Produção de etanol e hidrolisado protéico da casca de soja

A casca de soja, sendo um residuo lignocelulosico, contem celulose (38-51%) que pode ser convertida a etanol. Alem disso, contem 9-14% de proteinas, que uma vez hidrolisadas por endoproteases podem liberar de forma especifica oligopeptideos com aplicacoes nutricionais. Ligacoes glicosidicas e peptidicas podem ser rompidas por hidrolise acida, sendo hemicelulose mais susceptivel a que celulose. O ataque acido ao material permitiria assim reduzir o conteudo de hemicelulose da biomassa, tornando a celulose que permanece na fracao solida insoluvel mais acessivel as enzimas hidroliticas. Neste trabalho, foram estudadas diferentes rotas de solubilizacao de proteinas, hemicelulose lignina presentes na casca de soja, visando obtencao de etanol da fracao solida e oligopeptideos na fracao liquida. A recuperacao de proteinas na forma de peptideos foi feita hidrolisando-se a biomassa inicialmente com extrato comercial de endoprotease Novo-ProD, a 60oC, pH 9, por 5h, nas concentracoes enzimaticas de 1, 2 e 4% (m/m). Foi tambem testada, na sequencia da hidrolise com Novo-ProD1%, nova hidrolise com quimotripsina 1% (m/m). Os resultados mostraram que a hidrolise proteolitica com 1% de Novo-ProDpermitiu remocao de 56,9% da proteina presente na casca. Contudo, foi tambem verificado que e possivel remover 45,6% das proteinas nas mesmas condicoes, na ausencia de enzima, a pH9,0, possivelmente devido a liberacao de unidades agregadas apenas fisicamente. Um aumento da concentracao de endoproteases de 1% para 2% elevou a remocao para 74% de proteinas, nao se observando aumento significativo na extracao de proteinas aumentandose de 2 para 4%.. Com uso da enzima mais especifica, a quimotripsina, que opera em condicoes mais brandas, foi possivel remover 44% das proteinas, com uma maior remocao de lignina do material, comparando-se com Novo-ProD. Hidrolise acida de casca in natura ou sequencial a hidrolise com Novo-ProD1% sequencial permitiu atingir uma remocao total de aproximadamente 90%de proteinas O material solido remanescente e a casca in natura foram submetidos a hidrolise acida com H2SO4 3% (v/v), razao 1:4 (solido/liquido), 120oC, 20 min. Em todos os casos, as analises de carboidratos mostraram que foi possivel remover aproximadamente 46,7% de hemicelulose, maior parte na forma de xilose. Durante o tratamento acido ocorreu a remocao de quase toda a proteina da casca de soja, sem perda expressiva de celulose, o que se observou ocorrer na hidrolise acida da casca in natura. Assim, seja para obter-se um hidrolisado proteico de composicao controlada, seja para producao de etanol da fracao solida remanescente e recomendavel a remocao enzimatica previa de proteinas. A composicao quimica do material solido apos hidrolises proteolitica e acida sequenciais mostrou teores de celulose similares para todas as amostras (aproximadamente 49 %). Assim, o solido pre-tratado com 1% de Novo Pro-D, foi utilizado para a obtencao dos acucares fermentesciveis usando o complexo enzimatico Acellerase 1500 a 50oC, pH 4,8 e razao enzima/substrato de 7 FPU/g celulose durante 72 h. Nestas mesmas condicoes foram hidrolisadas as amostras de casca in natura pre-tratada com acido, a casca in natura e a casca apos hidrolise proteolitica. A conversao enzimatica de celulose em glicose foi em torno de 40%, tanto para as amostras pre-tratadas com Novo- ProD como para a amostra submetida so a hidrolise acida. Com um aumento de carga enzimatica para 20 FPU/g celulose foi possivel atingir uma conversao de 55% para amostras pre-tratadas com 1% de Novo-ProDe hidrolise acida sequenciais. A suplementacao do complexo enzimatico Acellerase 1500 com 120 UI de β-glicosidase e 1%(m/m) de pectinase nao produziu aumento na conversao enzimatica. A hidrolise da celulose proveniente de pre-tratado por organossolve usando etanol 50% a 170oC por 1 hora resultou em 52% de conversao com uma menor remocao de hemicelulose mostrando que a lignina estava causando o maior impedimento para o ataque enzimatico. A conversao de glicose em etanol foi similar para as amostras pre-tratadas por hidrolise acida e com as hidrolises proteoliticas (1% Novo-ProD) e acidas sequenciais chegando a uma concentracao aproximada de 13 g/L.Soybean hull is a lignocellulosic biomass that contains 38-51% of cellulose, which could be converted to ethanol. In addition, contains 9-14% of protein that can be hydrolyzed by endoproteases, releasing oligopeptides with nutritional applications. Although glycoside and peptide linkages can be hydrolyzed by acids, the cellulose molecules are more resistant than the protein and hemicellulose molecules. In this way, the acid treatment of the biomass would reduce its hemicellulose content, and the remnant cellulose into solid fraction would be more susceptible to hydrolytic enzymes. In this work, different routes of protein, hemicellulose, and lignin solubilization were evaluated, intending to obtain ethanol and soluble oligopeptides from the soybean hull. The protein was recovered as oligopeptides by hydrolysis of the lignocellulosic biomass using the commercial endoprotease Novo-Pro DR at 60oC, pH 9.0, 5 h, and different enzyme concentrations (1, 2, and 4%, m/m). A sequential hydrolysis using chymotrypsin and Novo-Pro DR, both at 1% (m/m) enzyme concentration, was also evaluated. The results showed that hydrolysis with 1% Novo-Pro DR allowed solubilization of 56.9% of protein from the soybean hulls. Nonetheless, at same temperature and pH, in absence of enzyme, was possible to solubilized 45.6% of the proteins. This solubilization is probably due to liberation of the physically aggregated units. The increase of endoprotease concentration from 1 to 2% increased the protein removal to 74%. However, the increase from 2 to 4% not increased significantly the protein solubilization. The use of chymotrypsin, an enzyme with high specificity and that work at mild conditions, allowed a solubilization of 44% protein. Nonetheless, the removal of lignin using chymotrypsin was higher than that using Novo-Pro DR. When in-nature soybean hull was hydrolyzed by acid or protease (1% Novo-Pro DR) followed by acid, the protein removal was around of 90%. The lignocellulosic biomass was hydrolyzed with 3% (v/v) H2SO4, solid:liquid ratio of 1:4, 120oC, and 20 min. 20 min. Carbohydrate analyses showed that the acid treatment allowed to hemicellulose removal around of 46.7% in the xylose form. The protein content of the soybean hull was almost totally solubilized during the acid hydrolysis, without significant loss of cellulose. On the contrary, large cellulose loss was observed during the acid hydrolysis of in-nature soybean hull. In this way, if it is intended to produce a protein hydrolysate containing controlled composition or ethanol from remnant solid fraction, is strongly recommended the previous enzymatic solubilization of the proteins. The chemical composition of the solid biomass after sequential hydrolyses with protease and acid showed cellulose content around of 49% for all samples. So, the biomass treated with 1% (m/m) Novo-Pro DR was saccharified with Acellerase 1500 at 50oC, pH 4.8, and enzyme/substrate ratio of 7 FPU/g of cellulose for 72 h. Under the same conditions, soybean hull in-nature, pretreated with acid, and pretreated with protease were submitted to cellulolytic hydrolyses. The cellulose-to-glucose conversion was around of 40% for the last two biomass. The increase of the enzymatic load to 20 FPU/g of cellulose allowed a cellulose conversion of 55% for biomass pretreated with 1% (m/m) Novo-Pro DR, followed by acid hydrolysis. The supplementation of the Acellarase 1500 with 120 IU of β-glucosidase and 1% (m/m) of pectinase not produced any increased in the cellulose conversion. The biomass was pretreated by organossolv method (50% ethanol, 170oC, and 1h) and saccharified with Acellerase 1500 under the same conditions described above. This procedure yielded a cellulose conversion of 52%, with less removal of hemicellulose. This result showed that lignin was causing greater steric hindrances to the enzymatic attack. The biomass pretreated with acid and with protease (1% Novo-Pro D) followed by acid yielded the same glucose-toethanol conversion, reaching an ethanol concentration around of 13 g/L