Orientador: Ranulfo Monte Alegre / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-16T22:59:40Z (GMT). No. of bitstreams: 1 Meirelles_RenataMiterhof_M.pdf: 815729 bytes, checksum: 9edb1e9645d9423ee9870568496b2ffd (MD5) Previous issue date: 2010 / Resumo: O mercado consumidor da frutose tem aumentado significativamente nos últimos anos pela sua utilização cada vez maior em substituição à sacarose em virtude do seu poder edulcorante 70% superior e dos benefícios fisiológicos importantes, como metabolismo independente da insulina, sendo adequada para alimentos fabricados especificamente para diabéticos. Sua maior aplicação tecnológica encontra-se no uso de xaropes enriquecidos com frutose em vários segmentos industriais como alimentício, farmacêutico e químico. Comercialmente, a obtenção de frutose envolve um processo de alto custo sendo interessante o desenvolvimento de um processo que combine a produção por via enzimática e a utilização da sacarose como substrato para obtenção de frutose de alto grau de pureza. O objetivo geral deste trabalho foi estudar e otimizar a hidrólise da levana para obtenção de frutose livre. Para tal, levana foi previamente obtida pela levanassacarase durante fermentação da cepa mutante de Zymomonas mobilis CCT 4494 em substrato a base de sacarose. Kluyveromyces marxianus NRRL Y-8281 foi selecionada dentre três linhagens da espécie Kluyveromyces marxianus (CCT4294, NRRL Y-8281 e NRRL Y-610) devido à sua maior produção de frutana ß-frutosidase. Foram realizados delineamentos experimentais tendo como variáveis temperatura, pHinicial, concentrações iniciais de levana, extrato de levedura e peptona. A enzima agiu exohidroliticamente obtendo apenas frutose como produto, e não foi observado inibição da reação pelo produto. A frutana ß-frutosidase de Kluyveromyces marxianus NRRL Y-8281 foi caracterizada parcialmente quanto ao pH e temperatura ótimos (4,4 e 50 ºC), estabilidade térmica e pH de pré incubação, além dos parâmetros cinéticos da Equação de Michaelis-Mentem, Km e Vmáx (61,5 µmol/mL e 0,0112 µmol/mL.min, respectivamente) para o substrato levana / Abstract: The market for fructose consumption has increased significantly in the last years by increasing its use in place of sucrose, because of its sweetening power 70% higher than the sucrose and the physiological benefits like independent metabolism of insulin, which is, therefore, suitable for food made specifically for diabetics. His greatest technological application is the use of enriched fructose syrups in various industries like food, pharmaceutical and chemical ones. Commercially, the obtainment of fructose involves a high cost, being interesting to develop a process that combines the production of fructose by an enzyme, using sucrose as substrate to obtain fructose of high purity. Therefore, the objective of this work was to study and optimize the hydrolysis of levan to obtain free fructose. To accomplish this, the levan was previously obtained by levansucrase during fermentation of mutant strain of Zymomonas mobilis CCT 4494 in sucrose substrate. Kluyveromyces marxianus NRRL Y-8281 was selected among three strains of the species Kluyveromyces marxianus (CCT4294, NRRL Y-8281 and NRRL Y-610) by the increased production of fructan ß-frutosidase. Experimental designs were performed having as variables temperature, initial pH, initial concentrations of levan, yeast extract and peptone. The enzyme acted in a exohydrolytically fashion, getting only fructose as released product, and it was not observed inhibition by product reaction. The fructan exo-ß-frutosidase of Kluyveromyces marxianus NRRL Y-8281 was partially characterized for optimum pH and temperature (4.4 and 50 °C), thermal and pH stability, besides the kinetic parameters of the Michaelis-Mentem equation, Km and Vmáx (61,5 µmol/mL and 0,0112 µmol/mL.min, respectively) to the substrate levan / Mestrado / Mestre em Engenharia de Alimentos

Orientador: Francisco Maugeri Filho / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-05T17:05:43Z (GMT). No. of bitstreams: 1 YepezSilva-Santisteban_BernardoOnagar_D.pdf: 1839103 bytes, checksum: 44dfdd6f4dc2a025d2bcdbcf149d2ef1 (MD5) Previous issue date: 2006 / Doutorado / Doutor em Engenharia de Alimentos

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2015-11-06T14:41:38Z No. of bitstreams: 1 texto completo.pdf: 1116633 bytes, checksum: fc318cc7ff97c79f0496b3e69c56e3fa (MD5) / Made available in DSpace on 2015-11-06T14:41:38Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1116633 bytes, checksum: fc318cc7ff97c79f0496b3e69c56e3fa (MD5) Previous issue date: 2013-03-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A levedura Kluyveromyces marxianus tem sido considerada uma candidata hospedeira para a síntese industrial de biomoléculas. Apesar de seu potencial, são poucos os estudos que relatam a expressão de proteínas heterólogas utilizando esta levedura. Neste trabalho, foi relatado pela primeira vez, a expressão da proteína do vírus da dengue em K. marxianus. O gene que codifica a proteína não estrutural (NS1) do vírus da dengue-1 foi integrado no genoma da levedura K. marxianus UFV-3 no locus LAC4, utilizando um vetor integrativo adaptado projetado para expressão de proteínas recombinantes em Kluyveromyces lactis. O gene de dengue-1 NS1 foi otimizado utilizando os códons preferenciais para aumentar os níveis de expressão de proteínas em leveduras. O gene sintético foi clonado “in frame” com o peptídeo sinal (mating-α-factor) de K. lactis e o plasmídeo recombinante obtido foi utilizado para transformar K. marxianus UFV-3 por eletroporação. As células transformantes selecionadas em YPD (yeast extract peptone dextrose) contendo 200 ug mL-1 de geneticina foram mitoticamente estáveis. A análise das linhagens recombinantes por meio de RT-PCR e a detecção da proteína utilizando Dot-blot confirmou a transcrição e a expressão dos peptídeos extracelulares. Após a indução com galactose, a proteína NS1 foi analisada por SDS-PAGE e Western blot. A produção da proteína foi investigada sob duas condições: com pulso de galactose e biotina com intervalos de 24 horas durante 96 horas após a indução e sem pulso de galactose e biotina. A atividade proteolítica não foi detectada no sobrenadante das culturas. Nossos resultados indicam que células recombinantes de K. marxianus podem ser consideradas boas hospedeiras para a produção de proteínas do vírus de dengue, que têm um potencial para aplicações em diagnósticos. / The yeast Kluyveromyces marxianus has been considered a candidate host for industrial synthesis of biomolecules. Despite its potential, there are few studies reporting the expression of heterologous proteins using this yeast. Here, it was reported for the first time a dengue viral protein expression in K. marxianus. The dengue virus type 1 nonstructural protein 1 (NS1) was integrated into the K. marxianus UFV-3 genome at the LAC4 locus using adapted integrative vector designed for high-level expression of recombinant protein in Kluyveromyces lactis. The gene of dengue-1 NS1 was optimized using preferential codons to increase the levels of proteins expression in yeast. The synthetic gene was cloned in frame with K. lactis mating-α-factor signal peptide and the recombinant plasmid obtained was used to transform K. marxianus UFV-3 by electroporation. The transformants cells selected in Yeast Extract Peptone Dextrose (YPD) containing 200 μg mL-1 Geneticin were mitotically stable. The analysis of recombinant strains by RT-PCR technique and the protein detection using blot analysis have confirmed both transcription and expression of the extracellular peptides. After induction with galactose, the NS1 protein was analyzed by SDS-PAGE and immunogenic detection. The protein production was investigated under two conditions: with galactose and biotin pulse at 24 hours intervals during 96 hours of induction and without galactose and biotin pulse. Protease activity was not detected into the medium. Our results indicate that the constructed recombinant K. marxianus can be considered good host for the production of dengue virus proteins, which have a potential for diagnostic applications.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2015-11-09T09:20:33Z No. of bitstreams: 1 texto completo.pdf: 1613973 bytes, checksum: 68e6cb9b2db2276aab154d130130c331 (MD5) / Made available in DSpace on 2015-11-09T09:20:33Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1613973 bytes, checksum: 68e6cb9b2db2276aab154d130130c331 (MD5) Previous issue date: 2011-04-25 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / O Ácido L-ascórbico (L-AA), popularmente conhecido como vitamina C é naturalmente sintetizado pelas plantas a partir de D-glicose por uma via de 10 etapas. L- galactose é o intermediário chave para a biossíntese de L-ascórbico, cuja via de biossíntese foi recentemente elucidada. As leveduras produzem um composto análogo ao L-AA, o ácido D-eritroascórbico, mas em presença de um de seus precursors tais como L-galactose, L galactono-1,4-lactona, ou L-gulono-1 ,4-lactona, as leveduras são capazes de sintetizar o L-AA. Para evitar alimentar a cultura de levedura com o "L" enantiômero, a levedura Kluyveromyces lactis CBS2359 foi engenheirada com genes da via de biossíntese de L-galactose: GDP-manose-3 ,5-epimerase (GME), GDP-L- galactose fosforilase (VTC2) e L-galactose-1-fosfato fosfatase (VTC4) isolados de Arabidopsis thaliana. Com este objetivo plasmídeos foram construídos visando a integração por recombinação homóloga dos cassetes de expressão no Locus LAC4 (beta- galactosidase) Após o processo de transformação, o promotor do gene LAC4 promove a transcrição do gene GME, enquanto que genes VTC2 e VTC4 estão sob o controle dos promotores GPD1 e ADH1 respectivamente provenientes da levedura S. cerevisiae. A expressão dos genes da via de biossíntese de L-galactose em K. lactis foi determinada por RT-PCR e western blot. As leveduras recombinantes foram capazes de produzir cerca de 23 mg.L-1 de ácido L-ascórbico após 48 horas de cultivo, quando cultivados em meio YP suplementado com 2% (p/v) de D-galactose. A biossíntese de L-AA foi também realizada quando as linhagens recombinantes foram cultivadas em meio de soro de queijo, fonte alternativa rica em lactose proveniente da indústria de laticínios. Este trabalho é um dos primeiros relatos de engenharia metabólica na levedura K. lactis visando a biossíntese de ácido L-ascórbico por um processo fermentativo sem a adição de intermediários precursores no meio de cultura. / L-ascorbic acid is naturally synthesized in plants from D-glucose via a ten-step pathway. The branch pathway to synthesize L-galactose, the key intermediate for L- ascorbic biosynthesis, has been recently elucidated. Budding yeast is only able to synthesize L-ascorbic acid if it is cultivated in the presence of one of its precursors: L- galactose, L-galactono-1,4-lactone, or L-gulono-1,4-lactone extracted from plants or animals. To avoid feeding the yeast culture with this “L” enantiomer, we engineered Kluyveromyces lactis with L-galactose biosynthesis pathway genes: GDP-mannose-3,5- epimerase (GME), GDP-L-galactose phosphorylase (VTC2) and L-galactose-1- phosphate phosphatase (VTC4) isolated from Arabidopsis thaliana. Plasmids were constructed to target the cloned plant genes to the K. lactis LAC4 Locus by homologous recombination and the expression was associated to the growth of the cells on D- galactose or lactose. Upon K. lactis transformation, GME was under the control of the native LAC4 promoter while VTC2 and VTC4 genes were transcribed by the S. cerevisiae promoters GPD1 and ADH1 respectively. The expression in K. lactis of the endogenous L-galactose biosynthesis plant genes was determined by RT-PCR and western blotting. The recombinant yeasts were able to produce about 23 mg.L-1 of L- ascorbic acid in 48 hours of cultivation when cultured on rich medium with 2% (w/v) D-galactose. We have also successfully evaluated the L-AA production culturing recombinant strains in cheese whey as an alternative source of lactose and which is a waste product during cheese production. This work is the first attempt to engineering K. lactis cells for L-ascorbic acid biosynthesis through a fermentation process without any trace of “L” isomers precursors in the culture medium.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2015-11-09T13:45:39Z No. of bitstreams: 1 texto completo.pdf: 600665 bytes, checksum: ee26156d6dc7124f99efde31caf1f198 (MD5) / Made available in DSpace on 2015-11-09T13:45:39Z (GMT). No. of bitstreams: 1 texto completo.pdf: 600665 bytes, checksum: ee26156d6dc7124f99efde31caf1f198 (MD5) Previous issue date: 2010-04-12 / Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A permease de lactose de Kluyveromyces lactis, Lac12, media o transporte de lactose e o de galactose de baixa afinidade. Aqui é apresentado o estudo do efeito de fontes de carbono na internalização de Lac12 através do uso de linhagens contendo o gene quimérico LAC12-GFP. Quando células de K. lactis pré-cultivadas em galactose ou lactose foram transferidas para um novo meio, Lac12-GFP foi removida da membrana plasmática e localizada intracelularmente. Surpreendentemente, mesmo a presença de galactose ou lactose no novo meio de transferência causou essa internalização, e a resposta celular foi diferente para esse dois açúcares. Os resultados obtidos revelam que o processo de internalização é dependente do tipo de açúcar presente e de sua concentração. A internalização de Lac12-GFP causou redução nas taxas de captação de lactose[C14] e também foi observada em uma linhagem mutante Klsnf1; portanto, esse evento independe da atividade de KlSnf1. Evidências indicam que glicose-6-fosfato é o sinal intracelular, uma vez que a internalização foi induzida por 2-deoxiglicose, e a inibição da atividade da enzima fosfoglimutase por lítio impediu a internalização por galactose, mas não por lactose ou glicose. A internalização não ocorreu em 6-deoxiglicose, e, em ausência de síntese protéica, o evento foi irreversível. / Kluyveromyces lactis Lac12 permease mediates lactose and low-affinity galactose transports. In this study we have investigated the effects of carbon sources on internalization of Lac12 by using a LAC12-GFP fusion construct. When galactose- or lactose-grown cells are shifted to a fresh sugar medium, Lac12-GFP is removed from the plasma membrane and localized intracellularly. Surprisingly, even galactose or lactose in the new media caused the internalization, and cells responded differently to theses two sugars. Our results reveal that this process is dependent of sugar species and also sugar concentration. Lac12-GFP internalization causes reduction on [C14]lactose uptake rates and also occurs in a Klsnf1 mutant strain; thereby, it is independent of KlSnf1 activity. We suggest that glucose-6-phosphate is the intracellular signal, since internalization was induced by 2-deoxyglucose and inhibition of phosphoglucomutase by lithium prevented galactose- but not lactose- or glucose-induced internalization. Lac12-GFP internalization was not triggered by 6-deoxyglucose, and was irreversible in absence of protein synthesis.

Submitted by Nathália Faria da Silva (nathaliafsilva.ufv@gmail.com) on 2017-07-13T14:06:37Z No. of bitstreams: 1 texto completo.PDF: 366653 bytes, checksum: 8a45b52da863a243cba7f8275fc1ef4c (MD5) / Made available in DSpace on 2017-07-13T14:06:37Z (GMT). No. of bitstreams: 1 texto completo.PDF: 366653 bytes, checksum: 8a45b52da863a243cba7f8275fc1ef4c (MD5) Previous issue date: 2001-09-05 / Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A levedura Kluyveromyces marxianus var. lactis (K. lactis) foi cultivada em soro de queijo ultrafiltrado (SUF) em regimes de batelada e contínuo com o objetivo de investigar as condições fisiológicas que levam ao aumento e à queda da atividade de β-galactosidase na entrada da fase de desaceleração do crescimento. As fases fisiológicas do crescimento no cultivo em batelada foram caracterizadas, e observou-se que as concentrações iniciais de células de DO 600 0,1, 0,2 e 0,3 afetam a velocidade de crescimento, porém a delimitação das fases de crescimento é semelhante. A fase estacionária do crescimento foi apenas iniciada em 144 horas, o que permitiu uma longa fase de desaceleração do crescimento. O aumento e a queda na atividade de β-galactosidase foram acompanhados durante os cultivos, assim como a utilização de lactose e a formação e o consumo de etanol, além do perfil eletroforético da β- galactosidase intra e extracelular. Os picos de atividade máxima da enzima foram encontrados no final da fase log e no início da fase de desaceleração nas culturas conduzidas em regime de batelada e no cultivo contínuo na taxa de diluição de 0,09 h^-1. Nessas condições, as concentrações de lactose no meio não se correlacionaram com o máximo de atividade da enzima. Após a queda da atividade máxima, havia ainda lactose no meio e o etanol em concentrações crescentes. Desta forma, a queda na atividade não está relacionada com a exaustão de lactose no meio, nem com o crescimento diáuxico à custa do etanol, embora durante a fase de desaceleração do crescimento tenha sido observada diauxia quando a concentração de lactose era limitante. Outros picos de atividade foram evidenciados antes e após o pico máximo, onde foram obtidos os mesmos resultados. A β-galactosidase das amostras das culturas em batelada e contínua foi analisada em gel de poliacrilamida desnaturante e indicou a inexistência de uma relação direta entre a atividade da enzima e a concentração da proteína, com exceção nos tempos em que a atividade é máxima, quando houve aumento da intensidade da banda protéica no gel. Nas amostras de sobrenadante de ambas as bateladas e da cultura contínua submetidas à análise em gel, não se encontrou β-galactosidase, indicando que o etanol produzido não permeabilizou K. lactis. Cerca de 55 a 69% da lactose em ambos regimes de cultivo, foram convertidos em etanol. E, a variação cíclica da cinética de atividade durante o cultivo em regime de batelada e regime contínuo pode ser explicada pelos eventos de regulação da síntese e da atividade de β-galactosidase. / The yeast Kluyveromyces marxianus var. lactis (K. lactis) was cultivated in ultrafiltered cheese whey (UCW) in batch and continuous culture with the aim to investigate the physiological conditions that lead to the increase and decrease of the activity of β-galactosidase in the beginning of the late log phase of growth. The physiological growth phases were characterized in the batch culture, and it was observed that the initial cell concentration of OD 600 0,1, 0,2 e 0,3 affected the growt h velocity. However, the delimitation of the growth phases is similar. The stationary phase of growth started after 144 hours, which enabled a long late log phase of growth. The increase and the decrease in the β-galactosidase activity were monitored during the cultivation, as well as the use of lactose, the formation and consumption of ethanol as well as the electrophoretical profiles of intra cellular and extra cellular β- galactosidase. The peaks of maximum activity of the enzyme were found in the end of the log phase, and in the beginning of the late log phase in batch culture, and in the continuous culture at the dilution rate of 0,09 h^-1. Under these conditions, the lactose concentrations in the medium did not correlate with the maximum activity of the enzyme. After the maximum activity, there was still lactose in the medium and a rising concentration of ethanol. Therefore, the decrease of activity is not related to the exhaustion of lactose in the medium nor with the diauxic growth due to the ethanol. Albeit, during the late log phase of growth slight diauxic growth was observed when the lactose concentration was limiting. Other peaks of activity were noticed before and after the maximum peak, where the same results were obtained. β- galactosidase of the batch and continuous culture samples was analyzed in denaturing polyacrylamide gel and showed that a direct relationship between the enzyme activity and the protein concentration does not exist, except in the maximum activity times, when there was some increase in intensity in the proteic band of the gel. In the supernatant samples of both batch and continuous culture submitted to gel electrophoresis, β-galactosidase was not found, implying that the ethanol produced did not permeabilize K. lactis. Fifty five to sixty nine percent of the lactose in both cultures was converted to ethanol. And, the cyclical variation in the kinetic of the activity during the cultivation in batch and continuous culture can be explained through the regulation events of the synthesis and the activity.

This thesis was dedicated to the investigation of the interplay between signaling pathways governing cell wall biosynthesis, central carbohydrate metabolism and cytokinesis in two different yeast species. The first part of the thesis addressed cell wall biosynthesis, which forms an essential structure and determines both shape and integrity of fungal cells and hyphae. It also serves as a first barrier against changing and adverse environmental conditions. Cell wall synthesis and remodeling is primarily mediated by the cell wall integrity pathway in both the budding yeast Saccharomyces cerevisiae and the milk yeast Kluyveromyces lactis. The latter is a Crabtree-negative yeast with a similar life cycle to that of S. cerevisiae. Yet, K. lactis did not undergo a whole genome duplication, presenting a lower degree of genetic redundancy, which often avoids the need for multiple gene deletions. In this work the SNF1 kinase complex was identified as playing a role in cell wall synthesis. It belongs to the family of AMP-activated protein kinases (AMPKs), whose primary function is thought to be the regulation of energy balance in different organisms. Mutants with defects in this complex have a thinner cell wall than wild type and are hypersensitive to cell wall stress agents such as Caspofungin, Calcofluor white and Congo red. Epistasis analyses with mutants affecting cell wall integrity signaling suggested a parallel action of CWI- and SNF1 signaling in the two yeast species. Further genetic analyses indicated a known downstream effector of the SNF1 kinase complex, the transcriptional repressor Mig1, to mediate the signaling function in cell wall synthesis, too. Further epistasis analyses indicated that the hypersensitivity of the SNF1 complex mutants to the stress agents can be suppressed by an additional defect in the upper part of glycolysis. This has been attributed to the accumulation of the glycolytic intermediates glucose-6-phosphate and fructose-6-phosphate, which serve as precursors of cell wall polysaccharides. A function of the SNF1 complex in yeast cell wall synthesis has not been described, until now. In order to study the relation to cytokinesis, i.e. the last step of cell division, first some tools to follow this process in K. lactis had to be established in the second part of the thesis. Cytokinesis is also an essential feature of life since it ensures cell proliferation. In yeast and mammalian cells the concluding abscission of the plasma membrane is initiated by the construction of an actomyosin ring (AMR), accompanied by the formation of a primary septum, and followed by the deposition of secondary septa from both mother and daughter cells. Cytokinesis is a highly coordinated process and regulation is not yet fully explained. Proteins important for cytokinesis in S. cerevisiae were identified in K. lactis by in silico analyses and then genetically investigated. In contrast to S. cerevisiae, analysis of deletion mutants showed that deletion of the gene encoding K. lactis myosin II (KlMYO1) as a key component of the AMR is viable but temperature-sensitives and therefore dispensable for cytokinesis in K. lactis under normal growth conditions. Also different from its S. cerevisiae ortholog, a Klcyk3 deletion is lethal, while inn1 deletions are not viable in either yeast species. In contrast to the other genes studied, expression of INN1 does not cross-complement between the two yeast species, which could be narrowed down to a species specificity of the C2 domain in the Inn1 protein, which activates the chitin synthase II in S. cerevisiae. Deletions lacking the K. lactis chitin synthase II gene (KlCHS2) are also not viable. Fluorescently tagged versions of all proteins show a similar spatiotemporal localization at the bud neck as their counterparts in S. cerevisiae. The second part of this work thus provides insights into the regulation of cytokinesis in K. lactis and indicates that AMR constriction and its regulation could be more important in K. lactis than in S. cerevisiae, while the overall sequence of morphological events in cytokinesis is quite similar.

Le lactosérum et le perméat de lactosérum (WP) sont les principaux sous-produits du processus de fabrication du fromage et de la caséine. Ils sont considérés comme des polluants environnementaux en raison de leur charge organique élevée caractérisée par une haute demande biologique et chimique en oxygène. Ils créent un problème majeur d'élimination pour l'industrie laitière en raison des grands volumes de leur production annuelle. Par conséquent, il y a une demande constante de développer une approche durable pour leur utilisation afin d'éviter la pollution de l'environnement. Dans ce contexte, cette étude visait à comparer la technologie d'électro-activation (EA) à un processus d'isomérisation chimique, à alcalinité équivalente de la solution, pour produire du lactulose, qui est un prébiotique reconnu et éprouvé, en utilisant du lactose pur, du lactosérum et du perméat de lactosérum, comme matières premières sources de lactose, et de valoriser les produits électro-activés en produisant des métabolites à haute valeur ajoutée en utilisant une culture de kéfir et une culture pure de Kluyveromyces marxianus comme approche intégrée pour la valorisation complète de ces résidus de l'industrie laitière. La technologie d'électro-activation a été appliquée pour isomériser le lactose en lactulose dans un réacteur d'électro-activation modulé par des membranes échangeuses d'anions et de cations. L'électro-isomérisation du lactose en lactulose a été réalisée en utilisant des solutions de lactose (5, 10, 15 et 20 % p/v), de lactosérum (7, 14 et 21 % p/v) et de perméat de lactosérum (6, 12 et 18 % p/v) sous des intensités de courant électrique de 300, 600 et 900 mA pendant 60 min avec un intervalle d'échantillonnage de 5 min. L'isomérisation chimique conventionnelle a été réalisée à une alcalinité de la solution équivalente au KOH correspondant à celle mesurée dans les substrats électro-activés (lactose, lactosérum et perméat de lactosérum) à chaque intervalle de 5 min en utilisant de la poudre de KOH comme catalyseur à température ambiante (22 ± 2 °C). Les résultats obtenus ont montré que la production de lactulose en utilisant l'approche par électro-activation dépendait de l'intensité du courant électrique, de la concentration de la solution soumise à l'électro-activation et du temps de réaction. Les rendements les plus élevés de lactulose sont de 38 % en utilisant une solution de lactose de 10 % électro-activé pendant 40 min sous 900 mA, de 32 % en utilisant une solution de 7 % de lactosérum électro-activé sous 900 mA pendant 60 min et de 37 % en utilisant une solution de 6 % de perméat de lactosérum électro-activé sous 900 mA pendant 50 min. Parallèlement, les résultats ont montré qu'avec une approche chimique conventionnelle avec du KOH comme catalyseur, les rendements de lactulose étaient de ~27 % en utilisant une solution de 10 % de lactose pendant 60 min et de 25,47 % en utilisant une solution de 6 % de perméat de lactosérum pendant 50 min. Cependant, aucune formation de lactulose n'a été observée en utilisant du lactosérum dans le procédé chimique conventionnel à une alcalinité équivalente de la solution traitée par électro-activation. Les résultats de cette étude ont révélé que la technologie d'électro-activation est plus efficace pour la production du lactulose à partir du lactose pur, du lactosérum et du perméat de lactosérum par rapport au processus d'isomérisation chimique conventionnelle. Par la suite, la faisabilité d'utiliser les substrats à base de lactose électro-activé, du lactosérum électro-activé et du perméat de lactosérum électro-activé comme sources de carbone pour produire de la biomasse riche en protéines et métabolites à valeur commerciale élevée comme des acides organiques (lactique, acétique, citrique et propionique) et des biomolécules aux propriétés aromatiques et gustatives a été étudiée en utilisant une culture microbienne mixte provenant de grains de kéfir comme ferment et une culture pure de Kluyveromyces marxianus. La fermentation a été réalisée pendant 96 h à 30 °C en utilisant les substrats électro-activés et non électro-activés du lactose, du lactosérum et du perméat de lactosérum. Les résultats obtenus ont montré que les substrats électro-activés ont permis d'atteindre une croissance de la biomasse la plus élevée en un temps de fermentation réduit comparativement aux substrats non électro-activés en utilisant la culture de kéfir comme agent de fermentation. La croissance cellulaire la plus élevée (6,04 g/L) a été obtenue dans le lactosérum électro-activé après 72 h, qui était 1,7 fois supérieure à ce qui était obtenu dans le milieu clostridien renforcé (RCM). De plus, le lactosérum électro-activé a permis de produire un maximum de 8,46, 3,97, 0,60 et 1,02 g/L d'acide lactique, acétique, citrique et propionique, respectivement. De plus, le lactosérum électro-activé a permis la production de kéfiran la plus élevée de 2,99 g/L, suivi par le lactosérum (2,67 g/L), le perméat de lactosérum électro-activé (2,31 g/L), le perméat de lactosérum (1,88 g/L), le milieu RCM (1,42 g /L), le lactose électro-activé (1,37 g/L) et le lactose (0,91 g/L). Les résultats ont également démontré que divers composés aromatiques volatils étaient produits au cours de la fermentation du lactosérum électro-activé, ce qui peut améliorer les caractéristiques organoleptiques et la qualité sensorielle des produits fermentés. Également, K. marxianus a également montré une production satisfaisante de la biomasse dans tous les substrats utilisés et que le lactosérum électro-activé a permis d'atteindre une biomasse maximale (4,23 g/L) après 96 h de fermentation, suivie du milieu standard YM (4,85 g/L). La biomasse produite avait une teneur élevée en protéines et en lipides (24,43-57,83 et 15,44-25,64 %, respectivement) dépendamment des substrats utilisés et des conditions de fermentation. Plusieurs acides organiques majeurs comme les acides lactique, acétique, citrique et propionique ont été produits pendant la fermentation sur tous les milieux, avec des différences significatives entre les substrats électro-activés et non électro-activés. De plus, K. marxianus a produit divers composés aromatiques volatils aux propriétés organoleptiques appréciées. Le milieu de culture YM a entraîné la plus faible production d'éthanol (8,42 g/L à 48 h) tandis que la plus forte production d'éthanol a été produite dans le lactosérum non électro-activé (28,13 g/L à 48 h), suivi du lactose (27,85 g/L à 48 h), du lactose électro-activé (26,77 g/L à 36 h), du perméat de lactosérum (25,99 à 72 h), du perméat de lactosérum électro-activé (24,66 g/L à 36 h) et du lactosérum électro-activé(22,06 g/L à 48 h). De plus, un maximum de 393,85 à 988,22 mg/L de 2-phényléthanol a été atteint, selon les substrats utilisés. Par conséquent, les résultats de ce projet suggèrent que la technologie d'électro-activation peut être une approche durable émergente permettant d'atteindre le double objectif de production de lactulose, un prébiotique reconnu et éprouvé, et de valorisation intégrale du lactosérum et de ses dérivés en utilisant des bioprocédés à base de culture de kéfir et de K. marxianus pour produire des métabolites à valeur commerciale élevée pour différentes applications; y compris pour l'industrie de l'alimentation humaine et animale. Ainsi, les connaissances obtenues dans ce projet pourront servir à améliorer la valorisation du lactosérum. / Whey and whey permeate (WP) are the main agro-industrial by-products from cheese or casein production process that are regarded as environmental pollutants because of their high organic load (high biochemical and chemical oxygen demand) and are creating a major disposal problem for the dairy industry. Consequently, there is a serious demand of developing a sustainable approach for their utilization to evade environmental pollution. In this context, the study was intended to compare the electro-activation (EA) technology with a chemical isomerization process at equivalent solution alkalinity to produce a prebiotic lactulose using lactose, whey, and WP as feedstocks and to valorize the electro-activated materials into valuable metabolites using a whole Kefir culture and a pure culture of Kluyveromyces marxianus as an integrated approach for complete valorization of these waste products. The EA technique was applied to isomerize lactose into lactulose in an EA react or modulated by anion and cation exchange membranes. Electro-isomerization of lactose into lactulose was performed by using lactose (5, 10, 15, and 20%, w/v), whey (7, 14, and 21%, w/v), and WP (6, 12, and 18%, w/v) solutions under current intensities of 300, 600, and 900 mA during 60 min with a sampling interval of 5 min. The conventional chemical isomerization was carried out at the KOH-equivalent solution alkalinity corresponding to that measured in the electro-activated lactose (EA-lactose), electro-activated whey (EA-whey), electro-activated whey permeate (EA-WP) solutions at each 5 min interval using KOH powder as a catalyst at ambient temperature (22 ± 2 °C). The results showed that the production of lactulose using the EA approach was current intensity-, solution concentration-, and reaction time-dependent. The highest lactulose yields of 38 (at 40 min for a 900 mA and 10% lactose solution), 32 (at 60 min for a 900 mA and 7% whey solution), and 36.98% (at 50 min for a 900 mA and 6% WP solution) were achieved for lactose, whey, and WP, respectively. Whereas the maximum lactulose yields of ~27 (at 60 min for 10% lactose solution) and 25.47% (at 50 min for 6% WP solution) were obtained for lactose and WP, respectively. However, no lactulose was produced for whey using the chemical process at the equivalent solution alkalinity as in the EA technique. The outcomes of this study revealed that the EA technology is a more efficient technique for the enhanced production of lactulose from lactose, whey, and WP compared to the convention chemical isomerization process. Thereafter, the feasibility of using electro-activated whey-based substrates including EA-lactose, EA-whey, EA-WP as carbon sources to produce protein enriched biomass and valuable metabolites including organic acids (i.e., lactic, acetic, citric, and propionic acids) and biomolecules with aroma and flavor properties was studied using a mixed microbiota originated from whole kefir grains as a starter culture and a pure culture of Kluyveromyces marxianus ATCC 64884. Fermentation was performed for 96 h at 30 °C using both electro-activated (EA) and non-electroactivated (non-EA) substances of lactose, whey, and WP. The results showed that the EA-substrates achieved a higher biomass growth in a reduced fermentation time than their non-EA mediums using the kefir culture. The highest cell growth (6.04 g/L) was obtained for EA-whey after 72 h which was even 1.7-fold higher than a standard nutrition broth, the reinforced clostridial medium (RCM). Furthermore, EA-whey produced a maximum of 8.46, 3.97, 0.60, and 1.02 g/L of lactic, acetic, citric, and propionic acid, respectively. Moreover, EA-whey achieved the highest kefiran production of 2.99 g/L, followed by the whey (2.67 g/L), EA-WP (2.31 g/L), WP (1.88 g/L), RCM broth (1.42 g/L), EA-lactose (1.37 g/L), and lactose (0.91 g/L). The results also demonstrated that various aromatic volatile compounds were produced during the fermentation of EA-whey, which may increase the organoleptic characteristic/sensory quality of the fermented products. Nevertheless, K. marxianus also demonstrated a satisfactory biomass growth in all substrates used and EA-whey achieved a maximum biomass (4.23 g/L) at 96 h of fermentation followed by YM broth (4.85 g/L). The produced biomass had high protein and lipid content (24.43-57.83, and 15.44-25.64%) depending on the used substrates and fermentation conditions. Several major organic acids including lactic, acetic, citric, propionic acids were produced during the fermentation on all media, with significant differences between electro-activated and non-electro-activated substrates. Furthermore, K. marxianus produced various volatile aroma compounds with valued organoleptic properties. The YM-broth resulted in the lowest ethanol production (8.42 g/L at 48 h) while the highest ethanol was produced in the non-electro-activated whey (28.13 g/L at 48 h), followed by lactose (27.85 g/L at 48 h), EA-lactose (26.77 g/L at 36 h), WP (25.99 at 72 h), EA-WP (24.66 g/L at 36 h), EA-Whey (22.06 g/L at 48 h). Moreover, a maximum of 393.85 to 988.22 mg/L of 2-phenylethanol was achieved, depending on the substrates used. Therefore, the results of this work suggest that the EA technology can be an emergent sustainable technology for achieving dual objectives of prebiotic lactulose production and concurrent valorization of whey and its derivatives in Kefir culture and K. marxianus driven bioprocesses to produce valuable metabolites for different applications including in food and feed industry. Thus, this knowledge is not only helpful to reduce the production cost of dairy industries, but also provide an eco-friendly alternative for the disposal of whey/WP as a part of integrated approach for complete valorization.

Made available in DSpace on 2014-12-17T15:01:51Z (GMT). No. of bitstreams: 1 MariaVPR_TESE_1-170.pdf: 4608142 bytes, checksum: 9ed83cbe76a127f48714302cd74c674a (MD5) Previous issue date: 2010-11-22 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Recently, global demand for ethanol fuel has expanded very rapidly, and this should further increase in the near future, almost all ethanol fuel is produced by fermentation of sucrose or glucose in Brazil and produced by corn in the USA, but these raw materials will not be enough to satisfy international demand. The aim of this work was studied the ethanol production from cashew apple juice. A commercial strain of Saccharomyces cerevisiae was used for the production of ethanol by fermentation of cashew apple juice. Growth kinetics and ethanol productivity were calculated for batch fermentation with different initial sugar (glucose + fructose) concentration (from 24.4 to 103.1 g.L-1). Maximal ethanol, cell and glycerol concentrations (44.4 g.L-1, 17.17 g.L-1, 6.4 g.L-1, respectively) were obtained when 103.1 g.L-1 of initial sugar concentration were used, respectively. Ethanol yield (YP/S) was calculated as 0.49 g (g glucose + fructose)-1. Pretreatment of cashew apple bagasse (CAB) with dilute sulfuric acid was investigated and evaluated some factors such as sulfuric acid concentration, solid concentration and time of pretreatment at 121?C. The maximum glucose yield (162.9 mg/gCAB) was obtained by the hydrolysis with H2SO4 0.6 mol.L-1 at 121?C for 15 min. Hydrolysate, containing 16 ? 2.0 g.L-1 of glucose, was used as fermentation medium for ethanol production by S. cerevisiae and obtained a ethanol concentration of 10.0 g.L-1 after 4 with a yield and productivity of 0.48 g (g glucose)-1 and 1.43 g.L-1.h-1, respectively. The enzymatic hydrolysis of cashew apple bagasse treated with diluted acid (CAB-H) and alkali (CAB-OH) was studied and to evaluate its fermentation to ethanol using S. cerevisiae. Glucose conversion of 82 ? 2 mg per g CAB-H and 730 ? 20 mg per g CAB-OH was obtained when was used 2% (w/v) of solid and loading enzymatic of 30 FPU/g bagasse at 45 ?C. Ethanol concentration and productivity was achieved of 20.0 ? 0.2 g.L-1 and 3.33 g.L-1.h-1, respectively when using CAB-OH hydrolyzate (initial glucose concentration of 52.4 g.L-1). For CAB-H hydrolyzate (initial glucose concentration of 17.4 g.L-1), ethanol concentration and productivity was 8.2 ? 0.1 g.L-1 and 2.7 g.L-1.h-1, respectively. Hydrolyzates fermentation resulted in an ethanol yield of 0.38 g/g glucose and 0.47 g/g glucose, with pretreated CABOH and CAB-H, respectively. The potential of cashew apple bagasse as a source of sugars for ethanol production by Kluyveromyces marxianus CE025 was evaluated too in this work. First, the yeast CE025 was preliminary cultivated in a synthetic medium containing glucose and xylose. Results showed that it was able to produce ethanol and xylitol at pH 4.5. Next, cashew apple bagasse hydrolysate (CABH) was prepared by a diluted sulfuric acid pre-treatment. The fermentation of CABH was conducted at pH 4.5 in a batch-reactor, and only ethanol was produced by K. marxianus CE025. The influence of the temperature in the kinetic parameters was evaluated and best results of ethanol production (12.36 ? 0.06 g.L-1) was achieved at 30 ?C, which is also the optimum temperature for the formation of biomass and the ethanol with a volumetric production rate of 0.25 ? 0.01 g.L-1.h-1 and an ethanol yield of 0.42 ? 0.01 g/g glucose. The results of this study point out the potential of the cashew apple bagasse hydrolysate as a new source of sugars to produce ethanol by S. cerevisiae and K. marxianus CE025. With these results, conclude that the use of cashew apple juice and cashew apple bagasse as substrate for ethanol production will bring economic benefits to the process, because it is a low cost substrate and also solve a disposal problem, adding value to the chain and cashew nut production / Recentemente, a demanda mundial por etanol combust?vel tem se expandido de forma muito r?pida, sendo quase todo etanol combust?vel ? produzido por fermenta??o de sacarose no Brasil ou glicose de milho nos Estados Unidos, por?m, estas mat?rias-primas n?o ser?o suficientes para satisfazer a demanda internacional. Neste contexto, o objetivo deste trabalho foi avaliar a produ??o de bioetanol a partir do ped?nculo de caju. Para tal fim, inicialmente, estudou-se a produ??o de etanol utilizando o suco de caju como fonte de carbono, avaliando a influ?ncia da concentra??o inicial de substrato por Saccharomyces cerevisiae. Nessa etapa, os melhores resultados foram utilizando uma concentra??o inicial de a??car de 87,71 g.L-1 obtendo a concentra??o m?xima de etanol de 42,8 ? 3 g.L-1 com uma produtividade de 9,71 g.L-1.h-1 e rendimento de etanol de 0,49 g etanol/g glicose + frutose. Posteriormente, estudou-se a produ??o de etanol utilizando como material lignocelul?sico o baga?o de caju (CAB) que continha 20,9% celulose, 16,3% hemicelulose e 33,6% lignina + cinzas. Inicialmente estudou-se o pr?-tratamento do CAB com ?cido sulf?rico dilu?do avaliando-se diferentes par?metros, obtendo as maiores concentra??es dos a??cares glicose (22,8 ? 1,5 g.L-1) e xilarabin (arabinose + xilose plus, 29,2 ? 2,4 g.L-1), na fra??o l?quida (CAB-H), no pr?-tratamento conduzido em autoclave a 121?C por 15 min usando H2SO4 0,6 mol.L-1 e 30% m/v de CAB, com rendimentos de glicose, xilarabin e a??cares totais de 75,99 ? 5,0, 97,17 ? 8,1 e 173,16 ? 13,0 mg.(g de baga?o)-1, respectivamente. A convers?o obtida nesse pr?-tratamento com base na percentagem de celulose e hemicelulose do CAB foi 322,1 ? 20,1 mg glicose.(g celulose)-1 e 514,1 ? 43,1 mg xilarabin.(g hemicelulose)-1. Na fermenta??o do hidrolisado CAB-H por S. cerevisiae obteve-se 10 g.L-1 de etanol ap?s 4 horas de cultivo, com rendimento de 0,48 g.(g glicose)-1 e produtividade de 2,62 g.L-1h-1. Ap?s, estudou-se a hidr?lise enzim?tica do CAB ap?s pr?-tratamento com H2SO4 dilu?do (CAB-H) e alcalino (CAB-OH) e a fermenta??o dos hidrolisados por S. cerevisiae para produzir etanol. Uma convers?o de glicose de 82 ? 2 mg.(gCAB-H)-1 e 730 ? 20 mg.(gCAB-OH)-1 foi obtida utilizando 2% (m/v) de s?lidos e carga enzim?tica de 30 FPU.(g baga?o)-1 a 45?C. Na fermenta??o conduzida com o hidrolisado obtido da hidr?lise enzim?tica do CAB-OH, obteve-se uma concentra??o de etanol, produtividade e rendimento de 20,0 ? 0,2 g.L-1, 3,33 g.L-1.h-1 e 0,38 g.(g de glicose)-1, respectivamente. Para o hidrolisado da hidr?lise do CAB-H, a concentra??o de etanol foi 8,2 ? 0,1 g.L-1 com 2,7 g.L-1.h-1 de produtividade e rendimento de 0,47 g.(g glicose)-1 em 3 h de ensaio. O potencial do baga?o de caju como fonte de a??cares para a produ??o de etanol por Kluyveromyces marxianus CE025 tamb?m foi avaliado e verificou-se a influ?ncia da temperatura nos par?metros cin?ticos, sendo os ensaios conduzidos em batelada a pH 4,5, utilizado o hidrolisado (CAB-H) como fonte de carbono. Os melhores resultados para a produ??o de etanol foram a 30?C, coincidindo com a temperatura ?tima de crescimento, resultando em 12,36 ? 0,06 g.L-1 de etanol, com uma taxa volum?trica de produ??o de 0,26 ? 0,01 g.L-1.h-1 e rendimento de 0,42 ? 0,01 g.(g de glicose)- 1. Os resultados apresentados demonstram o potencial do ped?nculo de caju (suco e baga?o) como nova fonte de carbono para produzir etanol por S. cerevisiae e K. marxianus CE025

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A imobilizaÃÃo de β-galactosidase para hidrÃlise de lactose à uma proposta para agregar valor ao soro de leite com conseqÃente produÃÃo de galactose e glicose. O objetivo deste trabalho foi desenvolver biocatalisadores a partir de diferentes suportes orgÃnicos e protocolos de ativaÃÃo visando à hidrÃlise de lactose proveniente do soro de leite. Inicialmente, prepararam-se os suportes a serem aplicados no estudo como quitosana 2,5% (m/v) (sem e com prÃ-tratamento com dimetilformamida) e 2,0% (m/v), quitosana-alginato-epoxilado (QAE), bagaÃo de caju (BC) e fibra de casca de coco verde (CV), os quais foram ativados de diferentes formas, com glutaraldeÃdo, epicloridrina ou glicidol. Na primeira etapa, determinaram-se o rendimento de imobilizaÃÃo, atividade recuperada e atividade aparente dos diferentes derivados obtidos para assim determinar os seis melhores â quitosana 2,5% (m/v) ativada com glutaraldeÃdo (QUITGLU1), quitosana 2,0% (m/v) coagulada com KOH a 50ÂC ativada com glutaraldeÃdo (QUITGLU2) ou epicloridrina (QUITEPI) ou glicidol (QUITGLI), quitosana 2,5% (m/v) tratada com dimetilformamida ativada com epicloridrina (QUIT-DMFEPI) ou glicidol (QUIT-DMFGLI). Para segunda fase, os catalisadores (QUITGLU1, QUITGLU2, QUITEPI, QUITGLI) foram estudados quanto à estabilidade operacional com o uso de reator contÃnuo, assim como ensaios de carga mÃxima e efetividade. Baseado nestes ensaios determinou-se QUITGLU2 como melhor biocatalisador e realizaram-se os seguintes estudos: variaÃÃo do tempo de imobilizaÃÃo, determinaÃÃo da melhor temperatura e pH para atividade enzimÃtica, determinaÃÃo de parÃmetros cinÃticos, estocagem sob 10ÂC e estabilidade operacional com o uso de alta carga enzimÃtica usando soro de leite como substrato. Suportes como CV e BC nÃo apresentaram boa adequaÃÃo para imobilizaÃÃo de β-galactosidase de Kluyveromyces lactis, assim como o suporte QAE. Suportes com tratamento com dimetilformamida apresentaram baixos rendimentos de imobilizaÃÃo. Os resultados para o derivado QUITGLU2 apresentaram carga mÃxima de 75 mgProteÃna.g-1 de suporte e efetividade superiores aos demais. A estabilidade operacional para este derivado apresentou-se estÃvel, visto sua produÃÃo de glicose constante por 10 h de reaÃÃo. O tempo 3 h mostrou-se suficiente para imobilizaÃÃo. Os valores de Km e VmÃx tanto para enzima solÃvel (46,79 mM e 7.142,86 μmol.(mL.min)-1) quanto para o derivado (69,56 mM e 113,25 μmol.(g.min)-1). Durante os 120 dias de armazenamento sob 10ÂC, nÃo houve decrÃscimo da atividade hidrolÃtica do derivado, demonstrando Ãtima estabilidade à estocagem. Por fim, o biocatalisador mostrou bons resultados de estabilidade operacional quando utilizado em alta carga oferecida (255,9 mgProteÃna.g-1 de quitosana de carga teoricamente imobilizada) para hidrÃlise de soro de leite / β-galactosidase immobilization was studied seeking to add value to cheese whey trough lactose hydrolyze producing galactose and glucose. This work aimed to develop biocatalysts using different organic supports and activation protocols. Firstly, some supports were prepared as chitosan 2.5% (w/v) (with and without pretreatment with dimethylformamide) and 2.0% (w/v), chitosan-alginate-epoxide (QAE), cashew bagasse (BC) and coconut shell fiber (CV), which were activated in different ways with glutaraldehyde, epichlorohydrin or glycidol. Initially, it was determined the immobilization yield, couple yield and apparent activity from obtained catalysts, being chosen six derivatives according to better results parameters: 2.5% chitosan (w/v) glutaraldehyde activated (QUITGLU1), 2.0% chitosan (w/v) KOH coagulated at 50ÂC glutaraldehyde activated (QUITGLU2) and epichlorohydrin (QUITEPI) or glycidol (QUITGLI), chitosan 2.5% (w/v) dimethylformamide treated with epichlorohydrin (QUIT-DMFEPI) or glycidol (QUIT-DMFGLI). Thus, catalysts (QUITGLU1, QUITGLU2, QUITEPI, QUITGLI) were studied as operational stability by using a continuous reactor, as well as, maximum enzyme loading and effectiveness assays. Then, it was determined QUITGLU2 as the best biocatalyst and following studies were carried out: immobilization time, enzyme optimum temperature and pH, kinetic parameters using lactose as substrate at 37ÂC, storage at 10ÂC and operational stability using high load enzyme and cheese whey as substrate. CV and BC supports did not present good results for β-Kluyveromyces lactis galactosidase immobilization, as well as, QAE support. Supports treated with dimethylformamide presented low immobilization yields. The results for QUITGLU2 derivative presented maximum loading of 75 mgProtein.g-1support and higher effectiveness than others. The operational stability for this derivative remained stable, with constant glucose production for 10 h of reaction. Immobilization time of 3h proved enough for the process. The Km and VmÃx values were respectively: free enzyme (46.79 mM and 7,142.86 μmol.(mL.min)-1) and catalyst (69.56 mM and 113.25 μmol.(g.min)-1). During 120 days of storage at 10ÂC, no decrease derivative hydrolitic activity was noted, demonstrating satisfactory storage stability. Finally, the biocatalyst showed good results as operational stability when used high offered enzyme load (theoretically immobilized load 255.9 mgProtein.g-1chitosan) for cheese whey hydrolysis