Optimization of L-Asparaginase production by Bacillus subtilis
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/10773/30464 |
Resumo: | Currently the enzyme L-Asparaginase (L-ASNase) is used in both food and pharmaceutical industries due to its ability to catalyze the hydrolysis reaction of the amino acid L-Asparagine in ammonia and aspartic acid. In the food industry, this enzyme is used in a way to prevent the formation of cancerous compounds in foods, such as acrylamide. In the pharmaceutical industry, L-ASNase is used in the treatment of acute lymphoblastic leukemia (ALL) once it prevents cancer cells from growing as a result of the decrease in exogenous L-Asparagine. As cancer cells have low levels of the enzyme asparagine synthase, they are dependent on the absorption of this amino acid from the physiological environment to survive. Thus, the optimized production of L-ASNase, particularly with high purity, is highly desired, particularly for medical applications. In the production of this enzyme, several microorganisms are used, such being the most commonly used Escherichia coli and Erwinia sp. However, associated with these bacteria are the L-ASNase production with low yields and side effects, which leads to increased demand for other sources of production. This way, Bacillus subtilis appears as an alternative microorganism for the production of this therapeutic enzyme. This organism has been studied for several decades, being the prokaryote most understood in terms of molecular and cellular biology, playing an important role as a model for gram-positive bacteria research. On this work, several factors of the fermentation process were optimized such as inductor’s concentration (0.5, 1, 2, 3 and 5% (v/v) of xylose), temperature after induction (25, 30, 35 and 40ºC) and incubation times (8, 12, 18, 24 and 36h). In the optimum fermentation conditions (3% of xylose, 30ºC during 24h) it was obtained a L-ASNase, after cellular lysis, with an enzymatic activity of 0,756 U/mL, a specific activity of 0,107 U/mg and a purity of 21,97 %. |
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Optimization of L-Asparaginase production by Bacillus subtilisProductionBiopharmaceuticalsL-AsparaginaseSubmerged fermentationBacillus subtilisCurrently the enzyme L-Asparaginase (L-ASNase) is used in both food and pharmaceutical industries due to its ability to catalyze the hydrolysis reaction of the amino acid L-Asparagine in ammonia and aspartic acid. In the food industry, this enzyme is used in a way to prevent the formation of cancerous compounds in foods, such as acrylamide. In the pharmaceutical industry, L-ASNase is used in the treatment of acute lymphoblastic leukemia (ALL) once it prevents cancer cells from growing as a result of the decrease in exogenous L-Asparagine. As cancer cells have low levels of the enzyme asparagine synthase, they are dependent on the absorption of this amino acid from the physiological environment to survive. Thus, the optimized production of L-ASNase, particularly with high purity, is highly desired, particularly for medical applications. In the production of this enzyme, several microorganisms are used, such being the most commonly used Escherichia coli and Erwinia sp. However, associated with these bacteria are the L-ASNase production with low yields and side effects, which leads to increased demand for other sources of production. This way, Bacillus subtilis appears as an alternative microorganism for the production of this therapeutic enzyme. This organism has been studied for several decades, being the prokaryote most understood in terms of molecular and cellular biology, playing an important role as a model for gram-positive bacteria research. On this work, several factors of the fermentation process were optimized such as inductor’s concentration (0.5, 1, 2, 3 and 5% (v/v) of xylose), temperature after induction (25, 30, 35 and 40ºC) and incubation times (8, 12, 18, 24 and 36h). In the optimum fermentation conditions (3% of xylose, 30ºC during 24h) it was obtained a L-ASNase, after cellular lysis, with an enzymatic activity of 0,756 U/mL, a specific activity of 0,107 U/mg and a purity of 21,97 %.Atualmente a enzima L-Asparaginase (L-ASNase) é utilizada nas indústrias alimentar e farmacêutica devido à sua capacidade de catalisar a reação de hidrólise do aminoácido L-Asparagina em amoníaco e ácido aspártico. Na indústria alimentar esta enzima é utilizada de forma a evitar a formação de compostos cancerígenos nos alimentos, como a acrilamida. Por outro lado, na indústria farmacêutica, a L-ASNase é usada no tratamento da leucemia linfoblástica aguda (LLA) pelo facto de impedir as células cancerígenas de crescerem, como resultado do decréscimo de L-Asparagina exógena. Como estas células (células cancerígenas) têm baixos níveis da enzima asparagina sintetase, encontram-se dependentes da absorção da L-Asparagina do meio fisiológico para sobreviver. Assim, a produção otimizada da L-ASNase, nomeadamente com alta pureza, é muito desejada, principalmente para aplicações médicas. Na produção desta enzima são utilizados diversos microrganismos, sendo os mais comuns as bactérias Escherichia coli e Erwinia sp. Contudo, associadas a estas bactérias está a produção de L-ASNase com baixos rendimentos e efeitos colaterais, o que leva ao aumento da procura de outras fontes de produção. Deste modo, a bactéria Bacillus subtilis surge como um microrganismo alternativo para a produção desta enzima terapêutica. Esta bactéria tem sido estudada por várias décadas, sendo o procariota mais compreendido em termos de biologia molecular e celular, desempenhando um papel importante como modelo de pesquisa de bactérias gram-positiva. Neste trabalho otimizaram-se vários parâmetros do processo fermentativo de produção da L-ASNase, tais como a concentração de indutor (0,5; 1; 2; 3 e 5% (v/v) de xilose), temperatura de fermentação após indução (25, 30, 35 e 40ºC) e o período de fermentação após indução (8, 12, 18, 24 e 36h). Nas concentrações ótimas de fermentação (3% de indutor, 30ºC durante 24h) obteve-se, após lise celular, uma L-ASNase com uma atividade enzimática de 0,756 U/mL, uma atividade específica de 0,107 U/mg e uma pureza de 21,97 %.2022-12-30T00:00:00Z2020-12-18T00:00:00Z2020-12-18info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10773/30464engCastro, Daniel Figueiredo deinfo: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:10773/30464Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:02:34.580677Repositório 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 |
Optimization of L-Asparaginase production by Bacillus subtilis |
title |
Optimization of L-Asparaginase production by Bacillus subtilis |
spellingShingle |
Optimization of L-Asparaginase production by Bacillus subtilis Castro, Daniel Figueiredo de Production Biopharmaceuticals L-Asparaginase Submerged fermentation Bacillus subtilis |
title_short |
Optimization of L-Asparaginase production by Bacillus subtilis |
title_full |
Optimization of L-Asparaginase production by Bacillus subtilis |
title_fullStr |
Optimization of L-Asparaginase production by Bacillus subtilis |
title_full_unstemmed |
Optimization of L-Asparaginase production by Bacillus subtilis |
title_sort |
Optimization of L-Asparaginase production by Bacillus subtilis |
author |
Castro, Daniel Figueiredo de |
author_facet |
Castro, Daniel Figueiredo de |
author_role |
author |
dc.contributor.author.fl_str_mv |
Castro, Daniel Figueiredo de |
dc.subject.por.fl_str_mv |
Production Biopharmaceuticals L-Asparaginase Submerged fermentation Bacillus subtilis |
topic |
Production Biopharmaceuticals L-Asparaginase Submerged fermentation Bacillus subtilis |
description |
Currently the enzyme L-Asparaginase (L-ASNase) is used in both food and pharmaceutical industries due to its ability to catalyze the hydrolysis reaction of the amino acid L-Asparagine in ammonia and aspartic acid. In the food industry, this enzyme is used in a way to prevent the formation of cancerous compounds in foods, such as acrylamide. In the pharmaceutical industry, L-ASNase is used in the treatment of acute lymphoblastic leukemia (ALL) once it prevents cancer cells from growing as a result of the decrease in exogenous L-Asparagine. As cancer cells have low levels of the enzyme asparagine synthase, they are dependent on the absorption of this amino acid from the physiological environment to survive. Thus, the optimized production of L-ASNase, particularly with high purity, is highly desired, particularly for medical applications. In the production of this enzyme, several microorganisms are used, such being the most commonly used Escherichia coli and Erwinia sp. However, associated with these bacteria are the L-ASNase production with low yields and side effects, which leads to increased demand for other sources of production. This way, Bacillus subtilis appears as an alternative microorganism for the production of this therapeutic enzyme. This organism has been studied for several decades, being the prokaryote most understood in terms of molecular and cellular biology, playing an important role as a model for gram-positive bacteria research. On this work, several factors of the fermentation process were optimized such as inductor’s concentration (0.5, 1, 2, 3 and 5% (v/v) of xylose), temperature after induction (25, 30, 35 and 40ºC) and incubation times (8, 12, 18, 24 and 36h). In the optimum fermentation conditions (3% of xylose, 30ºC during 24h) it was obtained a L-ASNase, after cellular lysis, with an enzymatic activity of 0,756 U/mL, a specific activity of 0,107 U/mg and a purity of 21,97 %. |
publishDate |
2020 |
dc.date.none.fl_str_mv |
2020-12-18T00:00:00Z 2020-12-18 2022-12-30T00:00:00Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/masterThesis |
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masterThesis |
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publishedVersion |
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http://hdl.handle.net/10773/30464 |
url |
http://hdl.handle.net/10773/30464 |
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eng |
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eng |
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reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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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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