• JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
 
  Bookmark and Share
 
 
Doctoral Thesis
DOI
https://doi.org/10.11606/T.17.2020.tde-11022020-134919
Document
Author
Full name
Juliana da Conceição Infante
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2019
Supervisor
Committee
Polizeli, Maria de Lourdes Teixeira de Moraes (President)
Farinas, Cristiane Sanchez
Lubini, Greice
Pereira, Marita Gimenez
Title in Portuguese
Imobilização e caracterização bioquímica de mananases e pectinases produzidas por Aspergillus brasiliensis: potencial para aplicação na clarificação de sucos de frutas
Keywords in Portuguese
Aspergillus brasiliensis
Caracterização bioquímica
Cascas de soja
Imobilização
Mananase
Pectinase
Abstract in Portuguese
Mananases e pectinases são carboidrases que estão ganhando significativo interesse na área biotecnológica devido ao aumento da demanda pela utilização de fontes biorenováveis. Em vista disso, foram selecionados 4 fungos potenciais produtores de mananase (Talaromyces pinophilus, Aspergillus unguis, Aspergillus brasiliensis e Aspergillus clavatus), que também apresentaram elevadas atividades de pectinase. O fungo Aspergillus brasiliensis foi definido para dar continuidade ao estudo e apresentou melhor perfil de atividade de mananase a 70 °C em pH 4,0 (2,516 UI/mL) e de pectinase a 50 °C em pH 3,5 (4,168 UI/mL). Quando testados em relação a estabilidade ao pH a atividade de mananase foi ativada em 5 horas de incubação e se manteve estável durante 24 horas nos pHs 3,0 a 7,0. A pectinase se manteve estável durante 4 horas de incubação diminuindo sua eficiência após 5 horas em todos os pHs testados. A mananase apresentou termoestabilidade a 50 °C e 60 °C durante 24 horas e pectinase a 40 °C durante 24 horas. O extrato bruto extracelular (EbE) foi imobilizado em resinas de troca aniônica DEAE-Celulose e Q-Sefarose, no qual apresentou taxa de imobilização de mananase de 94,5 % em DEAE-Celulose e 93,8 % em Q-Sefarose e taxa de imobilização de pectinase de 75,2 % em DEAE-Celulose e 86,5 % em Q-Sefarose. HgCl2 inibiu as atividades de mananase e pectinase presentes no EbE e nos derivados DEAECelulose e Q-Sefarose, que pode indicar a presença de sítios tióis próximos ao sítio catalítico. O agente quelante EDTA hiperativou 210 % a pectinase no EbE apresentando característica de metaloproteína, porém inibiu a mananase no EbE e nos derivados DEAE-Celulose e Qsefarose. As mananases e pectinases foram avaliadas em relação a dessorção em diferentes concentrações de NaCl, no qual o derivado Q-Sefarose_Pectinase apresentou forte ligação ao suporte. Com intuito de analisar o potencial do efeito das enzimas imobilizadas nesse estudo (derivado QsMP), foram realizados testes no processo de clarificação de sucos de frutas. QsMP apresentou eficiência na redução da turbidez do suco de maçã na primeira aplicação e nos dois reusos (45 %, 60 % e 49 % respectivamente), no entanto no suco de uva apresentou uma redução de apenas 20 % somente no primeiro uso, perdendo sua eficiência nos reusos seguintes. Podemos concluir nesse estudo que a imobilização aumentou a atividade das mananases e pectinases estudadas, além do aumento da temperatura, pH, estabilidade ao pH e testes de reuso no processo de clarificação de sucos de frutas.
Title in English
Immobilization and biochemical characterization of mannanases and pectinases produced by Aspergillus brasiliensis: potential application in fruit juice clarification
Keywords in English
Aspergillus brasiliensis
Biochemical caractherization
Imobilization
Manannase
Pectinase
Soybean husk
Abstract in English
Mannanases and pectinases are carbohydrases that are gaining great biotechnological interest due to demand for biorenovable catalysts sources. In this context 4 mannanase-producing fungi (Talaromyces pinophilus, Aspergillus unguis, Aspergillus brasiliensis and Aspergillus clavatus), which were also identified as pectinase producers, were selected. The fungus Aspergillus brasiliensis was chosen to continue the study, as it presented a better mannanase production profile, with an activity of 2,51 IU / mL at 70 ° C and pH 4.0, and 4.16 IU / mL of pectinase at 50 ° C and pH 3.5. When tested for pH stability the mannanase activity was activated within 5 hours of incubation and remained stable for 24 hours in pHs 3.0 to 7.0. The pectinase was stable for 4 hours incubation, decreasing its efficiency after 5 hours in all pHs tested. The mannanase was thermostable at 50°C and 60°C for 24 hours and the pectinase at 40°C for 24 hours. The extracellular crude extract (EbE) was immobilized in DEAECellulose and Q-Sepharose anion exchange resins. The rate of EbE immobilization was 94.5% for mannanase in DEAE-Cellulose and 93.8% in Q-Sepharose. For pectinase immobilization the rate was 75% in DEAE-Cellulose and 86.5% in Q-Sepharose. HgCl2 inhibited the mannanase and pectinase activities in EbE and in DEAE and Q-Sepharose derivatives. This may indicate the presence of thiol sites near the catalytic site. EDTA chelating agent hyperactivated 210% the pectinase present in EbE, exhibiting metalloprotein characteristics, but inhibited the mannanase in EbE, DEAE and Q-Sepharose derivatives. The resulting derivatives were evaluated for desorption at different concentrations of NaCl, in which the Q-Sepharose_Pectinase derivative showed a strong binding to the support. In order to analyze a potential application for the pectinase and mannanase immobilized in QSepharose (QsMP derivative), were carried out tests in the clarifying fruit juices process. QsMP was efficient in the reduction of turbidity in apple juice in the first application and in two consecutive reuses (45%, 60% and 49% respectively), on the other hand, in grape juice it showed a turbidity reduction of only 20% in the first use, losing its efficiency in the following tests. We can conclude in this study that the immobilization improved mannanases and pectinases activities, in addition to the increase of temperature and pH, a better pH stability and the possibility of reuse in the clarification process of fruit juices.
 
WARNING - Viewing this document is conditioned on your acceptance of the following terms of use:
This document is only for private use for research and teaching activities. Reproduction for commercial use is forbidden. This rights cover the whole data about this document as well as its contents. Any uses or copies of this document in whole or in part must include the author's name.
Publishing Date
2020-04-29
 
WARNING: Learn what derived works are clicking here.
All rights of the thesis/dissertation are from the authors
CeTI-SC/STI
Digital Library of Theses and Dissertations of USP. Copyright © 2001-2021. All rights reserved.