IJISPM Editorial Vol. 7 No. 2

The present study describes a methodology of dosage of glycerol kinase (GK) from baker's yeast. The standardization of the activity of the glycerol kinase from baker's yeast was accomplished using the diluted enzymatic preparation containing glycerol phosphate oxidase (GPO) and glycerol kinase. The mixture was incubated at 60 degrees C by 15 min and the reaction was stopped by the SDS solution addition. A first set of experiments was carried out in order to investigate the individual effect of temperature (7), pH and substrate concentration (S), on GK activity and stability. The pH and temperature stability tests showed that the enzyme presented a high stability to pH 6.0-8.0 and the thermal stability were completely maintained up to 50 degrees C during 1 h. The K(m) of the enzyme for glycerol was calculated to be 2 mM and V(max) to be 1.15 U/mL. In addition, modeling and optimization of reaction conditions was attempted by response surface methodology (RSM). Higher activity values will be attained at temperatures between 52 and 56 degrees C, pH around 10.2-10.5 and substrate concentrations from 150 to 170 mM.This low cost method for glycerol kinase dosage in a sequence of reactions is of great importance for many industries, like food, sugar and alcohol. RSM showed to be an adequate approach for modeling the reaction and optimization of reaction conditions to maximize glycerol kinase activity. (C) 2007 Elsevier B.V. All rights reserved

Use of different carbon sources in cultivation of baker's yeast for production of glycerol-3-phosphate dehydrogenase

The physiological state of yeast cells changes during culture growth as a consequence of environmental changes (nutrient limitations, pH and metabolic products). Cultures that grow exponentially are heterogeneous cell populations made up of cells regulated by different metabolic and/or genetic control systems. The strain of baker's yeast selected by plating commercial compressed yeast was used for the production of glycerol-3- phosphate dehydrogenase. Glycerol-3-phosphate dehydrogenase (GPD) has been widely used in the enzyme assays with diverse compounds of industrial interest, such as glycerol or glycerol phosphate, as well as a number of important bioanalytical applications. Each cell state determines the level of key enzymes (genetic control), fluxes through metabolic pathways (metabolic control), cell morphology and size. The present study was carried out to determine the effects of environmental conditions and carbon source on GPD production from baker's yeast. Glucose, glycerol, galactose and ethanol were used as carbon sources. Glycerol and ethanol assimilations required agitation, which was dependent on the medium volume in the fermentation flask for the greatest accumulation of intracellular GPD. Enzyme synthesis was also affected by the initial pH of the medium and inoculum size. The fermentation time required for a high level of enzyme formation decreased with the inoculum size. The greatest amount of enzyme (0.45 U/ml) was obtained with an initial pH of 4.5 in the medium containing ethanol or glycerol. The final pH was maintained in YP-ethanol, but in the YP-glycerol the final pH increased to 6.9 during growth

Aspectos toxicológicos do bissulfito e outros agentes tóxicos sobre a síntese de glicerol-3-fosfato desidrogenase citoplasmática de levedura de panificação

A síntese de glicerol-3-fosfato desidrogenase intracelular (EC 1.1.1.8) foi investigada a partir de fermento de panificação em cultivos submersos, contendo glicose ou glicerol como únicas fontes de carbono. Agentes inibitórios da via glicolítica, do ciclo de Krebs e da Cadeia Respiratória inibiram a síntese da enzima quando adicionados em baixas concentrações até 7,5 x 10-4 mol/L. A repressão exercida pela glicose sobre a síntese da glicerol-3-fosfato desidrogenase em meio YP-glicose foi reduzida, com a adição de produtos de fermentação e de bissulfito de sódio. Observou-se aumentos de 22-110% na síntese da enzima. Entretanto, em meio YP-glicerol, a adição de bissulfito de sódio 0,06 % (p/v) reduziu a síntese da enzima em 29%, enquanto, o acetaldeído 0,012 % (v/v) estimulou a síntese de glicerol-3-fosfato desidrogenase em 12%. Palavras-chave: fermento de panificação; glicerol-3-fosfato desidrogenase; inibidores

Efeito da temperatura de estocagem de leveduras de panificação sobre a atividade da glicerol-3-fosfato desidrogenase

Níveis intracelulares de G-3-PDH (sn-glicerol-3-fosfato:NAD+ 2oxidoredutase, EC 1.1.1.8) de levedura de panificação foram acompanhados durante a estocagem sob três diferentes temperaturas. Semelhantes valores de biomassa final e de atividade específica da enzima foram obtidos após crescimento por 48 horas de duas linhagens de leveduras de panificação. O melhor meio (meio indutor) para obtenção de G-3-PDH foi: extrato de levedura (1%,p/v), peptona (2%,p/v), glicerol (3%,v/v) e etanol (1%,v/v). O choque osmótico com adição de NaCl 0,6 M provocou aumento da atividade de G-3-PDH de 1,2 vezes para leveduras crescidas em meio indutor por 48 horas e transferidas para o meio salino, por 2 horas. A estocagem (até 10 dias) da linhagem de levedura GD0, sob temperatura ambiente (27 ºC) estimulou a síntese da G-3-PDH de células propagadas no meio indutor, lavadas e liofilizadas. Estocagens em geladeira (temperatura de 4 - 5 ºC) ou em ''freezer'' (temperatura de -18 ºC) mantiveram a atividade da G-3-PDH por até 8 meses.Intracellular levels of glycerol-3-phosphate dehydrogenase (G-3-PDH) in baker's yeasts were monitored during storage at 3 different temperatures. Similar values for final biomass and specific activity of the enzyme were found, in each of two strains of baker's yeast, after 48 hours growth. The best medium tested, for the induction of G-3-PDH, contained: yeast extract (1% w/v), peptone (2% w/v), glycerol (3% w/v)and ethanol (1% w/v). Osmotic shock, provoked by suspending cells, after 48 hours growth in inducing medium, in 0.6 M NaCl solution for 2 hours, caused the activity to increase by a factor of 1.2. Cells of the GDO yeast strain, grown in inducing conditions, washed and lyophilized, exhibited a 35% rise in G-3-PDH activity during storage (10 days) at ambient temperature (about 27 ºC). Both refrigeration (4-5 ºC) and freezer storage (-18 ºC) maintained the G-3-PDH activity for up to 8 months.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Using Pichia pastoris to produce recombinant glycerol kinase

The methylotrophic yeast Pichia pastoris has been developed into an efficient expression system for the production of recombinant protein under the tight control of the methanol-induced alcohol oxidase promoter (pAOX1). In this study, a 2.5-liter culture system was developed for the growth of a P. pastoris strain bearing the GUT1 gene from Saccharomyces cerevisiae for the expression of recombinant glycerol kinase (GK). The best culture conditions to produce high levels of secreted GK were investigated by growing the recombinant strain of P. pastoris in shake flasks and a fermenter. Cell growth and enzyme production were found to be optimal after two days of growth. Enzyme production was affected by the nitrogen source, Difco peptone being the most appropriate for this purpose. Three different rates of air flow (1 to 3 L/min) were tested to observe their effect on cell growth and the secretion of GK into a medium containing 1% methanol as the sole carbon source. Increasing the rate of air bubbling in the culture medium enhanced both cell growth and GK activity, reaching a dry biomass of 7.84 mg/mL, cell viability of 98.4% and a maximal GK activity of 1.57 U/ mL, at a flow rate of 2.0 L/minute, at 30° C and pH 6.0. Moreover, the enzyme activity in the P. pastoris culture medium was 2.3 times higher under these conditions than in the shake-flask culture, demonstrating the significant influence of aeration on biomass production and GK activity secreted by P. pastoris.A levedura metilotrófica Pichia pastoris possui um sistema de expressão eficiente para a produção de proteínas recombinantes. A indução da produção da proteína de interesse é feita com metanol, que é capaz de ativar a transcrição do gene de interesse clonado sob controle do promotor do gene AOX1. Um meio de cultura de 2.5 litros foi elaborado para o crescimento da cepa Pichia pastoris construída com o gene GUT1 de Saccharomyces cerevisiae para expressar a enzima recombinante glicerol quinase (GK). As condições ideais de cultura, para alcançar altos níveis de expressão de GK foram investigados em crescimentos realizados em frascos e fermentador. Crescimento celular e produção de enzima atingiram valores ótimos em dois dias de cultura. A produção enzimática foi afetada pela fonte de nitrogênio no meio. Peptona da marca Difco foi a fonte de nitrogênio mais adequada para a expressão desta enzima. Três diferentes concentrações (1-3 L / min) de fluxo de ar foram analisados em ensaios de crescimento celular e secreção da GK, no meio contendo 1 % de metanol como única fonte de carbono. O aumento do fluxo do ar no meio de cultura produziu melhores resultados para o crescimento celular e atividade da GK, atingindo 7,84 mg / mL de biomassa seca e 98,4% de viabilidade. A máxima atividade de GK foi de 1,57 U / mL, com a concentração de fluxo de ar de 2,0 L / minuto a 30 ° C e pH 6.0. O aumento da atividade enzimática foi 2,3 vezes maior no meio de cultura da Pichia pastoris nestas condições, revelando a influência deste parâmetro na produção de biomassa e atividade da GK.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Isolamento de fungos produtores de enzimas pectinolíticas

Soil samples collected in the campus, UNESP, Araraquara, SP, were employed to isolate and characterize fungi strains with potential pectinolytic enzymes. These enzymes have arisen great interest due to its increasing application in the food industry. Two hundred forty six strains were isolated based on the appearance of colony on PDA medium, morphology (septate mycelia, nonseptate conidiophore, black conidia, and clublike spore-bearing head), after 48 h of growth at 30°C. Strains were selected in solid medium containing pectin citrus as sole carbon source and 0.5% rutenium red. The characterization of pectinolytic production was performed in solid culture and batch fermentation medium containing pectin citrus. The enzyme pectinolytic production was evaluated at 30°C, without agitation in 100 mL of medium containing 2% pectin citrus, 0.2% ammonium sulphate, 0.2% magnesium sulphate, and 0.05% potassium phosphate. The maximum pectinolytic activity (15U/mL) was observed in the medium after Aspergillus sp CFCF-0492 growth, while Aspergillus sp CFCF-CC1 showed the higher level of the final biomass. The pectinolytic activity is more preserved when the fungi-spores were maintained in agar-Czapeck medium

Colorimetric assay of ethanol using alcohol dehydrogenase from dry baker's yeast

Alcohol dehydrogenases (ADHs) are oxidoreductases present in animal tissues, plants, and microorganisms. These enzymes attract major scientific interest for the evolutionary perspectives, afforded by their wide occurrence in nature, and for their use in synthesis, thanks to their broad substrate specificity and stereoselectivity. In the present study, the standardization of the activity of the alcohol dehydrogenase from baker's yeast was accomplished, and the pH and temperature stability showed, that the enzyme presented a high stability to pH 6.0-7.0 and the thermal stability were completely maintained up to 50 degrees C during 1 h. The assays of ethanol (detection range 1-5 mM or 4.6 x 10(-2) to 23.0 x 10(-2) g/L) in different samples in alcoholic beverages, presented a maximum deviation of only 7.2%. The standard curve and the analytic curve of this method meet the conditions of precision, sensitivity, simplicity, and low cost, required for a useable analytical method. (c) 2006 Elsevier B.V. All rights reserved

Application of methylotrophic yeast Pichia pastoris in the field of food industry - A review

Since ancient times, the utilization of yeasts by the man has a great impact on the socio-economic development. After the advent of the technology of recombinant DNA, great advances have occurred due to the acquisition of strains of mutant yeasts in the field of applied research, and Saccharomyces cerevisiae has soon been outstanding as an interesting candidate for the expression of heterologous proteins of biotechnological interest. As the time goes by other alternative systems of expression have been shown because they have advantages over Saccharomyces cerevisiae. Among those new systems, Pichia pastoris is outstanding as methylotrophic yeast capable of growing in a culture medium containing methanol as the only source of carbon and energy. The induction of production of glycerol-3-phosphate dehydrogenase (GPD, NAD(+): oxido-redutase EC 1.1. 1.8) by Pichia pastoris was accomplished in the medium containing methanol. One of the most important key parameters in Pichia pastoris expression system is the methanol concentration. Bibliographic reviews on the Pichia pastoris production system have shown that the best culture conditions vary according to the strain used and/or kind of heterologous protein desired to be expressed. Therefore, we have sought to develop a system, involving expression of glycerol-3-phosphate dehydrogenase in the yeast Pichia pastoris, for generating sufficient quantities of the enzyme in order to asses its potential value for use in various food bioanalytical determination. Dehydrogenases have been widely used in the enzymatic assays of diverse composites of industrial interest, being enclosed among them glycerol and a number of important bioanalytical applications