Master's Dissertation
DOI
https://doi.org/10.11606/D.100.2022.tde-05052022-113742
Document
Author
Full name
Vandierly Sampaio de Melo
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2022
Supervisor
Committee
Chambergo Alcalde, Felipe Santiago (President)
Perpetuo, Elen Aquino
Salinas, Roberto Kopke
Zanphorlin, Letícia Maria
Title in Portuguese
Caracterização de glicosil hidrolases envolvidas na degradação de biomassa vegetal
Keywords in Portuguese
β-xilosidase
Endo-α-1,5-L-arabinanase
Enzimas termoestáveis
Etanol de segunda geração
GH43
Abstract in Portuguese
Em virtude da crescente demanda por energia e de problemas ambientais causados pelo consumo de combustíveis fósseis, tem-se aumentado a necessidade de produção de combustíveis renováveis. O etanol de segunda geração (2G) surgiu como uma alternativa promissora aos combustíveis fósseis devido ao uso de biomassa vegetal como matéria-prima. Para que haja o processo de hidrólise enzimática da biomassa vegetal e obtenção de açúcares fermentáveis é fundamental o uso de diversas enzimas, tais como endo-α-1,5-L-arabinanases e β-xilosidases. Dessa forma, este trabalho teve por objetivo caracterizar as enzimas glicosil hidrolases endo-α-1,5-L-arabinanase e β-xilosidase de Geobacillus sp. JS12 e Geobacillus thermodenitrificans, respectivamente. Os genes das enzimas putativas, endo-α-1,5-L-arabinanase (GeoARA) e β-xilosidase (GeoXIL) foram obtidos a partir de análises da base de dados CAZy e posteriormente sintetizados e clonados para a expressão das proteínas recombinantes em E. coli BL21. Análises de bioinformática foram realizadas da sequência de aminoácidos da GeoARA e GeoXIL. As enzimas foram purificadas por cromatografia de afinidade em coluna de níquel-sefarose e realizou-se a caracterização bioquímica e análises de termoestabilidade por dicroísmo circular. As enzimas GeoARA e GeoXIL foram agrupadas dentro da família de glicosil hidrolases 43 (GH43). A partir dos testes de atividade, notou-se que a GeoARA possui maior atividade em pH 7,0 e temperatura de 70 °C, enquanto a GeoXIL possui maior atividade em pH 5,0 e temperatura de 60 °C. Nos testes com íons metálicos e EDTA, ambas as enzimas foram estáveis. Verificou-se que a GeoARA possui uma boa estabilidade térmica em 70 °C, principalmente na presença do íon Co2+ após 48 horas, enquanto que a GeoXIL apresentou maior estabilidade a 50 °C por 4 horas. A GeoARA possui uma significativa atividade sobre a arabinana desramificada (226,67 U/mg) e eficiência catalítica de 53 mL/mg/s. Por sua vez, a GeoXIL apresentou atividade bifuncional de β-xilosidase e α- L-arabinofuranosidase, com maior atividade sobre o substrato p-nitrofenil-β-D-xilopiranosídeo. A atividade específica sobre o p-nitrofenil-β-D-xilopiranosídeo foi de 18,33 U/mg e eficiência catalítica de 20,21 /mM/s; comparável com outras β-xilosidases. A atividade em elevadas temperaturas, termoestabilidade e estabilidade a íons metálicos são características desejáveis a enzimas industriais. Nesse sentido, as enzimas GeoARA e GeoXIL apresentam características bioquímicas interessantes para aplicação na hidrólise enzimática de biomassa vegetal
Title in English
Characterization of glycosyl hydrolases involved in plant biomass degradation
Keywords in English
β-xylosidase
Endo-α-1,5-L-arabinanase
GH43
Second generation ethanol
Thermostable enzymes
Abstract in English
Due to the growing demand for energy and environmental problems caused by the consumption of fossil fuels, the need to produce renewable fuels has increased. Second generation ethanol (2G) has emerged as a promising alternative to fossil fuels due to the use of plant biomass as a feedstock. In order to enzymatic hydrolysis the plant biomass and obtain fermentable sugars, it is essential to use several enzymes, such as endo-α-1,5-L-arabinanases and β-xylosidases. Thus, this work aimed to characterize the glycosyl hydrolases enzymes endo-α-1,5-L-arabinanase and β-xylosidase from Geobacillus sp. JS12 and Geobacillus thermodenitrificans, respectively. The genes sequences corresponding to the putative endo-α-1,5-L-arabinanase (GeoARA) and β-xylosidase (GeoXIL) enzymes were recovered from analyzes of the CAZy database and later synthesized and cloned for the expression of recombinant proteins in E. coli BL21. Bioinformatics analysis was performed on the amino acid sequence of GeoARA and GeoXIL. The enzymes were purified by affinity chromatography on a nickel-sepharose column and biochemical characterization and thermostability analysis by circular dichroism were performed. The enzymes GeoARA and GeoXIL were grouped within the family of glycosyl hydrolases 43 (GH43). Through activity tests, it was found that GeoARA has greater activity at pH 7.0 and temperature of 70 °C while GeoXIL has greater activity at pH 5.0 and temperature of 60 °C. In tests with metal ions and EDTA, the enzymes were stable. It was found that GeoARA has a good thermal stability at 70 °C, mainly in the presence of the Co2+ ion after 48 hours, while GeoXIL showed greater stability at 50 °C for 4 hours. GeoARA has significant activity on debranched arabinan (226.67 U/mg) and catalytic efficiency of 53 mL/mg/s. In fact, GeoXIL showed bifunctional activity of β-xylosidase and α-L-arabinofuranosidase, with greater activity on the substrate p-nitrophenyl-β-D-xylopyranoside. The specific activity on p-nitrophenyl-β-D-xylopyranoside was 18.33 U/mg and catalytic efficiency of 20.21 /mM/s, comparable to other β-xylosidases. The activity at high temperatures, thermostability and stability to metal ions are desired characteristics for industrial enzymes. In this sense, the GeoARA and GeoXIL enzymes present interesting biochemical characteristics for application in the enzymatic hydrolysis of lignocellulosic biomass
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Publishing Date
2022-05-17