Enzimas lignocelulolíticas da microbiota ruminal de ovinos Morada Nova e seu potencial na sacarificação do bagaço de cana-de-açúcar.

Resumo: Os ruminantes sao fontes de microrganismos produtores de enzimas lignoceluloliticas, as quais podem ser utilizadas para diferentes aplicacoes biotecnologicas, como a degradacao da fibra do bagaco de cana-de-acucar (BCA) para uso alimentar ou para producao de bioetanol. Enzimas mais especificas sao obtidas quando o proprio substrato de interesse e utilizado na producao dos microrganismos. Nesse sentido, o presente trabalho direcionou a microbiota ruminal de ovinos Morada Nova para a degradacao do BCA objetivando extrair as proteinas aderidas a fibra (PAFs) e caracteriza-las quanto ao potencial de degradacao do BCA in natura e pre-tratado com 10, 20, 30, 40 ou 50% de NaOH. O conteudo ruminal foi obtido via fistula, filtrado, lavado com Solucao Salina Tamponada (PBS, pH 7,2 sem Ca2+ e Mg2+) e as PAFs extraidas com ureia 8 mmol.L-1 durante 15 minutos. Apos serem filtradas e dialisadas contra agua (cut off de 12 kDa) as PAFs foram liofilizadas. A atividade total de celulase foi determinada pelo ensaio do acido 3,5-dinitrosalicilico (DNS) em pH 3,99 e papel de filtro. As PAFs mostraram maior atividade enzimatica em pH 3,99. O pH 11,71 foi identificado como o de maxima extracao de proteina. A temperatura de 50 oC por 60 minutos manteve a atividade especifica de celulase total das PAFs e essas tambem resistiram ao aquecimento a 100 °C por 60 minutos. Em relacao aos substratos, as PAFs apresentaram maior atividade especifica para xilanase (7,01 ± 0,19 U.mg-1) seguida por endoglucanase (4,71 ± 1,65 U.mg-1), exoglucanase (2,68 ± 0,42 U.mg-1) e celulase total (0,62 ± 0,05 U.mg-1). Quanto aos substratos BCA in natura e pre-tratado, a atividade enzimatica das PAFs aumentou de forma proporcional a concentracao de NaOH utilizado no pre-tratamento, ao mesmo tempo em que se observou na fibra melhor solubilizacao da hemicelulose e lignina, e aumento da cristalinidade. As fibras de BCA in natura e pre-tratada foram utilizadas como matriz cromatografica e observou-se interacao das PAFs com as matrizes, com destaque para as matrizes de BCA pre-tratadas com 20% e 40% NaOH. Em conclusao, as enzimas ruminais de ovinos Morada Nova foram capazes de degradar o BCA, sendo sua eficiencia elevada ajustando-se o pH e a temperatura reacional, assim como realizando o pre-tratamento alcalino do BCA. Ainda, a propria fibra de BCA pre-tratada pode ser utilizada para isolar e caracterizar enzimas ruminais lignoceluloliticas. Abstract: Ruminants are sources of microorganisms that produce lignocellulolytic enzymes which in turn can be used for different biotechnological applications, such as in degrading sugarcane bagasse (SCB) fibers for food use or for producing bioethanol. More specific enzymes are obtained when the substrate of interest itself is used in producing the microorganisms. In this sense, the present work directed Morada Nova sheep ruminal microbiota to degrade SCB, aiming at extracting proteins adhered to the fibers (PAFs), characterizing them as to the potential degradation of the in natura SCB and those pretreated with 10, 20, 30, 40 or 50% NaOH. The ruminal content was obtained via fistula, filtered, washed with Phosphate Buffer Saline (pH 7.2 without Ca2+ and Mg2+) and the PAFs were extracted with 8 mmol.L-1 urea for 15 minutes. The PAFs were then lyophilized after filtration and dialysis against water (cut-off of 12 kDa). Total cellulase activity was determined by the 3,5-dinitrosalicylic acid (DNS) assay at pH 3.99 and filter paper. The PAFs showed higher enzymatic activity at pH 3.99. The pH of 11.71 was identified as the maximum protein extraction. A temperature of 50 oC for 60 minutes maintained the specific activity of PAFs total cellulase and they also resisted heating at 100 °C for 60 minutes. Regarding substrates, PAFs showed higher specific activity for xylanase (7.01 ± 0.19 U.mg-1), followed by endoglucanase (4.71 ± 1.65 U.mg-1), exoglucanase (2.68 ± 0.42 U.mg-1) and total cellulase (0.62 ± 0.05 U.mg-1). For the SCB substrates in natura and pretreated, the enzymatic activity of PAFs increased in proportion to the increased concentration of NaOH used in the pretreatment, while at the same time better hemicellulose and lignin solubilization were observed in the fiber, as well as increased crystallinity. In natura and pretreated SCB fibers were used as a chromatographic matrix and interaction of the PAFs with the matrices was observed, especially the pretreated SCB matrices with 20% and 40% NaOH. In conclusion, Morada Nova sheep ruminal enzymes were able to degrade SCB fibers, with higher efficiency by adjusting the pH and reaction temperature, as well as performing alkaline pretreatment of the SCB. In addition, the pretreated SCB fiber itself can be used to isolate and characterize lignocellulolytic ruminal enzymes.