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Previous issue date: 2011-09-16 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Enzymatic synthesis of peptides using proteases has attracted a great deal of attention in
recent years. One key challenge in peptide synthesis is to find supports for protease
immobilization capable of working in aqueous medium at high performance, producing watersoluble
oligopeptides. At present, few reports have been described using this strategy. Therefore,
the aim of this thesis was to immobilize proteases applying different methods (Immobilization by
covalent bound, entrapment onto polymeric gels of PVA and immobilization on glycidil
metacrylate magnetic nanoparticles) in order to produce water-soluble oligopeptides derived from
lysine. Three different proteases were used: trypsin, α-chymotrypsin and bromelain. According to
immobilization strategies associated to the type of protease employed, trypsin-resin systems
showed the best performance in terms of hydrolytic activity and oligopeptides synthesis.
Hydrolytic activities of the free and immobilized enzymes were determined
spectrophotometrically based on the absorbance change at 660 nm at 25 ?C (Casein method).
Calculations of oligolysine yield and average degree of polymerization (DPavg) were monitored
by 1H-NMR analysis. Trypsin was covalently immobilized onto four different resins
(Amberzyme, Eupergit C, Eupergit CM and Grace 192). Maximum yield of bound protein was 92
mg/g, 82 mg/g and 60 mg/g support for each resin respectively. The effectiveness of these
systems (Trypsin-resins) was evaluated by hydrolysis of casein and synthesis of water-soluble
oligolysine. Most systems were capable of catalyzing oligopeptide synthesis in aqueous medium,
albeit at different efficiencies, namely: 40, 37 and 35% for Amberzyme, Eupergit C and Eupergit
CM, respectively, in comparison with free enzyme. These systems produced oligomers in only 1
hour with DPavg higher than free enzyme. Among these systems, the Eupergit C-Trypsin system
showed greater efficiency than others in terms of hydrolytic activity and thermal stability.
However, this did not occur for oligolysine synthesis. Trypsin-Amberzyme proved to be more
successful in oligopeptide synthesis, and exhibited excellent reusability, since it retained 90% of
its initial hydrolytic and synthetic activity after 7 reuses. Trypsin hydrophobic interactions with
Amberzyme support are responsible for protecting against strong enzyme conformational
changes in the medium. In addition, the high concentration of oxirane groups on the surface
promoted multi-covalent linking and, consequently, prevented the immobilized enzyme from leaching. The aforementioned results suggest that immobilized Trypsin on the supports evaluated
can be efficiently used for oligopeptides synthesis in aqueous media / S?ntese enzim?tica de pept?deos usando proteases tem atra?do uma enorme aten??o nos
?ltimos anos. Um desafio chave na s?ntese de pept?deos ? encontrar suportes para imobiliza??o de
proteases capazes de apresentar um alto desempenho em meio aquoso, produzindo oligopept?deos
sol?veis em ?gua, j? que at? o presente momento, pouco tem sido descrito usando essa estrat?gia.
Dessa forma, o objetivo dessa tese foi imobilizar proteases usando diferentes m?todos
(imobiliza??o por liga??o covalente, aprisionamento em g?is polim?ricos de PVA e imobiliza??o
em nanopart?culas magn?ticas de Glicidil) para a produ??o de oligopept?deos derivados da lisina.
Tr?s proteases foram utilizadas: tripsina, α-quimotripsina e bromela?na. De acordo com as
estrat?gias de imobiliza??o associadas ao tipo de protease empregada, foi provado que os
sistemas tripsina-resinas mostraram os melhores desempenhos em termos de atividade hidrol?tica
e s?ntese de oligopept?deos. A atividade hidrol?tica das enzimas livres e imobilizadas foi
determinada por espectrofotometria com base na mudan?a de absorb?ncia em 660 nm ?
temperatura de 25 ?C (Casein method). O rendimento de oligolisina e o c?lculo do grau de
polimeriza??o m?dio foram monitorados por RMN H. A protease tripsina foi covalentemente
imobilizada em quatro diferentes resinas (Amberzyme, Eupergit C, Eupergit CM and Grace 192).
O m?ximo rendimento de prote?na imobilizada foi 92, 82, 60, e 71 mg/g de suporte para cada
resina, respectivamente. A efici?ncia desses sistemas (Tripsina-resinas) foi avaliada pela hidr?lise
do substrato case?na e a s?ntese de oligolisina em meio aquoso. A maioria dos sistemas foram
capazes de catalisar a s?ntese de oligopept?deos, entretanto com diferentes efici?ncias, tais como:
40, 37 e 35% para os suportes Amberzyme, Eupergit C e Eupergit CM, respectivamente, em
compara??o com a enzima livre. Esses sistemas produziram olig?meros em somente 1 hora com
grau de polimeriza??o m?dio mais alto que a enzima livre. Dentre esses sistemas, Eupergit CTripsina
mostrou ser mais eficiente que os outros sistemas em termos de atividade hidrol?tica e
estabilidade t?rmica, ao passo que n?o exibiu a mesma efici?ncia como era esperado para a
s?ntese de oligolisina. Tripsina-amberzyme provou ser mais eficiente para a s?ntese de
oligopept?deos, al?m de exibir um excelente reuso, mantendo 90% de sua atividade hidrol?tica e
sint?tica ap?s sete reusos. As intera??es hidrof?bicas da tripsina com o suporte Amberzyme s?o
respons?veis por proteger a enzima contra as fortes mudan?as conformacionais no meio
reacional. Al?m disso, a alta concentra??o de grupos oxiranos na superf?cie da resina promoveu
liga??es covalentes multipontuais e, consequentemente, preveniu a enzima imobilizada do processo de desor??o (Leaching process). Os resultados acima mencionados sugerem que a tripsina imobilizada nesses suportes pode ser eficientemente usada para a s?ntese de
oligopept?deos em meio aquoso
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufrn.br:123456789/17750 |
| Date | 16 September 2011 |
| Creators | Fagundes, Fabio Pereira |
| Contributors | CPF:18822851404, http://lattes.cnpq.br/7623147392470166, Uchoa, Adriana Ferreira, CPF:58024867320, http://lattes.cnpq.br/6644671747055211, Souto, Carlos Roberto Oliveira, CPF:28467809949, http://lattes.cnpq.br/5481020781565211, Pedrosa, Matheus de Freitas Fernandes, CPF:96728647449, http://lattes.cnpq.br/2929963416385218, Oliveira, Maria da Concei??o Ferreira de, CPF:97771430791, http://lattes.cnpq.br/9314709318730076, Porto, Andre Luiz Meleiro, CPF:18099859863, http://lattes.cnpq.br/2689760395534218, Costa, Marta |
| Publisher | Universidade Federal do Rio Grande do Norte, Programa de P?s-Gradua??o em Qu?mica, UFRN, BR, F?sico-Qu?mica; Qu?mica |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Repositório Institucional da UFRN, instname:Universidade Federal do Rio Grande do Norte, instacron:UFRN |
| Rights | info:eu-repo/semantics/openAccess |
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