Abordagem integrativa na prática de bioquímica: do laboratório à clínica / Integrative approach in biochemistry practice from laboratory to clinic

Objetivou-se relatar a experiência de discentes e docentes diante da aplicação de ferramentas de metodologia ativa para a integração dos temas de Bioquímica com os das disciplinas do eixo clínico-profissional. Inicialmente, as subturmas da aula prática foram divididas em equipes de trabalho. Os alunos receberam um protocolo contendo objetivos, princípios gerais e procedimentos na semana anterior à aula prática. Nos dias das aulas de "Preparo de Soluções" e "Capacidade Tamponante", um artigo científico relativo à Odontologia foi entregue para leitura e discussão em grupo. Como atividade prática, as equipes recebiam um desafio relacionado ao artigo e que exigiria aplicação dos objetivos de aula. Esta experiência demonstrou que a metodologia ativa pode funcionar como facilitadora para uma abordagem contextualizada e integrada da Bioquímica, refletindo em maior engajamento e rendimento dos alunos, além de contribuir para um aprendizado significativo (AU).

El objetivo fue relatar la experiencia de estudiantes y profesores en cuanto a la aplicación de herramientas metodológicas activas para la integración de los temas de Bioquímica con los de las disciplinas del eje clínico-profesional. Inicialmente, las subclases de la clase práctica se dividían en equipos de trabajo. Los estudiantes recibieron un protocolo con objetivos, principios generales y procedimientos en la semana anterior a la clase práctica. En los días de las clases de "Preparación de Soluciones" y "Capacidad Amortiguadora", se entregó un artículo científico relacionado con la Odontología para lectura y discusión en grupo. Como actividad práctica, los equipos recibieron un reto relacionado con el artículo y que requería la aplicación de los objetivos de clase. Esta experiencia demostró que la metodología activa puede funcionar como facilitadora de un abordaje contextualizado e integrado de la Bioquímica, reflejándose en un mayor compromiso y desempeño de los estudiantes, además de contribuir al aprendizaje significativo (AU).

The objective was to report the experience of students and professors regarding the application of active methodology tools aimed at integrating Biochemistry themes with those of the disciplines from the clinical-professional axis. Initially, subgroups forthe practical class were divided into work teams. The students received a protocol containing objectives, general principles and procedures the week before the practical class. On the days of the "Preparation of Solutions" and "Buffering Capacity" classes, a scientific article related to Dentistry was delivered for group reading and discussion. As a practical activity, the teams received a challenge related to the article,which would require application of the class objectives. This experience demonstrated that the active methodology can work as a facilitator for a contextualized and integrated approach to Biochemistry, reflecting in greater engagement and student performance, in addition to contributing to meaningful learning (AU).

Mango peel as a potential enzyme inducer in Trichoderma harzianum: a strategy for cariogenic biofilm degradation and reuse of industrial waste

Water-insoluble exopolysaccharides (I-EPS) are a virulence factor for dental biofilms. It has already been demonstrated that mango pulp induces the secretion of glucan-hydrolytic enzymes in the fungus Trichoderma harzianum, and that they have an effect on I-EPS from young biofilms. Aim: Evaluate the effect of mango peel as an enzyme inducer in T. harzianum, and the effect of enzymes secreted on mature biofilms. Methods: Fractions of the peel (PL) and ethanol-precipitated pulp (PP) of Tommy Atkins mangoes were sterilized and added to a culture medium containing T. harzianum for induction of hydrolytic enzymes. After 192 h, the culture medium was centrifuged and the supernatant (enzyme extract) was used as treatment on S. mutans biofilms (n=9): a) NaCl 0.9 %; b) 0.12 % chlorhexidine digluconate; and c) extract of enzymes induced by PL or PP. Acidogenicity, bacterial viability, quantification of insoluble polysaccharides, and three-dimensional analysis of the biofilm by scanning electron microscopy (SEM) was performed. Data were analyzed by ANOVA followed by the Tukey test (α=5 %). Results: The hydrolytic enzymes did not alter the metabolism or bacterial viability of the biofilm (p<0.05). Although the images obtained by SEM suggest some degree of matrix degradation, the quantification of I-EPS for the PL and PP groups did not differ from the control group (p>0.05), suggesting a slight effect on the disorganization of the mature S. mutans biofilm. Conclusion: The results suggest that mango peel fraction can induce secretion of mutanase by T. harzianum, however in an insufficient amount to generate significant degradation on cariogenic biofilm.

Production of short-chain fructooligosaccharides (scFOS) using extracellular ß-D-fructofuranosidase produced by Aspergillus thermomutatus.

Aspergillus thermomutatus produces an extracellular ß-D-fructofuranosidase when cultured in Khanna medium with sucrose as additional carbon source at 30°C under agitation for 72 hr. Addition of glucose and fructose in the culture medium affected the production of the enzyme negatively. The optimum hydrolytic activity was achieved at 60°C and pH 5.0, with half-life (T50) of 30 hr at 50°C and 62% of its activity maintained at pH 5.0 for 48 hr. The extracellular extract containing ß-D-fructofuranosidase was effective in producing fructooligosaccharides (FOS), mainly 1-kestose. The highest concentration of FOS was obtained at 30°C and 60°C, indicating the existence of at least two enzymes with transfructosylating activity. At 30°C, the maximal FOS concentration was obtained from 48 to 72 hr, while at 60°C, it was achieved only at 72 hr. The best production of FOS (86.7 g/L) was obtained using 500 g/L sucrose as substrate. PRACTICAL APPLICATION: Fructooligosaccharides (FOS) are linear oligomers of fructose units with important applications in the food industry as sweetening agents and biopreservatives. Due to the presence of ß-glycosidic bonds, they cannot be hydrolyzed by human enzymes, allowing the use of FOS-containing products by diabetics. FOS used in the preparation of dairy products imparts humectancy to soft baked products, lowers the freezing point of frozen desserts, provides crispness to low-fat cookies, and provides many other advantages. Diets containing FOS can reduce the levels of triglycerides and cholesterol and improve the absorption of ions, such as Ca2+ and Mg2+ . FOS also exhibit bifidogenic effect on Bifidobacterium and Lactobacillus strains in the colon. Industrially, FOS is produced during the transfructosylation reaction of sucrose catalyzed by ß-D-fructofuranosidase. Identifying new sources of ß-D-fructofuranosidase is an important challenge to meet its industrial demand.

Characterization of a novel sugar transporter involved in sugarcane bagasse degradation in Trichoderma reesei.

BACKGROUND: Trichoderma reesei is a saprophytic fungus implicated in the degradation of polysaccharides present in the cell wall of plants. T. reesei has been recognized as the most important industrial fungus that secretes and produces cellulase enzymes that are employed in the production of second generation bioethanol. A few of the molecular mechanisms involved in the process of biomass deconstruction by T. reesei; in particular, the effect of sugar transporters and induction of xylanases and cellulases expression are yet to be known. RESULTS: In our study, we characterized a novel sugar transporter, which was previously identified by our group through in silico analysis of RNA-seq data. The novel T. reesei 69957-sugar transport system (Tr69957) is capable of transporting xylose, mannose, and cellobiose using a T. reesei 69957-sugar transport system in Saccharomyces cerevisiae. The deletion of Tr69957 in T. reesei affected the fungal growth and biomass accumulation, and the sugar uptake in the presence of mannose, cellobiose, and xylose. Molecular docking studies revealed that Tr69957 shows reduced protein-ligand binding energy for interactions towards disaccharides in comparison with monosaccharides. Furthermore, the deletion of Tr69957 affected the gene expression of cellobiohydrolases (cel7a and cel6a), ß-glucosidases (cel3a and cel1a), and xylanases (xyn1 and xyn2) in the cultures of parental and mutant strains in the presence of cellobiose and sugarcane bagasse (SCB). CONCLUSION: The transporter Tr69957 of T. reesei can transport cellobiose, xylose, and mannose, and can affect the expression of a few genes encoding enzymes, such as cellulases and xylanases, in the presence of SCB. We showed for the first time that a filamentous fungus (T. reesei) contains a potential mannose transporter that may be involved in the degradation of cellulose.