Production of recombinant a-amylase from Bacillus subtilis by Kluyveromyces lactis

Detalhes bibliográficos
Ano de defesa: 2013
Autor(a) principal: Nascimento, Bruno Motta
Orientador(a): Thomaz-Soccol, Vanete, 1954-
Banca de defesa: Não Informado pela instituição
Tipo de documento: Dissertação
Tipo de acesso: Acesso aberto
Idioma: por
Instituição de defesa: Não Informado pela instituição
Programa de Pós-Graduação: Não Informado pela instituição
Departamento: Não Informado pela instituição
País: Não Informado pela instituição
Palavras-chave em Português:
Enzimas - Aplicações industriais
Amidos
Bacillus (Bacteria)
Link de acesso: https://hdl.handle.net/1884/36145
Resumo: Orientadora: Profa. Dra. Vanete Thomaz Soccol
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spelling Universidade Federal do Paraná. Setor de Tecnologia. Programa de Pós-Graduação em Engenharia de Bioprocessos e BiotecnologiaThomaz-Soccol, Vanete, 1954-Nascimento, Bruno Motta2024-04-15T19:20:43Z2024-04-15T19:20:43Z2013https://hdl.handle.net/1884/36145Orientadora: Profa. Dra. Vanete Thomaz SoccolDissertação (mestrado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia. Defesa: Curitiba, 02/2013Inclui referênciasÁrea de concentração: Agroindústria e biocombustíveisAbstract: Amylolytic enzymes catalyze the hydrolysis or the modification of starch structure. Many organisms are able to produce these enzymes, but only a few of them exhibit satisfactory characteristics for industrial application. The most common commercial a-amylases with desirable industrial characteristics are produced by bacteria from the genus Bacillus. Although the yield of a-amylase produced by these bacterial strains can be increased with culture optimization, the recombinant production of the enzyme could offer some advantages such as easier genetic manipulation/mutation, inducible expression, faster production, higher yields and easier purification steps. The present work proposes the production of a recombinant a-amylase in Kluyveromyces lactis. A Bacillus subtilis NRRL B-4212 strain provided the a-amylase gene, which was amplified and inserted in a pKLAC2 vector in four different ways: (i) with both the B. subtilis signal sequence and K. lactis leader sequence; (ii) with only the yeast leader or (iii) only the B. subtilis signal, and (iv) without any signal or leader sequence. The final constructs were linearized and the cassettes were used for K. lactis CBS 2359 cells transformation. After growth in a selective medium, the positive yeast colonies were assayed for enzymatic activity on agar plates and liquid medium. It was expected that cells with construct (i) and cells with construct (ii) would present extracellular enzymatic activity. On the other way, transformants with constructs (iii) and transformants with construct (iv) should present an intracellular activity. However, only constructs (ii) and (iv) behaved as expected. Construct (i) could not secrete the enzyme, indicating that the presence of both signals might interfere in the secretion machinery, and construct (iii) presented a high extracellular activity (1200 U/L), even though it did not have the yeast leader sequence, indicating that the yeast cell probably can also recognize the bacterial signal peptide. One sample from each construct was selected to determinate the enzyme’s optimal temperature and pH, obtaining results between 50 an 55 °C, which is consistent with other observed values for Bacillus amylases. Transformant LAC-EXAMY 4 was also cultured in different carbon sources to observe cellular growth and enzyme production. The recombinant yeast was able to quickly grow in the presence of glucose, sucrose and lactose. Even without the induction with lactose or galactose the enzyme was produced with a basal level (0.44 U/mL). However, the presence of these carbon inducers greatly enhances the production yield, achieving 2.21 U/mL with lactose and 2.55 U/mL with galactose as the carbon source.83f. : il. algumas color., grafs., tabs.application/pdfDisponível em formato digitalEnzimas - Aplicações industriaisAmidosBacillus (Bacteria)Production of recombinant a-amylase from Bacillus subtilis by Kluyveromyces lactisinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisporreponame:Repositório Institucional da UFPRinstname:Universidade Federal do Paraná (UFPR)instacron:UFPRinfo:eu-repo/semantics/openAccessORIGINALR - D - BRUNO MOTTA NASCIMENTO.pdfapplication/pdf4584226https://acervodigital.ufpr.br/bitstream/1884/36145/1/R%20-%20D%20-%20BRUNO%20MOTTA%20NASCIMENTO.pdf5d2639778d74e13ad41fcb557b108d87MD51open accessTEXTR - D - BRUNO MOTTA NASCIMENTO.pdf.txtExtracted Texttext/plain147054https://acervodigital.ufpr.br/bitstream/1884/36145/2/R%20-%20D%20-%20BRUNO%20MOTTA%20NASCIMENTO.pdf.txt7f73f5701f560eac0b3a5aae2b6538e3MD52open accessTHUMBNAILR - D - BRUNO MOTTA NASCIMENTO.pdf.jpgGenerated Thumbnailimage/jpeg1473https://acervodigital.ufpr.br/bitstream/1884/36145/3/R%20-%20D%20-%20BRUNO%20MOTTA%20NASCIMENTO.pdf.jpgb0c3f542a7395b019e5007c9e9db24f7MD53open access1884/361452024-04-15 16:20:43.304open accessoai:acervodigital.ufpr.br:1884/36145Repositório InstitucionalPUBhttp://acervodigital.ufpr.br/oai/requestinformacaodigital@ufpr.bropendoar:3082024-04-15T19:20:43Repositório Institucional da UFPR - Universidade Federal do Paraná (UFPR)false
dc.title.pt_BR.fl_str_mv Production of recombinant a-amylase from Bacillus subtilis by Kluyveromyces lactis
title Production of recombinant a-amylase from Bacillus subtilis by Kluyveromyces lactis
spellingShingle Production of recombinant a-amylase from Bacillus subtilis by Kluyveromyces lactis
Nascimento, Bruno Motta
Enzimas - Aplicações industriais
Amidos
Bacillus (Bacteria)
title_short Production of recombinant a-amylase from Bacillus subtilis by Kluyveromyces lactis
title_full Production of recombinant a-amylase from Bacillus subtilis by Kluyveromyces lactis
title_fullStr Production of recombinant a-amylase from Bacillus subtilis by Kluyveromyces lactis
title_full_unstemmed Production of recombinant a-amylase from Bacillus subtilis by Kluyveromyces lactis
title_sort Production of recombinant a-amylase from Bacillus subtilis by Kluyveromyces lactis
author Nascimento, Bruno Motta
author_facet Nascimento, Bruno Motta
author_role author
dc.contributor.other.pt_BR.fl_str_mv Universidade Federal do Paraná. Setor de Tecnologia. Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia
dc.contributor.advisor1.fl_str_mv Thomaz-Soccol, Vanete, 1954-
dc.contributor.author.fl_str_mv Nascimento, Bruno Motta
contributor_str_mv Thomaz-Soccol, Vanete, 1954-
dc.subject.por.fl_str_mv Enzimas - Aplicações industriais
Amidos
Bacillus (Bacteria)
topic Enzimas - Aplicações industriais
Amidos
Bacillus (Bacteria)
description Orientadora: Profa. Dra. Vanete Thomaz Soccol
publishDate 2013
dc.date.issued.fl_str_mv 2013
dc.date.accessioned.fl_str_mv 2024-04-15T19:20:43Z
dc.date.available.fl_str_mv 2024-04-15T19:20:43Z
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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dc.identifier.uri.fl_str_mv https://hdl.handle.net/1884/36145
url https://hdl.handle.net/1884/36145
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