Optimalni uvjeti za proizvodnju biomase i rekombinantne glicerol kinaze s pomoću kvasca Pichia pastoris

The extracellular glycerol kinase gene from Saccharomyces cerevisiae (GUT1) was cloned into the expression vector pPICZα A and integrated into the genome of the methylotrophic yeast Pichia pastoris X-33. The presence of the GUT1 insert was confirmed by PCR analysis. Four clones were selected and the functionality of the recombinant enzyme was assayed. Among the tested clones, one exhibited glycerol kinase activity of 0.32 U/mL, with specific activity of 0.025 U/mg of protein. A medium optimized for maximum biomass production by recombinant Pichia pastoris in shaker cultures was initially explored, using 2.31 % (by volume) glycerol as the carbon source. Optimization was carried out by response surface methodology (RSM). In preliminary experiments, following a Plackett-Burman design, glycerol volume fraction (φ(Gly)) and growth time (t) were selected as the most important factors in biomass production. Therefore, subsequent experiments, carried out to optimize biomass production, followed a central composite rotatable design as a function of φ(Gly) and time. Glycerol volume fraction proved to have a significant positive linear effect on biomass production. Also, time was a significant factor (at linear positive and quadratic levels) in biomass production. Experimental data were well fitted by a convex surface representing a second order polynomial model, in which biomass is a function of both factors (R²=0.946). Yield and specific activity of glycerol kinase were mainly affected by the additions of glycerol and methanol to the medium. The optimized medium composition for enzyme production was: 1 % yeast extract, 1 % peptone, 100 mM potassium phosphate buffer, pH=6.0, 1.34 % yeast nitrogen base (YNB), 4·10^–5 % biotin, 1 % methanol and 1 % glycerol, reaching 0.89 U/mL of glycerol kinase activity and 14.55 g/L of total protein in the medium after 48 h of growth.Gen za ekstracelularnu glicerol kinazu iz Saccharomyces cerevisiae (GUT1) kloniran je u ekspresijski vektor pPICZα A i integriran u genom metilotrofnog kvasca Pichia pastoris X-33. Prisutnost GUT1 potvrđena je PCR analizom. Izdvojena su četiri klona, u kojima je ispitana funkcionalnost rekombinantnog enzima. Jedan je od ispitanih klonova imao aktivnost glicerol kinaze od 0,32 U/mL i specifičnu aktivnost proteina od 0,0025 U/mg. Podloga za maksimalnu proizvodnju biomase na tresilici s pomoću rekombinantnog kvasca Pichia pastoris optimirana je uporabom glicerola, volumnog udjela od 2,31 %, kao izvora ugljika. Za optimiranje je upotrijebljena metoda odzivnih površina. U preliminarnim su ispitivanjima, primjenom Plackett-Burmanovog dizajna, određeni najvažniji čimbenici što utječu na proizvodnju biomase, a to su: volumni udio glicerola (φ(Gly)) i vrijeme uzgoja (t). Daljnji su eksperimenti provedeni radi optimiranja proizvodnje biomase, a pratili su centralno složeni dizajn kao funkciju volumnog udjela glicerola i vremena. Volumni je udio glicerola imao pozitivni linearni utjecaj na proizvodnju biomase. Vrijeme uzgoja je također bitno utjecalo (na razini linearno pozitivnih i kvadratnih zavisnosti) na proizvodnju biomase. Eksperimentalni su se podaci dobro uklapali u konveksnu funkciju koja opisuje polinom drugoga reda, u kojem je biomasa funkcija obaju faktora (R²=0,946). Prinos i specifična aktivnost glicerol kinaze ponajprije su ovisili o dodatku glicerola i metanola podlozi. Sastav optimirane podloge za proizvodnju enzima bio je: 1 % kvaščeva ekstrakta, 1 % peptona, 100 mM fosfatnog pufera (pH=6,0), 1,34 % podloge s kvascem i dušikom, 4·10^-5 % biotina, 1 % metanola i 1 % glicerola, pomoću kojih je dobivena aktivnost glicerol kinaze od 0,89 U/mL i koncentracija ukupnih proteina od 14,55 g/L u podlozi nakon 48 sati uzgoja

Use of sugarcane juice and syrup as adjunct of malted barley in the production of beers

Parte do malte pode ser substituído por adjuntos, que podem ser à base de cereais, ou de carboidratos de origem vegetal. A cana de açúcar constitui-se de uma matéria prima favorável à fermentação alcoólica por ser rica em carboidratos e micronutrientes, e pode ser utilizada como um adjunto do malte. Estudos de fermentação do mosto com este adjunto, na forma de caldo e de melado de cana, foram realizados na Planta Piloto de Bebidas da EEL-USP, e a partir daí, foram feitas análises físico-química, microbiológicas e sensoriais da cerveja obtida, com diferentes proporções de adjunto. As fermentações foram realizadas inicialmente em escala de bancada (1 L), utilizando mostos preparados com caldo ou melado de cana com ou sem diferentes tratamentos de clarificação, em duas concentrações diferentes (25% e 50% de caldo ou melado), além do mosto puro malte. Análises de concentração e viabilidade celular, pH, consumo de extrato e formação de etanol foram realizadas ao longo das fermentações e, também foi efetuado o cálculo de produtividade e rendimento. Os ensaios sem tratamento do caldo ou melado foram escolhidos para uma produção em uma escala de 5 L, para que análises físico-químicas e sensorial fossem realizadas. A cerveja elaborada com 25% de melado de cana sem tratamento foi a cerveja mais aceita e foi escolhida para a produção em escala piloto (200 L). Análises físico-químicas e de envelhecimento foram realizadas, e essa cerveja também foi avaliada sensorialmente juntamente com duas cervejas de mercado, onde novamente foi declarada como a mais aceita, mostrando que o melado de cana é um adjunto adequado na fabricação de cervejas, mostrando ser um potencial no mercado de bebidas. Finalmente, cervejas com melado de cana foram elaboradas na forma ale e lager em uma infra-estrutura bem equipada em um laboratório em uma universidade belga.Part of malted barley can be replaced by adjuncts, which can be from cereals, or carbohydrates of vegetable origin. Sugarcane is a raw material favorable to the alcoholic fermentation to be rich in carbohydrates and micronutrients, and can be used as a adjunct. The wort fermentation studies with this adjunct in the form of sugarcane juice and sugarcane syrup, were performed in the Beverage Pilot Plant EEL-USP, and from there, it were made physical-chemical, microbiological and sensorial analysis with beers with different proportion of adjuncts. The fermentations were initially carried out in laboratory scale (1 L) prepared with sugarcane juice or sugarcane syrup in different treatments and in two different concentrations (25% and 50%), and also 100% of malt wort. Analysis of cell concentration and viability, pH, extract consumption and ethanol formation were held throughout the fermentations and also calculation of the productivity and yield. Assays with sugarcane juice and syrup without treatment were selected for production on a slightly larger scale (5L) and physico-chemical and sensorial analyzes were performed. Beer made with 25% of sugarcane syrup without treatment was a beer more accepted and it was chosen for the production on a pilot scale (200 L). Physico-chemical and aging analyzes were performed, and that beer was also evaluated by sensory analyzis compared with two beer market, where again it was declared as the most accepted, showing that the sugarcane syrup is a very interesting adjunct in the manufacture of beer, showing a potential in the beverage market. Finally, beers with sugarcane syrupo were prepared in ale and lager form in a well-equipped infrastructure in a laboratory in a Belgian university

Produção de glicerol quinase em Pichia pastoris

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)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

Optimal Conditions for Biomass and Recombinant Glycerol Kinase Production Using the Yeast Pichia pastoris

The extracellular glycerol kinase gene from Saccharomyces cerevisiae (GUT]) was cloned into the expression vector pPICZ alpha. A and integrated into the genome of the methylotrophic yeast Pichia pastoris X-33. The presence of the GUT1 insert was confirmed by PCR analysis. Four clones were selected and the functionality of the recombinant enzyme was assayed. Among the tested clones, one exhibited glycerol kinase activity of 0.32 U/mL, with specific activity of 0.025 U/mg of protein. A medium optimized for maximum biomass production by recombinant Pichia pastoris in shaker cultures was initially explored, using 2.31 % (by volume) glycerol as the carbon source. Optimization was carried out by response surface methodology (RSM). In preliminary experiments, following a Plackett-Burman design, glycerol volume fraction (phi(Gly)) and growth time (t) were selected as the most important factors in biomass production. Therefore, subsequent experiments, carried out to optimize biomass production, followed a central composite rotatable design as a function of phi(Gly) and time. Glycerol volume fraction proved to have a significant positive linear effect on biomass production. Also, time was a significant factor (at linear positive and quadratic levels) in biomass production. Experimental data were well fitted by a convex surface representing a second order polynomial model, in which biomass is a function of both factors (R(2)=0.946). Yield and specific activity of glycerol kinase were mainly affected by the additions of glycerol and methanol to the medium. The optimized medium composition for enzyme production was: 1 % yeast extract, 1 % peptone, 100 mM potassium phosphate buffer, pH=6.0, 1.34 % yeast nitrogen base (YNB), 4.10(-5) % biotin, 1 %, methanol and 1 %, glycerol, reaching 0.89 U/mL of glycerol kinase activity and 14.55 g/L of total protein in the medium after 48 h of growth

Optimal Conditions for Biomass and Recombinant Glycerol Kinase Production Using the Yeast Pichia pastoris

The extracellular glycerol kinase gene from Saccharomyces cerevisiae (GUT1) was cloned into the expression vector pPICZα A and integrated into the genome of the methylotrophic yeast Pichia pastoris X-33. The presence of the GUT1 insert was confirmed by PCR analysis. Four clones were selected and the functionality of the recombinant enzyme was assayed. Among the tested clones, one exhibited glycerol kinase activity of 0.32 U/mL, with specific activity of 0.025 U/mg of protein. A medium optimized for maximum biomass production by recombinant Pichia pastoris in shaker cultures was initially explored, using 2.31 % (by volume) glycerol as the carbon source. Optimization was carried out by response surface methodology (RSM). In preliminary experiments, following a Plackett-Burman design, glycerol volume fraction (φ(Gly)) and growth time (t) were selected as the most important factors in biomass production. Therefore, subsequent experiments, carried out to optimize biomass production, followed a central composite rotatable design as a function of φ(Gly) and time. Glycerol volume fraction proved to have a significant positive linear effect on biomass production. Also, time was a significant factor (at linear positive and quadratic levels) in biomass production. Experimental data were well fitted by a convex surface representing a second order polynomial model, in which biomass is a function of both factors (R²=0.946). Yield and specific activity of glycerol kinase were mainly affected by the additions of glycerol and methanol to the medium. The optimized medium composition for enzyme production was: 1 % yeast extract, 1 % peptone, 100 mM potassium phosphate buffer, pH=6.0, 1.34 % yeast nitrogen base (YNB), 4·10^–5 % biotin, 1 % methanol and 1 % glycerol, reaching 0.89 U/mL of glycerol kinase activity and 14.55 g/L of total protein in the medium after 48 h of growth