Otimiza??o da produ??o de amilases por Rhizopus arrhizus I 1.2.1 e hidr?lise do amido dos gr?os de milho

Autor: Lopes, Paulo Henrique Silva
Přispěvatelé: Nelson, David Lee, Baffi, Milla Alves, Lucas, Rosymar Coutinho de, Benassi, Vivian Machado, Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM)
Jazyk: portugalština
Rok vydání: 2022
Předmět:
Zdroj: Repositório Institucional da UFVJM
Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM)
instacron:UFVJM
Popis: Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2022-09-16T20:06:23Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) paulo_henrique_silva_lopes.pdf: 4539808 bytes, checksum: c533c987c165d33df48327ac95554f8c (MD5) Approved for entry into archive by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2022-09-16T21:24:10Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) paulo_henrique_silva_lopes.pdf: 4539808 bytes, checksum: c533c987c165d33df48327ac95554f8c (MD5) Made available in DSpace on 2022-09-16T21:24:10Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) paulo_henrique_silva_lopes.pdf: 4539808 bytes, checksum: c533c987c165d33df48327ac95554f8c (MD5) Previous issue date: 2022 Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM) Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) A hidr?lise do pol?mero de amido ? realizado pelo sistema amilol?tico, formando diferentes produtos de hidr?lise, como a glicose e a maltose. Essas enzimas s?o, principalmente, sintetizadas por microrganismos, em especial, fungos filamentosos, no entanto, ? necess?rio otimizar os processos de produ??o e uso das enzimas para tornar os processos de hidr?lise mais vi?veis. O objetivo desse trabalho foi otimizar a produ??o das amilases por Rhizopus arrhizus I 1.2.1 e caracterizar bioquimicamente as amilases, bem como realizar a hidr?lise do amido de milho para libera??o de a??cares pass?veis de fermenta??o. Realizou-se a identifica??o molecular e filogen?tica do fungo, al?m das melhores condi??es de cultivo. Os par?metros analisados foram o meio de cultivo e tempo de maior produ??o enzim?tica, fonte de nitrog?nio, sais, pH inicial do meio de cultivo, tamanho do in?culo em disco, fonte de carbono e suplementa??o com glicose, al?m da caracteriza??o das amilases brutas e a aplica??o na hidr?lise de diferentes substratos. Identificou-se que a cepa analisada pertence ? esp?cie Rhizopus arrhizus, cuja produ??o enzim?tica foi otimizada de 0,12 para 22,50 U.mL-?, com 6 dias de cultivo em meio submerso Carvalho-Peixoto, a 35?C, de forma estacion?ria, em presen?a de ureia, pH inicial 6,0, in?culo em disco de 0,5'' e casca de abacaxi acrescida de glicose. As amilases brutas apresentaram atividade ?tima aparente a 60?C e pH 6,0 (17,5 U.mL-?), com termoestabilidade por at? 240 minutos entre 50 e 55?C. Al?m de que sais contendo sulfato e pot?ssio aumentaram a a??o catal?tica em 26%. As enzimas foram capazes de hidrolisar diferentes substratos, bem como os gr?nulos de amido presentes nos gr?os de milho em temperaturas abaixo do seu ponto de gelatiniza??o, como foi observado pela presen?a de cavidades nas micrografias superficiais dos gr?nulos e pela cin?tica de hidr?lise com a libera??o de at? 0,443 mg de a??cares redutores em 24 horas e sem otimiza??o. Pelo exposto, o estudo demonstrou potencial aplicabilidade para a produ??o de amilases, uma vez que se utilizou fonte residual de carbono, fonte de nitrog?nio relativamente barata, e menor gasto de reagentes qu?micos na formula??o do meio de cultivo e in?culo. Al?m disso, as amilases produzidas demonstraram ser capazes de hidrolisar os gr?nulos de amido presentes no milho numa temperatura mais baixa que a utilizada a n?vel industrial, indicando menor gasto energ?tico na libera??o dos a??cares fermentesc?veis essenciais na produ??o do etanol de milho. Disserta??o (Mestrado) ? Programa de P?s-gradua??o em Biocombust?veis, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2022. The hydrolysis of the starch polymer is carried out by the amylolytic system, forming different hydrolysis products, such as glucose and maltose. These enzymes are mainly synthesized by microorganisms, especially filamentous fungi, however, it is necessary to optimize the production processes and use of enzymes to make the hydrolysis processes more viable. The objective of this work was to optimize the production of amylases by Rhizopus arrhizus I 1.2.1 and biochemically characterize the amylases, as well as perform the hydrolysis of corn starch to release sugars susceptible to fermentation. Molecular and phylogenetic identification of the fungus was carried out, in addition to the best cultivation conditions. The parameters analyzed were the culture medium and time of highest enzyme production, nitrogen source, salts, initial pH of the culture medium, disk inoculum size, carbon source, and glucose supplementation, in addition to the characterization of crude amylases and the application in the hydrolysis of different substrates. It was identified that the analyzed strain belongs to the species Rhizopus arrhizus, whose enzyme production was optimized from 0.12 to 22.50 U.mL-?, with 6 days of cultivation in Carvalho-Peixoto submerged medium, at 35?C, in a stationary way, in the presence of urea, initial pH 6.0, inoculum in a 0.5'' disk, and pineapple peel added with glucose. Crude amylases showed optimal apparent activity at 60?C and pH 6.0 (17.5 U.mL-?), with thermostability for up to 240 minutes between 50 and 55?C. In addition, salts containing sulfate and potassium increased the catalytic action by 26%. The enzymes were able to hydrolyze different substrates, as well as the starch granules present in corn grains at temperatures below their gelatinization point, as observed by the presence of cavities in the surface micrographs of the granules and by the kinetics of hydrolysis with the release of up to 0.443 mg of reducing sugars in 24 hours and without optimization. Based on the above, the study demonstrated potential applicability for the production of amylases, since it used a residual carbon source, a relatively cheap nitrogen source, and a lower expense of chemical reagents in the formulation of the culture medium and inoculum. In addition, the amylases produced were able to hydrolyze the starch granules present in corn at a lower temperature than that used at an industrial level, indicating lower energy expenditure in the release of essential fermentable sugars in the production of corn ethanol.
Databáze: OpenAIRE
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