Full text
| |
| Author(s): |
Suelen de Barros Sampaio
Total Authors: 1
|
| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Escola de Artes, Ciências e Humanidades (EACH) |
| Defense date: | 2019-11-12 |
| Examining board members: |
Felipe Santiago Chambergo Alcalde;
Sirlene Maria da Costa;
Marcia Zorello Laporta;
Roberto Kopke Salinas
|
| Advisor: | Felipe Santiago Chambergo Alcalde |
| Abstract | |
The increase in global demand for ethanol is linked to concerns about carbon imbalance in the atmosphere due to the use of fossil fuels, driven by the interest in renewable energy sources. Sugar cane sugar consists mainly of lignin, hemicellulose and cellulose, the latter two of which can be broken down and fermented in ethanol. During the hydrolysis of lignocellulosic biomass there is a synergistic action of an enzymatic complex to obtain soluble fermentable sugars, in which recent research has demonstrated the importance of oxidoreductase enzymes in lignin depolymerization. The objective of this study was to identify the filamentous fungus BBF210, to evaluate its use in the production of laccase in a culture medium containing sugarcane bagasse, to purify and characterize the oxidoreductase of this fungus, as well as to express and purify the recombinant lacase enzyme. By classical and molecular taxonomy analysis the filamentous fungus BBF210 was identified as a producer of oxidoreductase enzymes belonging to the species Cochliobolus sativus. The computational analysis of the C. sativus genome allowed the identification of the gene cDNA with 1575 bp, that encodes the putative 524 amino acid laccase enzyme and estimated molecular weight of 57,7 kDa. The gene was synthesized and cloned into vectors (pET, pTrc and pFLAG) for expression in E. coli. A recombinant lacase enzyme was expressed and purified. The fungus C. sativus was evaluated for its ability to produce oxidoreductases in MSB (mineral salts broth) culture medium containing 0,5% sugarcane bagasse and the results showed a higher activity of laccase enzyme with ABTS substrate when compared to peroxidases. After the 7th day of cultivation the enzyme was precipitated with 50% ethanol and purified in two steps of ion exchange chromatography (Q-sepharose column). The pure laccase fraction presented 3,264.32 U/L and 11,25 U/mg. The results suggest that the native protein is a isoenzyme with about 50 kDa, similar to the recombinant protein, as confirmed by western blot analysis. Native laccase activity was determined with ABTS substrate by zymogram and spectrophotometry, with catalytic constants Km = 0.094 mM and Vmax = 1.62 mM s-¹. The optimal pH and temperature of the enzyme were determined as being 4.0 and 30ºC, respectively, with thermal stability at 30°C and ideal temperature in the range between 10 and 50°C. Since laccase is a fundamental oxidoreductase in the degradation process of plant biomass, the present study provides information about enzyme expressed by C. sativus and whose production, purification and characterization of the native enzyme of this fungus is describes for the first time. Knowledge of their biochemical characteristics and properties may be important for further scientific investigations around the identification and characterization of enzymes and for the optimization of processes using enzymatic cocktail with oxidases, both for ethanol production using the 2G methodology and for other biotechnological processes (AU) | |
| FAPESP's process: | 16/23628-3 - Oxidoreductases enzymes from Cochliobolus sp |
| Grantee: | Suelen de Barros Sampaio |
| Support Opportunities: | Scholarships in Brazil - Master |