Na produção do etanol 2G, as celulases representam um componente importante do custo. Com base em informações da literatura e resultados obtidos no Laboratório de Engenharia Bioquímica da Escola Politécnica da USP, foi feito um estudo do custo de produção das celulases via fermentação em estado sólido (FES) em escala industrial. Foram consideradas as seguintes variáveis no processo de produção das celulases, denominadas cenários de produção: concentração das celulases no meio de cultura de 1 a 150 FPU/gms; produtividade em celulases em 0,11 e 0,45FPU/gms.h; atividade de celulase na hidrólise de bagaço de cana-de-açúcar entre 7 e 30 FPU/g de substrato seco; massa de bagaço de cana a ser hidrolisada entre 5 e 30% do bagaço gerado numa usina sucroalcooleira de referência (1.000.000 ton. de cana-de-açúcar /ano); custo dos substratos para a FES variando entre US$6,00 a US$12,00 por tonelada para o bagaço de cana-de-açúcar (80% da massa do meio) e US$80,00 a US$110,00 por tonelada para o farelo de trigo (20% da massa do meio); capacidade volumétrica dos reatores de FES variando entre 5 e 50 m3. Os impactos das variáveis consideradas foram: concentração de celulases no meio de cultura, acima de 45FPU/gms causam reduções menores que 5% no custo de produção; produtividades iguais em celulases (0,45FPU/gms.h), apresenta um processo 11% menos custoso para aquela com maior produção de enzimas, em relação ao tempo de fermentação; destinação de até 10% do bagaço gerado pela usina, reduz em 7% o custo de produção das celulases e a dosagem de celulase não causa redução significativa nos custos de produção, assim como o custo das matérias-primas; o aumento da capacidade dos reatores de FES causam redução de até 47% no custo de produção das celulases. A análise de regressão linear das variáveis apresentadas indica qual o impacto percentual dobre o custo de produção das celulases: quantidade de bagaço de cana-de-açúcar destinado à hidrólise (0,6%), atividade enzimática para a reação de hidrólise (0,3%), produção de celulase (73,4%), volume dos reatores de inóculo (0,5%), volume dos reatores de FES (2,7%), tempo de crescimento do inóculo (0,1%), tempo de fermentação (3,7%), custo do bagaço de cana-de-açúcar (0,9%), custo do farelo de trigo (0,6%) e outras variáveis não analisadas (17,2%). Conclui-se que, com os dados atuais sobre FES, a probabilidade de se obter um processo em escala industrial com custo de produção de celulase menor que US$0,50/100.000FPU é de 15%. Desenvolvimento de culturas microbianas mais produtivas em celulases e tecnologias mais avançadas de reatores de grande escala, são necessárias à viabilidade da produção das celulases via FES para aplicação na hidrólise de celulose. / In ethanol 2G production, cellulases are an important component of the cost. Based on information from literature and results obtained in the Labotatório de Engenharia Bioquímica da Escola Politécnica da USP, a study was made of the production cost of cellulases via solid state fermentation (SSF) on industrial scale. The following variables were considered in the production of cellulases, called \"production scenarios\": the concentration of cellulase in the culture medium from 1 to 150 FPU / gms; productivity cellulases in 0.11 and 0.45 FPU / gms.h; cellulase activity in the hydrolysis of bagasse from sugar cane between 7 and 30 FPU / g substrate dry mass; cane bagasse hydrolyzed to be between 5 and 30% of sugarcane bagasse generated in a reference plant (1,000,000 ton of cane sugar per year) and the cost of substrates for SSF ranging from $ 6.00 to $ 12.00 per ton for the sugarcane bagasse (80% by weight of medium) and $ 80, 00 to $ 110.00 per ton for wheat bran (20% of the mass medium); volumetric capacity of the reactors of FES ranged between 5 and 50 m3. The impacts of the variables considered were: concentration of cellulase in the culture medium above 45FPU/gms cause reductions of less than 5% of the cost of production; for equal productivities of cellulose (0.45 FPU / gms.h) shows a process 11% less costly when the production of enzymes is higher in relation to fermentation time; using up to 10% of sugarcane bagasse generated by the reference plant, reduces by 7% the cost of production of cellulases and cellulase dosage does not cause significant reduction in production costs as well as the cost of raw materials; increasing the capacity of the reactors of FES cause a reduction of up to 47% on cost of production of cellulases. The linear regression analysis of the variables presented indicates what percentage impact the production cost of cellulases: The amount of bagasse cane sugar for the hydrolysis (0.6%), enzymatic activity for the hydrolysis reaction (0, 3%), production of cellulase (73.4%), the reactor volume of inoculum (0.5%), volume of the reactors of FES (2.7%), time of growth of the inoculum (0.1%) fermentation time (3.7%), cost of sugarcane bagasse (0.9%), cost of wheat bran (0.6%) and other variables not analyzed (17.2%). It was concluded that with current data on FES, the probability of obtaining a process on an industrial scale with production cost of cellulase less than $ 0.50/100.000FPU is 15%. Development of microbial cultures more productive in cellulases and more advanced technologies for large-scale reactors are necessary for the viability of the production of cellulases for FES for application in the hydrolysis of cellulose.

O processamento da uva para produção de suco e vinho engloba diferentes processos tecnológicos, como por exemplo, o tratamento enzimático, onde usualmente são utilizadas preparações de enzimas comerciais, buscando-se aprimorar processos como clarificação e extração. Entretanto, esse é um tratamento que demanda elevado tempo de processamento, sendo relevante a busca por novas tecnologias a fim de apurar o processo. O ultrassom pode ser utilizado como uma alternativa para reduzir o tempo de processamento e acrescer qualidade ao produto, pois além de ser uma técnica sustentável, pode proporcionar benefícios no uso combinado com enzimas, favorecendo as reações em decorrência de seus efeitos. Neste trabalho, o uso em conjunto de tratamento enzimático (ET), ultrassom (US), e agitação mecânica (MS) foi avaliado na extração do mosto da uva Cabernet Sauvignon, a fim de compreender os efeitos das técnicas combinadas. Inicialmente, nove preparados enzimáticos comerciais foram caracterizados segundo suas atividades enzimáticas e aplicados para extração do mosto, avaliando a variação de temperatura (40, 50 e 60 °C), tempo (15 e 30 min), e concentração de enzima (0,01 a 2,0 U.g-1). Os melhores resultados foram obtidos com o preparado Zimopec PX5® nas condições de 1,0 U.g-1 de pectinase a 50 °C por 30 min. A extração do mosto apresentou aumento significativo nos parâmetros de qualidade avaliados (cor, sólidos solúveis totais, rendimento de extração, capacidade antioxidante, antocianinas totais) quando os três métodos de extração (US, MS e ET) foram combinados, principalmente na presença do ultrassom. Tendo em vista o efeito sinérgico obtido entre a aplicação enzimática e o ultrassom na extração do mosto, um novo estudo foi conduzido buscando investigar a influência da sonicação na eficiência catalítica das enzimas pectinase (PE), xilanase (XLN) e celulase (CE). Para isso, as atividades enzimáticas foram avaliadas em diferentes pHs e em amostras de enzima e substratos submetidas a um tratamento prévio de sonicação por tempos determinados (0 – 30 min). Os parâmetros cinéticos e termodinâmicos foram estimados, apresentando um efeito positivo do US na atividade das enzimas. A sonicação prévia do substrato contribuiu significativamente para o aumento da atividade de XLN, enquanto a sonicação prévia da enzima melhorou a atividade da CE e PE. Os resultados apresentaram um aumento na velocidade de hidrólise das enzimas com o uso do US, assim como um aumento da eficiência catalítica (Vmax/Km), mostrando o potencial da aplicação do US para ativação das enzimas. De acordo com os resultados obtidos, pode-se afirmar que o ultrassom proporcionou melhorias na extração do mosto, podendo ser utilizado como uma técnica alternativa de processo, bem como apresentou efeitos benéficos sobre as atividades enzimáticas. / Grape processing for juice and wine production encompasses different technological processes, such as enzymatic treatment, that usually uses commercial enzyme preparations to improve clarification and extraction processes. However, this is a treatment that demands high processing time, being relevant the search for new technologies in order to improve the whole process. Ultrasound (US) can be used as an alternative to reduce the processing time and improve the quality of the product, moreover, it can provide benefits in the combined use with enzymes, favoring the enzymatic reactions. In this work, the combined use of enzymatic treatment (ET), ultrasound (US), and mechanical agitation (MS) was studied in order to understand the effects of the techniques on Cabernet Sauvignon must extraction. Initially, nine commercial enzyme preparations were characterized according to their enzymatic activities and applied to must extraction, evaluating temperature (40, 50 and 60 °C), time (15 and 30 min), and enzyme concentration (0.01 to 2.0 Ug-1). The best results were obtained for the preparation Zimopec PX5® under conditions of 1.0 U.g-1 of pectinase at 50 °C for 30 min. A significant increase on quality parameters (color, °Brix, yield, antioxidant capacity, total anthocyanins) was obtained to extraction combining the three methods (US, MS and ET), mainly when US were applied. Based on these previous results, a new study was conducted to investigate the influence of sonication on the catalytic efficiency of the enzymes pectinase (PE), xylanase (XLN) and cellulase (CE). The enzymatic activities were evaluated at different pH and in samples of enzyme and substrates submitted to a previous treatment of sonication by determined times (0 - 30 min). The kinetic and thermodynamic parameters were estimated, showing a positive effect of the ultrasonic treatment on the enzymatic activity. Previous sonication of the substrate contributed significantly to the increase in XLN activity, whereas the previous sonication of the enzyme improved the activity of CE and PE. This research showed the potential benefits of ultrasound treatment on reaction rate and catalytic efficiency (Vmax/Km) improvement of enzymes, attesting that US can be used to increase the catalytic efficiency of pectinase, xylanase and cellulase enzymes. According to the obtained results, it can be affirmed that ultrasound improved the extraction process, and can be applied as an alternative technique, as well as provided beneficial effects on the enzymatic activity.

Atualmente os processos biotecnológicos têm conquistado um lugar de destaque no desenvolvimento tecnológico mundial, tendo as enzimas como as celulases, grande importância econômica e diferentes aplicações industriais. O objetivo deste trabalho foi à obtenção de fungos isolados e selecionados do ambiente agroindustrial, com a capacidade de hidrolisar a fração celulósica do bagaço de cana-de-açúcar. Um total de 46 isolados foram obtidos de bagaço de cana coletados em 3 destilarias. Quinze apresentaram a formação de halo pela coloração com vermelho Congo, os quais foram avaliados quanto a atividade celulolítica (F1 a F15), tendo como referência o fungo Trichoderma ressei 9414. Os isolados foram cultivados em bagaço de canade- açúcar 1% e conduziu-se a determinação da atividade de endoglucanase (CMCase) empregando-se carboximetilcelulose (CMC) como substrato, e da atividade da celulase total (FPase) empregando papel de filtro como substrato. Após 21 dias de cultivo em bagaço de canade- açúcar nenhum dos isolados apresentou atividade da celulase total maior que T. ressei QM9414. Destes isolados, F9 apresentou maior valor para a atividade da celulase total após 7 dias de cultivo. Com relação a atividade da endoglucanase, no sétimo dia de cultivo o isolado F27 obteve melhor resultado que T. ressei QM 9414. Quanto à cinética da atividade de endoglucanase os isolados 9, 23 e 27 apresentaram atividades enzimáticas muito próximas do fungo referência (QM9414). Os resultados mostram o grande potencial da biodiversidade existente em nichos industriais, na busca de linhagens com potencialidade na hidrólise enzimática de substrato lignocelulósico, como o bagaço de cana- de-açúcar. / Biotechnological processes are achieving increased relevance in today\'s technological development, being enzymes such as celullases, of great economical importance in different industrial applications. The objective of this work was the isolation and selection of fungi from agroindustrial environment, with capacity of hydrolysing the cellulosic fraction of sugar cane bagasse. Forty six isolates were evatuated for their cellulolytic activity using CMC as carbon source and Cong Red as staining indicator . Fifteen isolates showed halo formation by red Congo staining, and they were evaluated for cellulolytic activity (F1 to F15), using Trichoderma ressei 9414 as a reference. The isolates were cultivated in 1% sugar cane bagasse and the cellulolytic activities were assessed by the determination of endoglucanase activity (CMCase) using carboximetilcelulose (CMC) as substrate, and the total celullase activity (FPase) applying filter paper as substrate. After 21 days of cultivation in sugar cane bagasse neither of the isolates showed total cellulase activity higher than T. ressei QM9414. From these isolates, F9 showed higher total cellulase activity after 7 cultivation days. For endoglucanase activity, the isolate F27 showed better result than T. ressei QM 9414 in the seventh cultivation day. For the endoglucanase activity kinetics, the isolates 9, 23 and 27 showed enzymatic activities very close to the reference fungus (QM9414). These results allow to suggest a great biotechnological potential for the biodiversity found in industrial niches, regarding the enzymatic hydrolysis of lignocellulosics substrates, namely the sugar cane bagasse.

Atualmente os processos biotecnológicos têm conquistado um lugar de destaque no desenvolvimento tecnológico mundial, tendo as enzimas como as celulases, grande importância econômica e diferentes aplicações industriais. O objetivo deste trabalho foi à obtenção de fungos isolados e selecionados do ambiente agroindustrial, com a capacidade de hidrolisar a fração celulósica do bagaço de cana-de-açúcar. Um total de 46 isolados foram obtidos de bagaço de cana coletados em 3 destilarias. Quinze apresentaram a formação de halo pela coloração com vermelho Congo, os quais foram avaliados quanto a atividade celulolítica (F1 a F15), tendo como referência o fungo Trichoderma ressei 9414. Os isolados foram cultivados em bagaço de canade- açúcar 1% e conduziu-se a determinação da atividade de endoglucanase (CMCase) empregando-se carboximetilcelulose (CMC) como substrato, e da atividade da celulase total (FPase) empregando papel de filtro como substrato. Após 21 dias de cultivo em bagaço de canade- açúcar nenhum dos isolados apresentou atividade da celulase total maior que T. ressei QM9414. Destes isolados, F9 apresentou maior valor para a atividade da celulase total após 7 dias de cultivo. Com relação a atividade da endoglucanase, no sétimo dia de cultivo o isolado F27 obteve melhor resultado que T. ressei QM 9414. Quanto à cinética da atividade de endoglucanase os isolados 9, 23 e 27 apresentaram atividades enzimáticas muito próximas do fungo referência (QM9414). Os resultados mostram o grande potencial da biodiversidade existente em nichos industriais, na busca de linhagens com potencialidade na hidrólise enzimática de substrato lignocelulósico, como o bagaço de cana- de-açúcar. / Biotechnological processes are achieving increased relevance in today\'s technological development, being enzymes such as celullases, of great economical importance in different industrial applications. The objective of this work was the isolation and selection of fungi from agroindustrial environment, with capacity of hydrolysing the cellulosic fraction of sugar cane bagasse. Forty six isolates were evatuated for their cellulolytic activity using CMC as carbon source and Cong Red as staining indicator . Fifteen isolates showed halo formation by red Congo staining, and they were evaluated for cellulolytic activity (F1 to F15), using Trichoderma ressei 9414 as a reference. The isolates were cultivated in 1% sugar cane bagasse and the cellulolytic activities were assessed by the determination of endoglucanase activity (CMCase) using carboximetilcelulose (CMC) as substrate, and the total celullase activity (FPase) applying filter paper as substrate. After 21 days of cultivation in sugar cane bagasse neither of the isolates showed total cellulase activity higher than T. ressei QM9414. From these isolates, F9 showed higher total cellulase activity after 7 cultivation days. For endoglucanase activity, the isolate F27 showed better result than T. ressei QM 9414 in the seventh cultivation day. For the endoglucanase activity kinetics, the isolates 9, 23 and 27 showed enzymatic activities very close to the reference fungus (QM9414). These results allow to suggest a great biotechnological potential for the biodiversity found in industrial niches, regarding the enzymatic hydrolysis of lignocellulosics substrates, namely the sugar cane bagasse.

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.

<p>The enzymatic degradation of cellulose is an important process in nature. This thesis has focused on the degradation of cellulose by enzymes from two cellulose-degrading fungi, <i>Hypocrea jecorina</i> and <i>Phanerochaete chrysosporium</i>, including both the action of the individual enzymes and their synergistic interplay. </p><p>The end-preference of cellobiohydrolases on crystalline cellulose was studied. Cellobiohydrolases belonging to glycosyl hydrolase (GH) family 7 were found to hydrolyse cellulose processively, starting from the reducing end of the cellulose chain. End-labelled cellulose can serve as a tool for functional classification of cellulases.</p><p>The synergy mechanism between endoglucanases and cellobiohydrolases was studied using substrates with different physical properties derived from bacterial cellulose. A new mechanism for synergism between endo- and exoacting enzymes was proposed whereby endoglucanases, in addition to creating nicks in amorphous parts of cellulose, thereby making new starting-points for processively acting cellobiohydrolases, also “polish” the cellulose surface by removing shorter chains from cellulose surface.</p><p>A new small endoglucanase belonging to the GH12 family was isolated and characterised. The proposed role of this enzyme is to make the cellulose in wood more accessible to other cellulases.</p><p>Oxygen conversion by cellobiose dehydrogenase was studied. Hydrogen peroxide produced by cellobiose dehydrogenase can be decomposed even by traces of certain metal ions into a hydroxyl radical and a hydroxyl ion. As an example, reduced metal ions will be continuously regenerated by cellobiose dehydrogenase, which thus stimulates the degradation.</p><p>Interactions between GH7 family cellobiohydrolases and o-nitrophenyl cellobioside were studied by fluorescence spectroscopy and kinetic tests. o-nitrophenyl cellobioside was used as indicator ligand to determine the dissociation constants for cellobiose binding to catalytically inactive Cel7A mutants by displacement binding experiments.</p>

The enzymatic degradation of cellulose is an important process in nature. This thesis has focused on the degradation of cellulose by enzymes from two cellulose-degrading fungi, Hypocrea jecorina and Phanerochaete chrysosporium, including both the action of the individual enzymes and their synergistic interplay. The end-preference of cellobiohydrolases on crystalline cellulose was studied. Cellobiohydrolases belonging to glycosyl hydrolase (GH) family 7 were found to hydrolyse cellulose processively, starting from the reducing end of the cellulose chain. End-labelled cellulose can serve as a tool for functional classification of cellulases. The synergy mechanism between endoglucanases and cellobiohydrolases was studied using substrates with different physical properties derived from bacterial cellulose. A new mechanism for synergism between endo- and exoacting enzymes was proposed whereby endoglucanases, in addition to creating nicks in amorphous parts of cellulose, thereby making new starting-points for processively acting cellobiohydrolases, also “polish” the cellulose surface by removing shorter chains from cellulose surface. A new small endoglucanase belonging to the GH12 family was isolated and characterised. The proposed role of this enzyme is to make the cellulose in wood more accessible to other cellulases. Oxygen conversion by cellobiose dehydrogenase was studied. Hydrogen peroxide produced by cellobiose dehydrogenase can be decomposed even by traces of certain metal ions into a hydroxyl radical and a hydroxyl ion. As an example, reduced metal ions will be continuously regenerated by cellobiose dehydrogenase, which thus stimulates the degradation. Interactions between GH7 family cellobiohydrolases and o-nitrophenyl cellobioside were studied by fluorescence spectroscopy and kinetic tests. o-nitrophenyl cellobioside was used as indicator ligand to determine the dissociation constants for cellobiose binding to catalytically inactive Cel7A mutants by displacement binding experiments.

Bacillus sp VLSH08 screened from sea wetland in Nam Dinh province produces an extracellular endo-1,4-beta-glucanase. According to the results of the classified Kit API 50/CHB as well as sequence of 1500 bp fragment coding for 16S rRNA gene of the Bacillus sp VLSH 08 strain showed that the taxonomical characteristics between the strain VLSH 08 and Bacillus amyloliquefaciene JN999857 are similar of 98%. Culture supernatant of this strain showed optimal cellulase activity at pH 5.8 and 60 Celsius degree and that was enhanced 2.03 times in the presence of 5 mM Co2+. Moreover, the gene encoding endo-1,4-beta-glucanase from this strain was cloned in Escherichia coli using pCR2.1 vector. The entire gene for the enzyme contained a 1500-bp single open reading frame encoding 500 amino acids, including a 29-amino acid signal peptide. The amino acid sequence of this enzyme is very close to that of an EG of Bacillus subtilis (EU022560.1) and an EG of Bacillus amyloliquefaciene (EU022559.1) which all belong to the cellulase family E2. A cocktail of enzyme containing this endo-1,4-beta-glucanase used for biomass hydrolysis indicated that the cellulose conversion attained to 72.76% cellulose after 48 hours. / Chủng vi khuẩn Bacillus sp VLSH08 được tuyển chọn từ tập hợp chủng vi khuẩn phân lập ở vùng ngập mặn tỉnh Nam Định có khả năng sinh tổng hợp enzyme endo-1,4-beta-glucanase ngoại bào. Kết quả phân loại chủng vi khuẩn Bacillus sp VLSH08 bằng Kit hóa sinh API 50/CHB cũng như trình tự gen mã hóa 16S rRNA cho thấy độ tương đồng của chủng Bacillus sp VLSH08 và chủng Bacillus amyloliquefaciene JN999857 đạt 98%. Dịch lên men của chủng được sử dụng làm nguồn enzyme thô để nghiên cứu hoạt độ tối ưu của enzyme ở pH 5,8 và nhiệt đô 60oC. Hoạt tính enzyme tăng 2,03 lần khi có mặt 5 mM ion Co2+. Đồng thời, gen mã hóa cho enzyme endo-1,4-betaglucanase cũng được tách dòng trong tế bào Escherichia coli sử dụng vector pCR 2.1. Gen mã hóa cho enzyme này có chiều dài 1500 bp, mã hóa cho 500 axit amin, bao gồm 29 axit amin của chuỗi peptid tín hiệu. So sánh cho thấy trình tự gen endo-1,4-beta-glucanase của chủng Bacillus sp VLSH08 có độ tương đồng cao với enzyme này của chủng Bacillus subtilis (EU022560.1) và của chủng Bacillus amyloliquefaciene (EU022559.1). Tất cả các enzyme nhóm này đều thuộc họ cellulase E2. Enzyme của chủng này cũng đã được phối trộn với các enzyme khác tạo thành cocktail để thủy phân sinh khối cho kết quả cellulose bị thủy phân 72,76% sau 48 giờ.

No description available.

Lignocellulosic biomass is under extensive investigation as a bioethanol and bio-based materials feedstock. However, the complex structural and chemical mechanisms of lignocellulosic plant, which cause resistance to deconstruction during saccharification, require a pretreatment process. In this study, raw materials (corn bran, wheat bran and wheat straw) were selected because of their production and consumption in Turkey and also their accessibilities to be used as bioethanol source. Microfluidization pretreatment (high-pressure fluidization), which stands as a new approach for nano-cellulosic fibers production, was studied at 500 bar and 2000 bar to observe the qualitative and quantitative modifications in enzymatic hydrolysis depending on its effects on lignocellulosic structure. Optimum cellulase concentrations were determined for microfluidized samples as 4.5 U/g dry biomass for wheat bran, corn bran and 6.0 U/g dry biomass for wheat straw samples for the first 150 min interval. Effective usage of solid loads were found as 5.0 %, 2.5 %, and 7.5 % (dw/v) for wheat bran, wheat straw and corn bran, respectively. X-ray diffraction and SEM results of the microfluidized samples have indicated that the pretreatment has increased crystallinity index of all the samples and resulted in a scattered structure. Comparisons with other methods (softening, dilute-acid and lime pretreatments) have shown that microfluidization is advantageous over others by reducing the time required for enzymatic hydrolysis and thus can be a promising alternative pretreatment.