tissues, such as the thylakoid membrane, for containment, creating a codon-optimised synthetic gene that better mimics the codon usage of the plant to be used for expression, or placing the expression under a reactive cascade that is only activated upon exposure to an external trigger. Heterologous expression of the full length gene for E1 from A. cellulolyticus was successful. Chloroplast homology vectors under the constitutive promoter Prrn, and the inducible promoter T7, were constructed and these were used to successfully transform N. tabacum cv. Petit Havana chloroplasts. Stable transgenic plants were produced and evaluated by a variety of means, with the heterologously expressed enzyme showing activity against the soluble substrate analogue MUC of up to 3122 ± 466 pmol 4-MU/mg TSP/min and an E1 accumulation level of up to 0.35% ± 0.06 of the total soluble protein. Lastly, chloroplast transformation was combined with nuclear transformation to create novel dual-transgenic plants simultaneously expressing E1 from both the nuclear and chloroplast genomes. The combination of these technologies was very successful, with the heterologously expressed enzyme showing activity against the soluble substrate analogue MUC of up to 35706 ± 955 pmol 4-MU/mg TSP/min and an E1 accumulation level of up to 4.78% ± 0.13 of the total soluble protein, and provides a new approach for increasing the accumulation levels of plant-produced cellulase enzymes.

Made available in DSpace on 2014-06-11T19:35:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2005Bitstream added on 2014-06-13T18:46:07Z : No. of bitstreams: 1 adalberto_pr_dr_araiq.pdf: 786247 bytes, checksum: cf55691544529062c4d2288dd8709c7a (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A β-D-galactopiranosídeo hidrolase é uma enzima com ampla aplicação tecnológica, clínica e experimental. A enzima possui um íon de metal bivalente coordenado. Para se obter informações a respeito do centro metálico da enzima, algumas espécies de fungos filamentosos foram testadas para a produção de β−galactosidase, ente as quais, a linhagem de Trichoderma reesei FTKO foi selecionada. O extrato enzimático, ao passar pelo processo de purificação, revelou ao menos duas isoformas de β- galactosidase identificadas como BGT I e BGT II. As enzimas são termofílicas e termotolerantes. A dependência do pH para a atividade, os valores de medidas cinéticas e a atividade revelada em gel de poliacrilamida distinguem as isozimas. Para avaliar a necessidade de metais bivalentes foi padronizada uma metodologia de desativação da enzima, apoiado na competição entre o agente seqüestrante e a enzima pelos metais. A metodologia foi aplicada ao o extrato comercial Lactozym 3000 (Novozyme Latin America Ltd.) revelou a necessidade de metal para a sua atividade com a desativação seguida de reativação pelos íons Mg2+, Mn2+, Co 2+, Ni2+, mas não por Zn2+ e Eu3+. A enzima BGT I (pH ótimo de 4,5) mostrou-se, contudo, resistente à desativação, pois se mantém ativa mesmo em altas concentrações de EDTA. / The β-D-galactopiranosyde-hidrolase is an enzyme with large technological, clinic, and scientific application. The enzyme coordinates a bivalent metal ion. In order to investigate the metal center of the enzyme of moulds, some strains were tested for β−galactosidase production. Among then, Trichoderma reesei FTKO were selected. The partially purified enzymatic extract reveals the production of two isoforms of β-galactosidase, at least. This enzymes are identified as BGT I e BGT II. Both are termophylic and thermotolerant. The pH dependence, the measured kinetic values and the activity revealed in polyacrylamide electrophoresis gels distinguished the isozymes. The metal assistance of enzymatic activity was evaluated in a standard deactivation methodology of enzyme by competition for the metal by chelating agent and metal site of enzyme. When this methodology was applied in the deactivation of commercial extract Lactozym 3000 (Novozyme Latin America Ltd.), revealed the metal importance for activity. The deactivation was reverted by metals ions as Mg2+, Mn2+, Co2+, Ni2+, but not by Zn2+ or Eu3+. On the other hand BGT I enzyme (optimal pH of 4,5) maintained the activity, even in high concentration of EDTA.

Resumo: A β-D-galactopiranosídeo hidrolase é uma enzima com ampla aplicação tecnológica, clínica e experimental. A enzima possui um íon de metal bivalente coordenado. Para se obter informações a respeito do centro metálico da enzima, algumas espécies de fungos filamentosos foram testadas para a produção de β−galactosidase, ente as quais, a linhagem de Trichoderma reesei FTKO foi selecionada. O extrato enzimático, ao passar pelo processo de purificação, revelou ao menos duas isoformas de β- galactosidase identificadas como BGT I e BGT II. As enzimas são termofílicas e termotolerantes. A dependência do pH para a atividade, os valores de medidas cinéticas e a atividade revelada em gel de poliacrilamida distinguem as isozimas. Para avaliar a necessidade de metais bivalentes foi padronizada uma metodologia de desativação da enzima, apoiado na competição entre o agente seqüestrante e a enzima pelos metais. A metodologia foi aplicada ao o extrato comercial Lactozym 3000 (Novozyme Latin America Ltd.) revelou a necessidade de metal para a sua atividade com a desativação seguida de reativação pelos íons Mg2+, Mn2+, Co 2+, Ni2+, mas não por Zn2+ e Eu3+. A enzima BGT I (pH ótimo de 4,5) mostrou-se, contudo, resistente à desativação, pois se mantém ativa mesmo em altas concentrações de EDTA. / Abstract: The β-D-galactopiranosyde-hidrolase is an enzyme with large technological, clinic, and scientific application. The enzyme coordinates a bivalent metal ion. In order to investigate the metal center of the enzyme of moulds, some strains were tested for β−galactosidase production. Among then, Trichoderma reesei FTKO were selected. The partially purified enzymatic extract reveals the production of two isoforms of β-galactosidase, at least. This enzymes are identified as BGT I e BGT II. Both are termophylic and thermotolerant. The pH dependence, the measured kinetic values and the activity revealed in polyacrylamide electrophoresis gels distinguished the isozymes. The metal assistance of enzymatic activity was evaluated in a standard deactivation methodology of enzyme by competition for the metal by chelating agent and metal site of enzyme. When this methodology was applied in the deactivation of commercial extract Lactozym 3000 (Novozyme Latin America Ltd.), revealed the metal importance for activity. The deactivation was reverted by metals ions as Mg2+, Mn2+, Co2+, Ni2+, but not by Zn2+ or Eu3+. On the other hand BGT I enzyme (optimal pH of 4,5) maintained the activity, even in high concentration of EDTA. / Orientador: Antonio Carlos Massabni. / Coorientador: Rubens Monti. / Doutor

Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-09-19T13:51:14Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-09-19T13:51:26Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) / Made available in DSpace on 2018-09-19T13:51:26Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) Previous issue date: 2014-05-06 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cellulolytic enzymes are highly specific biocatalysts. These are classified according to the place where they work in the cellulosic fiber: endoglucanases initiate hydrolysis exoglucanases act on reducing terminals produced by endoglucanases and β-glucosidases act on the product of catalysis of exoglucanases releasing glucose monomers . Penicillium fungi are widely reported as a great producer of cellulolytic enzymes. Aiming to contribute to the catalytic process and obtaining new sources of enzymes , this study evaluated the physicochemical properties of enzymes produced cellulolytic fungus Penicillium citrinum isolated from bagasse cane sugar mill Jayoro of S / A, located in the municipality of Presidente Figueiredo -AM - Brazil . And the enzyme production profile was repaired during submerged fermentation with the strain of Trichoderma reesei QM9414 which is widely reported as a great producer of cellulolytic enzymes. P. citrinum showed equivalent performance to T. reesei QM9414 in volumetric enzyme activity (U/mL) and superior performance in specific activities (U/mg). About properties of endoglucanases and β-glucosidase from P. citrinum, the optimum temperature for activity was 60 ° C for both enzymes. The optimum pH for endoglucanases activity was pH 4.2 and the activity of β-glucosidase pH 6.0. P. citrinum produced at least two endoglucananes with apparent molecular masses of ~ 50kDa and ~ 35kDa , visualized on SDS-PAGE gels and in gel activity ( zymogram ) . An elution fraction obtained from size exclusion chromatography, showed only β-glucosidase activity and revealed bands of approximately 100 kDa . The performance and characteristics of these endo and β- glucosidases demonstrate that P. citrinum represents a new alternative for production of cellulolytic enzymes for hydrolysis of fiber. / As enzimas celuloliticas são biocatalizadores altamente específicos. Estas, são classificadas de acordo com o local em que atuam na fibra celulósica: endoglucanases iniciam a hidrólise, exoglicanases agem nos terminais redutores produzidos pelas endoglicanases e as beta-glicosidases atuam no produto da catálise das exoglicanases liberando monômeros de glicose. Fungos do gênero Penicillium são amplamente reportados como ótimos produtores de enzimas celuloliticas. Visando contribuir para os processos biocataliticos e obtenção de novas fontes de enzimas, neste trabalho avaliou-se as propriedades físico-químicas de enzimas celuloliticas produzidas fungo Penicillium citrinum isolado do bagaço de cana-de-açucar da Usina Jayoro S/A, localizada no município de Presidente Figueiredo-AM-Brasil. E comparou-se o perfil de produção enzimática durante a fermentação submersa com o da estirpe Trichoderma reesei QM9414 que é amplamente reportado como um otimo produtor de enzimas celulolíticas. P. citrinum apresentou desempenho equivalente à T. reesei QM9414 nas atividades enzimáticas volumétricas (U/mL) e desempenho superior nas atividades especificas (U/mg). Quanto as propriedades das endoglicanases e beta-glicosidase de P. citrinum, a temperatura ótima de atividade foi 60ºC para ambas as enzimas. O pH ótimo para a atividade endoglicanásica foi pH 4,2 e para a atividade de beta-glicosidase pH 6,0. P. citrinum produziu pelo menos duas endoglicananes com massas moleculares aparente de ~ 50kDa e ~ 35kDa, visualizadas em gel de SDS-PAGE e em gel de atividade (zimograma). Uma fração obtida da eluição de cromatografia de exclusão molecular, apresentou somente atividade de beta-glicosidase e revelou bandas de aproximadamente 100kDa. O desempenho e as características dessas endo e beta-glicosidades demonstram que P. citrinum representa uma alternativa para produção de novas enzimas para hidrolise da fibra celulolitica.

No description available.

Dans le contexte énergétique et climatique mondial, le coût élevé des enzymes Cellulolytiques (cellulases) freine le développement des bioraffineries lignocellulosiques, pour produire des biocarburants et composés chimiques à partir d'une matière première végétale renouvelable. L'objectif de ce travail est de caractériser et de modéliser le métabolisme du micro-organisme Trichoderma reesei, afin d'optimiser le protocole industriel de production de cellulases. Cette étude a été réalisée sur des milieux modèles représentatifs de ceux attendus à l'échelle industrielle. Tout d'abord, la stoechiométrie des réactions de croissance et de production a été établie, puis une étude cinétique a été menée pour mesurer précisément le comportement du micro-organisme à forte induction de la production de cellulases. Le modèle résultant a été utilisé pour optimiser le protocole industriel de production. Ensuite, l'intégration de cette étape dans une bioraffinerie lignocellulosique a été étudiée, avec l'effet sur le métabolisme i) des mélanges de sucres disponibles, ii) des composés inhibiteurs issus de la dégradation de la lignocellulose, et iii) du changement d'échelle. Ces travaux ont fait progresser de façon substantielle les connaissances du métabolisme de T. reesei en ce qui concerne la production de cellulases, et les modèles développés sont des outils d'aide rationnelle à la définition d'un procédé de production de cellulases intégré dans une bioraffinerie lignocellulosique. / In the global energetic and climatic context, the high cost of the cellulolytic enzymes (cellulases) postpones the development of lignocellulosic biorefineries, dedicated to produce biofuels and chemical compounds from renewable vegetable feedstocks. The aim of this work was to measure and model the metabolism of the micro-organism Trichoderma reesei, in order to optimize the industrial protocol for the production of cellulase. This study was carried out using synthetic media representative of industrial ones. First, the stoichiometries of growth and protein production reactions were determined. Then, a kinetic study was conducted to precisely measure the specific rates of T. reesei at high induction of cellulase production. The resulting model was used to optimize the industrial production protocol. Finally the integration of this step in a lignocellulosic biorefinery was studied by determining the impacts on the metabolism of i) available sugar mixtures, ii) inhibitory compounds from lignocellulosic biomass degradation, and iii) scale-up. These results significantly contributed to improve the knowledge of T. reesei metabolism on cellulase production. The developed models are rational tools for the optimization of a cellulase production protocol suited to lignocellulosic biorefineries.

La production d’éthanol à partir de biomasse lignocellulosique est reconnue comme une des voies possibles de réduction des émissions de gaz à effet de serre et de remplacement partiel des énergies fossiles. Pour être compétitif, la production d'enzymes à bas coûts est nécessaire. Ces enzymes sont produites par le champignon filamenteux Trichoderma reesei, qui présente, à forte concentration, un comportement fortement rhéofluidifiant pouvant entrainer des limitations de mélange et de transfert de matière lors du changement d'échelle. Dans ce travail, il est proposé de compléter les données de la littérature concernant le temps de mélange, la puissance dissipée et le transfert de matière gaz-liquide (global et local) par des mesures à plusieurs échelles dans des fluides modèles de rhéologie similaire aux milieux biologiques visés. Les modèles et corrélations développés qui en résultent sont directement exploitables pour le design des fermenteurs industriels. Afin d’étudier plus en détail le mélange, le taux de cisaillement et la turbulence, une étude par PIV a été menée sur des milieux transparents. La caractérisation fine de l'hydrodynamique repose sur la dissociation des différentes composantes du mouvement à l’aide de la POD. L'évolution des grandeurs mesurées avec les conditions opératoires permet de fournir des indications précieuses pour l'extrapolation des fermenteurs mettant en œuvre des micro-organismes potentiellement sensibles au cisaillement / Ethanol made from cellulosic biomass is recognized as a promising substitute for fossil fuel and thus as a way to reduce greenhouse gas emissions. To be competitive, low cost cellulosic enzymes produced by the filamentous fungus Trichoderma reesei are required. At high biomass concentration, the culture broth becomes so highly shear-thinning that mixing and mass transfer limitations may be encountered when the process is scaled up.In this study, we propose to complete data available in the literature for mixing times, power draw, and mass transfer (local and global) with measurements at several scales in model fluids (shear thinning) that mimic the rheology of biological media. Models and correlations that derive from this work can be used directly for industrial fermentor design. In order to study mixing, local shear rate and turbulence in detail, PIV is performed in transparent model fluids. The refined hydrodynamic characterisation relies on the dissociation of instantaneous velocity by means of the POD method. The change of key parameters with operating conditions gives relevant information for the scale-up of shear-sensitive micro-organisms.

Dans le contexte actuel, il devient nécessaire de rendre les alternatives au pétrole, tel que le bioéthanol 2G, disponibles à grande échelle. Cependant, l’étape d’hydrolyse par les enzymes de Trichoderma reesei reste un verrou à un procédé économiquement stable et rentable. Ces travaux de thèse, s'intègrent dans le cadre du projet Futurol et ont pour objectifs d'identifier et de caractériser de nouvelles enzymes fongiques pour améliorer l'hydrolyse de la biomasse lignocellulosique. A partir des données protéomiques disponibles pour Podospora anserina et Fusarium verticillioides, une douzaine d'enzymes candidates ont été identifiées dans leurs sécrétomes. Ce travail de thèse s'est plus particulièrement focalisé sur les AA9s « Lytic Polysaccharide Monooxygenases » (LPMOs) de P. anserina. Parmi les LPMOs étudiées, PaLPMO9A, PaLPMO9E et PaLPMO9H, qui possèdent un CBM1, sont les plus actives sur la cellulose. La détermination de la régiosélectivité d'action a mis en évidence que PaLPMO9A et PaLPMO9H clivent la cellulose en position C1 et C4 alors que la PaLPMO9E génère uniquement des produits oxydés en C1. La PaLPMO9H est la plus versatile puisqu’elle est active sur les cello-oligosaccharides solubles et sur les polysaccharides hémicellulosiques liés en β-(1,4) (i.e., xyloglucane, glucomannane). La supplémentation du cocktail de T. reesei avec PaLPMO9E ou PaLPMO9H a permis de doubler les rendements d'hydrolyse du miscanthus prétraité. Les travaux réalisés au cours de cette thèse ont permis de démontrer l'importance de ces enzymes oxydatives dans les phénomènes de déconstruction de la lignocellulose chez les champignons filamenteux. / In the current context, it becomes essential to make alternative to oil, such as the 2G bioethanol, available at large scale. However, the hydrolysis step by Trichoderma reesei enzymes remains the major bottleneck for an economically sustainable process. The present work is part of the Futurol project, and aims at identifying and characterizing new fungal enzymes to improve the hydrolysis of lignocellulosic biomass. From the proteomic data available for Podospora anserina and Fusarium verticillioides, a dozen of interesting enzymes were identified in their secretomes. This work focuses, mainly, on the AA9s « Lytic Polysaccharide Monooxygenases » (LPMOs) from P. anserina. Among all the LPMOs studied, PaLPMO9A, PaLPMO9E and PaLPMO9H that harbored a CBM1 were the most active on cellulose. Investigation of their regioselective mode of action revealed that PaLPMO9A and PaLPMO9H oxidatively cleaved at both C1 and C4 positions while PaLPMO9E released only C1-oxidized products. PaLPMO9H that was the most versatile in terms of substrate specificity as it also displayed activity on cello-oligosaccharides and β-(1,4)-linked hemicellulose polysaccharides (e.g., xyloglucan, glucomannan). The hydrolysis yield of the pretreated miscanthus was significantly improved up to 2 fold, when the PaLPMO9E, or PaLPMO9H were supplemented to the T. reesei cocktail. This work demonstrated the importance of these oxidative enzymes for lignocellulose deconstruction by fungi. These biocatalysts open new prospects to improve the enzymatic conversion of plant biomass for 2G bioethanol production.