Kinetics of glucose isomerization to fructose by immobilized glucose isomerase: anomeric reactivity of D-glucose in kinetic model

The substrate specificity of immobilized D-glucose isomerase (EC 5.3. 1.5) is investigated with an immobilized enzyme-packed reactor. A series of isomerization experiments with alpha-, beta-, and equilibrated D-glucose solutions indicates that beta anomer as well as alpha anomer is a substrate of the glucose isomerase at pH 7.5 and 60 degrees C. For substrate concentration of 0.028 mol l(-1) (1% w/v), the initial conversion rate of alpha-D-glucose was 43% higher than that with equilibrated glucose at the same concentration and 113% higher than beta-D-glucose conversion rate. This anomeric reactivity of glucose isomerase is mathematically described with a set of kinetic equations based on the reaction steps complying with Briggs-Haldane mechanism and the experimentally determined kinetic constants. The proposed reaction mechanism includes the mutarotation and the isomerization reactions of alpha- and beta-D-glucose with different rate constants.     

Biotransformation in a microreactor: New method for production of hexanal

In this study, enzymatic oxidation of hexanol to hexanal (green note fragrance) using NAD⁺ dependent commercial alcohol dehydrogenase from S. cerevisiae was conducted in continuously operated tubular microreactors with internal volumes of 6 and 13 μL and in a tubular microreactor with a volume of 2 μL that was equipped with internal micromixers. Flow profiles in microchannel were observed in experiments in which the aqueous phase was stained brilliant blue and the hexane was kept colourless. The effects of enzyme and coenzyme inlet concentrations and flow ratios of the immiscible phases on the conversion of hexanol and the volumetric productivity of hexanal were analyzed. Significant improvement in the conversion of hexanol when compared to the classical macroscale process was obtained for c _i,hexanol = 5.5 mmol/L, c _i,NAD+ = 0.55 mmol/L, and γ _i,ADH = 0.092 g/L. In the 6 μL microreactor 11.78% conversion of hexanol was attained after 72 sec, while in the macroscale process 5.3% conversion of hexanol was reached after 180 sec.     

The production of ethanol from lactose in a tubular reactor by immobilized cells of Kluyveromyces fragilis

Summary An immobilized-cell tubular reactor for the continuous fermentation of lactose by Kluyveromyces fragilis was developed. Two types of supporting media were successfully tested; beechwood cubes and activated charcoal pellets. Ethanol productivity of 17.2 g/l/h was achieved from a 15% whey-lactose solution using K. fragilis immobilized on charcoal pellets, with a final ethanol concentration of 18 g/l. The use of two reactors in series demonstrated that it is possible to obtain up to 50 g/l of ethanol in the final product. No decrease in biological activity of the immobilized yeast cells occurred over a period of up to 31 days of continuous operation.     

Influence of substrate concentration on the stability and yield of continuous biohydrogen production

The effect of substrate concentration (sucrose) on the stability and yield of a continuous fermentative process producing hydrogen was studied. High substrate concentrations are attractive from an energy standpoint as they would minimise the energy required for heating. The reactor was a CSTR; temperature was maintained at 35 degrees C; pH was controlled between 5.2 and 5.3, and the hydraulic retention time (HRT) was 12 h. Online measurements were taken for ORP, pH, temperature, %CO2, gas output and %H2, and data logged using a MatLAB data acquisition toolbox. Steady-state operation was obtained at 10, 20 and 40 g/L of sucrose in the influent, but a subsequent step change to 50 g/L was unsustainable. The hydrogen content ranged between 50% and 60%. The yield of hydrogen decreased as the substrate concentration increased from 1.7 +/- 0.2 mol/mol hexose added at 10 g/L, to 0.8 +/- 0.1 mol/mol at 50 g/L. Sparging with nitrogen improved the hydrogen yield by at least 35% at 40 g/L and at least 33% at 50 g/L sucrose. Sparging also enabled steady-state operation at 50 g/L sucrose. Addition of an extra 4 g/L of n-butyric acid to the reactor operating at 40 g/L sucrose increased the butyrate concentration from 9,830 to 18,900 mg/L, immediately stopping gas production and initiating the production of propionate, whilst the addition of 2 g/L taking the butyrate concentration to 12,200 mg/L did not do so. It was shown that operation at 50 g/L sucrose in a CSTR in butyrate fermentation is possible.     

An ultrafiltration membrane bioreactor for the lipolysis of olive oil in reversed micellar media

The enzymatic hydrolysis of olive oil using Chromobacterium viscosum lipase B encapsulated in reversed micelles of dioctyl sodium sulfosuccinate (AOT) in isooctane was investigated in an ultrafiltration ceramic membrane reactor of tubular type, operating in a batch mode. Water concentration was found to be a critical parameter in the enzyme kinetics and hydrolysis yield of the reaction. The size of micelles, recirculation rate, and substrate concentration were found to be the major factors affecting the separation process. A correlation that enables the prediction of final conversion degrees in this bioreactor from the initial reaction conditions was established. (c) 1993 Wiley & Sons, Inc.     

Experimental investigation of G6P production and simultaneous ATP regeneration by conjugated enzymes in an ultrafiltration hollow-fiber reactor

Enzymatic synthesis of glucose 6-phosphate from glucose and ATP catalyzed by glucokinase from B. stearothermophilus and enzymatic regeneration of ATP from ADP and acetyl phosphate catalyzed by acetatekinase from B. stearothermophilus were simultaneously performed in an Ultrafiltration hollow-fiber reactor of the multitubular heat-exchanger type. Experimental results of space-time yield, the conversion, and ATP recycle number were in good agreement with the theoretical predictions based on the simple analytical model developed in the preceding article. The best results for space-time yield and conversion were Y(s) = 1.97 mol/m(3) h and X = 92.8%, respectively, under the same conditions, and the best result for the ATP recycle number was N(R) = 2130 under conditions different from those above. However, Y(s) = 1.72 mol/m(3) h, X = 81.2%, and N(R) = 1620 were the results when the space-time yield, conversion, and recycle number were at the highest in combination under the same conditions. Results of long-term operation showed that the apparent remaining activity of the enzyme system was ca. 55% after continuous operation for 16 days, the decrease in the enzyme activity being faster than that expected from their half-life times determined individually in the homogeneous system.     

Simulation of a cross-flow shrinking-bed reactor for the hydrolysis of lignocellulosics.

An idealized model is developed for the case in which biomass slurry is conveyed through an annulus, with water or steam entering through an inner porous wall and liquid product leaving through an outer porous wall. It is assumed that the ratio of occluded liquid to solid in the slurry is a constant, Rws, and that non-occluded water is immediately removed from the reactor. The goal of > 90% sugar yield with > 10% sugar in the product is almost reached (88% glucose yield, 91% xylose yield, 47 g/l glucose and 45 g/l xylose) at 240 degrees C, 1% acid. Rws = 1 and a radial wash water flow of three times the initial mass flow of solids to the reactor per meter of reactor length per g/l of sugar concentration in the occluded water. If Rws is limited to 3, the yield falls to 85% and the total sugar concentration to 61 g/l. Even without cross-flow wash, the yields can be increased by about 16 percentage points, compared to plug flow, by extracting excess liquid through the outer wall as it is formed. At 200 degrees C, where one might prefer to operate for ease of control and concern about the possibility of making fermentation inhibitors at higher temperatures, the maximum glucose yield in a plug-flow reactor is low (12-13%) whereas in a cross-flow reactor, at a high cross-flow wash rate, it can still be quite high (60-83%) but at a very low concentration (0.57-1.47%). In these simulations it is assumed that one-half of the inerts is solubilized. The formation of oligomers is neglected.     

Enzymatic Production of Pure D-Mannitol at High Productivity

An enzymatic, NAD(H)-dependent process for the efficient production of D-mannitol from D-fructose as one single product is described and optimized with respect to productivity at high substrate conversion. Stereospecific reduction of D-fructose is catalyzed by recombinant mannitol dehydrogenase from Pseudomonas fluorescens DSM 50106, overexpressed in Escherichia coli. Regeneration of NADH is accomplished by formate dehydrogenase-mediated oxidation of formate into CO₂, thus avoiding byproduct formation and yielding total turnover numbers for the coenzyme of approximately 1000 for a single round of D-fructose conversion. In optimized batchwise reduction of D-fructose, a D-mannitol productivity of 2.25 g/(L h) was obtained for a final product concentration of 72g/L and a D-fructose conversion of 80%. D-Mannitol was crystallized from the ultrafiltered product solution in 97% purity and 85% recovery, thus also allowing reuse of enzymes for repeated batchwise production of D-mann!itol!.     

Pilot-plant production of prednisolone using calcium alginate immobilized Arthrobacter simplex

The maximum conversion of hydrocortisone suspensions at initial substrate concentrations greater than 4 g/L by immobilized Arthrobacter simplex in a batch reactor was 80-85%. By feeding hydrocortisone suspensions continuously to either a fed-batch-operated stirred tank reactor or to a continuous-flow airlift loop reactor, at a rate such that the soluble hydrocortisone concentration in the reactor remained ca. 0.05 g/L, 95% conversion of substratewas obtained at final product concentrations exceeding 4 g/L.     

Mathematical modelling of the dehydrogenase catalyzed hexanol oxidation with coenzyme regeneration by NADH oxidase

The hexanol oxidation catalyzed by alcohol dehydrogenase from baker's yeast (YADH) has been investigated with two different forms of the biocatalyst: the isolated YADH as well as the YADH in the permeabilized whole cells. It was found that in this reaction, equilibrium is shifted to the reduction side. Hence, to increase the conversion it was necessary to regenerate NAD⁺. For that purpose, enzyme NADH oxidase isolated from Lactobacillus brevis was used. All biocatalysts were kinetically characterized. The overall reaction rate was described by the mathematical model which consisted of kinetics and balance equations. Due to the deactivation of NADH oxidase, only 50–58% hexanol was converted to hexanal in the batch reactor where the hexanol oxidation was catalyzed by isolated YADH. In the case of permeabilized baker's yeast cells, no enzyme deactivation occurred and 100% hexanol conversion in the hexanoic acid was detected.     

Kinetics of hydrogen production with continuous anaerobic cultures utilizing sucrose as the limiting substrate

In this study, local sewage sludge was acclimated to establish H2-producing enrichment cultures, which were used to convert sucrose to H2 with continuously stirred anaerobic bioreactors. The steady-state behaviors of cell growth, substrate utilization, and product formation were closely monitored. Kinetic models were developed to describe and predict the experimental results from the H2-producing cultures. Operation at dilution rates (D) of 0.075-0.167 h(-1) was preferable for H2 production, resulting in a H2 concentration of nearly 0.02 mol/l. The optimal hydrogen production rate was 0.105 mol/h occurring at D=0.125 h(-1). The major volatile fatty acid produced was butyric acid (HBu), while acetic acid and propionic acid were also produced in lesser quantities. The major solvent product was ethanol, whose concentration was only 15% of that of HBu, indicating that the metabolic flow favors H2 production. The proposed model was able to interpret the trends of the experimental data. The maximum specific growth rate (mu(max)), Monod constant (Ks), and yield coefficient for cell growth (Y(x/s)) were estimated as 0.172 h(-1), 68 mg COD/l, and 0.1 g/g, respectively. The model study also suggests that product formation in the continuous hydrogen-producing cultures was essentially a linear function of biomass concentration.     

Production of high-content fructo-oligosaccharides by an enzymatic system from Penicillium rugulosum

Summary The production of high-content fructo-oligosaccharides from sucrose by a crude FTF from a new strain of Penicillium isolated in our Laboratory was investigated. The optimum conditions for the production of the enzyme and for the enzymic reaction have been determined. It has been demonstrated that the crude enzyme acts as a mixed enzyme system of fructosyl transferase (FTF; Class 2 of Enzyme Nomenclature) and glycosidases (Class 3 of Enyme Nomenclature). Under optimum conditions: pH 5.5, temperature 55°C, sucrose concentration 750 g/l, enzyme concentration 5 FTF units/g sucrose, conversion yield up to 80% were obtained and high concentration of nystose (412 g/l) and fructofuranosyl-nystose (176 g/l) were accumulated.     

Continuous production of 3-fluoro-L-alanine with alanine dehydrogenase

Alanine dehydrogenase catalyzed the conversion of 3-fluoropyruvate into 3-fluoro-L-alanine in the presence of NADH and ammonia. The optimum pH of the reaction was 7.8. The K(m) values of the enzyme for 3-fluoropyruvate, polyethylene glycol-bound NADH, and ammonia were 2.94, 0.56, and 105mM, respectively. 3-Fluoro-L-alanine was selectively and continuously produced from 3-fluoropyruvate and ammonium formate in an enzyme membrane reactor by the multienzyme reaction system of alanine dehydrogenase and formate dehydrogenase with a simultaneous coenzyme regeneration. The average conversion and the space-time yield were 73% and 75 g/L day, respectively, with operation of the reactor for 4 days. Alanine dehydrogenase and formate dehydrogenase consumed were 11, 370 and 22, 950 units/kg 3-fluoro-L-alanine, respectively. The cycle number was 3150 mol/mol NAD.     

Kinetics of the enzymatic hydrolysis of cellulose: 1. A mathematical model for a batch reactor process

A mathematical model for enzymatic cellulose hydrolysis, based on experimental kinetics of the process catalysed by a cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] preparation from Trichoderma longibrachiatum has been developed. The model takes into account the composition of the cellulase complex, the structural complexity of cellulose, the inhibition by reaction products, the inactivation of enzymes in the course of the enzymatic hydrolysis and describes the kinetics of d-glucose and cellobiose formation from cellulose. The rate of d-glucose formation decelerated through the hydrolysis due to a change in cellulose reactivity and inhibition by the reaction product, d-glucose. The rate of cellobiose formation decelerated due to inhibition by the product, cellobiose, and inactivation of enzymes adsorbed on the cellulose surface. Inactivation of the cellobiose-producing enzymes as a result of their adsorption was found to be reversible. The model satisfactorily predicts the kinetics of d-glucose and cellobiose accumulation in a batch reactor up to 70–80% substrate conversion on changing substrate concentration from 5 to 100 g l^?1and the concentration of the enzymic preparation from 5 to 60 g l^?1.     

基因工程酶法生产N-氨基甲酰-D-对羟基苯甘氨酸反应条件的优化(英文)

为了实现利用生物酶转化法生产D 对羟基苯甘氨酸 ,以工程菌E .coliBL2 1 pMD T7 dht细胞作酶源 ,对底物对羟基苯海因到中间体N 氨基甲酰 D 对羟基苯甘氨酸的酶转化条件进行了优化 .酶转化的最适温度为 37℃ ,最适pH为 9 0 .在Tris HCl、磷酸盐、碳酸盐和硼酸盐 4种缓冲体系中 ,底物对羟基苯海因的转化率相近 .菌体细胞经适当冻融后 ,底物对羟基苯海因的转化率被提高 .水溶性有机溶剂DMSF、DMF和Tween 80使对羟基苯海因的转化率降低 .转化时底物和菌体的合适比例为 30g L对羟基苯海因和 10g L湿菌体 .经工程菌E .coliBL2 1 pMD T7 dht细胞催化 ,底物的转化率在 13h内可达到 96 % .所制备的产物熔点、旋光性和红外光谱等与标准品一致     

Enzymatic hydrolysis of willow treated with a steam burst without preliminary water extraction with a high concentration of substrate

A laboratory reactor equipped with a screw press was used for hydrolysis of steam-SO2 exploded willow Salix caprea by a composition of Trichoderma reesei and Aspergillus foetidus enzyme preparations at high substrate concentrations. Optimal conditions providing the maximal volume of hydrolysis syrup with maximal sugar concentrations were determined. Two different hydrolysis procedures were developed in order to exclude initial washing of steam-pretreated plant raw material by large volumes of water, which is necessary to eliminate the inhibitory effect of explosion by-products on enzymatic hydrolysis. The first procedure included a one-hour-long enzymatic prehydrolysis of the substrate, then separation of sugar syrup containing 40-60 g/l of glucose, 20-25 g/l of xylose, and up to 10% of disaccharides, as well as up to 35% of the initial enzymatic activity, then addition of a diluted acetate buffer (pH 4.5), and subsequent hydrolysis of the substrate by the adsorbed enzymes leading to the final accumulation of up to 140 g/l glucose and up to 15 g/l xylose. In the second scenario, the exploded willow was initially adjusted by alkali to pH 4.5 and then hydrolyzed directly by added enzymes for 24 hours. This procedure resulted in a nearly total polysaccharide hydrolysis and accumulation of up to 170 g/l glucose and 20 g/l xylose. The reasons of inhibition of enzymatic hydrolysis are discussed.     

Production of 2,3-butanediol in a membrane bioreactor with cell recycle

Summary The production of 2,3-butanediol by Enterobacter aerogenes DSM 30053 was studied in a cell recycle system with a microfiltration module. Emphasis was put on the influence of oxygen supply, cell residence time, dilution rate, and pH. Under optimal conditions a productivity as high as 14.6 g butanediol + acetoin/l per hour was achieved with a product concentration of 54 g/l and a product yield of 88%. This productivity is three times higher than that of an ordinary continuous culture. The achievable final product concentration of a cell recycle system was limited by the accumulation of the inhibiting by-product acetic acid, which increased very rapidly at low dilution rate. To maximize product concentration a fed-batch fermentation was carried out with stepwise pH adaption at high cell density. A final product concentration of 110 g/l was obtained with a productivity of 5.4 g/l per hour and a yield of 97%.     

A comparative study of the regioselectivity of the β-galactosidases from Kluyveromyces lactis and Bacillus circulans in the enzymatic synthesis of N-Acetyl-lactosamine in aqueous media

The enzymatic synthesis of N-acetyl-lactosamine (LacNAc) was studied in aqueous media with high substrate concentrations using the transgalactosylation of N-acetyl-D-glucosamine (GlcNAc), starting from lactose as a galactosyl donor. The efficiency and regioselectivity of the β-galactosidases from Kluyveromyces lactis (KlβGal) and Bacillus circulans (BcβGal) were compared. The reaction was optimized by varying the experimental conditions (pH, catalytic activity concentration, and mass concentration ratio of the substrates), which enhanced the synthesis yields with both enzymes and especially with BcβGal. BcβGal catalyzed the formation of the maximal LacNAc concentration obtained (101 mM or 39 g L(-1), corresponding to a yield of 11% on the basis of GlcNAc conversion), after 5 h at pH 6.5 and for a substrate mass concentration ratio of 1. This enzyme also gave an optimal synthesis yield of about 17.5%. No change in regioselectivity was observed when using KlβGal, whereas the regioselectivity of BcβGal proved to be subject to variations, the 1-4 and 1-6 linkages being favored under kinetic and thermodynamic control conditions, respectively. Finally, it was demonstrated that the N-acetyl-allolactosamine synthesized during the GlcNAc transgalactosylation catalyzed by BcβGal was a thermodynamic product and did not result from a chemical and/or enzymatic isomerization of LacNAc.     

Enzymatic Synthesis of Hexyl Glycosides from Lactose At Low Water Activity and High Temperature Using Hyperthermostable β-glycosidases

Optimization of hexyl-g-glycoside synthesis from lactose in hexanol at low water activity and high temperature was investigated using g-glycosidases from hyperthermophilic organisms: Sulfolobus solfataricus (LacS) and Pyrococcus furiosus (CelB). The method for water activity adjustment by equilibration with saturated salt solutions was adapted for use at high temperature. The influence of enzyme immobilization (on XAD-4, XAD-16, or Celite), addition of surfactants (AOT or SDS), substrate concentration, water activity, and temperature (60-90°C) on enzymatic activity and hexyl-g-glycoside yield were examined. Compared to other g-glycosidases in lactose conversion into alkyl glycoside, these enzymes showed high activity in a hexanol one-phase system and synthesized high yields of both hexyl-g-galactoside and hexyl-g-glucoside. Using 32 λg/l lactose (93 λmM), LacS synthesized yields of 41% galactoside (38.1 λmM) and 29% glucoside (27.0 λmM), and CelB synthesized yields of 63% galactoside (58.6 λmM) and 28% glucoside (26.1 λmM). With the addition of SDS to the reaction it was possible to increase the initial reaction rate of LacS and hexyl-g-galactoside yield (from 41 to 51%). The activity of the lyophilized enzyme was more influenced by the water content in the reaction than the enzyme on solid support. In addition, it was concluded that for the lyophilized enzyme preparation the enzymatic activity was much more influenced by the temperature when the water activity was increased. A variety of different glycosides were prepared using different alcohols as acceptors.     

Bacterial cellulose production under oxygen-enriched air at different fructose concentrations in a 50-liter, internal-loop airlift reactor

Bacterial cellulose (BC) production by Acetobacter xylinum subsp. sucrofermentans BPR2001 was carried out in a 50-1 internal-loop airlift reactor in air at an initial fructose concentration of 40 g/l. The BC production rate was 0.059 g/l per h. When oxygen-enriched air was supplied instead of air, the BC production rate increased to 0.093 g/l per h, and the BC yield was enhanced from 11% in air to 18%. When the initial fructose concentrations were varied from 30 to 70 g/l, the highest BC yield (35%) the highest production rate (0.22 g/l x per h), and the highest concentration of BC produced (10.4 g/l) were observed at 60-70 g/l fructose. From the carbon mass balance calculated at the final stage of cultivation, it was observed that enhanced BC production was reflected as a decrease in both CO2 evolution and the concentration of other unknown substances, suggesting the efficient utilization of energy for BC synthesis despite O2 limitation.