Production, Obtaining And Characterization Of Glucanases (β-1,4) By Trichoderma Virens

Nelson Rosa Ferreira^1,2*, Gilson Chagas Júnior¹, Alberdan Silva Santos²

¹Graduate Program in Food Science and Technology, Institute of Technology, Federal University of Pará (UFPA), 66075-110, (BRAZIL)
²Laboratories of Systematic Research in Biotechnology and Molecular Biodiversity, ICEN, Federal University of Pará (UFPA), 66075-110, (BRAZIL)

PAGE NO: 109-119

ABSTRACT – DOI: https://dx.doi.org/10.47204/EBR.1.2.2021.109-119

Submerged cultivation of Trichoderma virens was carried out in a conical flask system with agitation containing microcrystalline cellulose as a cellulosic substrate. Quadrangular fragments of mycelium obtained from the cultivation of T. virens on glucose agar with carboxymethylcellulose (CMC) were used as inoculum of the submerged culture. The enzyme concentrate was used in the cellulolytic evaluation activities like FPase (Filter Paper Activity), CMCase (Carboxymethyl Cellulase Activity), protein content, reducing sugar content and proteinase activity. The Concentrated Enzyme Pool (CEP) was obtained by centrifuging, lyophilization, resuspending, and dialysis of the liquid culture. Molecular exclusion chromatography (MEC) and PAGE electrophoresis was performed on the CEP. The main enzymes present in the electrophoresis gel were identified by mass spectrometry (MALDI-TOF-TOF-MS). Enzyme concentrates (EC) were obtained by precipitation in an organic solvent at low temperatures. The maximum cellulolytic activity (0.45UmL^-1) occurred in 72 h of submerged culture. The SDS-PAGE electrophoresis technique was performed, in which eleven bands were identified, two of which were more evident. The analysis of these main proteins performed using the MALDI-TOF-TOF-MS system made it possible to identify an endocellulase (β-1,4-endoglucanase) and an exocellulase (β-1,4-cellobiohydrolase). The fractions obtained in the MEC (133) showed the formation of the three most evident peaks at 280 nm, with FPase activity showing four peaks. The fractionation with organic solvent increased the specific activities FPase and CMCase. Thus, from the results, it was observed that the strategy used to obtain and treat the enzyme concentrate made it possible to evaluate the stability of the enzymes, as well as the identification of two glucanases..