β-frutofuranosidase de Cunninghamella echinulata PA3S12MM: purificação, caracterização bioquímica e aplicação na produção de açúcar invertido
Autor(a) principal: | |
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Data de Publicação: | 2020 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Biblioteca Digital de Teses e Dissertações do UNIOESTE |
Texto Completo: | http://tede.unioeste.br/handle/tede/5049 |
Resumo: | In the most diverse industrial sectors, the use of enzymes has been increasingly required, replacing several conventional chemical processes. Although they are increasingly used, the high cost of commercial enzymes often ends up making essential steps more expensive, making it necessary to search for new enzyme sources. In this context, fungi stand out as promising microorganisms, since they are major producers of several enzymes with high added value. Among the enzymes produced by these organisms, there is β-fructofuranosidase (β-FFase), which catalyzes the hydrolysis of the α1↔2β glycosidic link in sucrose. This enzyme has applications in several industrial sectors, such as in the production of inverted sugar, equimolar mixture of glucose and fructose. Obtained from a process known as inversion, inverted sugar has greater sweetening properties than sucrose, as well as better solubility and greater resistance to crystallization and the growth of microorganisms, being used in the manufacture of cakes, breads, ice cream, jams and soft-centered candies. The production of inverted sugar through the enzyme β-FFase, although it originates a high quality product, presents considerable costs, being of fundamental importance the search for new sources and methodologies that make enzymatic catalysis feasible. Thus, the objectives of this work consisted of enzymatic screening, optimization, purification, characterization and application of Cunninghamella echinulata PA3S12MM β-FFase in inverted sugar production. From enzymatic screening assays, it was found that the fungus C. echinulata produces the enzymes polygalacturonase (20.8 and 16.06 U mL-1 ) and β-FFase (133.65 and 194.31 U mL-1 ) in extra and intracellular extracts, respectively. The highest production of β-FFase was verified in Vogel medium and the cultivation conditions were optimized through the central rotational compound design, where the maximum production of this enzyme was achieved when the fungus was cultivated for 8 days at 28 ° C and supplemented with 1% apple peel, reaching 1054.85 U mL-1 . The best conditions for pH and temperature of the enzyme present in the crude extract were pH 5.0 and 60 °C. The β-FFase was purified through ion exchange and molecular exclusion columns and presented a relative molecular mass of 89.2 kDa, maximum activity at pH 6.0 and temperature of 60 °C, in addition to presenting stability in alkaline pH and thermoactivation at 50 °C. It was found that the enzyme is multifunctional, since it hydrolyzed different substrates, and its activity increased in the presence of Mn2+ and DTT. The apparent values of Km, Vmax and Kcat for sucrose hydrolysis were 173.8 mmol L-1 , 908.7 mmol L-1 min-1 and 1388.79 s-1 , respectively. The carbohydrate content of the purified enzyme was 83.13% and the analysis of the hydrolysis and transfrutosylation products indicated that the enzyme was effective in the production of glucose and fructose, but did not show fructosyltransferase activity. The enzyme immobilized in calcium alginate showed good results in the conversion of sucrose, showing promise for industrial applications. The results suggest that C. echinulata PA3S12MM is an excellent producer of β-FFase, with high catalytic activity, stability in alkaline pHs, multifunctional character, in addition to showing promising results in the sucrose inversion. |
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Maller, Alexandrehttp://lattes.cnpq.br/8153318875076127Maller, Alexandrehttp://lattes.cnpq.br/8153318875076127Kadowaki, Marina Kimikohttp://lattes.cnpq.br/1819723253019762Heinen, Paulo Ricardohttp://lattes.cnpq.br/1492513157306177http://lattes.cnpq.br/1052476608712520Rasbold, Letícia Mara2020-10-21T16:35:05Z2020-05-05RASBOLD, Letícia Mara. β-frutofuranosidase de Cunninghamella echinulata PA3S12MM: purificação, caracterização bioquímica e aplicação na produção de açúcar invertido. 2020. 55 f. Dissertação (Programa de Pós-Graduação em Ciências Farmacêuticas) - Universidade Estadual do Oeste do Paraná, Cascavel - PR.http://tede.unioeste.br/handle/tede/5049In the most diverse industrial sectors, the use of enzymes has been increasingly required, replacing several conventional chemical processes. Although they are increasingly used, the high cost of commercial enzymes often ends up making essential steps more expensive, making it necessary to search for new enzyme sources. In this context, fungi stand out as promising microorganisms, since they are major producers of several enzymes with high added value. Among the enzymes produced by these organisms, there is β-fructofuranosidase (β-FFase), which catalyzes the hydrolysis of the α1↔2β glycosidic link in sucrose. This enzyme has applications in several industrial sectors, such as in the production of inverted sugar, equimolar mixture of glucose and fructose. Obtained from a process known as inversion, inverted sugar has greater sweetening properties than sucrose, as well as better solubility and greater resistance to crystallization and the growth of microorganisms, being used in the manufacture of cakes, breads, ice cream, jams and soft-centered candies. The production of inverted sugar through the enzyme β-FFase, although it originates a high quality product, presents considerable costs, being of fundamental importance the search for new sources and methodologies that make enzymatic catalysis feasible. Thus, the objectives of this work consisted of enzymatic screening, optimization, purification, characterization and application of Cunninghamella echinulata PA3S12MM β-FFase in inverted sugar production. From enzymatic screening assays, it was found that the fungus C. echinulata produces the enzymes polygalacturonase (20.8 and 16.06 U mL-1 ) and β-FFase (133.65 and 194.31 U mL-1 ) in extra and intracellular extracts, respectively. The highest production of β-FFase was verified in Vogel medium and the cultivation conditions were optimized through the central rotational compound design, where the maximum production of this enzyme was achieved when the fungus was cultivated for 8 days at 28 ° C and supplemented with 1% apple peel, reaching 1054.85 U mL-1 . The best conditions for pH and temperature of the enzyme present in the crude extract were pH 5.0 and 60 °C. The β-FFase was purified through ion exchange and molecular exclusion columns and presented a relative molecular mass of 89.2 kDa, maximum activity at pH 6.0 and temperature of 60 °C, in addition to presenting stability in alkaline pH and thermoactivation at 50 °C. It was found that the enzyme is multifunctional, since it hydrolyzed different substrates, and its activity increased in the presence of Mn2+ and DTT. The apparent values of Km, Vmax and Kcat for sucrose hydrolysis were 173.8 mmol L-1 , 908.7 mmol L-1 min-1 and 1388.79 s-1 , respectively. The carbohydrate content of the purified enzyme was 83.13% and the analysis of the hydrolysis and transfrutosylation products indicated that the enzyme was effective in the production of glucose and fructose, but did not show fructosyltransferase activity. The enzyme immobilized in calcium alginate showed good results in the conversion of sucrose, showing promise for industrial applications. The results suggest that C. echinulata PA3S12MM is an excellent producer of β-FFase, with high catalytic activity, stability in alkaline pHs, multifunctional character, in addition to showing promising results in the sucrose inversion.Nos mais diversos setores industriais, o uso de enzimas tem sido cada vez mais requerido, substituindo diversos processos químicos convencionais. Embora sejam cada vez mais empregadas, o elevado custo de enzimas comerciais muitas vezes acaba encarecendo etapas essenciais, tornando necessária a busca por novas fontes enzimáticas. Neste contexto, os fungos se destacam como microrganismos promissores, uma vez que são grandes produtores de diversas enzimas de alto valor agregado. Dentre as enzimas produzidas por estes organismos, encontra-se a β-frutofuranosidase (β-FFase), que catalisa a hidrólise da ligação glicosídica α1↔2β da sacarose. Esta enzima apresenta aplicações em diversos setores industriais, como na produção de açúcar invertido, mistura equimolar de glicose e frutose. Obtido a partir de um processo conhecido como inversão, o açúcar invertido possui propriedades adoçantes superiores à da sacarose, assim como melhor solubilidade e maior resistência a cristalização e ao crescimento de microrganismos, sendo utilizado na fabricação de bolos, pães, sorvetes, geleias e bombons de recheio pastoso. A produção de açúcar invertido através da enzima β-FFase, embora origine um produto de elevada qualidade, apresenta custos consideráveis, sendo de fundamental importância a busca por novas fontes e metodologias que viabilizem a catálise enzimática. Deste modo, os objetivos deste trabalho consistiram no screening enzimático, otimização, purificação, caracterização e aplicação da β-FFase de Cunninghamella echinulata PA3S12MM na produção de açúcar invertido. A partir dos ensaios de screening enzimático, verificou-se que o fungo C. echinulata produz as enzimas poligalacturonase (20,8 e 16,06 U mL-1 ) e β-FFase (133,65 e 194,31 U mL-1 ) nos extratos extra e intracelular, respectivamente. A maior produção de β-FFase foi verificada em meio Vogel e as condições de cultivo foram otimizadas através do delineamento composto central rotacional, onde a máxima produção desta enzima foi alcançada quando o fungo foi cultivado por 8 dias à 28 °C e suplementado com 1% de casca de maçã, alcançando 1054,85 U mL-1 . As melhores condições para pH e temperatura da enzima presente no extrato bruto foram pH 5,0 e 60 °C. A β-FFase foi purificada através de colunas de troca iônica e exclusão molecular e apresentou massa molecular relativa de 89,2 kDa, atividade máxima em pH 6,0 e temperatura de 60 °C, além de apresentar estabilidade em pHs alcalinos e termoativação à 50 °C. Foi verificado que a enzima é multifuncional, uma vez que hidrolisou diferentes substratos, e sua atividade aumentou na presença de Mn2+ e DTT. Os valores aparentes de Km, Vmáx e Kcat para hidrólise da sacarose foram 173,8 mmol L-1 , 908,7 mmol L-1 min-1 e 1388,79 s-1 , respectivamente. O conteúdo de carboidratos da enzima purificada foi de 83,13% e a análise dos produtos de hidrólise e transfrutosilação indicaram que a enzima foi eficaz na produção de glicose e frutose, mas não apresentou atividade frutosiltransferase. A enzima imobilizada em alginato de cálcio exibiu bons resultados na conversão da sacarose, mostrando-se promissora para aplicações industriais. Os resultados sugerem que C. echinulata PA3S12MM é um excelente produtor de β-FFase, com elevada atividade catalítica, estabilidade em pHs alcalinos, caráter multifuncional, além de exibir bons resultados na inversão da sacarose.Submitted by Neusa Fagundes (neusa.fagundes@unioeste.br) on 2020-10-21T16:35:05Z No. of bitstreams: 2 Letícia_Rasbold2020.pdf: 2365746 bytes, checksum: 78a872f70110812d128bce268a126164 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Made available in DSpace on 2020-10-21T16:35:05Z (GMT). No. of bitstreams: 2 Letícia_Rasbold2020.pdf: 2365746 bytes, checksum: 78a872f70110812d128bce268a126164 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2020-05-05Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPESapplication/pdfpor6588633818200016417500Universidade Estadual do Oeste do ParanáCascavelPrograma de Pós-Graduação em Ciências FarmacêuticasUNIOESTEBrasilCentro de Ciências Médicas e Farmacêuticashttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccessInvertaseEnzima multifuncionalHidrólise da sacaroseImobilização em alginato de cálcioDelineamento composto central rotacionalScreening enzimáticoInvertaseMultifunctional enzymeSucrose hydrolysisCalcium alginate immobilizationComposite central rotational designEnzymatic screeningCIENCIAS DA SAUDE::FARMACIAβ-frutofuranosidase de Cunninghamella echinulata PA3S12MM: purificação, caracterização bioquímica e aplicação na produção de açúcar invertidoβ-fructofuranosidase from Cunninghamella echinulata PA3S12MM: purification, biochemical characterization and application on inverted sugar productioninfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesis7878055067573953101600600600600-894043971338784926769976364134497549962075167498588264571reponame:Biblioteca Digital de Teses e Dissertações do UNIOESTEinstname:Universidade Estadual do Oeste do Paraná (UNIOESTE)instacron:UNIOESTEORIGINALLetícia_Rasbold2020.pdfLetícia_Rasbold2020.pdfapplication/pdf2365746http://tede.unioeste.br:8080/tede/bitstream/tede/5049/5/Leti%CC%81cia_Rasbold2020.pdf78a872f70110812d128bce268a126164MD55CC-LICENSElicense_urllicense_urltext/plain; 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dc.title.por.fl_str_mv |
β-frutofuranosidase de Cunninghamella echinulata PA3S12MM: purificação, caracterização bioquímica e aplicação na produção de açúcar invertido |
dc.title.alternative.eng.fl_str_mv |
β-fructofuranosidase from Cunninghamella echinulata PA3S12MM: purification, biochemical characterization and application on inverted sugar production |
title |
β-frutofuranosidase de Cunninghamella echinulata PA3S12MM: purificação, caracterização bioquímica e aplicação na produção de açúcar invertido |
spellingShingle |
β-frutofuranosidase de Cunninghamella echinulata PA3S12MM: purificação, caracterização bioquímica e aplicação na produção de açúcar invertido Rasbold, Letícia Mara Invertase Enzima multifuncional Hidrólise da sacarose Imobilização em alginato de cálcio Delineamento composto central rotacional Screening enzimático Invertase Multifunctional enzyme Sucrose hydrolysis Calcium alginate immobilization Composite central rotational design Enzymatic screening CIENCIAS DA SAUDE::FARMACIA |
title_short |
β-frutofuranosidase de Cunninghamella echinulata PA3S12MM: purificação, caracterização bioquímica e aplicação na produção de açúcar invertido |
title_full |
β-frutofuranosidase de Cunninghamella echinulata PA3S12MM: purificação, caracterização bioquímica e aplicação na produção de açúcar invertido |
title_fullStr |
β-frutofuranosidase de Cunninghamella echinulata PA3S12MM: purificação, caracterização bioquímica e aplicação na produção de açúcar invertido |
title_full_unstemmed |
β-frutofuranosidase de Cunninghamella echinulata PA3S12MM: purificação, caracterização bioquímica e aplicação na produção de açúcar invertido |
title_sort |
β-frutofuranosidase de Cunninghamella echinulata PA3S12MM: purificação, caracterização bioquímica e aplicação na produção de açúcar invertido |
author |
Rasbold, Letícia Mara |
author_facet |
Rasbold, Letícia Mara |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Maller, Alexandre |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/8153318875076127 |
dc.contributor.referee1.fl_str_mv |
Maller, Alexandre |
dc.contributor.referee1Lattes.fl_str_mv |
http://lattes.cnpq.br/8153318875076127 |
dc.contributor.referee2.fl_str_mv |
Kadowaki, Marina Kimiko |
dc.contributor.referee2Lattes.fl_str_mv |
http://lattes.cnpq.br/1819723253019762 |
dc.contributor.referee3.fl_str_mv |
Heinen, Paulo Ricardo |
dc.contributor.referee3Lattes.fl_str_mv |
http://lattes.cnpq.br/1492513157306177 |
dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/1052476608712520 |
dc.contributor.author.fl_str_mv |
Rasbold, Letícia Mara |
contributor_str_mv |
Maller, Alexandre Maller, Alexandre Kadowaki, Marina Kimiko Heinen, Paulo Ricardo |
dc.subject.por.fl_str_mv |
Invertase Enzima multifuncional Hidrólise da sacarose Imobilização em alginato de cálcio Delineamento composto central rotacional Screening enzimático |
topic |
Invertase Enzima multifuncional Hidrólise da sacarose Imobilização em alginato de cálcio Delineamento composto central rotacional Screening enzimático Invertase Multifunctional enzyme Sucrose hydrolysis Calcium alginate immobilization Composite central rotational design Enzymatic screening CIENCIAS DA SAUDE::FARMACIA |
dc.subject.eng.fl_str_mv |
Invertase Multifunctional enzyme Sucrose hydrolysis Calcium alginate immobilization Composite central rotational design Enzymatic screening |
dc.subject.cnpq.fl_str_mv |
CIENCIAS DA SAUDE::FARMACIA |
description |
In the most diverse industrial sectors, the use of enzymes has been increasingly required, replacing several conventional chemical processes. Although they are increasingly used, the high cost of commercial enzymes often ends up making essential steps more expensive, making it necessary to search for new enzyme sources. In this context, fungi stand out as promising microorganisms, since they are major producers of several enzymes with high added value. Among the enzymes produced by these organisms, there is β-fructofuranosidase (β-FFase), which catalyzes the hydrolysis of the α1↔2β glycosidic link in sucrose. This enzyme has applications in several industrial sectors, such as in the production of inverted sugar, equimolar mixture of glucose and fructose. Obtained from a process known as inversion, inverted sugar has greater sweetening properties than sucrose, as well as better solubility and greater resistance to crystallization and the growth of microorganisms, being used in the manufacture of cakes, breads, ice cream, jams and soft-centered candies. The production of inverted sugar through the enzyme β-FFase, although it originates a high quality product, presents considerable costs, being of fundamental importance the search for new sources and methodologies that make enzymatic catalysis feasible. Thus, the objectives of this work consisted of enzymatic screening, optimization, purification, characterization and application of Cunninghamella echinulata PA3S12MM β-FFase in inverted sugar production. From enzymatic screening assays, it was found that the fungus C. echinulata produces the enzymes polygalacturonase (20.8 and 16.06 U mL-1 ) and β-FFase (133.65 and 194.31 U mL-1 ) in extra and intracellular extracts, respectively. The highest production of β-FFase was verified in Vogel medium and the cultivation conditions were optimized through the central rotational compound design, where the maximum production of this enzyme was achieved when the fungus was cultivated for 8 days at 28 ° C and supplemented with 1% apple peel, reaching 1054.85 U mL-1 . The best conditions for pH and temperature of the enzyme present in the crude extract were pH 5.0 and 60 °C. The β-FFase was purified through ion exchange and molecular exclusion columns and presented a relative molecular mass of 89.2 kDa, maximum activity at pH 6.0 and temperature of 60 °C, in addition to presenting stability in alkaline pH and thermoactivation at 50 °C. It was found that the enzyme is multifunctional, since it hydrolyzed different substrates, and its activity increased in the presence of Mn2+ and DTT. The apparent values of Km, Vmax and Kcat for sucrose hydrolysis were 173.8 mmol L-1 , 908.7 mmol L-1 min-1 and 1388.79 s-1 , respectively. The carbohydrate content of the purified enzyme was 83.13% and the analysis of the hydrolysis and transfrutosylation products indicated that the enzyme was effective in the production of glucose and fructose, but did not show fructosyltransferase activity. The enzyme immobilized in calcium alginate showed good results in the conversion of sucrose, showing promise for industrial applications. The results suggest that C. echinulata PA3S12MM is an excellent producer of β-FFase, with high catalytic activity, stability in alkaline pHs, multifunctional character, in addition to showing promising results in the sucrose inversion. |
publishDate |
2020 |
dc.date.accessioned.fl_str_mv |
2020-10-21T16:35:05Z |
dc.date.issued.fl_str_mv |
2020-05-05 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
RASBOLD, Letícia Mara. β-frutofuranosidase de Cunninghamella echinulata PA3S12MM: purificação, caracterização bioquímica e aplicação na produção de açúcar invertido. 2020. 55 f. Dissertação (Programa de Pós-Graduação em Ciências Farmacêuticas) - Universidade Estadual do Oeste do Paraná, Cascavel - PR. |
dc.identifier.uri.fl_str_mv |
http://tede.unioeste.br/handle/tede/5049 |
identifier_str_mv |
RASBOLD, Letícia Mara. β-frutofuranosidase de Cunninghamella echinulata PA3S12MM: purificação, caracterização bioquímica e aplicação na produção de açúcar invertido. 2020. 55 f. Dissertação (Programa de Pós-Graduação em Ciências Farmacêuticas) - Universidade Estadual do Oeste do Paraná, Cascavel - PR. |
url |
http://tede.unioeste.br/handle/tede/5049 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.relation.program.fl_str_mv |
7878055067573953101 |
dc.relation.confidence.fl_str_mv |
600 600 600 600 |
dc.relation.department.fl_str_mv |
-8940439713387849267 |
dc.relation.cnpq.fl_str_mv |
6997636413449754996 |
dc.relation.sponsorship.fl_str_mv |
2075167498588264571 |
dc.rights.driver.fl_str_mv |
http://creativecommons.org/licenses/by-nc-nd/4.0/ info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
http://creativecommons.org/licenses/by-nc-nd/4.0/ |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Universidade Estadual do Oeste do Paraná Cascavel |
dc.publisher.program.fl_str_mv |
Programa de Pós-Graduação em Ciências Farmacêuticas |
dc.publisher.initials.fl_str_mv |
UNIOESTE |
dc.publisher.country.fl_str_mv |
Brasil |
dc.publisher.department.fl_str_mv |
Centro de Ciências Médicas e Farmacêuticas |
publisher.none.fl_str_mv |
Universidade Estadual do Oeste do Paraná Cascavel |
dc.source.none.fl_str_mv |
reponame:Biblioteca Digital de Teses e Dissertações do UNIOESTE instname:Universidade Estadual do Oeste do Paraná (UNIOESTE) instacron:UNIOESTE |
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Universidade Estadual do Oeste do Paraná (UNIOESTE) |
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UNIOESTE |
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UNIOESTE |
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Biblioteca Digital de Teses e Dissertações do UNIOESTE |
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Biblioteca Digital de Teses e Dissertações do UNIOESTE |
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http://tede.unioeste.br:8080/tede/bitstream/tede/5049/5/Leti%CC%81cia_Rasbold2020.pdf http://tede.unioeste.br:8080/tede/bitstream/tede/5049/2/license_url http://tede.unioeste.br:8080/tede/bitstream/tede/5049/3/license_text http://tede.unioeste.br:8080/tede/bitstream/tede/5049/4/license_rdf http://tede.unioeste.br:8080/tede/bitstream/tede/5049/1/license.txt |
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Biblioteca Digital de Teses e Dissertações do UNIOESTE - Universidade Estadual do Oeste do Paraná (UNIOESTE) |
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biblioteca.repositorio@unioeste.br |
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