A forced-flow membrane reactor for transfructosylation using ceramic membrane

A forced-flow membrane reactor system for transfructosylation was investigated using several ceramic membranes having different pore sizes. beta-Fructofuranosidase from Aspergillus niger ATCC 20611 was immobilized chemically to the inner surface of a ceramic membrane activated by a silane-coupling reagent. Sucrose solution was forced through the ceramic membrane by crossflow filtration while transfructosylation took place. The saccharide composition of the product, which was a mixture of fructooligosaccharides (FOS), was a function of the permeate flux, which was easily controlled by pressure. Using 0.2 micrometer pore size of symmetric ceramic membrane, the volumetric productivity obtained was 3.87 kg m(-3) s(-1), which was 560 times higher than that in a reported batch system, with a short residence time of 11 s. The half-life of the immobilized enzyme in the membrane was estimated to be 35 days by a long-term operation.     

Ceramic membrane microfilter as an immobilized enzyme reactor.

This study investigated the use of a ceramic microfilter as an immobilized enzyme reactor. In this type of reactor, the substrate solution permeates the ceramic membrane and reacts with an enzyme that has been immobilized within its porous interior. The objective of this study was to examine the effect of permeation rate on the observed kinetic parameters for the immobilized enzyme in order to assess possible mass transfer influences or shear effects. Kinetic parameters were found to be independent of flow rate for immobilized penicillinase and lactate dehydrogenase. Therefore, neither mass transfer nor shear effects were observed for enzymes immobilized within the ceramic membrane. Both the residence time and the conversion in the microfilter reactor could be controlled simply by regulating the transmembrane pressure drop. This study suggests that a ceramic microfilter reactor can be a desirable alternative to a packed bed of porous particles, especially when an immobilized enzyme has high activity and a low Michaelis constant.     

A forced flow membrane enzyme reactor for sucrose inversion using molasses

We have developed a bioreactor which uses enzyme immobilized within a ceramic membrane support (1 mm thickness). Substrate is forced through the membrane by cross-flow filtration with the reaction taking place during the process of crossing the membrane. The bioreactor is termed forced-flow membrane enzyme reactor, FFMER. Invertase, which uses sucrose to form glucose and fructose, was tested in this system. The immobilized invertase membrane converted 100% of the sucrose in a feed stream made up of a 50% molasses solution. Because molasses contains many substances besides sucrose, this method is applicable to processes using substrates present in impure feeds.     

Inversion of sucrose with β-d-fructofuranosidase (invertase) immobilized on bead DEAHP-cellulose: Batch process

The inversion of sucrose with β-d-fructofuranosidase (EC 3.2.1.26) immobilized by an ionic bond on bead cellulose containing weak basic N,N-diethylamino-2-hydroxypropyl groups has been investigated. The immobilized enzyme is strongly bound at an ionic strength up to 0.1 M in the pH range 3–6. The amount adsorbed is proportional to porosity and to the exchange capacity of the ion exchange cellulose, reaching values up to 200 mg/g dry carrier, with an activity in 10% sucrose solution at 30°C, pH 5, >8000 μmol min^?1 g^?1. The inversion of sucrose with immobilized β-d-fructofuranosidase was carried out in a stirred reactor. The dependence of activity on pH (3–7), temperature (0–70°C) and concentration of the substrate (2–64 wt%) were determined, and the inversion was compared with that obtained using non-immobilized enzyme under similar conditions. The rate of inversion at low substrate concentration (2–19 wt%) was described by Michaelis-Menten kinetics.     

Inversion of sucrose with β- -fructofuranosidase (invertase) immobilized on bead DEAHP-cellulose: Batch process

The inversion of sucrose with β- -fructofuranosidase (EC 3.2.1.26) immobilized by an ionic bond on bead cellulose containing weak basic N,N-diethylamino-2-hydroxypropyl groups has been investigated. The immobilized enzyme is strongly bound at an ionic strength up to 0.1 M in the pH range 3–6. The amount adsorbed is proportional to porosity and to the exchange capacity of the ion exchange cellulose, reaching values up to 200 mg/g dry carrier, with an activity in 10% sucrose solution at 30°C, pH 5, >8000 μmol min⁻¹ g⁻¹. The inversion of sucrose with immobilized β- -fructofuranosidase was carried out in a stirred reactor. The dependence of activity on pH (3–7), temperature (0–70°C) and concentration of the substrate (2–64 wt%) were determined, and the inversion was compared with that obtained using non-immobilized enzyme under similar conditions. The rate of inversion at low substrate concentration (2–19 wt%) was described by Michaelis-Menten kinetics.     

Application of immobilized invertase to continuous hydrolysis of concentrated sucrose solutions

Invertase immobilized onto corn grits was utilized in the hydrolysis of highly concentrated sucrose solutions producting liquid sugar solutions containing glucose and fructose. Comparisons of conversion efficiencies of this immobilized invertase in a continuous stirredtank reactor and a plug-flow reactor indicated that the plug-flow reactor has an higher efficiency. Continuous sucrose hydrolysis was then performed in 0.1- and 1-L tubular reactors. This tenforld scaling-up was achieved without any noticeable loss in efficiency. This process thus was scaled-up to a 17.6-L pilot reactor set in a cane sugar refinery. This reactor was fed with highly concentrated sucrose solutions [71% (w/w)] to produce invert sugar syrup with the desired inversion degree. It allows a productivity equal to 9.1 kg sucrose hydrolyzed/h in the case of a 69% (w/w) sucrose initial concentration with a 72% conversion rate.     

Inversion of sucrose in a continuous process with β-d-fructofuranosidase (invertase) immobilized on bead DEAHP-cellulose

Inversion of sucrose with β-d-fructofuranosidase (EC 3.2.1.26) immobilized by the ionic bond on bead DEAHP-cellulose has been studied under flow conditions. Under these conditions, the inversion of sucrose is affected by the concentration and flow rate of the substrate and by the reaction temperature. The effect of substrate concentration on the reaction was investigated in the range 19.5–64.2 wt %; the effect of flow rate was examined in the range 0.25–5.57 g solution per min, and the temperature range used was 25–50°C. It was found that the activities of immobilized β-d-fructofuranosidase in stirred and flow reactors were the same. The lower activities of β-d-fructofuranosidase in the case of concentrated solutions, and of immobilized β-d-fructofuranosidase compared with the native enzyme are attributed to more difficult diffusion through the beads of the ion exchanger, especially of the strongly viscous substrate. A long-term investigation of the enzyme activity over a period of three months demonstrated the stability of the β-d-fructofuranosidase immobilized by the ionic bond on bead DEAHP-cellulose; the half-life of the enzyme was 215 days. It was also found that the immobilization of the enzyme on a carrier was more effective under flow conditions, i.e. through an ion exchanger in the column, than under the equilibrium conditions of a stirred reactor.     

L-DOPA production from tyrosinase immobilized on nylon 6,6

The production of L-DOPA immobilized on chemically modified nylon 6,6 membranes was studied in a batch reactor. Tyrosinase was immobilized on nylon using glutaraldehyde as a crosslinking agent. The effects of membrane pore size and glutaraldehyde concentration upon enzyme uptake and L-DOPA production were investigated. Enzyme uptake was unaffected by glutaraldehyde concentration; approximately 70% uptake was observed when 25% w/v (group 1), 5% (group 2), and 3% (group 3) glutaraldehyde were used, indicating that glutaraldehyde was in excess. Similarly, uptake was the same for membranes with 0.20 and 10 mum pore sizes.Membranes produced using different levels of glutaraldehyde exhibited dramatically different capacities for L-DOPA production, despite the fact that enzyme uptake was equivalent. Membranes from groups 2 and 3 (5% and 3% glutaraldehyde) produced L-DOPA at a rate of 1.70 mg L(-1) h(-1) over 170 h in a 500-mL batch reactor. However, no free L-DOPA was detected when group 1 membranes were used. Experimental evidence suggests that L-DOPA was produced, but remained bound to these membranes via excess glutaraldehyde left over from the immobilization process. Membrane pore size also effected L-DOPA production; less production was observed when 10-mum membranes were used, despite equivalent enzyme uptake. The observed difference in production may be due to differences in the pore density on the two types of membranes which could affect the access of the substrate to the immobilized enzyme.The results of these studies indicate that tyrosinase can be effectively immobilized on nylon 6,6. L-DOPA production was optimal when 0.20-mum-pore-size membranes were activated with 3-5% glutaraldehyde. Stability studies indicated a 20% reduction in activity over 14 days when the immobilized enzyme was used under turnover conditions. (c) 1996 John Wiley & Sons, Inc.     

Continuous inversion of sucrose by gel-entrapped yeast cells

The feasibility of immobilizing invertase (β-d-fructofuranosidase; EC 3.2.1.26) from Saccharomyces cerevisiae cells by various methods was examined. The yeast cells were adapted for maximal invertase activity by growth in a medium containing 0.2% glucose and 1% lactate. There was no permeability barrier for the enzyme in the whole cells. Entrapment in acrylamide polymerized by gamma-rays (200 kR) was observed to be most effective, with retention of 85% of the activity. The evaluation of the properties of the immobilized invertase indicated that the kinetic values were not appreciably altered despite a broad pH optimum. The enzyme was more stable to both heat and gamma-radiation. The immobilized cells could be used repeatedly in a packed bed reactor system for inversion of sucrose without observable loss in activity for over one month.     

Immobilization of acetyl-CoA:arylamine N-acetyltransferase and the preparation of an enzyme reactor for the synthesis of N-[11C]acetylserotonin

The enzyme arylamine acetyltransferase (acetyl-CoA:arylamine N-acetyltransferase, EC 2.3.1.5) from pigeon liver is immobilized onto differently derivatized controlled pore glass beads. Different silanes, spacer arms and reactive end-groups were tested, and immobilized enzyme stability tests were performed. From these experiments, the method of choice was selected: immobilization on controlled pore glass beads (24 nm pore size, 75-125 microns particle size) derivatized with gamma-aminopropyl and glutaraldehyde as the reactive end group. The kinetic properties of an enzyme reactor were investigated and optimized. The goal was to obtain a rapid high-yield conversion of 0.5-1 mumol acetyl-CoA to N-acetylserotonin, so that the reactor is useful for the 11C-labelling of N-acetylserotonin. Using an enzyme reactor (9.8 x 0.5 cm i.d.) containing 4.6 U active arylamine acetyltransferase immobilized onto 930 mg carrier, a 70% conversion of acetyl-CoA was obtained within 4 min.     

Hydrolysis of concentrated sucrose syrups by invertase immobilized on anion exchanger waste cotton thread.

Yeast invertase was immobilized on polyethyleneimine-coated cotton thread by adsorption followed by crosslinking with glutaraldehyde. The thread-bound invertase was used as an easily retrievable system for the hydrolysis of 80% w/v commercial sucrose syrups. The immobilized enzyme was stable for over 90 days to a temperature of 50 degrees C, only when stored in 80% sucrose solution. Above this temperature, inactivation of enzyme was observed. The cotton threads were used in a batch reactor for hydrolysis of sucrose in about 30 batches carried out over a period of 50 days without loss in activity. The threads could also be used in a packed bed reactor (1.51) for 97% hydrolysis of 80% sucrose syrups at 50 degrees C at a rate of about 360 kg per month for a period of 3 months.     

Inversion of sucrose in a continuous process with β- -fructofuranosidase (invertase) immobilized on bead DEAHP-cellulose

Inversion of sucrose with β- -fructofuranosidase (EC 3.2.1.26) immobilized by the ionic bond on bead DEAHP-cellulose has been studied under flow conditions. Under these conditions, the inversion of sucrose is affected by the concentration and flow rate of the substrate and by the reaction temperature. The effect of substrate concentration on the reaction was investigated in the range 19.5–64.2 wt %; the effect of flow rate was examined in the range 0.25–5.57 g solution per min, and the temperature range used was 25–50°C. It was found that the activities of immobilized β- -fructofuranosidase in stirred and flow reactors were the same. The lower activities of β- -fructofuranosidase in the case of concentrated solutions, and of immobilized β- -fructofuranosidase compared with the native enzyme are attributed to more difficult diffusion through the beads of the ion exchanger, especially of the strongly viscous substrate. A long-term investigation of the enzyme activity over a period of three months demonstrated the stability of the β- -fructofuranosidase immobilized by the ionic bond on bead DEAHP-cellulose; the half-life of the enzyme was 215 days. It was also found that the immobilization of the enzyme on a carrier was more effective under flow conditions, i.e. through an ion exchanger in the column, than under the equilibrium conditions of a stirred reactor.     

Production of palatinose using<Emphasis Type="Italic"> Serratia plymuthica</Emphasis> cells immobilized in chitosan

In recent decades, the production of palatinose has aroused great interest since this structural isomer of sucrose has interesting potential. We describe a simple and effective method of immobilizing Serratia plymuthica cells in chitosan. The sucrose isomerase activity of immobilized preparations was enhanced many times by activation with fresh nutrient medium and subsequent drying. The preparations obtained were physically very stable with high enzyme activity and excellent operational stability. The effect of temperature, pH and substrate concentration on enzyme activity of the immobilized cells was investigated. Using immobilized cells, a complete conversion of sucrose (40% solution) into palatinose was achieved in 4 h in a "batch"-type enzyme reactor. The use of free or immobilized cells had no effect on the composition of the solution, in particular the sugar content. The palatinose content was 80% and that of trehalulose 7%.     

Control of an ethanol fermentation carried out with alginate entrapped Saccharomyces cerevisiae

Process control of different reactor models for continuous production of ethanol from sucrose with immobilized yeast has been studied. An enzyme thermistor with immobilized invertase recorded the concentration of sucrose continuously. Ethanol was recorded by a membrane gas sensor with a SnO(2) semiconductor used as detector. A process computer controlled the substrate feed to keep substrate as well as ethanol concentration at preset values by using algorithms of varying complexity. It was thereby demonstrated that PID regulators as well as more advanced algorithms (Otto-Smith regulator, state feedback from a Kalman filter, and cascade control) are useful alternatives to maintain a constant concentration in the fermentor effluents. The time required for the system to return to predetermined conditions after various kinds of disturbances has been especially studied. It was shown that the more advanced regulator used the shorter time.     

Enantioselective esterification of butanol-2 in an enzyme membrane reactor

The enzymatic esterification of octanoic acid with racemic butanol-2 was investigated. Esterifications of the acid were performed in a forced flow enzyme membrane reactor. The used membrane was prepared by a phase inversion process in polyamide-6 solution followed by the chemical immobilization of a lipase-catalyst. Influences of water content and pH were estimated. Their optimum values are equal to 0.5% w/w and pH 8. The reaction rate (at 303 K) of 5.1 × 10^?5 mol/h·cm² of the membrane area, and at least 85% enantiomeric excess in the produced ester mixture were obtained. The activity of immobilized lipase in the membrane process is about two times higher than that of the native lipase in the esterification performed in a tank reactor.     

Comparative study of controlled pore glass, silica gel and poraver for the immobilization of urease to determine urea in a flow injection conductimetric biosensor system

This study compared the responses of three enzyme reactors containing urease immobilized on three types of solid support, controlled pore glass (CPG), silica gel and Poraver. The evaluation of each enzyme reactor column was done in a flow injection conductimetric system. When urea in the sample solution passed though the enzyme reactor, urease catalysed the hydrolysis of urea into charged products. A lab-built conductivity meter was used to measure the increase in conductivity of the solution. The responses of the enzyme reactor column with urease immobilized on CPG and silica gel were similar and were much higher than that of Poraver. Both CPG and silica gel reactor columns gave the same limit of detection, 0.5 mM, and the response was still linear up to 150mM. The analysis time was 4-5 min per sample. The enzyme reactor column with urease immobilized on CPG gave a slightly better sensitivity, 4% higher than the reactor with silica gel. The life time of the immobilized urease on CPG and silica gel were more than 310h operation time (used intermittently over 7 months). Good agreement was obtained when urea concentrations of human serum samples determined by the flow injection conductimetric biosensor system was compared to the conventional methods (Fearon and Berthelot reactions). These were statistically shown using the regression line and Wilcoxon signed rank tests. The results showed that the reactor with urease immobilized on silica gel had the same efficiency as the reactor with urease immobilized on CPG.     

Process control of an ethanol fermentation with an enzyme thermistor as a sucrose sensor

Summary An enzyme thermistor was used to monitor and control the sucrose concentration in a conversion of sucrose to ethanol with immobilized yeast. A continuous stirred tank reactor containing calcium alginate to entrap Saccharomyces cerevisiae was used. The enzyme thermistor was continuously measuring the sucrose concentration in the fermenter with an on-line arrangement giving stable and reproducible heat signals. The control of the sucrose concentration level was performed with an analogue PI-controller.     

Immobilization of lipase by ultrafiltration and cross-linking onto the polysulfone membrane surface

In this study, a biphasic enzymatic membrane reactor was made by immobilizing Candida Rugosa lipase onto the dense surface of polysulfone ultrafiltration membrane by filtration and then cross-linking with glutaraldehyde solution. The reactor was further applied for the hydrolysis of olive oil, the performance of which was evaluated in respect of apparent reaction rate based on the amount of fatty acids extracted into the aqueous phase per minute and per membrane surface. It was found that the ultrafiltration and cross-linking process greatly improved the reaction rate per unit membrane area and the enzyme lifetime. The highest reaction rate reached 0.089 micromol FFA/min cm2 when the enzyme loading density was 0.098 mg/cm2. The results also indicated that the performance of lipase immobilized on the membrane surface was superior to that immobilized in the pores, and the apparent reaction rate and stability of immobilized lipases were improved greatly after cross-linking. It suggested that immobilization of enzymes by filtration and then cross-linking the enzymes onto the membrane surface is a simple and convenient way to prepare a high-activity immobilized enzyme membrane.     

Immobilization of glucoamylase on ceramic membrane surfaces modified with a new method of treatment utilizing SPCP-CVD

Glucoamylase, as a model enzyme, was immobilized on a ceramic membrane modified by surface corona discharge induced plasma chemical process-chemical vapor deposition (SPCP-CVD). Characterizations of the immobilized enzyme were then discussed. Three kinds of ceramic membranes with different amounts of amino groups on the surface were prepared utilizing the SPCP-CVD method. Each with 1-time, 3-times and 5-times surface modification treatments and used for supports in glucoamylase immobilization. The amount of immobilized glucoamylase increased with the increase in the number of surface modification treatments and saturated to a certain maximum value estimated by a two-dimensional random packing. The operational stability of the immobilized glucoamylase also increased with the increase in the number of the surface treatment. It was almost the same as the conventional method, while the activity of immobilized enzyme was higher. The results indicated the possibility of designing the performance of the immobilized enzyme by controlling the amount of amino groups. The above results showed that the completely new surface modification method using SPCP was effective in modifying ceramic membranes for enzyme immobilization.