RuBisCOs’ extraction and purification: from residual biomass to value added protein
Autor(a) principal: | |
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Data de Publicação: | 2020 |
Tipo de documento: | Dissertação |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10773/30462 |
Resumo: | With the growth of the world population, new sources of proteins are needed. Vegetable biomass is a promising source, as it allows the use of agricultural crop residues with the ability to be a continuous source of biomass without harming existing ecosystems, and the extraction of biomass proteins while still fresh. Currently, with the methods used, a final product is obtained that cannot be applied in the food industry. Furthermore, the methods developed until today are not selective and do not allow simultaneously reaching purities and extraction efficiencies close to 100%. RuBisCO (Ribulose-1,5-bisphosphate carboxylase/oxygenase) is the most abundant protein (enzyme) on the planet and can be applied in the most diverse areas, from pharmaceuticals to food and feed. Thus, in this master's thesis, a new method of extracting RuBisCO and subsequent separation and purification was developed. For this purpose, aqueous solutions of biocompatible ionic liquids (ILs) (glycine-betaine analogues and cholinium derivatives) were used in the solid-liquid extraction of RuBisCO from the spinach leaf. Through the response surface methodology, the extraction conditions (pH, solid-liquid ratio, and IL concentration) were optimized. Under optimum conditions, a maximum extraction of 2.00 mg/ mL of RuBisCO and a yield of 10.93 mg of RuBisCO/ g of biomass for cholinium acetate ([Ch][Acetate]) and 1.86 mg/ mL and a yield of 10.12 mg RuBisCO / g biomass for cholinium chloride ([Ch]Cl). The optimum points determined were: solid-liquid ratio of 0.184, IL concentration of 2,68 M and a pH of 9.09 for [Ch]Cl and 11.2 for [Ch][Acetate]. After extraction, the separation and purification of RuBisCO were performed through the application of aqueous biphasic systems (ABSs). The extracts from the solid-liquid extraction with [Ch]Cl and [Ch][Acetate] containing IL and RuBisCO were individually mixed with polypropylene glycol 400 g.mol-1 (PPG 400) or polyethylene glycol 1000 g.mol - 1 (PEG 1000) and dipotassium phosphate (K2HPO4) forming two types of ABSs composed of IL and polymer or salt. After this process, it was possible to obtain a RuBisCO extraction efficiency of 100% for the IL phase in the system composed of PPG400 + [Ch][Acetate] and K2HPO4 + IL ([Ch][Acetate] and [Ch]Cl), however, it was not possible to separate RuBisCO from the remaining proteins, having demonstrated that these ABSs (IL + polymer and IL + salt) are not selective for the purification of RuBisCO. |
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RuBisCOs’ extraction and purification: from residual biomass to value added proteinRibulose-1,5-biphosphate carboxylase/oxygenaseSolid-liquid extractionAqueous biphasic systemsIonic liquidsExtractionPurificationWith the growth of the world population, new sources of proteins are needed. Vegetable biomass is a promising source, as it allows the use of agricultural crop residues with the ability to be a continuous source of biomass without harming existing ecosystems, and the extraction of biomass proteins while still fresh. Currently, with the methods used, a final product is obtained that cannot be applied in the food industry. Furthermore, the methods developed until today are not selective and do not allow simultaneously reaching purities and extraction efficiencies close to 100%. RuBisCO (Ribulose-1,5-bisphosphate carboxylase/oxygenase) is the most abundant protein (enzyme) on the planet and can be applied in the most diverse areas, from pharmaceuticals to food and feed. Thus, in this master's thesis, a new method of extracting RuBisCO and subsequent separation and purification was developed. For this purpose, aqueous solutions of biocompatible ionic liquids (ILs) (glycine-betaine analogues and cholinium derivatives) were used in the solid-liquid extraction of RuBisCO from the spinach leaf. Through the response surface methodology, the extraction conditions (pH, solid-liquid ratio, and IL concentration) were optimized. Under optimum conditions, a maximum extraction of 2.00 mg/ mL of RuBisCO and a yield of 10.93 mg of RuBisCO/ g of biomass for cholinium acetate ([Ch][Acetate]) and 1.86 mg/ mL and a yield of 10.12 mg RuBisCO / g biomass for cholinium chloride ([Ch]Cl). The optimum points determined were: solid-liquid ratio of 0.184, IL concentration of 2,68 M and a pH of 9.09 for [Ch]Cl and 11.2 for [Ch][Acetate]. After extraction, the separation and purification of RuBisCO were performed through the application of aqueous biphasic systems (ABSs). The extracts from the solid-liquid extraction with [Ch]Cl and [Ch][Acetate] containing IL and RuBisCO were individually mixed with polypropylene glycol 400 g.mol-1 (PPG 400) or polyethylene glycol 1000 g.mol - 1 (PEG 1000) and dipotassium phosphate (K2HPO4) forming two types of ABSs composed of IL and polymer or salt. After this process, it was possible to obtain a RuBisCO extraction efficiency of 100% for the IL phase in the system composed of PPG400 + [Ch][Acetate] and K2HPO4 + IL ([Ch][Acetate] and [Ch]Cl), however, it was not possible to separate RuBisCO from the remaining proteins, having demonstrated that these ABSs (IL + polymer and IL + salt) are not selective for the purification of RuBisCO.Com o crescimento da população mundial são necessárias novas fontes de proteínas. A biomassa vegetal é uma fonte promissora, pois permite a utilização de resíduos de culturas agrícolas com a capacidade de ser uma fonte contínua de biomassa sem prejudicar os ecossistemas já existentes, e a extração de proteínas de biomassa ainda fresca. Atualmente, com os métodos utilizados, é obtido um produto final que não pode ser aplicado na indústria alimentar além de que, os métodos até hoje desenvolvidos não são seletivos nem permitem atingir simultaneamente purezas e eficiências de extração próximas de 100 %. A RuBisCO (Ribulose-1,5-bifosfato carboxilase/oxigenase) é a proteína (enzima) mais abundante no planeta e pode ser aplicada nas mais diversas áreas, desde a farmacêutica à alimentar e de rações. Deste modo, nesta tese de mestrado foi desenvolvido um novo método de extração da RuBisCO e posterior separação e purificação. Para tal, foram usadas soluções aquosas de líquidos iónicos (LIs) biocompatíveis (análogos da glicina-betaína e derivados de colínio) na extração sólido-líquido da RuBisCO da folha do espinafre. Através da metodologia de superfície de resposta, foram otimizadas as condições da extração (pH, razão sólido-líquido e a concentração de LI). Nas condições ótimas, foi obtida uma extração máxima de 2.00 mg /mL de RuBisCO e um rendimento de 10.93 mg de RuBisCO /g de biomassa para o acetato de colínio ([Ch][Acetato]) e 1.86 mg /mL e um rendimento de 10.12 mg de RuBisCO /g de biomassa para o cloreto de colínio ([Ch]Cl). Os pontos ótimos determinados foram: razão sólido-líquido de 0.184, concentração de LI de 2.68 M e um pH de 9.09 para o [Ch]Cl e 11.2 para o [Ch][Acetato]. Após a extração, a separação e a purificação da RuBisCO foi realizada através da aplicação de sistema aquoso bifásicos (SABs). Os extratos provenientes da extração sólido-líquido com [Ch]Cl e [Ch][Acetato] que contêm LI e RuBisCO foram misturados individualmente com polipropileno glicol 400 g.mol-1 (PPG 400) ou polietileno glicol 1000 g.mol-1 (PEG 1000) e fosfato dipotássico (K2HPO4) formando-se dois tipos de SABs compostos por LI e polímero ou sal. Após este processo foi possível obter uma eficiência de extração da RuBisCO de 100% para a fase de LI no sistema composto por PPG400+ [Ch][Acetato] e por K2HPO4 + LI ([Ch][Acetato] e [Ch]Cl), no entanto, não foi possível separar a RuBisCO das restantes proteínas, tendo demonstrado estes SABs (LI+polímero e LI+sal) não serem seletivos para a purificação da RuBisCO.2022-12-30T00:00:00Z2020-12-17T00:00:00Z2020-12-17info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10773/30462engValente, Ana Isabel Bastosinfo:eu-repo/semantics/embargoedAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-02-22T11:58:54Zoai:ria.ua.pt:10773/30462Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:02:34.452695Repositório 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 |
RuBisCOs’ extraction and purification: from residual biomass to value added protein |
title |
RuBisCOs’ extraction and purification: from residual biomass to value added protein |
spellingShingle |
RuBisCOs’ extraction and purification: from residual biomass to value added protein Valente, Ana Isabel Bastos Ribulose-1,5-biphosphate carboxylase/oxygenase Solid-liquid extraction Aqueous biphasic systems Ionic liquids Extraction Purification |
title_short |
RuBisCOs’ extraction and purification: from residual biomass to value added protein |
title_full |
RuBisCOs’ extraction and purification: from residual biomass to value added protein |
title_fullStr |
RuBisCOs’ extraction and purification: from residual biomass to value added protein |
title_full_unstemmed |
RuBisCOs’ extraction and purification: from residual biomass to value added protein |
title_sort |
RuBisCOs’ extraction and purification: from residual biomass to value added protein |
author |
Valente, Ana Isabel Bastos |
author_facet |
Valente, Ana Isabel Bastos |
author_role |
author |
dc.contributor.author.fl_str_mv |
Valente, Ana Isabel Bastos |
dc.subject.por.fl_str_mv |
Ribulose-1,5-biphosphate carboxylase/oxygenase Solid-liquid extraction Aqueous biphasic systems Ionic liquids Extraction Purification |
topic |
Ribulose-1,5-biphosphate carboxylase/oxygenase Solid-liquid extraction Aqueous biphasic systems Ionic liquids Extraction Purification |
description |
With the growth of the world population, new sources of proteins are needed. Vegetable biomass is a promising source, as it allows the use of agricultural crop residues with the ability to be a continuous source of biomass without harming existing ecosystems, and the extraction of biomass proteins while still fresh. Currently, with the methods used, a final product is obtained that cannot be applied in the food industry. Furthermore, the methods developed until today are not selective and do not allow simultaneously reaching purities and extraction efficiencies close to 100%. RuBisCO (Ribulose-1,5-bisphosphate carboxylase/oxygenase) is the most abundant protein (enzyme) on the planet and can be applied in the most diverse areas, from pharmaceuticals to food and feed. Thus, in this master's thesis, a new method of extracting RuBisCO and subsequent separation and purification was developed. For this purpose, aqueous solutions of biocompatible ionic liquids (ILs) (glycine-betaine analogues and cholinium derivatives) were used in the solid-liquid extraction of RuBisCO from the spinach leaf. Through the response surface methodology, the extraction conditions (pH, solid-liquid ratio, and IL concentration) were optimized. Under optimum conditions, a maximum extraction of 2.00 mg/ mL of RuBisCO and a yield of 10.93 mg of RuBisCO/ g of biomass for cholinium acetate ([Ch][Acetate]) and 1.86 mg/ mL and a yield of 10.12 mg RuBisCO / g biomass for cholinium chloride ([Ch]Cl). The optimum points determined were: solid-liquid ratio of 0.184, IL concentration of 2,68 M and a pH of 9.09 for [Ch]Cl and 11.2 for [Ch][Acetate]. After extraction, the separation and purification of RuBisCO were performed through the application of aqueous biphasic systems (ABSs). The extracts from the solid-liquid extraction with [Ch]Cl and [Ch][Acetate] containing IL and RuBisCO were individually mixed with polypropylene glycol 400 g.mol-1 (PPG 400) or polyethylene glycol 1000 g.mol - 1 (PEG 1000) and dipotassium phosphate (K2HPO4) forming two types of ABSs composed of IL and polymer or salt. After this process, it was possible to obtain a RuBisCO extraction efficiency of 100% for the IL phase in the system composed of PPG400 + [Ch][Acetate] and K2HPO4 + IL ([Ch][Acetate] and [Ch]Cl), however, it was not possible to separate RuBisCO from the remaining proteins, having demonstrated that these ABSs (IL + polymer and IL + salt) are not selective for the purification of RuBisCO. |
publishDate |
2020 |
dc.date.none.fl_str_mv |
2020-12-17T00:00:00Z 2020-12-17 2022-12-30T00:00:00Z |
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/10773/30462 |
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http://hdl.handle.net/10773/30462 |
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eng |
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eng |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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RCAAP |
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RCAAP |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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