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Made available in DSpace on 2020-12-12T01:35:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-10-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Abstract: Arabinanases from glycoside hydrolase family GH93 are enzymes with exo-activity that hydrolyze the α-1,5 bonds between arabinose residues present on arabinan. Currently, several initiatives aiming to use byproducts rich in arabinan such as pectin and sugar beet pulp as raw material to produce various compounds of interest are being developed. However, it is necessary to use robust enzymes that have an optimal performance under pH and temperature conditions used in the industrial processes. In this work, the first GH93 from the thermophilic fungus Thermothielavioides terrestris (Abn93T) was heterologously expressed in Aspergillus nidulans, purified and biochemically characterized. The enzyme is a thermophilic glycoprotein (optimum activity at 70 °C) with prolonged stability in acid pHs (4.0 to 6.5). The presence of glycosylation affected slightly the hydrolytic capacity of the enzyme, which was further increased by 34% in the presence of 1 mM CoCl2. Small-angle X-ray scattering results show that Abn93T is a globular-like-shaped protein with a slight bulge at one end. The hydrolytic mechanism of the enzyme was elucidated using capillary zone electrophoresis and molecular docking calculations. Abn93T has an ability to produce (in synergism with arabinofuranosidases) arabinose and arabinobiose from sugar beet arabinan, which can be explored as fermentable sugars and prebiotics. Key points: • Thermophilic exo-arabinanase from family GH93 • Molecular basis of arabinan depolymerization. Department of Biotechnology Lorena School of Engineering University of São Paulo Departamento de Física e Ciências Aplicadas Instituto de Física de São Carlos Universidade de São Paulo Departamento de Bioquímica Instituto de Biologia Universidade Estadual de Campinas Programa de Processos Tecnológicos e Ambientais Universidade de Sorocaba Instituto de Pesquisas Tecnológicas do Estado de São Paulo Diretoria de Operações e Negócios Núcleo de Bionanomanufatura Department of Physics – Institute of Biosciences Humanities and Exact Sciences São Paulo State University (UNESP) Department of Physics – Institute of Biosciences Humanities and Exact Sciences São Paulo State University (UNESP) FAPESP: 2014/06923-6 FAPESP: 2014/18714-2 FAPESP: 2017/00525-0 FAPESP: 2019/01165-0 CNPq: 2019/06663-8 FAPESP: 2019/06663-8 FAPESP: 2019/22284-7 CNPq: 302627/2018-9 CNPq: 443916/2014-4

Lytic polysaccharide monooxygenases (LPMOs), monocopper enzymes that oxidatively cleave recalcitrant polysaccharides, have important biotechnological applications. Thermothelomyces thermophilus is a rich source of biomass-active enzymes, including many members from auxiliary activities family 9 LPMOs. Here, we report biochemical and structural characterization of recombinant TtLPMO9H which oxidizes cellulose at the C1 and C4 positions and shows enhanced activity in light-driven catalysis assays. TtLPMO9H also shows activity against xyloglucan. The addition of TtLPMO9H to endoglucanases from four different glucoside hydrolase families (GH5, GH12, GH45 and GH7) revealed that the product formation was remarkably increased when TtLPMO9H was combined with GH7 endoglucanase. Finally, we determind the first low resolution small-angle X-ray scattering model of the two-domain TtLPMO9H in solution that shows relative positions of its two functional domains and a conformation of the linker peptide, which can be relevant for the catalytic oxidation of cellulose and xyloglucan.

A biomassa lignocelulósica é uma fonte abundante de açúcares simples passíveis de serem fermentados em uma variedade de bioprodutos de maior valor agregado, além do etanol de segunda geração. Tal diversidade é relevante ao desenvolvimento e aprimoramento do conceito de biorrefinarias e da bioeconomia, em um viés mais amplo. Todavia, a elevada recalcitrância dos lignocelulósicos dificulta a sua sacarificação enzimática, resultando em bioprocessos mais onerosos. Por isso, coquetéis com diferentes enzimas ativas em carboidrato (CAZymes) são desenvolvidos, em busca de uma maior eficiência e melhor relação custo/benefício, para processos em larga escala. Dentre as CAZymes estudadas, encontram-se as mono-oxigenases líticas de polissacarídeo (LPMOs), tendo em vista a sua atestada capacidade de otimizar a hidrólise da lignocelulose, quando em sinergismo com diversas hidrolases. Levando isto em conta, selecionou-se, ao atual estudo, o ascomiceto termofílico Thermothelomyces thermophila (anteriormente denominado Myceliophthora thermophila), pois este tem se mostrado capaz de expressar e secretar ampla gama de LPMOs ativas em diferentes substratos. Objetivando-se estudar duas LPMOs derivadas deste organismo, as mesmas foram expressas, heterologamente, por Aspergillus nidulans linhagem A773, utilizando-se o vetor de expressão pEXPYR construído para viabilizar a secreção de altas concentrações de proteínas recombinantes. As proteínas heterólogas aqui analisadas foram denominadas TtLPMO1A9 e TtLPMO2A9. Embora ambas tenham sido capazes de gerar peróxido de hidrogênio na presença de oxigênio molecular e de um doador de elétrons, apenas TtLPMO2A9 apresentou atividade contra substratos celulósicos e bagaço de cana pré-tratado hidrotermicamente, atuando, em associação com hidrolases homemade e o preparo enzimático comercial Celluclast 1.5L, a degradação de tais materiais. The lignocellulosic biomass is an abundant source of simple sugars that can be fermented to various value-added bio-based products. This diversity is seen as relevant to the improvement of biorefinery and bioeconomy concept. Nevertheless, the significant recalcitrance of lignocellulose imposes dificulties to its enzymatic saccharification, resulting in onerous bioprocessing. This scenario stimulates studies based on the development of efficient and cost-effective customizable carbohydrate-active enzyme (CAZymes) cocktails for large-scale processes. Among the available CAZymes, there are the lytic polysaccharide monooxygenases (LPMOs), a set of oxidative proteins capable of optimizing the lignocellulose hydrolysis, when acting in synergism with various hydrolases. Based on this fact, in the current study, the thermophilic ascomycete Thermothelomyces thermophila (previously known as Myceliophthora thermophila) was adopted, because of its ability of expressing and secreting large amounts of LPMOs. Thus, two LPMOs derived from this fungus was heterologously produced by an expression system composed by Aspergillus nidulans strain A773 and the vector pEXPYR: an expression vector built to increase the secretion of recombinant proteins. The heterologous proteins herein analysed were termed as TtLPMO1A9 and TtLPMO2A9. Although both enzymes were able to produce hydrogen peroxide in the presence of molecular oxygen and an electron donor, only the second one was active in reactions with cellulosic substrates and hydrothermally pre-treated sugar cane bagasse. When tailor-made hydrolases and the commercial enzymatic mixture Celluclast 1.5L were supplemented with TtLPMO2A9, it was noticed na improvement of the deconstruction of the aforementioned substrates.

The increase in the development of new molecular biology techniques and tools, mainly due to the number of genomes available at databanks together with bioinformatics and some engineering methodologies, has emerged in synthetic biology science. Since then, many research groups and companies around the world have worked on the development of microorganisms with desirable industrial characteristics, such as the capacity to synthesize and also secrete a chemical block that could be employed in the production of complex molecules and polymers. In this chapter we provide information about the expectations and future of synthetic biology applications for sustainability, through the increase in the portfolio of chemicals and blocks that can be produced in biorefineries.