Continuous production ofL-malic acid by immobilizedBrevibacterium ammoniagenes cells

Summary Continuous production ofL-malic acid from fumaric acid using immobilized microbial cells was investigated. Several microorganisms having fumarase activity were immobilized into a polyacrylamide gel lattice. Among the microorganisms tested, immobilizedBrevibacterium ammoniagenes IAM 1645 showed the highest enzyme activity, but produced an unwanted by-product, succinic acid. Conditions for suppression of this side reaction were investigated, and bile extract treatment of immobilized cells was found to be effective.The bile extract treatment of immobilized cells also resulted in a marked increase of reaction rate forL-malic acid formation.No difference was observed between the native enzyme and immobilized cells in optimal pH and temperature of the enzyme reaction.The effect of temperature on the reaction rate and the stability of fumarase activity of an immobilized cell column were investigated under conditions of continuous enzyme reaction. The decay of enzyme activity during continuous enzyme reaction was expressed by an exponential relationship. Half-life of the fumarase activity of the immobilized cell column at 37°C was calculated to be 52.5 days.Presented at the Annual Meeting of the Society of Fermentation Technology, Japan, Osaka, Japan, October 30, 1975.     

Engineering analysis of continuous production of L-aspartic acid by immobilized Escherichia coli cells in fixed beds

The reaction mechanism and decay behavior of aspartase activity for immobilized Escherichia coli cells were investigated by using a sectional packed column. Reaction within the immobilized cell column proceeded at zero-order on substrate solutions ranging in concentration from 0.1 to 1.0M, and the initial reaction rate was found to be 1.556 × 10^?2 mol/min/liter of immobilized cells. The effect of temperature on the reaction rate constant was investigated. The Arrhenius plot was straight line at temperatures below 43°C, and the activation energy for immobilized cells was calculated to be 12.36 kcal/mol. Asparatase activity in the immobilized cell column decayed exponentially and uniformly in all sections of a column. Its half-life was approximately 120 days. The rate of formation of L-aspartic acid was shown to be independent of column dimensions.     

Immobilization of Brevibacterium flavum with carrageenan and its application for continuous production of l-malic acid

Extensive experiments were carried out to improve the productivity ofl-malic acid from fumaric acid using Brevibacterium flavum immobilized with carrageenan. The most favourable preparation for the continuous production ofl-malic acid was obtained when 16 g of B. flavum cells was entrapped in 100 ml 3.4% carrageenan gel. However, the immobilized cells produced an unwanted by-product, succinic acid. Treatment of the immobilized cells with 0.6% bile extract suppressed the side reaction and gave the highest operational stability of fumarase activity. By the immobilization of intact cells, the optimal temperature of the enzyme reaction shifted to 10°C higher, the optimal pH became broader, and the operational stability of fumarase activity increased. The effect of temperature on the stability of fumarase activity in the immobilized cell column was investigated under conditions of continuous enzyme reaction. The decay of fumarase activity during continuous enzyme reaction was expressed by an exponential relationship. The productivity of the immobilized B. flavum using carrageenan was as high as 5.2 times that of the conventional immobilized B. ammoniagenes using polyacrylamide.     

Continuous production of urocanic acid by immobilized Achromobacter liquidum cells

Several microorganisms having higher L -histidine ammonia-lyase activity were immobilized into polyacrylamide gel lattice. The yield of enzyme activity by immobilization was highest in Achromobacter liquidum IAM 1667. As A. liquidum has urocanase activity, the cells were heat-treated at 70°C for 30 min to inactivate the urocanase. Enzymatic properties of the immobilized A. liquidum cells were investigated and compared with those of the intact cells. No difference was observed between the pH activity curve and optimal temperature for the intact and immobilized cells. The permeability of substrate or product through the cell wall was increased by immobilization of the cells. When an aqueous solution of 0.25M L -histidine (pH 9.0) containing 1mM Mg²⁺ was passed through a column packed with the immobilized A. liquidum cells at a flow rate of SV = 0.06 at 37°C, L -histidine was completely converted to urocanic acid. The L -histidine ammonia-lyase activity of the immobilized cell column was stable over 40 days at 37°C. From the effluent of the immobilized cell column, Urocanic acid was easily obtained in a good yield.     

A Deuterium Effect in Insect Attraction

Brevibacterium flavum cells obtained from different growth phases were immobilized with κ-carrageenan and the stability of the fumarase activity was investigated. The stability of fumarase activity of the immobilized preparation of cells of the stationary growth phase was highest. The highest stability of the immobilized cells seemed to be correlated to the high stability of fumarase activity in free cells of the stationary phase. High rigidity of the cell wall and membrane of B. flavum cells of the stationary phase and firm binding of fumarase protein to the cell membrane were suggested from several lines of evidence obtained on treatment of the cells with lysozyme and detergents or sonication of the cells. Electronmicrographs showed that the cells of the stationary phase retained the original shape after repeated batch reactions. Solubilized fumarase prepared from cells of the stationary phase showed the highest stability. Experiments using the partially purified enzyme strongly suggested the existence of fumarase-stabilizing components in the cells.     

Study of an enzyme membrane reactor with immobilized fumarase for production of L-malic acid

The conversion of fumaric acid into L-malic acid by fumarase immobilized in a membrane reactor was analyzed experimentally. The enzyme was entrapped in asymmetric capillary membranes made of polysulfone. The performance of the reactor was evaluated in terms of conversion degree, reaction rate, and stability. The influence of operating conditions, such as amount of immobilized enzyme, substrate concentration, residence time, and axial flow rate, were investigated. The kinetic parameters K(m), V(max), and k(+2) were also measured. The stability of the immobilized enzyme was very good, showing no activity decay during more than 2 weeks of continuous operation.     

Some properties of a papain–agarose reactor provided with a pH monitor

A small reactor of immobilized papain was used to gain some knowledge about the effect of immobilization upon the reactivity of the enzyme towards one substrate and various types of inhibitors. A buffer solution containing benzoyl–arginine ethyl ester as substrate was run through a small column of papain immobilized by attachment to agarose beads. The pH of the effluent was measured continuously and provided the data used to calculate the substrate conversion during passage through the reactor. The operation of the system was checked by determining the substrate conversion as a function of flow rate. It proved to operate as theory demanded. The rate and extent of inhibition were measured after addition of various inhibitors to the buffer–substrate solution. The following quantities of immobilized papain were found to be equal within ±20% to those of the free enzyme in solution: the overall activity, the K_m of benzoyl–arginine ethyl ester, the K_i of the competitive inhibitor benzoylamino-acetonitrile, the rate of inactivation by chloroacetic acid and by chloroacetamide, the rate of activation by cysteine of the mixed disulfide of papain and cysteine, and the rate of spontaneous reactivation of the KCNO–papain adduct. The inactivation by KCNO proved to be strongly pH dependent. This may explain why the rate of the latter reaction is only 66% of the rate with free enzyme. It is concluded that the rates and equilibrium constants measured in the present reactor system are within ±20% of the values of the dissolved enzyme, provided that the reactions are not strongly pH dependent. Calculation showed there was no diffusion limitation.     

A Bioreactor-Crystallizer for L-Malic Acid Production

A bioreactor with associated crystallizer for the accumulation of a highly concentrated slurry product has been developed and investigated. The transformation of Ca-fumarate to Ca-L-malate by the action of the fumarase of immobilized Brevibacterium flavum cells focussed on the performance of this newly-devised bioreactor-crystallizer system.

The following results were obtained

(1) The fumarase reaction in the bioreactor proceeded at a rate that was first-order in apparent substrate concentration.

(2) The reaction rate increased with the addition of Na₂-fumarate to the substrate solution.

(3) The reaction rate was independent of the substrate circulation rate and the initial substrate concentration in the crystallizer.

(4) Fumarase activity of immobilized B. flavum cells was stable after 10 repeated uses over a period of 10 days.

(5) Maximum concentration of the product, final conversion ratio of the substrate and the productivity of the bioreactor-crystallizer system were much higher than those for a conventional bioreactor using solubilized Ca-fumarate as a substrate.     

Continuous production of L-aspartic acid by immobilized aspartase

Various methods were tried for the immobilization of aspartase, and the preparation having the highest activity was obtained when partially purified aspartase from Escherichia coli was entrapped into polyacrylamide gel Iattice. Enzymatic properties of the immobilized aspartase were investigated and compared with those of the native aspartase. With regard to optimum pH, temperature, concentration of Mn⁺⁺, kinetic constants and heat stability, no marked difference was observed between the native and immobilized aspartases. By employing an enzyme column packed with the immobilized aspartase, conditions for continuous production of L -aspartic acid from ammonium fumarate were investigated. When a solution of 1M ammonium fumarate (pH 8.5, containing 1mM MnCl₂) was passed through the aspartase column at the flow rate of SV = 0.08 at 37°C, the highest rate of reaction was attained. From the column effluents, L-aspartic acid was obtained in a good yield.     

Screening of microorganisms having high fumarase activity and their immobilization with carrageenan

Summary To develop an efficient method for continuous production of L-malic acid from fumaric acid using immobilized microbial cells, screening of microorganisms having high fumarase activity was carried out and cultural conditions of selected microorganisms were investigated. As a result of screening microorganisms belonging to the genera Brevibacterium, Proteus, Pseudomonas, and Sarcina were found to produce fumarase in high levels. Among these microorganisms Brevibacterium ammoniagenes, B. flavum, Proteus vulgaris, and Pseudomonas fluorescens were further selected for their high fumarase levels in the cultivation on several media. These 4 microorganisms were entrapped into a k-carrageenan gel lattice, and the resultant immobilized B. flavum showed the highest fumarase activity and operational stability.Cultural conditions for the fumarase formation and the operational stability of fumarase activity of immobilized B. flavum are detailed. Productivity for L-malic acid using immobilized B. flavum with k-carrageenan was 2.3 fold of that using immobilized B. ammoniagenes with polyacrylamide.Presented at the Annual Meeting of the Agricultural Chemical Society of Japan, Nagoya, April 3, 1978     

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.     

Continuous production of NADP by immobilized Achromobacter aceris cells.

Several microorganisms having higher nicotinamide adenine dinucleotide kinase (NAD kinase, EC 2.7.1.23) activity were immobilized into polyacrylamide gel lattices. The enzyme activity field by immobilization was highest in Achromobacter aceris AKU 0120. By the incubation of the immobilized A. aceris cells at pH 4.0, the NAD kinase activity increased and the adenosine triphosphate (ATP)-degradation activity disappeared completely. Enzymatic properties of the immobilized A. aceris cells were investigated and compared with those of intact cells. The optimal pH and the optimal temperature of immobilized cells were the same as those of intact cells. Immobilized cell NAD kinase was more stable than that of intact cells. The operational half-life of immobilized cells was 20 days when the substrate solution was passed through a column packed with immobilized cells at a flow rate which gives a space velocity (SV) of 0.1 hr-1 at 37 degrees C. On the other hand, the half-life of the intact cells was only 6 hr.     

Improvement of production of l-aspartic acid using immobilized microbial cells

Summary In our laboratory, EAPc-7 a strain having higher aspartase activity was derived from Escherichia coli ATCC 11303. For the improvement of l-aspartic acid productivity using EAPc-7 cells immobilized in -carrageenan, it was necessary to eliminate the fumarase activity which converts fumaric acid to l-malic acid. Several treatments for specifically eliminating fumarase activity from EAPc-7 cells were tested and it was found that when EAPc-7 cells were treated in a culture broth (pH 4.9) containing 50 mM l-aspartic acid at 45° C for 1 h, fumarase activity was almost completely eliminated without inactivation of the aspartase.The treated cells, immobilized in -carrageenan, were used for continuous production of l-aspartic acid from ammonium fumarate. The formation of l-malic acid was negligible and the half-life of the immobilized preparation was 126 days.Productivity of immobilized preparation of treated EAPc-7 cells in l-aspartic acid production was six times of that of the parent cell preparation.     

Yeast alcohol dehydrogenase immobilized in a glutaraldehyde–albumin matrix: Kinetics and cofactor diffusional effects*

This study was carried out to define how the overall rate of reaction would be influenced by different degrees of diffusional resistance to cofactor transport within an oxidoreductase membrane matrix. To accomplish this, 0.7–6.6μM yeast alcohol dehydrogenase was immobilized in an albumin matrix crosslinked with 2.5 or 5.0% glutaraldehyde to give 102–1685 μM thick membranes. The enzyme half-life was at least doubled at pH 7.5 or 8.8 on immobilization. Values of the kinetic constants for the soluble and immobilized enzyme were determined at 25°C and pH 8.8 over the range of 0.01–1.0M bulk solution concentration of ethanol as substrate and 140–1000μM bulk solution concentration of nicotinamide adenine dinucleotide (NAD⁺) as cofactor, to give essentially single substrate kinetics in NAD⁺. Equilibrium partitioning of ethanol and NAD⁺ between the solution and membrane was measured and used in the data analysis. The four kinetic constants for the soluble enzyme agreed with literature values; and all increased with immobilization of the enzyme. The Michaelis constants for NAD⁺ and for ethanol were greater for the immobilized enzyme. The diffusional resistance to NAD⁺ transport, presented in terms of the Thiele modulus, showed that the overall rate of reaction was decreased by about 50% even at values of the modulus as low as 2.0.     

Enhancement and stabilization of the production of glucoamylase by immobilized cells of Aureobasidium pullulans in a fluidized-bed reactor

Summary Glucoamylase production by Aureobasidium pollulans A-124 was compared in free-living cells, cells immobilized in calcium alginate gel beads aerated on a rotary shaker (agitation rate 150 rpm), and immobilized cells aerated in an air bubble column reactor. Fermentation conditions in the bioreactor were established for bead concentration, substrate (starch) concentration, calcium chloride addition to the fermentation medium, and rate of aeration. Production of glucoamylase was optimized at approximately 1.5 units of enzyme activity/ml medium in the bioreactor under the following conditions: aeration rate, 2.0 vol air per working volume of the bioreactor (280 ml) per minute; gel bead concentration, 30% of the working volume; substrate (starch) concentration, at 0.3% (w/v); addition of calcium chloride to the medium at a final concentration of 0.01 M. Productivity levels were stabilized through the equivalent of ten batches of medium with the original inoculum of immobilized beads. Offprint requests to: M. Petruccioli     

l-Malic acid formation by immobilized Saccharomyces cerevisiae amplified for fumarase

The yeast Saccharomyces cerevisiae was amplified for the enzyme fumarase by cloning the single nuclear gene downstream of a strong promoter. The overproducing strain converted fumaric acid to l-malic acid at a rate of 65 mM g⁻¹ h⁻¹ in free cell experiments, and approximately 87% of the fumaric acid was converted to l-malic acid within 45 min. Activity was dependent on the addition of surfactant to the medium, and minimal activity was seen with the wild-type yeast strain. The constructed strain was immobilized in agarose beads (2.4 mm mean diameter) and within agarose microspheres (193 and 871 μm mean diameter). The rate of bioconversion increased with decreasing bead diameter, with similar rates observed with the 193-μm diameter microspheres to that achieved with the free cells. The presence of surfactant was essential for initial activity of the immobilized cells; however, high activity was observed in subsequent experiments in the absence of surfactant. Stable activities over a 48-h period were maintained within the large-diameter agarose beads, while decreasing activities were observed within the agarose microspheres.     

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.     

Modeling of a Catalyzed Reaction Using Lipase Immobilized in a Poly(vinyl alcohol) Membrane

A mathematical model for the hydrolysis reaction of p‐nitro phenol laurate catalyzed by a lipase immobilized in a membrane was developed. In an earlier study this model reaction was found to show very different reaction rates when it was performed in aqueous micellar solution with free enzyme and with membrane immobilized enzyme. It was assumed that a local accumulation of substrate in the membrane is responsible for the observed rate enhancement. The conversion of p‐nitro phenol ester within the membrane was modeled by considering a combination of the convective flow through poly(vinyl alcohol) membrane pores, concentration polarization of substrate containing micelles at the membrane surface and the kinetics of the reaction with free enzymes. It was demonstrated that the model offered a comprehensive understanding of the interaction of the involved phenomena. The modeling results are in good agreement with the experimental data from 10 runs with different enzyme and substrate concentrations. The substrate concentration at the membrane surface increased by up to a factor of 3 compared to the feed concentration. This effect explains the observed rate enhancement. Moreover, the model was used to determine the unknown parameters, i.e., the intrinsic retention and the mass transfer coefficient, by fitting the model to the experimental data. The model may also be used to calculate the optimum operating conditions and design parameters of such a reactor.     

Synthesis of cinnamyl acetate catalysed by highly reusable cotton-immobilized Pseudomonas fluorescens lipase

Cinnamyl acetate as an important fragrance ingredient could be synthesized by lipase-catalysed transesterification in organic systems, but enzyme proteins tended to denature and inactivate for no water lubrication. To improve the non-aqueous stability of lipases, absorbent cotton was taken as an alternative “water” phase to stabilize enzyme proteins. In a mass ratio of 1:1, Pseudomonas fluorescens lipase was immobilized on cotton fibres by physical absorption in a column glass bottle, forming a facile cotton-lipase bioreactor in which the transesterification between cinnamyl alcohol and vinyl acetate processed efficiently. From the molar conversions after reaction for 2?h at 37?°C and 160?rpm, the ability of cotton-lipase to transform substrate was more than 5-folds of native lipase. And even in static state and at 4?°C, the conversion of reaction catalysed by cotton-PFL had 11-fold increase relative to native lipase after 8?h. Recycles showed that the cotton-lipase had an extra-long half-life of activity (t_1/2?=?693?h) and a negligible decay rate in the ability to transform substrate (D_r?=?0.08% h^?1). All these showed that this lipase had been effectively activated and stabilized by cotton fibres for the numerous hydroxyl groups and fluffy structure.     

Application of Urethane Prepolymers to Immobilization of Biocatalysts: Δ1Dehydrogenation of Hydrocortisone by Arthrobacter simplex Cells Entrapped with Urethane Prepolymers

Acetone-dried cells of Arthrobacter simplex having appreciable steroid Δ¹-dehydrogenase activity were immobilized by mixing the cell suspension with water-miscible urethane prepolymers synthesized from toluene diisocyanate and polyether diols. The entrapped cell activity in the transformation of hydrocortisone to prednisolone was affected by the properties of urethane prepolymers, such as the isocyanate group content in prepolymers, the molecular weight of polyether diols and the ethylene oxide content in diols. The addition of 10% of organic solvents, such as methanol and glycols, to the aqueous reaction mixture enhanced the solubility of the substrate greatly and the reaction rate of the immobilized cells. The activity of immobilized cells remained high even in the system containing 30% of methanol, which drastically inhibited the activity of free cells. The presence of an electron acceptor, phenazine methosulfate or 2,6-dichlorophenolindophenol, significantly stimulated the steroid conversion with entrapped cells, as well as free cells. The stability of the cells over repeated reactions was greatly improved by immobilization.