Imobilização da enzima β-Galactosidase de origem microbiana em diferentes suportes

A β-galactosidase é empregada pelas indústrias de laticínios no desenvolvimento de produtos lácteos com baixos teores de lactose. Entretanto, estes biocatalisadores possuem algumas limitações que dificultam sua aplicação industrial. Estas podem ser superadas através da imobilização enzimática, pois permite a reutilização da enzima e um maior controle reacional. O Immobead 150 é um suporte comercial formado de polímeros de metacrilato que contém grupamentos epóxi na sua superfície permitindo a ligação com grupos funcionais. Entre os suportes de baixo custo que podem ser utilizados na imobilização enzimática está o colágeno, uma proteína pouco solúvel na sua forma natural, organizada em fibras resistentes. A celulose microcristalina é outro material promissor para este processo, pois além de ser abundante e barata, possui excelentes propriedades físicas. O objetivo geral desse trabalho foi estudar o processo de imobilização das β-galactosidases de Aspergillus oryzae e Kluyveromyces lactis utilizando diferentes suportes. As matrizes utilizadas no processo foram o colágeno em pó, a celulose microcristalina e o Immobead. Estudos de cargas foram realizados a fim de determinar a melhor proporção enzima-suporte, além de diferentes tratamentos para funcionalizar essas matrizes. Os derivados obtidos que apresentaram melhores rendimentos e eficiências foram avaliados quanto a sua estabilidade térmica e ao armazenamento, condições operacionais de pH e temperatura e ciclos de reuso. Além disso, seus parâmetros cinéticos foram comparados com os da enzima livre. As cargas da enzima de A. oryzae na imobilização em Immobead, colágeno e celulose foram de 100, 1000 e 100 mg de proteína/g de suporte, respectivamente. Já para a β-galactosidase de K. lactis a carga de 100 foi a melhor nos três suportes. Em relação aos parâmetros cinéticos e as condições ótimas de catálise, diferentes comportamentos foram observados entre as diferentes enzimas e suportes. A aplicação em batelada da enzima imobilizada de A. oryzae nas soluções de substrato com pHs ácidos (solução de lactose e permeado) pode ser realizada por aproximadamente 35, 60 e 30 vezes quando imobilizada em Immobead, colágeno e celulose, respectivamente. A β-galactosidase de K. lactis por sua vez, resistiu por 35 ciclos de reuso, em soro de queijo e/ou leite, quando imobilizada em Immobead e 30 em colágeno e celulose. Com base nos resultados obtidos nesse trabalho, verificou-se que o suporte comercial Immobead, e outros dois materiais, colágeno e celulose, demonstram potencial como materiais para imobilização de β-galactosidases microbianas.β-galactosidase is used by the dairy industries in the development of dairy products with low lactose content. However, these biocatalysts have some limitations that hinder their industrial application. These can be overcome through enzymatic immobilization, as it allows the reuse of the enzyme and a greater reaction control. Immobead 150 is a commercial support formed of methacrylate polymers containing epoxy groups on its surface allowing attachment to functional groups. Among the low-cost supports that can be used in enzymatic immobilization is collagen, a poorly soluble protein in its natural form, organized into resistant fibers. Microcrystalline cellulose is another promising material for this process, as it is abundant and inexpensive and has excellent physical properties. The general objective of this project was to study the immobilization process of the β-galactosidases of Aspergillus oryzae and Kluyveromyces lactis using different supports. The matrices used in the process were collagen powder, microcrystalline cellulose and Immobead. Load studies were performed in order to determine the best enzyme-support ratio, as well as different treatments to functionalize these matrices. The obtained derivatives that presented better yields and efficiencies were evaluated for their thermal and storage stability, pH and temperature operating conditions and reuse cycles. In addition, their kinetic parameters were compared with those of the free enzyme. The protein loads of the immobilization of A. oryzae on Immobead, collagen and cellulose were 100, 1000 and 100 mg protein/g support, respectively. For K. lactis β-galactosidase, the loading of 100 was the best in all three supports. Regarding the kinetic parameters and the optimal conditions of catalysis, different behaviors were observed between the different enzymes and supports. Batch application of the A. oryzae immobilized enzyme in substrate solutions with acid pHs (lactose solution and permeate) could be carried out for approximately 35, 60 and 30 times when immobilized on Immobead, collagen and cellulose, respectively. The β-galactosidase of K. lactis, in turn, resisted for 35 cycles of reuse in cheese and/or milk, when immobilized on Immobead and 30 on collagen and cellulose. Based on the results obtained in this work, the commercial support Immobead, and other two materials, collagen and cellulose, have shown potential as materials for the immobilization of microbial β-galactosidases

Tungsten Fabricated by Laser Powder Bed Fusion

AbstractAdditive Manufacturing (AM) is the process that allows the production of complex geometry and lightweight components. Thanks to the high density of refractory metals, AM could be a possible solution for their application in the aerospace field and for biomedical or future nuclear fusion devices. Yet, Laser Powder Bed Fusion (LPBF) of refractory metals as Ta, Mo, and W faces some challenges due to their main properties: high melting point, heat conductivity, and susceptibility to cracks.The purpose of this study is to optimize the process parameters in order to produce high-density Tungsten parts by LPBF on an EOS M100 (maximum power of 170 W). The characterization is performed through physical properties measurements and microstructural analysis. Single Scan Tracks (SSTs) are produced on the top surfaces of Tungsten blocks to evaluate the process parameters that give regular-shape and continuous melt-pools. Both analytical and experimental optimizations of process parameters were performed. Micro-hardness measurements were done for dense bulk specimens. Finally, a description of susceptibility to cracks of additively manufactured Tungsten was performed

CARACTERÍSTICAS FÍSICO-QUÍMICAS DE SOROS DE QUEIJO E RICOTA PRODUZIDOS NO VALE DO TAQUARI, RS

O Rio Grande do Sul é um dos maiores produtores de leite do país e o Vale do Taquari contribui com aproximadamente 7% da produção do estado. O queijo e a ricota são alguns dos principais derivados do leite produzidos na região, que geram nos seus processos de fabricação quantidades elevadas de soros de queijo e de ricota, respectivamente. Por sua elevada carga orgânica estes soros devem ser tratados antes do lançamento em corpos hídricos, no entanto estudos indicam que esses subprodutos podem ser reaproveitados. Portanto, o objetivo desse trabalho foi avaliar as características físico-químicas de soros de queijo e de ricota coletados em uma indústria de laticínios do Vale do Taquari visando o aproveitamento na elaboração de novos produtos alimentícios. As amostras foram submetidas às determinações de acidez, pH, cinzas, umidade, proteínas, gordura, demanda química de oxigênio, carbono e nitrogênio total e lactose. Os parâmetros avaliados apresentaram variações, provavelmente em função dos soros serem provenientes de processos de fabricação de diferentes tipos de queijo e de ricota. Além disso, o soro de queijo apresentou maior carga orgânica. As concentrações de proteínas e lactose dos soros indicam que estes podem ser reaproveitados no desenvolvimento de novos produtos e processos

Stabilization study of tetrameric kluyveromyces lactis β-galactosidase by immobilization on immobead: thermal, physico-chemical, textural and catalytic properties

We investigated the immobilization of a tetrameric Kluyveromyces lactis β-galactosidase (EC: 3.2.1.23) (KL-Gal) on Immobead 150 using different support modification strategies. Immobead support was modified using an acid solution of H2SO4:HNO3 (3:1) (Immobead-Ac) or 5 % (v/v) glutaraldehyde (Immobead-Glu). Its unmodified form (Immobead) was also tested. Immobilization yields and efficiencies were evaluated by testing protein loads from 10 to 200 mg.g-1 support. The thermal, physico-chemical, textural and catalytic properties of the supports (modified and unmodified) and their derivatives (Immobead-KL-Gal, Immobead-Ac-KL-Gal and Immobead-Glu-KL-Gal) were analyzed. The highest immobilization yields and efficiencies were achieved with a protein load of 100 mg.g-1 support. Surface and pore areas of the Immobead support were greatly decreased after modification. Michaelis constant of the immobilized β-galactosidase increased in the derivatives. Maximum velocity decreased approximately 2.8 times for Immobead-KL-Gal and Immobead-Glu-KL-Gal, and approximately 1.4 times for Immobead-Ac-KL-Gal. In batch processes, the three derivatives could be reused successfully at least 15 times, maintaining high residual enzymatic activity during the lactose hydrolysis (in both cheese whey and milk). The tetrameric K. lactis β-galactosidase immobilized on Immobead supports via the tested treatments was stabilized and is an alternative tool for lactose hydrolysis in the dairy industry

Synthesis of magnetic nanoparticles functionalized with histidine and nickel to immobilize His-tagged enzymes using β-galactosidase as a model

The aim of this study was to synthesize iron magnetic nanoparticles functionalized with histidine and nickel (Fe3O4-His-Ni) to be used as support materials for oriented immobilization of His-tagged recombinant enzymes of high molecular weight, using β-galactosidase as a model. The texture, morphology, magnetism, thermal stability, pH and temperature reaction conditions, and the kinetic parameters of the biocatalyst obtained were assessed. In addition, the operational stability of the biocatalyst in the lactose hydrolysis of cheese whey and skim milk by batch processes was also assessed. The load of 600 Uenzyme/gsupport showed the highest recovered activity value (~50%). After the immobilization process, the recombinant β-galactosidase (HisGal) showed increased substrate affinity and greater thermal stability (~50×) compared to the free enzyme. The immobilized β-galactosidase was employed in batch processes for lactose hydrolysis of skim milk and cheese whey, resulting in hydrolysis rates higher than 50% after 15 cycles of reuse. The support used was obtained in the present study without modifying chemical agents. The support easily recovered from the reaction medium due to its magnetic characteristics. The iron nanoparticles functionalized with histidine and nickel were efficient in the oriented immobilization of the recombinant β-galactosidase, showing its potential application in other high-molecular-weight enzymes

TUNGSTEN AND MOLYBDENUM FABRICATED BY LASER POWDER BED FUSION

Additive Manufacturing (AM) is the process that allows the production of complex geometry and lightweight components. According to refractory metals’ high density and their possible applications in the aerospace field, and for biomedical or future nuclear fusion devices, AM could be a good solution. However, selective laser melting of refractory metals as Ta, Mo, and W faces some challenges due to their main properties: high melting point, heat conductivity and susceptibility to cracks. The purpose of this study is to optimize the process parameters in order to produce high-density Tungsten and Molybdenum parts by SLM on an EOS M100 (maximum power of 170 W). Characterization is performed through physical properties measurements and microstructural analysis. Single Scan Tracks (SSTs) are produced on the top surfaces of Tungsten blocks to evaluate the process parameters that give regular-shape and continuous melt-pools. Both analytical and experimental optimization of process parameters were performed. Micro-hardness measurements were done for dense bulk specimens. Finally, a description of susceptibility to cracks of both additively manufactured Molybdenum and Tungsten was performed