Avaliação do efeito de isoforma de lacase produzida por fungos endofíticos na formação de ligações cruzadas em córneas
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Data
2021-03-01
Autores
Carvalho, Taleesa [UNIFESP]
Orientadores
Bersanetti, Patrícia Alessandra [UNIFESP]
Tipo
Trabalho de conclusão de curso de graduação
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Resumo
O ceratocone é uma doença degenerativa que afeta a córnea, tornando-a
mais cônica (ectasia), devido ao afrouxamento das fibrilas de colágeno, o que resulta
em uma perda de acuidade visual. Um método de tratamento existente é o
crosslinking (CXL), no qual novas ligações cruzadas são formadas no estroma, a
partir da estimulação da riboflavina com radiação ultravioleta de 365 nm. Esse
processo resulta em um aumento da força biomecânica e resistência da córnea, com
consequente diminuição da taxa de progressão do ceratocone. Porém, esse
tratamento apresenta alguns inconvenientes, como: o desconforto causado pela
retirada do epitélio hidrofóbico para penetração da riboflavina e danos aos
queratócitos, pela aplicação direta de radiação UV na córnea.
Em projetos de IC anteriores, observou-se que a enzima comercial lacase de
Trametes versicolor, principalmente na presença da molécula de catequina, é capaz
de promover a formação de ligações cruzadas na córnea, conforme verificado pela
diminuição da taxa de degradação enzimática, mudança no perfil de desnaturação
térmica e aumento no módulo de elasticidade.
Assim, o presente estudo teve o objetivo de produzir, purificar e analisar a
eficiência de uma isoforma de lacase produzida por fungos endofíticos na formação
in vitro de ligações cruzadas em córneas.
Inicialmente, foi realizada uma análise de resistência à digestão enzimática
dos dois extratos de lacase produzidos pelos fungos Xylaria sp e Sordariomycetes
sp. O extrato de Xylaria sp selecionado foi purificado por precipitação salina e, em
seguida, por cromatografia de troca-iônica. Nos experimentos de promoção de
ligações cruzadas, os olhos de porco de abatedouro foram desepitelizados,
trepanados e divididos em grupos. O controle foi mantido à 36° C por 24 horas
apenas em uma mistura (1:1) de tampão acetato 10 mM pH=5 e de DMEM/F12 com
10% de dextran pH 7,0. As amostras do grupo lacase/catequina foram deixadas à
36° C por 24 horas em uma solução composta de 250 μL do extrato de Xylaria sp.,
250 μL de meio DMEM/F12 com dextran e 5,0 mg/mL de catequina. Posteriormente,
todas as amostras foram mantidas por 24 horas em geladeira no meio DMEM/F12
com dextran e, após esse período, foram submetidas à análise por calorimetria
exploratória diferencial (DSC), análise mecânica e digestão enzimática.
Os resultados de caracterização térmica mostraram que o grupo submetido à
ação da enzima apresentou uma maior temperatura de desnaturação em relação ao
controle, o que pode ser explicado pela desidratação das fibras de colágeno do
estroma que acompanha o crosslinking. Dos resultados de resistência à degradação
enzimática temos, que as córneas do grupo tratado com a enzima foram mais
resistentes à degradação do que o grupo controle. As córneas também foram
caracterizadas por ensaios mecânicos, e os resultados obtidos mostram que o grupo
tratado com lacase na presença de catequina apresentou um aumento na resistência
mecânica da córnea.
Desta forma, a enzima lacase produzida pelo fungo endofítico Xylaria sp, na
presença de catequina, foi capaz de promover a formação de ligações cruzadas in
vitro em córneas de abatedouro.
The keratoconus is a degenerative disease that affects the cornea becoming it more conical (ectasia) due to the loosening of collagen fibrils, resulting in a loss of visual acuity. An existing treatment method is the crosslinking (CXL), this method induces new stromal crosslinks from the stimulation of riboflavin with ultraviolet radiation from 365nm. The result of this process is the increase in the biomechanical strength and resistance of the cornea with a consequent decrease in the rate of progression of keratoconus. However, this treatment has some drawbacks such as: the discomfort caused by the removal of the hydrophobic epithelium for the penetration of riboflavin and damage to keratocytes because of the direct application of UV radiation to the cornea. In previous scientific initiation projects, it was observed that the commercial enzyme laccase of Trametes versicolor, mainly in the presence of the catechin molecule is capable of promoting the formation of crosslinks in the cornea, as verified by the decrease in the rate of enzymatic degradation, change in the thermal denaturation profile and increase in the elasticity module. Thus, the present study aimed to produce, purify and analyze the efficiency of a laccase isoform produced by endophytic fungi in vitro formation of crosslinking in corneas. Initially, we carried out an analysis of resistance to enzymatic digestion of the two laccase extracts produced by the fungi Xylaria sp and Sordariomycetes sp. The extracts of Xylaria sp selected were purified by saline precipitation and then and then by ion exchange chromatography. In cross-link promotion experiments, slaughterhouse pig eyes were de-epithelialized, trepanned and apportion in groups. The control was maintained at 36 °C for 24 hours only in a mixture (1:1) of acetate buffer 10 mM pH=5 and DMEM/F12 with 10% de dextran pH 7,0. The samples from the laccase /catechin group were left at 36 °C for 24 hours in a solution composed of 250 μL of the extract to Xylaria sp., 250 μL of DMEM/F12 with dextran and 5,0 mg/mL of catechin. Posteriorly, all groups were maintained for 24 hours in the refrigerator in the DMEM/F12 with 10% de dextran and after that period they were submitted to analysis by differential exploratory calorimetry, mechanical analysis and enzymatic digestion. The results of thermal characterization showed that the group submitted to the action of the enzyme presented a higher temperature of denaturation in relation to the control, which can be explained by the dehydration of the stroma collagen fibrils that accompanies the crosslinking. From the results of resistance to enzymatic degradation, we have that the corneas of the group treated with the enzyme were more resistant to degradation than the control group. The corneas were also characterized by mechanical tests, and the results obtained show that the group treated with laccase in the presence of catechin showed an increase in the mechanical resistance of the cornea. In this way, the laccase enzyme produced by the endophytic fungus Xylaria sp, in the presence of catechin, was able to promote the formation of cross-links in vitro in slaughterhouse corneas.
The keratoconus is a degenerative disease that affects the cornea becoming it more conical (ectasia) due to the loosening of collagen fibrils, resulting in a loss of visual acuity. An existing treatment method is the crosslinking (CXL), this method induces new stromal crosslinks from the stimulation of riboflavin with ultraviolet radiation from 365nm. The result of this process is the increase in the biomechanical strength and resistance of the cornea with a consequent decrease in the rate of progression of keratoconus. However, this treatment has some drawbacks such as: the discomfort caused by the removal of the hydrophobic epithelium for the penetration of riboflavin and damage to keratocytes because of the direct application of UV radiation to the cornea. In previous scientific initiation projects, it was observed that the commercial enzyme laccase of Trametes versicolor, mainly in the presence of the catechin molecule is capable of promoting the formation of crosslinks in the cornea, as verified by the decrease in the rate of enzymatic degradation, change in the thermal denaturation profile and increase in the elasticity module. Thus, the present study aimed to produce, purify and analyze the efficiency of a laccase isoform produced by endophytic fungi in vitro formation of crosslinking in corneas. Initially, we carried out an analysis of resistance to enzymatic digestion of the two laccase extracts produced by the fungi Xylaria sp and Sordariomycetes sp. The extracts of Xylaria sp selected were purified by saline precipitation and then and then by ion exchange chromatography. In cross-link promotion experiments, slaughterhouse pig eyes were de-epithelialized, trepanned and apportion in groups. The control was maintained at 36 °C for 24 hours only in a mixture (1:1) of acetate buffer 10 mM pH=5 and DMEM/F12 with 10% de dextran pH 7,0. The samples from the laccase /catechin group were left at 36 °C for 24 hours in a solution composed of 250 μL of the extract to Xylaria sp., 250 μL of DMEM/F12 with dextran and 5,0 mg/mL of catechin. Posteriorly, all groups were maintained for 24 hours in the refrigerator in the DMEM/F12 with 10% de dextran and after that period they were submitted to analysis by differential exploratory calorimetry, mechanical analysis and enzymatic digestion. The results of thermal characterization showed that the group submitted to the action of the enzyme presented a higher temperature of denaturation in relation to the control, which can be explained by the dehydration of the stroma collagen fibrils that accompanies the crosslinking. From the results of resistance to enzymatic degradation, we have that the corneas of the group treated with the enzyme were more resistant to degradation than the control group. The corneas were also characterized by mechanical tests, and the results obtained show that the group treated with laccase in the presence of catechin showed an increase in the mechanical resistance of the cornea. In this way, the laccase enzyme produced by the endophytic fungus Xylaria sp, in the presence of catechin, was able to promote the formation of cross-links in vitro in slaughterhouse corneas.
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Citação
CARVALHO, Taleesa Souza. Avaliação do efeito de isoforma de lacase produzida por fungos endofíticos na formação de ligações cruzadas em córneas. São Paulo, 2021. 58 p. Trabalho de Conclusão de Curso (Graduação em Tecnologia Oftálmica) - Universidade Federal de São Paulo. São Paulo, 2021.