Produção, purificação e caracterização de celulases e hemicelulases do fungo da podridão-branca Pycnoporus sanguineus PF-2

• Autor(a) principal: Rodrigues, Rosilene Souza
• Data de Publicação: 2014
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/339
• topic: Biocombustíveis; Biomassas lignocelulósicas; Pycnoporus sanguineus; Chrysoporthe cubensis; Biofuels; Lignocellulosic biomass; Pycnoporus sanguineus; Chrysoporthe cubensis; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA

In this work, the process of cellulases and hemicellulases production by white-rot fungus Pycnoporus sanguineus PF- 2 targeting its application in sugarcane bagasse saccharification was evaluated. Initially, the culture medium for the production of cellulase and xylanase was optimized using methods of Fractional Factorial 2 5-1 (Resolution V) followed by a Rotational Central Composite Design (RCCD). For maximum production of endoglucanase (16.42 U/mL), FPase (0.38 U/ml) and xylanase (111,03 U/ml), the optimum values for the factors forage concentration, peptone concentration and ZnSO4 concentration were 4 % (w/v), 2 % (w/v) and 12.5 mg/L, respectively. The optimal values for the cultivation time were 186.1 h for endoglucanase, 180 h for FPase and 185.1 h for xylanase. For cellobiase activity, the highest yield (0.35 U/ml) comes when the forage concentration, concentration peptone and cultivation time were 4 % (w/v), 2 % (w/v) and 60 h. The endoglucanase, cellobiase and xylanase activities produced presented maximal activity between 55 and 65°C and pH between 4.0 and 5.5. Endoglucanase and xylanase retained more than 50 % of residual activity after 13 h of incubation at 60°C. The optimized crude extract was applied to the sugarcane bagasse saccharification, converting 17.4 % of cellulose into glucose after 76 h. Following, in order to obtain higher yields in the hydrolysis of sugarcane bagasse, the enzymatic extract of P. sanguineus produced in Submerged Fermentation (SF) and enzyme extract of C. cubensis produced in Solid State Fermentation (SSF) were blended in different proportions and evaluated in relation to the appearance of synergism between the enzymes present in these extracts. The cocktails made from a simple enzymatic extracts mixture of these fungi showed no positive synergistic effect. However, when this mixture was carried out in an unconventional way, i.e., during the enzymatic extraction of enzymes produced in the FES, we observed large synergistic effects on the activities of enzymes FPase, exoglicanase, cellobiase, β- glucosidase, xylanase, β-xylosidase and α-arabinofuranosidase. The increases obtained in the enzymes activities were 52.2 %, 9.2 %, 62 %, 26.5 %, 12.2 %, 56.2 % and 18.4 %, respectively. The cocktail produced from this unconventional mixture of P. sanguineus and C. cubensis enzymatic extracts was applied to the sugarcane bagasse saccharification and high conversion yields of glucan and xylan were achieved. When used an enzymatic load of 10 FPU/g, conversion yields of 85 % and 95 % for glucan and xylan, respectively, were obtained. When an enzymatic load was 5 FPU/g, the conversion yields were 68 % and 85 % for glucan and xylan, respectively. Also, a new cellulasic complex CMCase produced by the P. sanguineus fungus was purified and characterized kinetically and biochemically. The purified CMCase complex consists of two endoglucanases, one GH10 (EG1) and another with partial homology to TLs proteins and unrated (EG2), and two cellobiohydrolases, GH6 and GH7. The EG1 endoglucanase has a molecular mass of 35.9 kDa, the EG2 endoglucanase and the GH6 cellobiohydrolase have molecular masses of 42 kDa, and the cellobiohydrolase GH7 has molecular mass of 52.7 kDa. The CMCase complex was very thermostable, with optimal temperature of activity at 70 °C and half-life of 57.2 h at 50 °C. Furthermore, showed high activities in a broad range of pH (3.5-5.5) and temperature (50-75°C). The values of KM and Vmax were 45.72 mg/ml and 7 μmol/min, respectively. It was found that the metallic ions Fe3+, Ag3+, Hg2+ and Cu2+ and the reagents SDS, furfural, HMF and acetic acid have inhibitory effect on the enzymatic activity of the CMCase complex. It was also observed that the CMCase complex has high specificity for the barley-β-glucan substrate and the birchwood xylan and locust bean gum hemicelluloses, also showing high activity on this substrate. Furthermore, the CMCase complex was applied to the hydrolysis of CMC and a large glucose and cellobiose formation was observed during the time, suggesting an asymmetric cleavage of the larger oligosaccharides produced, in other words, the CMCase complex cleaves preferentially at the outer regions of the larger oligosaccharides chain.