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Previous issue date: 2008-02-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / ?-D-glucosidase (EC 3.2.1.21) is one of the most interesting glycosidases, especially for hydrolysis cellobiose releasing glucose, is last step degradation of cellulose. This function makes the ?-D-glucosidase is of great interest as a versatile industrial biocatalyst, being critical to various bio-treatment / biorefinery processes, such as bioethanol production. Hen in the report, a ?-D-glucosidase was extracts from protein extracted of the invertebrate marine Artemia franciscana was purified and characterized with a combination of precipitation with ammonium sulfate (0 - 30%, 30 to 50%, 50 to 80%), the fraction saturated in the range of 30 to 50% (called F-II) was applied in a molecular exclusion chromatography, in Sephacryl S-200, the fractions corresponding to the first peak of activity of ?-D-glucosidase were gathered and applied in a chromatography of ion exchange in Mono Q; the third peak this protein obtained chromatography, which coincides with the peak of activity of ?-D-glucosidase was held and applied in a gel filtration chromatography Superose 12 where the first peak protein, which has activity of ?-D-glucosidase was rechromatography on Superose 12. This enzyme is probably multimerica, consisting of three subunit molecular mass of 52.7 kDa (determined by SDS-PAGE) with native molecular mass of 157 kDa (determined by gel filtration chromatography on Superose 12 under the system FPLC). The enzyme was purified 44.09 times with a recovery of 1.01%. Using up p-nitrophenyl-?-D-glucopiranoside as substrate obtained a Km apparent of 0.229 mM and a Vmax of 1.109 mM.60min-1.mL-1mM. The optimum pH and optimum temperature of catalysis of the synthetic substrate were 5.0 and 45 ?C, respectively. The activity of the ?-D-glucosidase was strongly, inhibited by silver nitrate and N- etylmaleimide, this inhibition indicates the involvement of radical sulfidrila the hydrolysis of synthetic substrate. The ?-D-glucosidase of Artemia franciscana presented degradativa action on celobiose, lactose and on the synthetic substrate ?-nitrophenyl-?-D-glucopiranoside indicating potential use of this enzyme in the industry mainly for the production of bioethanol (production of alcohol from the participating cellulose), and production hydrolysate milk (devoid of milk lactose) / ?-D-glicosidase (EC 3.2.1.21) ? uma das mais interessantes glicosidases, especialmente por hidrolisar celobiose liberando glicose, ?ltimo passo de degrada??o de celulose. Esta fun??o faz com que a ?-D-glicosidase seja de grande interesse como um vers?til biocatalizador industrial, sendo critica para v?rios processos de bio-tratamento / biorrefinaria, como produ??o de bioetanol. Neste trabalho, uma ?-D-glicosidase extra?da de extratos prot?icos do invertebrado marinho Artemia franciscana foi purificada e caracterizada com uma combina??o de precipita??o com sulfato de am?nio (30; 50; 80%), a fra??o saturada na faixa de 50% (chamada F-II) foi aplicada em uma cromatografia de exclus?o molecular em Sephacryl S-200, as fra??es correspondentes ao primeiro pico de atividade da ?-D-glicosidase foram reunidas e aplicadas numa cromatografia de troca i?nica em Mono Q; o terceiro pico prot?ico obtido nessa cromatografia, o qual coincide com o pico de atividade da ?-D-glicosidase, foi reunido e aplicado numa cromatografia gel filtra??o Superose 12 onde o primeiro pico prot?ico, o qual possui atividade da ?-D-glicosidase, foi recromatografado em Superose 12. Esta enzima provavelmente ? multimerica, constitu?da por tr?s subunidades, de massa molecular de 52,7 kDa (determinado por SDS-PAGE) e com massa molecular nativa de 157 kDa (determinado por cromatografia gel filtra??o em Superose 12 sob o sistema FPLC). A enzima foi purificada 44,09 vezes com uma recupera??o de 1,01%. Utilizando-se ?-nitrofenil-?-D-glicopiranos?deo como substrato obtivemos uma Km aparente de 0,229 mM (12,7 ?M de produto/cm/hora) e Vm?x de 1,109 mM.60min-1.mL-1. O pH ?timo e a temperatura ?tima de cat?lise do substrato sint?tico foram 5,0 e 45?C, respectivamente. A atividade ?-D-glicosid?sica foi fortemente inibida por nitrato de prata e N-etilmaleimida, esta inibi??o indica o envolvimento de radicais sulfidrila na hidr?lise do substrato sint?tico. A ?-D-glicosidase de Artemia franciscana apresentou a??o degradativa sobre celobiose, lactose e sobre o substrato sint?tico ?-nitrofenil-?-D-glicopiranos?deo, indicando potencial uso desta enzima na ind?stria principalmente para produ??o de bioetanol (produ??o de ?lcool a parti de celulose) e produ??o de leite hidrolisado (leite destitu?do de lactose)
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufrn.br:123456789/12520 |
| Date | 27 February 2008 |
| Creators | Nascimento, Rob?rio Medeiros do |
| Contributors | CPF:27785858500, http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4723470U6, Franco, Oct?vio Luiz, CPF:75523400378, http://lattes.cnpq.br/8598274096498065, Sales, Maur?cio Pereira de, CPF:29706106391, http://lattes.cnpq.br/7890362793618911, Abreu, Luiz Roberto Diz de |
| Publisher | Universidade Federal do Rio Grande do Norte, Programa de P?s-Gradua??o em Bioqu?mica, UFRN, BR, Bioqu?mica; Biologia Molecular |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| 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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