Biosynthesis, Isolation and Magnetization of gellan spheres for biorecognition of therapeutic His-tag proteins
Autor(a) principal: | |
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Data de Publicação: | 2017 |
Tipo de documento: | Dissertação |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10400.6/6687 |
Resumo: | The bacterium Sphingomonas paucimobilis ATCC 31461 has the ability to produce a low cost anionic polysaccharide gellan gum, composed of a tetrasaccharide structure of glucose, glucuronic acid and rhamnose units. This biopolymer has many applications in the food, pharmaceutical and cosmetic industries. Typically, for gellan aggregation, the presence of divalent ions is required to decrease the electrostatic repulsion between helices, allowing the cross-link. Curiously, the affinity interaction between transition metal ions and histidine has been used as a site-specific, noncovalent method for the purification and immobilization of recombinant proteins bearing three to ten consecutive histidine amino acids at their amino- or carboxyl-terminus. Thus, the present project intends to formulate and magnetize biosynthetic gellan spheres to extract COMT protein from a lysate sample. Gellan gum production was improved by using two different media, N medium and S medium. The fermentation procedure was performed with a stirring of 250 rpm at 30 ºC for 48 hours, and a higher production of gellan gum was achieved through the S medium. Then, in order to recover the gellan gum produced by fermentation, different processes, like filtration, dialysis, washes with acetone and ether and dissolution in distilled water, were tested. The best method to recover gellan gum with a high degree of purity, comparatively with the commercial gellan gum was the filtration with acetone and ether and dissolution with distilled water. All the samples obtained from the different procedures of recovery were analyzed by nuclear magnetic resonance and Fourier transform infrared spectroscopy. The gellan spheres were prepared by water-in-oil emulsion technique. For that, the gellan gum was dissolved in distilled water with a stirring of 300 rpm at 90 ºC during 30 minutes and this solution was transferred to a syringe and trickled in individual drops into a 100 % vegetable cooking oil solution with a 750 rpm stirring, at 100 ºC. The mixture was transferred to different solutions of 200 mM BaCl2, CaCl2, CoCl2, CuCl2 and NiCl2 with a stirring of 750 rpm for 30 minutes, at room temperature. The spheres with nickel as a cross-linker were magnetized by the chemical co-precipitation method. All the spheres with different cross-linkers and magnetized spheres were characterized morphologically and chemically by scanning electron microscopy and energy-dispersive X-Ray spectroscopy. Finally, the gellan spheres were used to capture model proteins, BSA and lysozyme, and a more complex protein, SCOMT, through the batch method. Thus, different conditions to bind and elute the protein were study, through the variation of the amount of spheres, the presence of urea in the lysate, the pH and ionic strength of the binding and elution solutions. So, the best conditions for the capture of the total protein from the complex lysate are the equilibrium of 10 mL of spheres (with or without magnetization) at acidic pH, binding step with urea in the SCOMT lysate and elution step with the increase of pH and then the ionic strength. However, to selectively isolate the SCOMT, it could be more adequate to explore the equilibrium and binding conditions with a pH around 7.5 and the elution condition by decreasing the pH to the protein isoelectric point with magnetic spheres. |
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Biosynthesis, Isolation and Magnetization of gellan spheres for biorecognition of therapeutic His-tag proteinsBiossínteseCatechol-O-MethyltransferaseGoma de GelanaMagnetizaçãoSphingomonas PaucimobilisDomínio/Área Científica::Ciências Médicas::Ciências BiomédicasThe bacterium Sphingomonas paucimobilis ATCC 31461 has the ability to produce a low cost anionic polysaccharide gellan gum, composed of a tetrasaccharide structure of glucose, glucuronic acid and rhamnose units. This biopolymer has many applications in the food, pharmaceutical and cosmetic industries. Typically, for gellan aggregation, the presence of divalent ions is required to decrease the electrostatic repulsion between helices, allowing the cross-link. Curiously, the affinity interaction between transition metal ions and histidine has been used as a site-specific, noncovalent method for the purification and immobilization of recombinant proteins bearing three to ten consecutive histidine amino acids at their amino- or carboxyl-terminus. Thus, the present project intends to formulate and magnetize biosynthetic gellan spheres to extract COMT protein from a lysate sample. Gellan gum production was improved by using two different media, N medium and S medium. The fermentation procedure was performed with a stirring of 250 rpm at 30 ºC for 48 hours, and a higher production of gellan gum was achieved through the S medium. Then, in order to recover the gellan gum produced by fermentation, different processes, like filtration, dialysis, washes with acetone and ether and dissolution in distilled water, were tested. The best method to recover gellan gum with a high degree of purity, comparatively with the commercial gellan gum was the filtration with acetone and ether and dissolution with distilled water. All the samples obtained from the different procedures of recovery were analyzed by nuclear magnetic resonance and Fourier transform infrared spectroscopy. The gellan spheres were prepared by water-in-oil emulsion technique. For that, the gellan gum was dissolved in distilled water with a stirring of 300 rpm at 90 ºC during 30 minutes and this solution was transferred to a syringe and trickled in individual drops into a 100 % vegetable cooking oil solution with a 750 rpm stirring, at 100 ºC. The mixture was transferred to different solutions of 200 mM BaCl2, CaCl2, CoCl2, CuCl2 and NiCl2 with a stirring of 750 rpm for 30 minutes, at room temperature. The spheres with nickel as a cross-linker were magnetized by the chemical co-precipitation method. All the spheres with different cross-linkers and magnetized spheres were characterized morphologically and chemically by scanning electron microscopy and energy-dispersive X-Ray spectroscopy. Finally, the gellan spheres were used to capture model proteins, BSA and lysozyme, and a more complex protein, SCOMT, through the batch method. Thus, different conditions to bind and elute the protein were study, through the variation of the amount of spheres, the presence of urea in the lysate, the pH and ionic strength of the binding and elution solutions. So, the best conditions for the capture of the total protein from the complex lysate are the equilibrium of 10 mL of spheres (with or without magnetization) at acidic pH, binding step with urea in the SCOMT lysate and elution step with the increase of pH and then the ionic strength. However, to selectively isolate the SCOMT, it could be more adequate to explore the equilibrium and binding conditions with a pH around 7.5 and the elution condition by decreasing the pH to the protein isoelectric point with magnetic spheres.A bactéria Sphingomonas paucimobilis ATCC 31461 tem a capacidade de produzir um polissacarídeo aniónico de baixo custo, a goma de gelana, constituído por um tetrassacarídeo composto por unidades de glucose, ácido glucurónico e ramnose. Este biopolímero possui diversas aplicações na alimentação e na indústria farmacêutica e cosmética. Tipicamente, a presença de iões divalentes é necessária para diminuir a repulsão electroestática entre as hélices de gelana, permitindo a ligação entre cadeias e comsequente agregação. Curiosamente, a interação por afinidade que se promove entre os iões metálicos de transição e os aminoácidos de histidina tem explorada como um método não covalente de purificação e imobilização de proteínas recombinantes, contendo três a dez unidades consecutivas de histidina no seu terminal amino ou carboxil. Assim, o presente trabalho pretende formular e magnetizar esferas de gelana biossintética para extrair a proteína COMT de uma amostra de lisado. A produção de goma de gelana pela bactéria Sphingomonas paucimobilis ATCC 31561 foi otimizada usando dois meios de cultura diferentes, o meio N e o meio S. A fermentação foi realizada com uma agitação de 250 rpm, a 30 ºC durante 48 horas, e uma maior produção de gelana foi conseguida através do meio S. De seguida, de forma a recuperar a goma de gelana produzida por fermentação, foram testados diferentes processos, como a filtração, a diálise, lavagens com acetona e éter e dissolução em água destilada. O melhor método para recuperar a goma de gelana com um maior grau de pureza, comparativamente à goma de gelana comercial, foi a filtração com acetona e éter e dissolução em água destilada. Todas as amostras obtidas através dos diferentes procedimentos de recuperação foram analisadas por ressonância magnética nuclear e por espetroscopia de infravermelho da transformada de Fourier. As esferas de gelana foram preparadas pela técnica de emulsão de água-em-óleo. Para isso, a goma de gelana foi dissolvida em água destilada com uma agitação de 300 rpm, a 90 ºC durante 30 minutos, e esta solução foi transferida para uma seringa e libertada gota a gota para uma solução de óleo de cozinha 100% vegetal com uma agitação de 750 rpm, a 100 ºC. A mistura foi transferida para diferentes soluções de 200 mM de BaCl2, CaCl2, CoCl2, CuCl2 e NiCl2 com uma agitação de 750rpm, durante 30 minutos à temperatura ambiente. As esferas com níquel como ligando foram magnetizadas pelo método químico de co-precipitação. Todas as esferas com diferentes ligandos assim como as esferas magnetizadas foram caracterizadas morfologicamente e quimicamente por microscopia eletrónica de varrimento e por espetroscopia de energia dispersiva de raios-X. Finalmente, as esferas de gelana foram usadas para capturar proteínas modelo, BSA e lisozima, e uma proteína mais complexa, SCOMT, através do método de batch. Assim, foram estudadas diferentes condições para ligar e eluir a proteína, através da variação da quantidade de esferas, da presença de ureia no lisado, do pH e da força iónica das soluções de ligação e eluição. Deste modo, as melhores condições para a captura de proteína total de um lisado complexo são o equilíbrio de 10 mL de esferas (com ou sem magnetização) a pH ácido, o passo de ligação com ureia no lisado de SCOMT e o passo de eluição com um aumento de pH e, de seguida, da força iónica. Contudo, de forma a isolar seletivamente a SCOMT, poderia ser mais adequado explorar as condições de equilíbrio e de ligação com um pH de cerca de 7.5 e a condição de eluição pela diminuição do pH para o ponto isoelétrico com esferas magnetizadas.Passarinha, Luís António PaulinoSousa, Ângela Maria Almeida deuBibliorumCoelho, Joana Filipa da Silva2019-06-26T00:30:17Z2017-6-272017-07-132017-07-13T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10400.6/6687TID:202107566enginfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-12-15T09:45:24Zoai:ubibliorum.ubi.pt:10400.6/6687Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T00:47:21.330246Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Biosynthesis, Isolation and Magnetization of gellan spheres for biorecognition of therapeutic His-tag proteins |
title |
Biosynthesis, Isolation and Magnetization of gellan spheres for biorecognition of therapeutic His-tag proteins |
spellingShingle |
Biosynthesis, Isolation and Magnetization of gellan spheres for biorecognition of therapeutic His-tag proteins Coelho, Joana Filipa da Silva Biossíntese Catechol-O-Methyltransferase Goma de Gelana Magnetização Sphingomonas Paucimobilis Domínio/Área Científica::Ciências Médicas::Ciências Biomédicas |
title_short |
Biosynthesis, Isolation and Magnetization of gellan spheres for biorecognition of therapeutic His-tag proteins |
title_full |
Biosynthesis, Isolation and Magnetization of gellan spheres for biorecognition of therapeutic His-tag proteins |
title_fullStr |
Biosynthesis, Isolation and Magnetization of gellan spheres for biorecognition of therapeutic His-tag proteins |
title_full_unstemmed |
Biosynthesis, Isolation and Magnetization of gellan spheres for biorecognition of therapeutic His-tag proteins |
title_sort |
Biosynthesis, Isolation and Magnetization of gellan spheres for biorecognition of therapeutic His-tag proteins |
author |
Coelho, Joana Filipa da Silva |
author_facet |
Coelho, Joana Filipa da Silva |
author_role |
author |
dc.contributor.none.fl_str_mv |
Passarinha, Luís António Paulino Sousa, Ângela Maria Almeida de uBibliorum |
dc.contributor.author.fl_str_mv |
Coelho, Joana Filipa da Silva |
dc.subject.por.fl_str_mv |
Biossíntese Catechol-O-Methyltransferase Goma de Gelana Magnetização Sphingomonas Paucimobilis Domínio/Área Científica::Ciências Médicas::Ciências Biomédicas |
topic |
Biossíntese Catechol-O-Methyltransferase Goma de Gelana Magnetização Sphingomonas Paucimobilis Domínio/Área Científica::Ciências Médicas::Ciências Biomédicas |
description |
The bacterium Sphingomonas paucimobilis ATCC 31461 has the ability to produce a low cost anionic polysaccharide gellan gum, composed of a tetrasaccharide structure of glucose, glucuronic acid and rhamnose units. This biopolymer has many applications in the food, pharmaceutical and cosmetic industries. Typically, for gellan aggregation, the presence of divalent ions is required to decrease the electrostatic repulsion between helices, allowing the cross-link. Curiously, the affinity interaction between transition metal ions and histidine has been used as a site-specific, noncovalent method for the purification and immobilization of recombinant proteins bearing three to ten consecutive histidine amino acids at their amino- or carboxyl-terminus. Thus, the present project intends to formulate and magnetize biosynthetic gellan spheres to extract COMT protein from a lysate sample. Gellan gum production was improved by using two different media, N medium and S medium. The fermentation procedure was performed with a stirring of 250 rpm at 30 ºC for 48 hours, and a higher production of gellan gum was achieved through the S medium. Then, in order to recover the gellan gum produced by fermentation, different processes, like filtration, dialysis, washes with acetone and ether and dissolution in distilled water, were tested. The best method to recover gellan gum with a high degree of purity, comparatively with the commercial gellan gum was the filtration with acetone and ether and dissolution with distilled water. All the samples obtained from the different procedures of recovery were analyzed by nuclear magnetic resonance and Fourier transform infrared spectroscopy. The gellan spheres were prepared by water-in-oil emulsion technique. For that, the gellan gum was dissolved in distilled water with a stirring of 300 rpm at 90 ºC during 30 minutes and this solution was transferred to a syringe and trickled in individual drops into a 100 % vegetable cooking oil solution with a 750 rpm stirring, at 100 ºC. The mixture was transferred to different solutions of 200 mM BaCl2, CaCl2, CoCl2, CuCl2 and NiCl2 with a stirring of 750 rpm for 30 minutes, at room temperature. The spheres with nickel as a cross-linker were magnetized by the chemical co-precipitation method. All the spheres with different cross-linkers and magnetized spheres were characterized morphologically and chemically by scanning electron microscopy and energy-dispersive X-Ray spectroscopy. Finally, the gellan spheres were used to capture model proteins, BSA and lysozyme, and a more complex protein, SCOMT, through the batch method. Thus, different conditions to bind and elute the protein were study, through the variation of the amount of spheres, the presence of urea in the lysate, the pH and ionic strength of the binding and elution solutions. So, the best conditions for the capture of the total protein from the complex lysate are the equilibrium of 10 mL of spheres (with or without magnetization) at acidic pH, binding step with urea in the SCOMT lysate and elution step with the increase of pH and then the ionic strength. However, to selectively isolate the SCOMT, it could be more adequate to explore the equilibrium and binding conditions with a pH around 7.5 and the elution condition by decreasing the pH to the protein isoelectric point with magnetic spheres. |
publishDate |
2017 |
dc.date.none.fl_str_mv |
2017-6-27 2017-07-13 2017-07-13T00:00:00Z 2019-06-26T00:30:17Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
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masterThesis |
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publishedVersion |
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http://hdl.handle.net/10400.6/6687 TID:202107566 |
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TID:202107566 |
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eng |
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