Orientador: Edwil Aparecida de Lucca Gattás / Banca: Eleonora Cano Carmona / Banca: Rubens Monti / Resumo: A levedura Pichia pastoris vem sendo largamente utilizada como um eficiente sistema de expressão para a produção de proteínas heterólogas, pois é um sistema seguro, fácil e mais barato que sistemas de expressão de outros eucariotos. Neste trabalho, a enzima de interesse é a glicerol quinase (GK), que cataliza a transferência do fosfato terminal do ATP para o glicerol originando glicerol-3-fosfato e ADP. Esta reação pode ser utilizada na determinação da concentração de glicerol, subproduto da fermentação alcoólica. A leitura do consumo de glicerol é realizada pela determinação espectrofotométrica do NADH gerado na reação de oxido-redução catalizada pela enzima glicerol-3-fosfato desidrogenase. Este estudo de indução foi realizado em diferentes condições de crescimento da levedura Pichia pastoris. Os resultados mostraram a seleção do melhor clone da levedura Pichia pastoris para a expressão extracelular da enzima glicerol quinase, e a determinação das melhores condições do meio de cultura para a produção da enzima de interesse foram: concentração do meio de cultura BMMY (20 vezes), densidade inicial de célula (0,1 mg/mL), concentração de metanol na fase de indução (1%), natureza do tampão (fosfato de potássio), pH (6,0), suplementação de glicerol no meio BMMY (1%), peptona (marca Difco), sem adição de sulfato de amônio, caseína e glicina, uso do meio BMMY e liofilização do mesmo. Estudos de parâmetros cinéticos foram realizados e a atividade máxima da GK foi obtida em pH 9,8, a 50ºC e 2,5 μM de substrato, por metodologia clássica, além da presença de sulfato de magnésio e diluição da enzima de 30 vezes. A enzima apresentou alta estabilidade térmica ― a atividade foi completamente... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The yeast Pichia pastoris has been widely used as an efficient expression system for production of heterologous proteins because it is a safe, easy and cheaper than expression systems in other eukaryotes.In this studie, the enzyme of interest is glycerol kinase (GK), which catalizes the transfer of terminal phosphate from ATP to glycerol resulting glycerol-3-phosphate and ADP. This reaction can be used in determining the concentration of glycerol, a byproduct of fermentation. The reading of the consumption of glycerol is carried out by spectrophotometric determination of NADH generated in the redox reaction catalyzed by the enzyme glycerol-3-phosphate dehydrogenase. This study of induction was performed in different conditions of growth of the yeast Pichia pastoris. The results show that selecting the best clone of the yeast Pichia pastoris for the expression of extracellular enzyme glycerol kinase, and determining the best conditions of the culture medium for producing the enzyme of interest were: concentration of the culture medium BMMY (20 times), initial cell density (0.1 mg/mL), methanol concentration in the induction phase (1%), nature of buffer (potassium phosphate), pH (6.0), glycerol supplementation in BMMY medium (1%), peptone (Difco), without addition of ammonium sulfate, casein and glycine in BMMY and lyophilized medium. Studies of kinetic parameters were conducted and the GK maximum activity was obtained at pH 9.8 at 50°C and 2.5 μM substrate by conventional method, besides the presence of magnesium sulfate and diluting the enzyme 30 times. The enzyme showed high thermal stability - the activity was fully maintained up to 50°C for one hour - and at pH 7.0 for 7 days and kept under refrigeration, freeze-dried extract showed a decrease in enzymatic activity. Calculated by... (Complete abstract click electronic access below) / Mestre

There has been progress towards elucidating the mechanism of Escherichia coli glycerol kinase (EcGK) control by its allosteric effectors fructose-1,6-bisphosphate (FBP) and IIAglc (a member of the phosphoenolpyruvate:glycose phosphotransferase system). Determining the mechanism requires analysis of the interaction between these effectors and the substrates of EcGK. In this study, a structural and kinetic approach was used to determine inhibition by both the effectors. For this work, the use of fluorescence anisotropy to observe ligand binding was investigated. Also, a foundation was laid for future NMR experiments with EcGK. For fluorescence studies, E36C EcGK was labeled with fluorescein and tested for changes in anisotropy in the presence of different ligands. To ensure that E36C was an appropriate representative of wildtype protein, initial velocity, inhibition, and heterotropic coupling assays were performed. Groundwork for future NMR experiments required analyzing substitutions of the native EcGK cysteines by initial velocity and inhibition studies. By comparing wildtype enzyme and E36C (variant of wildtype with an engineered cysteine residue at position 36), it was found that E36C is a suitable substitute and was not drastically affected by labeling with fluorescein. Anisotropy values differed upon binding of different ligands and enabled titrations of the enzyme substrate complexes with both effectors to obtain dissociation constants. This supports using the stopped-flow method to assess the on- and off- rates of substrates and to obtain values for Q coupling. Furthermore, the results for FBP showed that inhibition by FBP is K-type (affects affinity) with respect to ATP and V-type (affects enzyme velocity) with respect to ADP. The findings presented also showed that native cysteine substitutions effect some of the catalytic and allosteric parameters of EcGK and would be powerful reporters for ligand binding in NMR. However, the enzymes are unstable and new protocols for protein isolation will need to be drafted.

Made available in DSpace on 2014-06-11T19:23:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-12Bitstream added on 2014-06-13T18:09:34Z : No. of bitstreams: 1 aizemberg_r_me_arafcf.pdf: 685654 bytes, checksum: d058342c2d66649275266dc34dcb968d (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Universidade Estadual Paulista (UNESP) / A levedura Pichia pastoris vem sendo largamente utilizada como um eficiente sistema de expressão para a produção de proteínas heterólogas, pois é um sistema seguro, fácil e mais barato que sistemas de expressão de outros eucariotos. Neste trabalho, a enzima de interesse é a glicerol quinase (GK), que cataliza a transferência do fosfato terminal do ATP para o glicerol originando glicerol-3-fosfato e ADP. Esta reação pode ser utilizada na determinação da concentração de glicerol, subproduto da fermentação alcoólica. A leitura do consumo de glicerol é realizada pela determinação espectrofotométrica do NADH gerado na reação de oxido-redução catalizada pela enzima glicerol-3-fosfato desidrogenase. Este estudo de indução foi realizado em diferentes condições de crescimento da levedura Pichia pastoris. Os resultados mostraram a seleção do melhor clone da levedura Pichia pastoris para a expressão extracelular da enzima glicerol quinase, e a determinação das melhores condições do meio de cultura para a produção da enzima de interesse foram: concentração do meio de cultura BMMY (20 vezes), densidade inicial de célula (0,1 mg/mL), concentração de metanol na fase de indução (1%), natureza do tampão (fosfato de potássio), pH (6,0), suplementação de glicerol no meio BMMY (1%), peptona (marca Difco), sem adição de sulfato de amônio, caseína e glicina, uso do meio BMMY e liofilização do mesmo. Estudos de parâmetros cinéticos foram realizados e a atividade máxima da GK foi obtida em pH 9,8, a 50ºC e 2,5 μM de substrato, por metodologia clássica, além da presença de sulfato de magnésio e diluição da enzima de 30 vezes. A enzima apresentou alta estabilidade térmica ― a atividade foi completamente... / The yeast Pichia pastoris has been widely used as an efficient expression system for production of heterologous proteins because it is a safe, easy and cheaper than expression systems in other eukaryotes.In this studie, the enzyme of interest is glycerol kinase (GK), which catalizes the transfer of terminal phosphate from ATP to glycerol resulting glycerol-3-phosphate and ADP. This reaction can be used in determining the concentration of glycerol, a byproduct of fermentation. The reading of the consumption of glycerol is carried out by spectrophotometric determination of NADH generated in the redox reaction catalyzed by the enzyme glycerol-3-phosphate dehydrogenase. This study of induction was performed in different conditions of growth of the yeast Pichia pastoris. The results show that selecting the best clone of the yeast Pichia pastoris for the expression of extracellular enzyme glycerol kinase, and determining the best conditions of the culture medium for producing the enzyme of interest were: concentration of the culture medium BMMY (20 times), initial cell density (0.1 mg/mL), methanol concentration in the induction phase (1%), nature of buffer (potassium phosphate), pH (6.0), glycerol supplementation in BMMY medium (1%), peptone (Difco), without addition of ammonium sulfate, casein and glycine in BMMY and lyophilized medium. Studies of kinetic parameters were conducted and the GK maximum activity was obtained at pH 9.8 at 50°C and 2.5 μM substrate by conventional method, besides the presence of magnesium sulfate and diluting the enzyme 30 times. The enzyme showed high thermal stability - the activity was fully maintained up to 50°C for one hour - and at pH 7.0 for 7 days and kept under refrigeration, freeze-dried extract showed a decrease in enzymatic activity. Calculated by... (Complete abstract click electronic access below)

Previous studies on inhibition of the Escherichia coli glycerol kinase enzyme have suggested that subunit-subunit or domain bridging interactions form part of the network in communicating ligand binding to inhibition. In this study, five amino acids were identified to be in close proximity to an Arg369 residue which is a domain bridging residue. Three of the amino acid residues (Q37, Y39 and Q104) are in domain I of the enzyme subunit, while the other two (M308 and Q314) are in domain II of the enzyme subunit. To evaluate the importance of each domain bridging residue in IIAGlc inhibition, alanine substitutions were made of the residues, and the kinetic properties characterized with respect to IIAGlc inhibition. Kinetic parameters obtained for each variant glycerol kinase enzyme was compared to values obtained for the Wild Type enzyme to assess the importance of the amino acid residue in IIAGlc inhibition. The effects of the substitutions on FBP inhibition as well as catalysis of the enzyme were also analyzed by obtaining kinetic parameters for each of the variant enzymes. The results from this study indicate that the domain I bridging interactions with Arg369 are important in IIAGlc regulation of the E. coli glycerol kinase enzyme. The domain II bridging interactions appear to be unimportant in regulating IIAGlc inhibition. Two of the domain I bridging residues studied were also found to be important in FBP inhibition. These results indicate that some the domain bridging residues seen to be involved in IIAGlc regulation also appear to be involved in FBP regulation. In catalysis, with the exception of Q314, the rest of the domain I and II bridging residues appear to be important for substrate binding and/or catalysis.

Glycerol kinase (GK) from Escherichia coli is allosterically controlled by fructose 1,6-bisphosphate (FBP) and the glucose-specific phosphocarrier protein IIAGlc of the phosphotransferase system. These controls allow glucose to regulate glycerol utilization. Fluorescence spectroscopic and enzyme kinetic methods are applied to investigate these allosteric controls in this study. The linkage between FBP binding and GK tetramer assembly is solved by observation of homo-fluorescence energy transfer of the fluorophore Oregon Green (OG) attached specifically to an engineered surface cysteine in GK. FBP binds to tetramer GK with an affinity 4000-fold higher than to dimeric GK. A region named the coupling locus that plays essential roles in the allosteric signal transmission from the IIAGlc binding site to the active site was identified in GK. The relationship between the coupling locus sequence in Escherichia coli or Haemophilus influenzae GK variants and the local flexibility of the IIAGlc binding site is established by fluorescence anisotropy determinations of the OG attached to the engineered surface cysteine in each variant. The local flexibility of the IIAGlc binding site is influenced by the coupling locus sequence, and in turn affects the binding affinity for IIAGlc. Furthermore, the local dynamics of each residue in the IIAGlc binding site of GK is studied systematically by the fluorescence anisotropy measurements of OG individually attached to each position of the IIAGlc binding site. The fluorescence steady-state anisotropy measurement provides a valid estimation of the local flexibility and correlates well with the crystallographic B-factors. Steady-state kinetics of FBP inhibition shows that the data are best described by a model in which the partial inhibition and FBP binding stoichiometry are taken into account. Kinetic viscosity effects show that the product-release step is not the purely rate-limiting step in the GK-catalyzed reaction. Viscosity effects on FBP inhibition are also discussed.

Glycerol kinase (GK) from Escherichia coli is allosterically controlled by fructose 1,6-bisphosphate (FBP) and the glucose-specific phosphocarrier protein IIAGlc of the phosphotransferase system. These controls allow glucose to regulate glycerol utilization. Fluorescence spectroscopic and enzyme kinetic methods are applied to investigate these allosteric controls in this study. The linkage between FBP binding and GK tetramer assembly is solved by observation of homo-fluorescence energy transfer of the fluorophore Oregon Green (OG) attached specifically to an engineered surface cysteine in GK. FBP binds to tetramer GK with an affinity 4000-fold higher than to dimeric GK. A region named the coupling locus that plays essential roles in the allosteric signal transmission from the IIAGlc binding site to the active site was identified in GK. The relationship between the coupling locus sequence in Escherichia coli or Haemophilus influenzae GK variants and the local flexibility of the IIAGlc binding site is established by fluorescence anisotropy determinations of the OG attached to the engineered surface cysteine in each variant. The local flexibility of the IIAGlc binding site is influenced by the coupling locus sequence, and in turn affects the binding affinity for IIAGlc. Furthermore, the local dynamics of each residue in the IIAGlc binding site of GK is studied systematically by the fluorescence anisotropy measurements of OG individually attached to each position of the IIAGlc binding site. The fluorescence steady-state anisotropy measurement provides a valid estimation of the local flexibility and correlates well with the crystallographic B-factors. Steady-state kinetics of FBP inhibition shows that the data are best described by a model in which the partial inhibition and FBP binding stoichiometry are taken into account. Kinetic viscosity effects show that the product-release step is not the purely rate-limiting step in the GK-catalyzed reaction. Viscosity effects on FBP inhibition are also discussed.

Made available in DSpace on 2014-06-11T19:23:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-07-28Bitstream added on 2014-06-13T19:29:24Z : No. of bitstreams: 1 aragon_cc_me_arafcf.pdf: 397051 bytes, checksum: e29ac6a4f5baf629041e6e9442044f9e (MD5) / Universidade Estadual Paulista (UNESP) / No presente trabalho, a atividade da enzima glicerol quinase (GK; EC 2.7.1.30; ATP: glicerol 3-fosfotransferase), proveniente de extratos de levedura seca de panificação, foi otimizada. A melhor preparação enzimática da GK foi obtida por rompimento celular com esferas de vidro, durante sete minutos, com lise de 54,2% das células. O extrato celular foi parcialmente purificado com 1% de sulfato de estreptomicina, antes da precipitação com igual volume de solução a 30% (m/v) de polietilenoglicol 3350, e posteriormente dialisado. A atividade máxima da GK foi obtida em pH 10,0, a 60ºC e 50mM de substrato, por metodologia clássica. A enzima apresentou alta estabilidade térmica ― a atividade foi completamente mantida até 50ºC, durante uma hora ― e em pH entre 6,0 e 8,0. Além disso, manteve-se estável, por quatro meses, a 4°C, na presença de azida de sódio 0,05% e cloreto de cobalto 10mM, e, por até oito meses, com o extrato liofilizado. Calculados pelos métodos de Lineweaver-Burk, Hanes-Woolf e Eadie-Hofstee, o valor da constante de Michaelis (Km) da enzima variou entre 1,99mM e 3,11mM, e a Vmax, entre 1,14U/mL e 1,19U/mL. Utilizou-se a metodologia de superfície de resposta (MSR) para melhor definição dos parâmetros da reação enzimática, observando-se valores ótimos de atividades a temperaturas entre 52ºC e 56ºC, pH entre 10,2 e 10,5 e concentração de substrato de 150mM a 170mM. A MSR mostrou-se adequada para modelar a reação e maximizar a atividade da glicerol quinase. Este método, de baixo custo, dosa a glicerol quinase em uma seqüência de reações, sendo de grande importância para diversas indústrias, como a de alimentos, açúcar e álcool. / In the present study, the activity of the enzyme glycerol kinase (GK; EC 2.7.1.30; ATP: glycerol 3-phosphotransferase) from dry baker´s yeast, was optimized. The best enzymatic preparation of GK was obtained by cell disruption with glass beads, for seven minutes, with 54.2% of lysed cells. Cell extract was partially purified with 1% of streptomycin sulphate, before the precipitation with equal volume of a 30% solution (m/v) of polyethylene glycol 3350, and then it was dialyzed. The maximum activity of GK was obtained with pH 10.0, at 60ºC and 50mM of substrate, by the classic methodology. The enzyme presented high thermal stability ― the activity was completely maintained up to 50ºC, during one hour ― and at pH between 6.0 and 8.0. Besides, it was stable, for four months, at 4°C, in the presence of sodium azide 0.05% and cobalt chloride 10mM, and, for up to eight months, with the lyophilized extract. The value of the Michaelis constant (Km) of the enzyme was calculated by the methods of Lineweaver-Burk, Hanes-Woolf and Eadie-Hofstee,and it varied between 1.99mM and 3.11mM, and Vmax, between 1.14U/mL and 1.19U/mL. Response surface methodology (RSM) was used for better definition of the parameters of the enzymatic reaction, being observed higher activity values at temperatures between 52ºC and 56ºC, pH between 10.2 and 10.5 and substrate concentration from 150mM to 170mM. RSM showed to be an adequate approach for modeling the reaction and maximizing the glycerol kinase activity. This low cost method doses glycerol kinase in a sequence of reactions, being of great importance for many industries, like food, sugar and alcohol.

Resumo: No presente trabalho, a atividade da enzima glicerol quinase (GK; EC 2.7.1.30; ATP: glicerol 3-fosfotransferase), proveniente de extratos de levedura seca de panificação, foi otimizada. A melhor preparação enzimática da GK foi obtida por rompimento celular com esferas de vidro, durante sete minutos, com lise de 54,2% das células. O extrato celular foi parcialmente purificado com 1% de sulfato de estreptomicina, antes da precipitação com igual volume de solução a 30% (m/v) de polietilenoglicol 3350, e posteriormente dialisado. A atividade máxima da GK foi obtida em pH 10,0, a 60ºC e 50mM de substrato, por metodologia clássica. A enzima apresentou alta estabilidade térmica ― a atividade foi completamente mantida até 50ºC, durante uma hora ― e em pH entre 6,0 e 8,0. Além disso, manteve-se estável, por quatro meses, a 4°C, na presença de azida de sódio 0,05% e cloreto de cobalto 10mM, e, por até oito meses, com o extrato liofilizado. Calculados pelos métodos de Lineweaver-Burk, Hanes-Woolf e Eadie-Hofstee, o valor da constante de Michaelis (Km) da enzima variou entre 1,99mM e 3,11mM, e a Vmax, entre 1,14U/mL e 1,19U/mL. Utilizou-se a metodologia de superfície de resposta (MSR) para melhor definição dos parâmetros da reação enzimática, observando-se valores ótimos de atividades a temperaturas entre 52ºC e 56ºC, pH entre 10,2 e 10,5 e concentração de substrato de 150mM a 170mM. A MSR mostrou-se adequada para modelar a reação e maximizar a atividade da glicerol quinase. Este método, de baixo custo, dosa a glicerol quinase em uma seqüência de reações, sendo de grande importância para diversas indústrias, como a de alimentos, açúcar e álcool. / Abstract: In the present study, the activity of the enzyme glycerol kinase (GK; EC 2.7.1.30; ATP: glycerol 3-phosphotransferase) from dry baker's yeast, was optimized. The best enzymatic preparation of GK was obtained by cell disruption with glass beads, for seven minutes, with 54.2% of lysed cells. Cell extract was partially purified with 1% of streptomycin sulphate, before the precipitation with equal volume of a 30% solution (m/v) of polyethylene glycol 3350, and then it was dialyzed. The maximum activity of GK was obtained with pH 10.0, at 60ºC and 50mM of substrate, by the classic methodology. The enzyme presented high thermal stability ― the activity was completely maintained up to 50ºC, during one hour ― and at pH between 6.0 and 8.0. Besides, it was stable, for four months, at 4°C, in the presence of sodium azide 0.05% and cobalt chloride 10mM, and, for up to eight months, with the lyophilized extract. The value of the Michaelis constant (Km) of the enzyme was calculated by the methods of Lineweaver-Burk, Hanes-Woolf and Eadie-Hofstee,and it varied between 1.99mM and 3.11mM, and Vmax, between 1.14U/mL and 1.19U/mL. Response surface methodology (RSM) was used for better definition of the parameters of the enzymatic reaction, being observed higher activity values at temperatures between 52ºC and 56ºC, pH between 10.2 and 10.5 and substrate concentration from 150mM to 170mM. RSM showed to be an adequate approach for modeling the reaction and maximizing the glycerol kinase activity. This low cost method doses glycerol kinase in a sequence of reactions, being of great importance for many industries, like food, sugar and alcohol. / Orientador: Maristela de Freitas Sanches Peres / Coorientador: Edwil Aparecida de Lucca Gattás / Banca: Luis Henrique Souza Guimarães / Banca: Rubens Monti / Mestre

O aproveitamento biotecnológico do glicerol representa uma alternativa para a redução dos problemas ambientais derivados do acúmulo de glicerol originado do processo de produção de biodiesel. O glicerol bruto (70,6% p/p) resultante do processo de transesterificação do óleo de soja e metanol foi submetido a tratamento com ácidos inorgânicos, com o objetivo de remover impurezas e reduzir a alcalinidade resultante do excesso de catalisador (KOH). A fração glicerínica resultante foi caracterizada quanto à concentração de glicerol, ésteres e íons metálicos; e empregada como fonte de carbono e energia para crescimento de bactérias probióticas. Os probióticos são organismos vivos que, quando administrados em quantidades adequadas, conferem benefício à saúde do hospedeiro. Quinze estirpes de Lactobacillus, com características probióticas, foram avaliadas quanto à capacidade de crescimento em meio contendo glicerol de biodiesel tratado (20 g/L) como principal fonte de carbono, sob condições de pHinicial=6,0 e 37 °C. Os resultados demonstraram a eficácia do ácido fosfórico para remoção de impurezas do glicerol bruto, o que permitiu a obtenção de uma fração contendo 900 a 964 g/L de glicerol. A avaliação de formulações de meios de cultivo contendo glicerol tratado revelou que treze estirpes de Lactobacillus mostraram capacidade de crescimento em glicerol de biodiesel, sendo os maiores rendimentos (YX/S) de 0,34, 0,28 e 0,26 g/g para as estirpes L. delbrueckii UFV-H2b20, L. acidophilus ATCC 4356 e L. plantarum ATCC 8014, respectivamente. A cinética de crescimento das estirpes selecionadas foi estudada em meio MRS modificado (ausência de glicose) contendo glicerol (10 g/L) suplementado ou não com citrato de amônio (2 g/L) e acetato de sódio (5,0 g/L), pH 6,0; 37 °C e 150 rpm. Os maiores rendimentos foram alcançados quando se utilizou meio MRS modificado contendo citrato de amônio e acetado de sódio; gerando valores de rendimentos correspondentes a 0,46, 0,38 e 0,46 g/g para L. delbrueckii UFV-H2b20, L. acidophilus ATCC 4356 e L. plantarum ATCC 8014, respectivamente. No tocante a atividade das enzimas envolvidas na assimilação de glicerol, glicerol quinase (EC. 2.7.1.30) e glicerol desidrogenase (EC.1.1.1.72), os resultados mostraram que, nos extratos livres de células de L. plantarum ATCC 8014, L. delbrueckii UFV-H2b20 e L. acidophilus ATCC 4356, os valores de atividade específica de glicerol quinase após 24 h de cultivo foram 91,1; 232,5 e 228,7 U/mg, respectivamente. Os valores da constante de Michaelis-Menten (Km) foram de 3,7; 1,2 e 2,5 mM para glicerol quinase e 19,2; 12,8; 33,3 e mM para glicerol desidrogenase de L. plantarum ATCC 8014, L. delbrueckii UFV-H2b20 e L. acidophilus ATCC 4356, respectivamente. Os valores de velocidades máximas (Vmáx) da reação foram de 46,4; 115,1 e 119,4 µM/min para glicerol quinase e 1,23; 1,03 e 2,7µM/min para glicerol desidrogenase de L. plantarum ATCC 8014, L. delbrueckii UFV-H2b20 e L. acidophilus ATCC 4356, respectivamente. A avaliação da técnica de encapsulamento de células de Lactobacillus probióticos em alginato-amido de banana verde (2%/2%) pela técnica de emulsificação em óleo vegetal e gelificação ionotrópica, permitiu a sobrevivência das células encapsuladas superior a 65%, na presença de fluído gástrico simulado, bem como sob condições de armazenagem a 4 °C. Os resultados do presente trabalho revelaram a potencialidade da utilização de glicerol de biodiesel como fonte de carbono e energia para o crescimento de bactérias lácticas que apresentam propriedades probióticas, visando a obtenção de um produto microencapsulado em matriz polimérica natural. / Biotechnological utilization of biodiesel-derived glycerol represents an alternative for the reduction of the environment concerns associated with the accumulation of this byproduct. Crude glycerol (70.6% w/w), obtained from the transesterification process of soybean oil and methanol; was treated with inorganic acids in order to remove impurities and decrease the alkalinity derived from the excess of catalyst (KOH). The glycerine fraction obtained was characterized regarding the final glycerol concentration, esters and metallic ions; and it was utilized as source of carbon and energy for growth of probiotic bacteria. Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. Fifteen probiotic bacterial strains were screened to evaluated their capabilities to assimilate treated-glycerol (20 g/L) as main carbon source, at pH=6.0 and 37 °C. The results showed the effectiveness of the phosphoric acid for the removal of impurities from crude glycerol; allowing to attain a glycerol fraction containing 964 g/L. The evaluation of media containing treated glycerol revealed that thirteen strains of Lactobacillus showed capability to grow in biodiesel-derived glycerol, with yieds (YX/S) of glycerol conversion of 0.34, 0.28 and 0.26 g/g for L. delbrueckii UFV-H2b20, L. acidophilus ATCC 4356 and L. plantarum ATCC 8014, respectively. Kinetics of growth of the selected strains was studied in modified MRS medium containing glycerol(10 g/L) supplemented with or in the absence of ammonium citrate (2 g/L) and sodium acetate (5 g/L), pH 6.0; 37 °C and 150 rpm. The highest yields were attained in modified MRS containing ammonium citrate and sodium acetate; with 0.46, 0.38 and 0.46 g/g for L. delbrueckii UFV-H2b20, L. acidophilus ATCC 4356 and L. plantarum ATCC 8014, respectively. The free-cell extract of L. plantarum ATCC 8014, L. delbrueckii UFV-H2b20 and L. acidophilus ATCC 4356 showed activity for glycerol kinase (EC.2.7.1.30) and glycerol dehydrogenase (EC1.1.1.72). The maximal glycerol kinase activity was attained at the late exponential phase of growth, with 91.1; 232.5 and 228.7 U/mg for L. plantarum ATCC 8014, L. delbrueckii UFV-H2b20 and L. acidophilus ATCC 4356, respectively. The Michaelis-Menten (Km) values were 3.7; 1.2 and 2.5 mM for glycerol kinase and 19.2; 12.4 and 33.2 mM for glycerol dehydrogenase of L. plantarum ATCC 8014; L. delbrueckii UFV-H2b20 and L. acidophilus ATCC 4356, respectively. The maximum reaction rates (Vmáx) were 46.5; 115.1 and 119.4 µM/min for glycerol kinase and 1.23; 1.03 and 270 µM/min for glycerol dehydrogenase of L. plantarum ATCC 8014; L. delbrueckii UFV-H2b20 and L. acidophilus ATCC 4356, respectively. Furthermore, the results of the evaluation of probiotic Lactobacillus cell encapsulation in alginate-unripe banana starch (2%/2%) obtained by emusification in soybean oil and ionotropic gelification with calcium chloride, showed a cell survival rate higher than 65%, regarding the initial cell concentration in simulated gastric fluid,and during 28 days stored at 4 °C. The results revealed that, glycerol from biodiesel production process represents a potential carbon and energy source for the growth of probiotic bacteria.

O aproveitamento biotecnológico do glicerol representa uma alternativa para a redução dos problemas ambientais derivados do acúmulo de glicerol originado do processo de produção de biodiesel. O glicerol bruto (70,6% p/p) resultante do processo de transesterificação do óleo de soja e metanol foi submetido a tratamento com ácidos inorgânicos, com o objetivo de remover impurezas e reduzir a alcalinidade resultante do excesso de catalisador (KOH). A fração glicerínica resultante foi caracterizada quanto à concentração de glicerol, ésteres e íons metálicos; e empregada como fonte de carbono e energia para crescimento de bactérias probióticas. Os probióticos são organismos vivos que, quando administrados em quantidades adequadas, conferem benefício à saúde do hospedeiro. Quinze estirpes de Lactobacillus, com características probióticas, foram avaliadas quanto à capacidade de crescimento em meio contendo glicerol de biodiesel tratado (20 g/L) como principal fonte de carbono, sob condições de pHinicial=6,0 e 37 °C. Os resultados demonstraram a eficácia do ácido fosfórico para remoção de impurezas do glicerol bruto, o que permitiu a obtenção de uma fração contendo 900 a 964 g/L de glicerol. A avaliação de formulações de meios de cultivo contendo glicerol tratado revelou que treze estirpes de Lactobacillus mostraram capacidade de crescimento em glicerol de biodiesel, sendo os maiores rendimentos (YX/S) de 0,34, 0,28 e 0,26 g/g para as estirpes L. delbrueckii UFV-H2b20, L. acidophilus ATCC 4356 e L. plantarum ATCC 8014, respectivamente. A cinética de crescimento das estirpes selecionadas foi estudada em meio MRS modificado (ausência de glicose) contendo glicerol (10 g/L) suplementado ou não com citrato de amônio (2 g/L) e acetato de sódio (5,0 g/L), pH 6,0; 37 °C e 150 rpm. Os maiores rendimentos foram alcançados quando se utilizou meio MRS modificado contendo citrato de amônio e acetado de sódio; gerando valores de rendimentos correspondentes a 0,46, 0,38 e 0,46 g/g para L. delbrueckii UFV-H2b20, L. acidophilus ATCC 4356 e L. plantarum ATCC 8014, respectivamente. No tocante a atividade das enzimas envolvidas na assimilação de glicerol, glicerol quinase (EC. 2.7.1.30) e glicerol desidrogenase (EC.1.1.1.72), os resultados mostraram que, nos extratos livres de células de L. plantarum ATCC 8014, L. delbrueckii UFV-H2b20 e L. acidophilus ATCC 4356, os valores de atividade específica de glicerol quinase após 24 h de cultivo foram 91,1; 232,5 e 228,7 U/mg, respectivamente. Os valores da constante de Michaelis-Menten (Km) foram de 3,7; 1,2 e 2,5 mM para glicerol quinase e 19,2; 12,8; 33,3 e mM para glicerol desidrogenase de L. plantarum ATCC 8014, L. delbrueckii UFV-H2b20 e L. acidophilus ATCC 4356, respectivamente. Os valores de velocidades máximas (Vmáx) da reação foram de 46,4; 115,1 e 119,4 µM/min para glicerol quinase e 1,23; 1,03 e 2,7µM/min para glicerol desidrogenase de L. plantarum ATCC 8014, L. delbrueckii UFV-H2b20 e L. acidophilus ATCC 4356, respectivamente. A avaliação da técnica de encapsulamento de células de Lactobacillus probióticos em alginato-amido de banana verde (2%/2%) pela técnica de emulsificação em óleo vegetal e gelificação ionotrópica, permitiu a sobrevivência das células encapsuladas superior a 65%, na presença de fluído gástrico simulado, bem como sob condições de armazenagem a 4 °C. Os resultados do presente trabalho revelaram a potencialidade da utilização de glicerol de biodiesel como fonte de carbono e energia para o crescimento de bactérias lácticas que apresentam propriedades probióticas, visando a obtenção de um produto microencapsulado em matriz polimérica natural. / Biotechnological utilization of biodiesel-derived glycerol represents an alternative for the reduction of the environment concerns associated with the accumulation of this byproduct. Crude glycerol (70.6% w/w), obtained from the transesterification process of soybean oil and methanol; was treated with inorganic acids in order to remove impurities and decrease the alkalinity derived from the excess of catalyst (KOH). The glycerine fraction obtained was characterized regarding the final glycerol concentration, esters and metallic ions; and it was utilized as source of carbon and energy for growth of probiotic bacteria. Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. Fifteen probiotic bacterial strains were screened to evaluated their capabilities to assimilate treated-glycerol (20 g/L) as main carbon source, at pH=6.0 and 37 °C. The results showed the effectiveness of the phosphoric acid for the removal of impurities from crude glycerol; allowing to attain a glycerol fraction containing 964 g/L. The evaluation of media containing treated glycerol revealed that thirteen strains of Lactobacillus showed capability to grow in biodiesel-derived glycerol, with yieds (YX/S) of glycerol conversion of 0.34, 0.28 and 0.26 g/g for L. delbrueckii UFV-H2b20, L. acidophilus ATCC 4356 and L. plantarum ATCC 8014, respectively. Kinetics of growth of the selected strains was studied in modified MRS medium containing glycerol(10 g/L) supplemented with or in the absence of ammonium citrate (2 g/L) and sodium acetate (5 g/L), pH 6.0; 37 °C and 150 rpm. The highest yields were attained in modified MRS containing ammonium citrate and sodium acetate; with 0.46, 0.38 and 0.46 g/g for L. delbrueckii UFV-H2b20, L. acidophilus ATCC 4356 and L. plantarum ATCC 8014, respectively. The free-cell extract of L. plantarum ATCC 8014, L. delbrueckii UFV-H2b20 and L. acidophilus ATCC 4356 showed activity for glycerol kinase (EC.2.7.1.30) and glycerol dehydrogenase (EC1.1.1.72). The maximal glycerol kinase activity was attained at the late exponential phase of growth, with 91.1; 232.5 and 228.7 U/mg for L. plantarum ATCC 8014, L. delbrueckii UFV-H2b20 and L. acidophilus ATCC 4356, respectively. The Michaelis-Menten (Km) values were 3.7; 1.2 and 2.5 mM for glycerol kinase and 19.2; 12.4 and 33.2 mM for glycerol dehydrogenase of L. plantarum ATCC 8014; L. delbrueckii UFV-H2b20 and L. acidophilus ATCC 4356, respectively. The maximum reaction rates (Vmáx) were 46.5; 115.1 and 119.4 µM/min for glycerol kinase and 1.23; 1.03 and 270 µM/min for glycerol dehydrogenase of L. plantarum ATCC 8014; L. delbrueckii UFV-H2b20 and L. acidophilus ATCC 4356, respectively. Furthermore, the results of the evaluation of probiotic Lactobacillus cell encapsulation in alginate-unripe banana starch (2%/2%) obtained by emusification in soybean oil and ionotropic gelification with calcium chloride, showed a cell survival rate higher than 65%, regarding the initial cell concentration in simulated gastric fluid,and during 28 days stored at 4 °C. The results revealed that, glycerol from biodiesel production process represents a potential carbon and energy source for the growth of probiotic bacteria.