Multienzymatic sucrose conversion into fructose and gluconic acid through fed-batch and membrane-continuous processes.

Multienzymatic conversion of sucrose into fructose and gluconic acid was studied through fed-batch and continuous (in a membrane reactor) processes. The law of substrate addition (sucrose or glucose) for the fed-batch process which led to a yield superior to 80% was the decreasing linear type, whose feeding rate (ϕ; L/h) was calculated through the equation: ϕ = ϕ(o) - k.t, where ϕ(o) (initial feeding rate, L/h), k (linear addition constant, L/h (2)), and t (reaction time, h). In the continuous process, the yield of conversion of sucrose (Y) was superior to 70% under the following conditions: dilution rate = 0.33 h(-1), total duration of 15 h, pH 5.0, 37 °C and initial sucrose concentration of 64 g/L (Y = 92%), 100 g/L (Y = 83%), or 150 g/L (Y = 76%).

Invertase, glucose oxidase and catalase for converting sucrose to fructose and gluconic acid through batch and membrane-continuous reactors / Invertase de glicose oxidase e catalase para a conversão de sacarose para ácido glucónico e frutose através de lote e de membrana-contínuas reactores

Conversion of sucrose into fructose and gluconic acid using invertase, glucose oxidase and catalase was studied by discontinuous (sequential or simultaneous addition of the enzymes) and continuous (simultaneous addition of the enzymes in a 100 kDa-ultrafiltration membrane reactor) processes. The following parameters were varied: concentration of enzymes, initial concentration of substrates (sucrose and glucose), pH, temperature and feeding rate (for continuous process). The highest yield of conversion (100 percent) was attained through the discontinuous (batch) process carried out at pH 4.5 and 37 ºC by the sequential addition of invertase (14.3 U), glucose oxidase (10,000 U) and catalase (59,000 U).

Neste trabalho estudou-se a conversão da sacarose em frutose e ácido glicônico, usando as enzimas invertase, glicose oxidase e catalase, através do emprego de processo descontínuo (com adição sequencial ou simultânea das enzimas) e contínuo (adição simultânea das enzimas em reator com membrana acoplado à membrana de ultrafiltração de 100 kDa). Os parâmetros variados foram: a concentração das enzimas, a concentração inicial dos substratos (sacarose e glicose), o pH, a temperatura e a vazão específica de alimentação (processo contínuo). Obteve-se rendimento de 100 por cento, quando a conversão foi conduzida por processo descontínuo em pH 4,5 e a 37 ºC com adição seqüencial das enzimas invertase (14,3 U), glicose oxidase (10.000 U) e catalase (59.000 U).

Inverted sugar syrup attained from sucrose hydrolysis using a membrane reactor / Xarope de açúcar invertido obtido a partir de hidrólise da sacarose utilizando um reator com membrana

Invertase, whether adsorbed on styrene-divinylbenzene copolymers or otherwise, was used for continuous sucrose hydrolysis using a cell-type membrane reactor (CTMR), coupled with an ultra (UF-100kDa), or a microfiltration (MF- pore diameter of 5 µm) membrane. In all tests, the pH (5.5), temperature (30 ºC), reaction volume (10 mL) and agitation (100 rpm) were set constant; whereas, variable parameters were: feeding rate (0.4, 0.8 and 1.6 h-1), inlet sucrose concentration (2.5, 6.5, 50 and 100 mM) and enzyme/resin ratio (1.64 mg or 3.28 mg of protein per 25, 50 or 100 mg of resin). The best result (yield of 100 percent, steady-state duration over 20h and specific reaction rate over 243 x 10-3 mmol/h.mE) was obtained when insoluble invertase (1.64 mg protein/100 mg resin) was used to convert 50 mM or 100 mM of sucrose solution at 0.4 h-1 using a UF-CTMR.

Invertase, na forma adsorvida ou não em copolímeros de estireno-divinilbenzeno, foi usada para a hidrólise contínua de sacarose utilizando um reator com membrana (RM), acoplado a uma membrana de ultrafiltração (UF-100kDa), ou de microfiltração (MF - um diâmetro de poro de 5µm). Em todos os testes, o pH (5,5), a temperatura (30ºC), o volume reacional (10mL) e a agitação (100 rpm) foram mantidas constantes; os parâmetros variados foram: a vazão de alimentação (0,4; 0,8 e 1,6 h-1), a concentração de sacarose alimentada (2,5; 6,5; 50 e 100 mM) e a relação enzima/resina (1,64 mg ou 3,28 mg de proteína por 25, 50 ou 100 mg de resina). O melhor resultado (um rendimento de 100 por cento, um período de estado estacionário acima de 20h e uma taxa de reação específica maior de 243 x 10-3 mmol/h.mE) foi obtido quando a invertase insolúvel (1,64 mg de proteína/100 de mg resina) foi usado para converter 50 mM ou 100 mM de solução de sacarose a 0,4 h-1 usando UF-RM.

Obtaining partial purified xylose reductase from Candida guilliermondii / Obtenção de xilose redutase de Candida guilliermondii parcialmente purificada

The enzymatic bioconversion of xylose into xylitol by xylose reductase (XR) is an alternative for chemical and microbiological processes. The partial purified XR was obtained by using the following three procedures: an agarose column, a membrane reactor or an Amicon Ultra-15 50K Centrifugal Filter device at yields of 40 percent, 7 percent and 67 percent, respectively.

A bioconversão enzimática da xilose em xilitol pela xilose redutase (XR) é uma alternativa para as vias química e microbiológica. Avaliouse a purificação parcial da XR, utilizando os três seguintes procedimentos: uma coluna de agarose, um reator com membrana ou tubos de ultracentrifugação Amicon Ultra-15 50K, com rendimento de 40 por cento, 7 por cento ou 67 por cento, respectivamente.

Obtaining partial purified xylose reductase from Candida guilliermondii.

The enzymatic bioconversion of xylose into xylitol by xylose reductase (XR) is an alternative for chemical and microbiological processes. The partial purified XR was obtained by using the following three procedures: an agarose column, a membrane reactor or an Amicon Ultra-15 50K Centrifugal Filter device at yields of 40%, 7% and 67%, respectively.

Bioconversão de sacarose em ácido glicônico e frutose usando reator com membrana / Sucrose bioconversion into gluconic acid and fructose using a membrane reactor

A conversão enzimática da sacarose pela ação sucessiva da invertase e da glicose oxidase (GOD), permite obter produtos de maior valor agregado, a saber, frutose e o ácido glicônico, dois produtos de amplo uso na indústria farmacêutica, alimentícia e química. Foi estudada a aplicação da invertase imobilizada em resinas aniônicas do tipo Dowex® (um copolímero de poliestireno-divinilbenzeno) sobre a hidrólise da sacarose bem como a oxidação da glicose pela glicose oxidase solúvel ou imobilizada no mesmo suporte em separado (sistema bifásico), utilizando-se um reator de membrana acoplado à membrana de ultrafiltração (100kDa) ou de microfitração (5µm). Posteriormente, avaliou-se o desempenho de ambas as formas de enzimas, solúveis ou imobilizadas num sistema monofásico empregando o mesmo reator...

The enzymatic conversion of sucrose through a successive action of invertase and glucose oxidase (GOO) allows the obtainment of products with higher commercial value, fructose and gluconic acid, which are widely used in pharmaceutical, food and chemical industries. Invertase and GOO immobilized on Dowex® anionic resin (a polystyrene divinylbenzene copolymer) as well as soluble GOD were used in a membrane bioreactor (MS) for sucrose hydrolysis and glucose oxidation. The MB was coupled with a UF-membrane (100kDa) or a MF-membrane (5µm). The bioconversion was conducted in two steps (biphasic system) as well as in one step (monophasic system). The bioconversion operated in a biphasic system permitted obtaining a fructose syrup with a concentration of about 70% through a separation of glucose and fructose using a cationic resin, 50W:8-100. As for the monophasic system, the yield of 96.6% and 67.4% for soluble and immobilized forms were attained respectively. No leakage of the enzymes from the support allowed the use of a microfiltration membrane, adding advantages to the membrane bioreactor operation.

Oxidation of glucose to gluconic acid by glucose oxidase in a membrane bioreactor.

Glucose oxidase (GO) (EC 1.1.3.4) was used as catalyst for oxidizing glucose into gluconic acid utilizing a 10-mL Bioengineering Enzyme Membrane Reactor or a 400-mL Millipore Stirred Ultrafiltration Cell (MSUC) coupled with a Millipore UF membrane (cutoff of 100 kDa) and operated for 12 h under an agitation of 100 rpm, pH 5.5, and 30 degrees C. The effect of feeding rate (0.10, 0.15, or 0.20 min-1), glucose (2.5 or 5.0 mM), and GO (1.0 or 2.0 mg/mL) concentrations on the catalysis were studied. A yield of about 75% was attained when the MSUC filled with 1.0 mg/mL of GO was fed with 2.5 mM glucose solution at a rate of 0.15 min-1.

Screening of Dowex anion-exchange resins for invertase immobilization.

Commercial yeast invertase (Bioinvert) was immobilized by adsorption on anion-exchange resins, collectively named Dowex(R) (1x8:50-400, 1x4:50-400, and 1x2:100-400). Optimal binding was obtained at pH 5.5 and 32 degrees C. Among different polystyrene beads, the complex Dowex-1x4-200/invertase showed a yield coupling and an immobilization coefficient equal to 100%. The thermodynamic and kinetic parameters for sucrose hydrolysis for both soluble and insoluble enzyme were evaluated. The complex Dowex/invertase was stable without any desorption of enzyme from the support during the reaction, and it had thermodynamic parameters equal to the soluble form. The stability against pH presented by the soluble invertase was between 4.0 and 5.0, whereas for insoluble enzyme it was between 5.0 and 6.0. In both cases, the optimal pH values were found in the range of the stability interval. The Km and Vmax for the immobilized invertase were 38.2 mM and 0.0489 U/mL, and for the soluble enzyme were 40.3 mM and 0.0320 U/mL.