Mutag?nese cl?ssica e melhoramento do perfil de assimila??o de nitrog?nio para a produ??o de amilase por Talaromyces trachyspermus

• Main Author: Veron, Nahyara Wandscheer
• Other Authors: Vanz?la, Ana Paula de Figueiredo Conte
• Language: Portuguese
• Published: UFVJM 2017
• Subjects: Amido; Enzimas amilol?ticas; Planejamento fatorial; Fungos; Starch; Amylolitic enzymes; Factorial planning; Fungi
• Online Access: http://acervo.ufvjm.edu.br/jspui/handle/1/1324

Submitted by Jos? Henrique Henrique ([e-mail]) on 2017-04-25T18:49:30Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) nahyara_wandscheer_veron.pdf: 2438141 bytes, checksum: 5dbb316d0dbe26eb1b8b02b31f860c2e (MD5) === Approved for entry into archive by Rodrigo Martins Cruz ([e-mail]) on 2017-05-09T17:31:51Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) nahyara_wandscheer_veron.pdf: 2438141 bytes, checksum: 5dbb316d0dbe26eb1b8b02b31f860c2e (MD5) === Made available in DSpace on 2017-05-09T17:31:51Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) nahyara_wandscheer_veron.pdf: 2438141 bytes, checksum: 5dbb316d0dbe26eb1b8b02b31f860c2e (MD5) Previous issue date: 2016 === Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) === As ?-amilases s?o essenciais para a hidr?lise industrial do amido e para a produ??o de etanol a partir de biomassas amil?ceas. Sua obten??o via importa??o impacta negativamente a ind?stria de biocombust?veis no Brasil, de modo que ? preciso impulsionar a produ??o de ?-amilases no mercado interno. Este trabalho teve por objetivo caracterizar e melhorar o perfil de utiliza??o de fontes de nitrog?nio por Talaromyces trachyspermus, um ascomiceto filamentoso, bem como apontar o efeito dessas fontes na produ??o de ?-amilase por processos submersos delineados por planejamento fatorial. Empregando mutag?nese com luz ultravioleta, foram induzidos e selecionados mutantes com perfil para a produ??o de ?-amilase a partir de fontes nitrogenadas mais baratas. A linhagem parental T. trachyspermus T10.5 utilizou todas as fontes de nitrog?nio avaliadas (extrato de levedura, glutamato, glutamina, ureia, sulfato de am?nio e nitrato de s?dio) mas a produ??o de ?-amilase foi inibida nos bioprocessos suplementados com fontes inorg?nicas (sulfato de am?nio e nitrato de s?dio). A an?lise do planejamento fatorial mostrou que extrato de levedura apresentou efeito estat?stico positivo (4,16 ap?s 72 h e 8,93 ap?s 96 h de cultivo) para a produ??o de ?-amilase pela linhagem parental, cuja produ??o atingiu 66,67 ? 0,18 U/mL ap?s 96h em meio suplementado com o n?vel mais elevado de extrato de levedura (3,7 g/L). Um ensaio de gel atividade e eletroforese prot?ica confirmaram a presen?a de uma enzima amilol?tica no filtrado, com peso molecular aproximado de 20 kDa. Dois mutantes foram selecionados: M12 e M15. A an?lise estat?stica apontou um efeito positivo do extrato de levedura (5,05 ap?s 48h e 104,8 ap?s 72 h de cultivo) para a produ??o de ?-amilase pelo mutante M12, que produziu 55,3 ? 0,2 U/mL ap?s 72 h. O mutante M12 produziu ?-amilase com atividade pr?xima ? do parental, mas o m?ximo de produ??o ocorreu 24 h antes. Ambos os mutantes produziram ?-amilase em meio suplementado com fontes inorg?nicas de nitrog?nio, mas a produ??o da linhagem M12 foi mais expressiva: 29,5 ? 0,2 U/mL ap?s 72 h em meio suplementado com sulfato de am?nio. Assim, foram melhorados o perfil de assimila??o de nitrog?nio e a produ??o de amilase a partir de fonte mais econ?mica, bem como foi reduzido o tempo de produ??o. Os resultados permitiram compreender o efeito das fontes de nitrog?nio e de carbono na produ??o de amilase por T. trachyspermus, gerando conhecimentos sobre fisiologia f?ngica, que ser?o posteriormente aplicados para a otimiza??o da produ??o pelas linhagens T10.5 e M12. === Disserta??o (Mestrado) ? Programa de P?s-gradua??o em Biocombust?veis, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016. === The enzymes ?-amylases are fundamental for the industrial hydrolysis of starch and for ethanol production from amylaceous biomasses. Their acquisition via importation impacts negatively the biofuels industry in Brazil, so that it is necessary to propel ?-amylase production in the internal market. This work aimed to characterize and improve the nitrogen source utilization profile of Talaromyces trachyspermus, a filamentous ascomycete, and to determine the effects of nitrogen sources on ?-amylase production in submerged processes designed by factorial planning. By means of mutagenesis with ultraviolet light, mutants with profile for ?-amylase production from more economical nitrogen sources were induced and selected. The parental strain T. trachyspermus T10.5 used all the nitrogen sources evaluated (yeast extract, glutamate, glutamine, urea, ammonium sulfate and sodium nitrate) but ?-amylase production was inhibited in bioprocesses supplemented with inorganic sources (ammonium sulfate and sodium nitrate). Factorial planning analysis showed that yeast extract exerted a positive statistical effect (4.16 after 72 h and 8.93 after 96 h of cultivation) on ?-amylase production by the parental strain, whose production reached 66.67 ? 0.18 U/mL after 96 h in media supplemented with yeast extract (3.7 g/L). A gel activity assay and protein electrophoresis confirmed the presence of an amylolitic enzyme in the filtrate, with an approximate molecular weight of 20 kDa. Two mutants were selected: M12 and M15. Statistical analysis revealed a positive effect of yeast extract (5.05 after 48 h e 104.8 after 72 h of cultivation) on ?-amylase production by the mutant M12, which produced 55.3 ? 0.2 U/mL after 72 h. Mutant M12 produced a ?-amylase activity similar to that of the parental, but the maximal production was anticipated in 24 h. Both mutants produced ?-amylase in media supplemented with inorganic nitrogen sources, but the production of strain M12 was more meaningful: 29.5 ? 0.2 U/mL after 72 h in medium supplemented with ammonium sulphate. Thus, the nitrogen assimilation profile and amylase production from a more economical source were improved, as well as it was reduced the production time. Results allowed understand nitrogen and carbon sources effect on amylase production by T. trachyspermus, and yielded knowledge about fungal physiology that will be further applied for optimization of production by strains T10.5 and M12.