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Study of Bacterial Glycoside Hydrolases for Biotechnological Applications: Bioprospection, Production and Immobilization

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Study of Bacterial Glycoside Hydrolases for Biotechnological Applications: Bioprospection, Production and Immobilization UNIVERSIDADE ESTADUAL DE CAMPINAS FACULDADE DE ENGENHARIA DE ALIMENTOS RICARDO RODRIGUES DE MELO STUDY OF BACTERIAL GLYCOSIDE HYDROLASES FOR BIOTECHNOLOGICAL APPLICATIONS: BIOPROSPECTION, PRODUCTION AND IMMOBILIZATION ESTUDO DE HIDROLASES GLICOSÍDICAS BACTERIANAS PARA APLICAÇÕES BIOTECNOLÓGICAS: BIOPROSPECÇÃO, PRODUÇÃO E IMOBILIZAÇÃO CAMPINAS - SP 2017 RICARDO RODRIGUES DE MELO STUDY OF BACTERIAL GLYCOSIDE HYDROLASES FOR BIOTECHNOLOGICAL APPLICATIONS: BIOPROSPECTION, PRODUCTION AND IMMOBILIZATION ESTUDO DE HIDROLASES GLICOSÍDICAS BACTERIANAS PARA APLICAÇÕES BIOTECNOLÓGICAS: BIOPROSPECÇÃO, PRODUÇÃO E IMOBILIZAÇÃO Thesis presented to the Faculty of Food Engineering of the University of Campinas for partial fulfillment of the requirements for the degree of Doctor in FOOD SCIENCE Tese apresentada à Faculdade de Engenharia de Alimentos da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Doutor em CIÊNCIA DE ALIMENTOS Supervisora/Orientadora: Dra. Hélia Harumi Sato Co-supervisor/Co-orientador: Dr. Roberto Ruller ESTE EXEMPLAR CORRESPONDE À VERSÃO FINAL DA TESE DEFENDIDA PELO ALUNO RICARDO RODRIGUES DE MELO E ORIENTADO PELA Dra. HÉLIA HARUMI SATO. CAMPINAS - SP 2017 Agência(s) de fomento e nº(s) de processo(s): CNPq, 140610/2014-6 Ficha catalográfica Universidade Estadual de Campinas Biblioteca da Faculdade de Engenharia de Alimentos Claudia Aparecida Romano - CRB 8/5816 Melo, Ricardo Rodrigues de, 1985- M491s Mel Study of bacterial glycoside hydrolases for biotechnological applications: Bioprospection, production and immobilization / Ricardo Rodrigues de Melo. – Campinas, SP : [s.n.], 2017. Mel Orientador: Hélia Harumi Sato. Mel Coorientador: Roberto Ruller. Mel Tese (doutorado) – Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos. M El 1. Hidrolases glicosídicas. 2. Streptomyces. 3. Genômica. 4. Proteômica. 5. Enzimas imobilizadas. I. Sato, Hélia Harumi. II. Ruller, Roberto. III. Universidade Estadual de Campinas. Faculdade de Engenharia de Alimentos. IV. Título. Informações para Biblioteca Digital Título em outro idioma: Estudo de hidrolases glicosídicas bacterianas para aplicações biotecnológicas: Bioprospecção, produção e imobilização Palavras-chave em inglês: Glycoside hydrolases Streptomyces Genomics Proteomics Immobilized enzymes Área de concentração: Ciência de Alimentos Titulação: Doutor em Ciência de Alimentos Banca examinadora: Hélia Harumi Sato [Orientador] Letícia Maria Zanphorlin Murakami Luciana Ferracini dos Santos Luciana Francisco Fleuri Marcela Pavan Bagagli Data de defesa: 30-10-2017 Programa de Pós-Graduação: Ciência de Alimentos BANCA EXAMINADORA Dra. Hélia Harumi Sato DCA-FEA-UNICAMP - Orientador(a) Dra. Letícia Maria Zanphorlin Murakami CTBE-CNPEM - Membro Titular Dra. Luciana Ferracini dos Santos UNIARARAS - Membro Titular Dra. Luciana Francisco Fleuri IBB - UNESP - Membro Titular Dra. Marcela Pavan Bagagli IFSP-Avaré - Membro Titular A ata de defesa com as respectivas assinaturas dos membros encontra-se no processo de vida acadêmica do aluno. AGRADECIMENTOS A DEUS por me dar vida, saúde e vigor para enfrentar os desafios e porque, sem Ele, não teria chegado até aqui. Aos meus pais pelo amor, constante apoio, e por não medirem esforços para que eu alcançasse meus objetivos. À Profa. Dra. Hélia Harumi Sato pela oportunidade desde o mestrado, pela orientação, incentivo e por acreditar no meu trabalho. Ao Dr. Roberto Ruller pela oportunidade única de desenvolver o trabalho no CTBE, pela co-orientação ao longo de todo o trabalho, pela coordenação das pesquisas no CTBE, e também por confiar no meu trabalho. Aos membros da banca por avaliarem e darem sugestões preciosas para o trabalho: Profa. Dra. Marcela Pavan Bagagli, Profa. Dra. Rosana Goldbeck, Profa. Dra. Luciana Francisco Fleuri, Profa. Dra. Luciana Ferracini dos Santos, Dra. Samantha Christine Santos, Dr. Clelton Aparecido dos Santos e Dra. Letícia Maria Zanphorlin Murakami. Aos amigos do CTBE/CNPEM pela amizade e tornarem o ambiente de trabalho mais agradável. Agradecimento em especial à ilha 2, pela grande amizade e troca de experiências. Aos amigos do laboratório de Bioquímica de Alimentos - DCA/FEA pela amizade e boa convivência. Aos funcionários da Pós-graduação pela paciência e por estarem sempre prontos a nos atender e tirar nossas dúvidas. À Universidade Federal de Viçosa pela excelente formação ao longo de cinco anos de graduação. À Universidade Estadual de Campinas pela oportunidade de cursar a pós-graduação em um centro de excelência em pesquisa, tanto no mestrado como agora no doutorado. Ao CNPq pelos recursos destinados à realização das pesquisas. Ao CNPq pela concessão da bolsa de doutorado. À todos que de alguma forma, direta ou indiretamente, contribuíram para a realização deste trabalho. À todos vocês, meu mais sincero, Muito Obrigado! RESUMO A biomassa lignocelulósica é um importante recurso renovável que está prontamente disponível, sendo uma fonte de polissacarídeos com alto potencial biotecnológico. Os polissacarídeos complexos que compõem a lignocelulose podem ser convertidos em monossacarídeos fermentescíveis, com grande aplicabilidade em diversos bioprocessos industriais. A degradação dos materiais lignocelulósicos pode ser realizada por uma diversidade de vias enzimáticas complexas, onde é requerido um número considerável de enzimas ativas sobre carboidratos. Entre elas, as famílias das celulases e hemicelulases, além de atuarem na hidrólise dos materiais lignocelulósicos, possuem um uso versátil em setores industriais, tais como, nas áreas alimentícia, bebidas e de biocombustíveis. A tese teve como principais objetivos o delineamento de estratégias para a produção de enzimas e coquetéis enzimáticos eficientes para o uso na hidrólise da biomassa vegetal e, a aplicação de técnicas de imobilização para ampliar a utilização de enzimas em escala comercial. Inicialmente, a bioprospecção de novos micro-organismos secretores de enzimas atuantes na biomassa lignocelulósica foi realizada, e dentre as oitenta linhagens de Streptomyces testadas, duas linhagens (F1 e F7) se destacaram por apresentarem elevadas atividades celulolíticas e hemicelulolíticas. Uma abordagem genômica dessas linhagens possibilitou a identificação de 85 hidrolases glicosídicas (GHs) distribuídas em 33 famílias diferentes na linhagem F1, e 100 GHs dispostas em 44 famílias na linhagem F7. Além disso, os dados genômicos das linhagens F1 e F7 também indicaram a presença de genes relacionados à degradação da lignina. Ferramentas estatísticas também foram aplicadas e possibilitaram a ampliação da produção de GHs pela linhagem F1. Com a otimização, elevadas concentrações de GHs foram alcançadas com meio nutriente adicionado de 16,4 g L-1 de farelo de trigo e 10,0 g L-1 de caseína, onde se obteve 9,27 U mL -1 de xilanase e 0,22 U mL -1 de celulase. Para confirmar a diversidade de GHs expressas pela linhagem F1, uma análise por espectrometria de massa foi realizada e observou-se que quanto maior a complexidade da fonte de carbono utilizada, maior foi a gama de proteínas expressas, incluindo vários tipos de celulases e hemicelulases. A eficiência do extrato enzimático produzido pela linhagem F1 foi estudada para a sacarificação da biomassa vegetal e possibilitou um aumento significativo na liberação de açúcares quando adicionado ao extrato celulolítico comercial, indicando que as enzimas secretadas pela Streptomyces sp. F1 podem ser aplicadas para o melhoramento dos atuais coquetéis comerciais. Foi estudada também a criação de métodos de imobilização de enzimas em condições neutras de pH. Os novos suportes produzidos com a agarose foram utilizados para a imobilização de enzimas monoméricas e multiméricas de grande importância biotecnológica. Um estudo mais detalhado explorando os novos suportes e o uso de técnicas de pós-imobilização foi também proposto. O processo desenvolvido com a aplicação do polímero polietilenimina (PEI) possibilitou a formação de um excelente sistema para imobilizar e estabilizar a β-glicosidase obtida de Exiguobacterium antarcticum. A β-glicosidase imobilizada apresentou uma melhora em suas características, incluindo estabilidade térmica e de armazenamento. Além disso, a β- glicosidase manteve sua atividade elevada mesmo após vários ciclos de hidrólise com celobiose como substrato. Palavras chave: Hidrolases glicosídicas; Streptomyces; Genômica; Proteômica; Imobilização de enzimas. ABSTRACT Lignocellulosic biomass is an important renewable resource that is readily available, being a source of polysaccharides with high biotechnological potential. The complex polysaccharides that compose lignocellulose can be converted into fermentable monosaccharides, with great applicability in several industrial bioprocesses. The degradation of lignocellulosic materials can be accomplished by a variety of complex enzymatic pathways, where a considerable number of carbohydrate active enzymes are required. Among them, the families of cellulases and hemicellulases, besides acting in the hydrolysis of lignocellulosic materials, have a versatile use in industrial sectors, such as in food, beverages and biofuels. The main aims of this thesis were the design of efficient strategies for the production of enzymes and enzymatic cocktails for use in plant biomass hydrolysis and the application of immobilization techniques to increase the use of enzymes in a commercial scale. Initially, bioprospection of new enzyme secreting microorganisms active in lignocellulosic biomass was performed, and among the
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