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| Author(s): |
Erica Aparecida Santos Bistratini
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São José do Rio Preto. 2016-12-13. |
| Institution: | Universidade Estadual Paulista (Unesp). Instituto de Biociências Letras e Ciências Exatas. São José do Rio Preto |
| Defense date: | 2016-11-18 |
| Advisor: | Heloiza Ferreira Alves Prado |
| Abstract | |
The Present work covers the study of the production of degradative enzymes from plant cell wall (EDPCP) produced by Rhizoctonia solani AG-1 IA isolated from rice, signalgrass and soybean. The enzymes studied initially were cellulases, β-glucosidase, xylanase, pectinase, amylase, lipase and proteases extracellularly secreted into the culture medium. It was evaluated 87 isolates of Rhizoctonia solani AG-1 IA and adopted cultivation in the solid state using wheat bran as the initial carbon source for inducing the production of extracellular enzymes and assess the potential isolates. The study of the effect of carbon source was performed with five isolates of each type of culture which were cultured on wheat bran, comminuted signalgrass, soybean straw, corn stover, corn cobs and rice straw. The best result results for xylanase activity was the isolated PA_B1F6 from cultures of signalgrass grown in wheat bran (15.82 U mL-1) to Avicelase and CMCase and the best results were obtained for the isolated of soybean MA_217 and TO_064 when grown corn straw (3.24 U ml-1) and soybean straw (2.91 U ml-1), respectively. For β-glucosidase the best results were obtained for the isolated of soybean TO_022 (2.75 U ml-1) grown in signalgrass. The amylase activity was higher in culture with wheat bran for isolated ROB4D7 from the signalgrass culture (142.88 U mL-1). The best pectinase activity was by the PA_B1F6 isolated signalgrass culture (17.69 U mL-1) when grown in signalgrass as substrate. The MT_S085 isolated from soybean showed better protease activity (UAP 1154.52 ml-1) in culture with wheat bran. For this same isolated, but when grown in signalgrass substrate was observed the best lipase activity (30.57 U mL-1). The five isolates of each type were grown in culture substrates that indicated better xylanase activity to evaluate the activity profile over time. Isolates obtained from signalgrass culture were those who generally had higher levels of xylanase activity. Crops using signalgrass and wheat bran as substrate presented the best xylanase activity and, in general, the cultivation time with greater enzymatic activity in 120h. The crude xylanase produced by rice culture isolates (RR_A23), signalgrass (PA_B1F6) and soybean (MT_S085) showed optimum temperature at 55 ° C. The optimal pH of these xylanase was pH 6.0 for isolated RR_A23, pH 6.5 for PA_B1F6, and pH 7.5 for MT_S085. The pH was stable at pH 4.0 to 7.5. It was possible to verify that the xylanases produced by these isolates differ with the type of substrate used in cultivation when subjected to polyacrylamide gel electrophoresis SDS-PAGE. They were identified 35 gene sequences for the xylanase in Rhizoctonia solani, however, there is not until now, gene sequence described for xylanase produced by Rhizoctonia solani AG-1 IA. It was found 4 gene sequences of Rhizoctonia solani AG-1 IA that showed greater similarity when compared to sequences of xylanase described for other groups of the same species. Thus, it was performed the purification of a xylanase produced by Rhizoctonia solani AG-1 IA isolated PA_B1F6 from the signalgrass culture. The cultivation in the solid state was carried out using wheat bran with xylan. For the purification of this xylanase concentration was performed by ultrafiltration, ethanol precipitation and ion exchange in Hitrap SP Sepharose FF, which can recover 4% of the initial activity. The molecular mass was estimated as 33.11 kDa. The purified xylanase showed optimal activity at 55 ° C and higher activity at pH 6.5. The enzyme was inhibited by ions and modifying agents and the kinetic parameters Km, Vmax, using xylan birch (beerchwood) as substrate were 1.42 mg mL-1, 1415 mol L-1 mim-1, respectively. (AU) | |
| FAPESP's process: | 13/03692-0 - Plant cell wall degradation enzymes from Rhizoctonia solani AG-1 IA: extracellular production study, characterization and sequencing. |
| Grantee: | Erica Aparecida Santos Bistratini |
| Support Opportunities: | Scholarships in Brazil - Doctorate |