Comparative study of reactor performance for the resolution of d,l-amino acids

The racemic resolution of l-valine and l-serine by fungal aminoacylase has been evaluated by comparing the performance of various reactor configurations including an anion exchange nylon tangential flow membrane reactor, a tubular reactor with aminoacylase adsorbed onto DEAE-Sephadex as support and a continuous stirred tank reactor with enzyme recycling using a flat ultrafiltration module (CSTR/UF). Among the substrates tested, the N-chloroacetyl-d,l-amino acids were the preferred substrates, showing the highest catalytic efficiency (V_m/K_m).Optimum reactor operational conditions obtained in discontinuous assays were selected to study the behaviour of the reactors in a continuous mode. DEAE-Sephadex loaded six-fold more enzyme than anion exchange nylon (60 and 10 gE/litre, respectively, related to reactor volume), whereas enzyme concentration within the CSTR/UF reactor was limited only by enzyme solubility.The tangential flow membrane reactor configuration with a 10 g/litre enzyme concentration produced higher productivity values (0·35 kg l-valine/litre per day, and 80% conversion degree) and operational stability (t = 161 days) than the CSTR/UF reactor (0·24 kg l-valine/litre per day, and 80% conversion degree) performing with the same enzyme concentration. The tubular reactor with the enzyme adsorbed onto DEAE-Sephadex (60 g/litre enzyme load) showed higher productivity values (1·9 kg l-valine/litre per day, and 80% conversion degree) and operational stability (t = 70 days) than the CSTR/UF reactor (1·05 kg l-valine/litre per day, and 80% conversion degree). However, the CSTR/UF reactor was the preferred configuration, as it had the highest enzyme load and productivity (1·95 kg l-valine/litre per day of reactor volume, and 80% conversion degree), a half-life of 55 days at 50°C, and the possibility of easy continuous enzyme addition.     

Improvement of the yield of physiologically active oligosaccharides in continuous hydrolysis of chitosan using immobilized chitosanases

The continuous production of chitosan oligosaccharides using a packed-bed enzyme reactor was investigated as to the effects of the operation conditions on the yield of pentamers and hexamers of chitosan oligosaccharides. A column reactor packed with immobilized chitosanases prepared by the multipoint attachment method was used for continuous hydrolysis of chitosan. In this reactor, the decrease of the yield of the target intermediate oligosaccharides due to axial mixing was negligible. The surface enzyme density of the support and flow rate of the substrate solution significantly affected the maximum yield of pentamers and hexamers. These effects were summarized as a correlation with the Damk?hler number (Da), defined as the ratio of the maximum reaction rate to the maximum mass transfer rate. The optimum condition was determined based on Da. Under the optimized condition (Da = 0.12), pentamers and hexamers could be produced continuously for a month with a yield of over 35% (7 kg/m(3) in concentration).     

Selective and stable production of physiologically active chitosan oligosaccharides using an enzymatic membrane bioreactor

We investigated the production of chitosan oligosaccharides by continuous hydrolysis of chitosan in an enzyme membrane bioreactor, with the goal of improving the yield of physiologically active oligosaccharides (pentamers and hexamers) and achieving operational stability. The bioreactor was a continuous-flow stirred-tank reactor equipped with an ultrafiltration membrane with a molecular weight cut-off of 2000 Da, and the hydrolysis was accomplished with chitosanase from Bacillus pumilus. After optimization of the reaction parameters, such as the amount of enzyme, the yield of the target oligosaccharides produced in the membrane bioreactor with free chitosanase reached 52% on the basis of the fed concentration of chitosan. An immobilized chitosanase prepared by the multipoint attachment method was used to improve the operational stability of the membrane bioreactor. Under the optimized conditions, pentameric and hexameric chitosan oligosaccharides were steadily produced at 2.3 g/L (46% yield) for a month. The half-life of the productivity of the reactor was estimated to be 50 d under the conditions examined.     

Oligosaccharide and alkyl β-galactopyranoside synthesis from lactose with Caldocellum saccharolyticum β-glycosidase

The synthetic utility of the thermostable β-glycosidase from Caldocellum saccharolyticum was investigated. The ability of the enzyme to catalyze oligosaccharide and β-galactopyranoside synthesis from lactose was compared with that of the readily commercially available, moderately thermostable β-galactosidase (β- -galactoside galactohydrolase, EC 3.2.1.23) from Aspergillus oryzae. Generally, the C. saccharolyticum enzyme showed significantly greater resistance to inactivation by heat and organic solvent and better yields of product. Although the A. oryzae enzyme gave better oligosaccharide yields at lower lactose concentrations, at higher concentrations (above 50% w/w) the C. saccharolyticum enzyme was significantly better, yielding a sugar mixture containing 42% by weight of tri- plus tetra-saccharides, from a 70% w/w lactose solution, compared with 31% by weight of oligosaccharides with the A. oryzae enzyme. In ethyl galactoside synthesis from ethanol and lactose, neither enzyme appeared to hydrolyze the product to any great extent. Under all conditions tested, the product yield did not peak, even at long reaction times, when most of the lactose had been consumed. The C. saccharolyticum enzyme, however, gave slightly higher product yields and could be used at higher ethanol concentrations without serious loss of activity.     

Production of toluene cis-diol by immobilized pseudomonas putida UV4 cells in barium alginate beads

In the present study, we have investigated the biotransformation of toluene to its cis-dihydrodiol (cis-diol) with immobilized Pseudomonas putida UV4 cells using different conditions of immobilization with a view to improving its production. The choice of alginate and its concentration for the immobilization of the cells were found to be the most important factors affecting the production of toluene cis-diol. The concentration of minerals and oxygen in the reaction medium and the methodology of substrate addition were investigated and the optimal conditions were defined. Once the optimal conditions for biotransformations and entrapment were determined, a packed-bed and fluidized-bed reactor were evaluated for the biotransformation process. The results using air as the gas supply showed an increase in the total production from 0.15 mol cis-diol · g⁻¹ dry cell weight (dcw) in the packed-bed reactor to 0.28 mol cis-diol · g⁻¹ dcw in the fluidized-bed reactor. When pure oxygen was used in place of air in the fluidized-bed reactor, a dramatic increase in total production up to a maximum of 6.1 mol cis-diol · g⁻¹ dcw using a medium flow rate of 100 ml min⁻¹ was achieved. Under optimal conditions, a maximum rate of production of 86.9 mmol cis-diol g⁻¹ dcw h⁻¹ was achieved for 48 h. This was seven times higher than the rate previously reported in the literature and for a much longer period of time; consequently, the overall production observed was more than 75 times higher than the values reported in the literature.     

A comparative study of free and immobilized soybean and horseradish peroxidases for 4-chlorophenol removal: protective effects of immobilization

Horseradish peroxidase (HRP) and soybean peroxidase (SBP) were covalently immobilized onto aldehyde glass through their amine groups. The activity yield and the protein content for the immobilized SBP were higher than for the immobilized HRP. When free and immobilized peroxidases were tested for their ability to remove 4-chlorophenol from aqueous solutions, the removal percentages were higher with immobilized HRP than with free HRP, whereas immobilized SBP needs more enzyme to reach the same conversion than free enzyme. In the present paper the two immobilized derivatives are compared. It was found that at an immobilized enzyme concentration in the reactor of 15 mg l(-1), SBP removed 5% more of 4-chlorophenol than HRP, and that a shorter treatment was necessary. Since immobilized SBP was less susceptible to inactivation than HRP and provided higher 4-chlorophenol elimination, this derivative was chosen for further inactivation studies. The protective effect of the immobilization against the enzyme inactivation by hydrogen peroxide was demonstrated.     

Production of galacto-oligosaccharides from lactose by Aspergillus oryzae beta-galactosidase immobilized on cotton cloth.

The production of galacto-oligosaccharides (GOS) from lactose by A. oryzae beta-galactosidase immobilized on cotton cloth was studied. The total amounts and types of GOS produced were mainly affected by the initial lactose concentration in the reaction media. In general, more and larger GOS can be produced with higher initial lactose concentrations. A maximum GOS production of 27% (w/w) of initial lactose was achieved at 50% lactose conversion with 500 g/L of initial lactose concentration. Tri-saccharides were the major types of GOS formed, accounting for more than 70% of the total GOS produced in the reactions. Temperature and pH affected the reaction rate, but did not result in any changes in GOS formation. The presence of galactose and glucose at the concentrations encountered near maximum GOS greatly inhibited the reactions and reduced GOS yield by as much as 15%. The cotton cloth as the support matrix for enzyme immobilization did not affect the GOS formation characteristics of the enzyme, suggesting no diffusion limitation in the enzyme carrier. The thermal stability of the enzyme increased approximately 25-fold upon immobilization on cotton cloth. The half-life for the immobilized enzyme on cotton cloth was more than 1 year at 40 degrees C and 48 days at 50 degrees C. Stable, continuous operation in a plugflow reactor was demonstrated for 2 weeks without any apparent problem. A maximum GOS production of 21 and 26% (w/w) of total sugars was attained with a feed solution containing 200 and 400 g/L of lactose, respectively, at pH 4.5 and 40 degrees C. The corresponding reactor productivities were 80 and 106 g/L/h, respectively, which are at least several-fold higher than those previously reported.     

Integration of purification with immobilization of Candida rugosa lipase for kinetic resolution of racemic ketoprofen

The two processes for the partial purification and for the immobilization of a crude lipase preparation (Candida rugosa Lipase OF) have been successfully integrated into one by simple adsorption of the enzyme onto a cation ion exchanger resin (SP-Sephadex C-50) at pH 3.5. Due to selective removal of the unfavorable lipase isoenzyme (L1), the enzyme components (mainly L2 and L3) that are tightly fixed on the resin displayed a significantly improved enantioselectivity (E value: 50 versus 13 with addition of Tween-80) in the biocatalytic hydrolysis of 2-chloroethyl ester of rac-ketoprofen. The activity yields of the immobilized lipase were 48 and 70%, respectively when emulsified and non-emulsified substrates were employed for enzyme assay. Moreover, the concentration of Tween-80 was found to be a factor affecting the lipase enantioselectivity. By using such an immobilized enzyme as biocatalyst, the process for preparing enantiopure (S)-ketoprofen becomes simpler and more practical as compared with the previously reported procedures and the product was obtained with >94% ee at 22.3% conversion in the presence of an optimal concentration (0.5 mg/ml) of Tween-80 at pH 3.5. Furthermore, the operational stability of the immobilized biocatalyst was examined in different types of reactors. In an air-bubbled column reactor, the productivity was much higher than that in a packed-bed column reactor, in spite of a slightly lower stability. Under optimal conditions, the air-bubbled column reactor could be operated smoothly for at least 350 h, remaining nearly 50% activity.     

Surfactant Effects on Lipase-Catalyzed Hydrolysis of Olive Oil in AOT/ISOOCTANE Reverse Micelles

The effects of surfactant concentration on the hydrolytic activity of Candida rugosa lipase in AOT/isooctane reverse micelles with olive oil as the substrate has been investigated. A noncompetitive inhibition by the surfactant on the enzyme was observed. Strong dependences of the kinetic constants k_cat and k_M, but not k_I on the water-to-surfactant ratio (R value) have been identified. The benefits of carrying out the hydrolysis at higher surfactant and water concentrations were demonstrated from the improvement of the initial rate and time course of conversion.     

A CSTR-hollow fiber system for continuous hydrolysis of proteins. Performance and kinetics

The effect of four operating variables (enzyme concentration, substrate concentration, flow rate, and reaction volume) on the performance of CSTR-hollow fiber membrane reactor was studied for the continuous hydrolysis of a soy protein isolate using Pronase. Based on a residence time distribution study, the reactor system was modeled as an ideal CSTR in combination with the Michaelis-Menten equation of enzyme kinetics. This kinetic model correlated conversion with a space-time parameter modified to include all four independent variables. An empirical model based on curvilinear regression analysis was also developed. Both models predicted conversion fairly well, although the kinetic model slightly underpredicts at high conversion.     

Compositional changes of cottonseed hull substrate during P. ostreatus growth and the effects on the feeding value of the spent substrate

The enzymic synthesis of alkyl-beta-glucosides by water-immiscible alcohols was studied in stirred flasks as well as in a tubular enzymatic reactor. In the first case, direct alkylation of beta-D-glucose from hexanol using immobilized beta-glycosidase gave a higher conversion yield and final product concentration than that using the enzyme in its free state (yield 10 against 8% mol/mol, concentration 2 against 1.6 g/l). Direct glycosylation of beta-D-glucose from hexanol resulted in a higher reaction performance (yield 10%) than that from octanol (yield 5%). However, the two different incubation temperatures tested (37 degrees C and 50 degrees C), showed no significant differences concerning final product concentrations. The more interesting results were obtained by transglycosylation of methyl-1-beta-glucose from hexanol, with a conversion yield of 21% mol/mol (product amount 4 g/l). However, the transgalactosylation of lactose from hexanol, catalyzed by a fungal beta-galactosidase, showed only a feeble reactivity. The feasibility of enzymic alkylation was also tested in a tubular enzymatic reactor; hexyl-1-beta-glucoside was produced via direct glycosylation from hexanol catalyzed by free beta-glycosidase with a final concentration 1.3-2.3 g/l and a yield varying between 11% and 20% mol/mol.     

Degradation of raffinose oligosaccharides in soymilk by immobilized alpha-galactosidase of Aspergillus oryzae

Alpha-galactosidase was immobilized in a mixture of k-carrageenan and locust bean gum. The properties of the free and immobilized enzyme were then determined. The optimum pH for both the soluble and immobilized enzyme was 4.8. The optimum temperature for the soluble enzymes was 50 degrees C, whereas that for the immobilized enzyme was 55 degrees C. The immobilized enzyme was used in batch, repeated batch, and continuous modes to degrade the raffinose-family sugars present in soymilk. Two hours of incubation with the free and immobilized alpha-galactosidases resulted in an 80% and 68% reduction in the raffinose oligosaccharides in the soymilk, respectively. In the repeated batch, a 73% reduction was obtained in the fourth cycle. A fluidized bed reactor was also designed to treat soymilk continuously and the performance of the immobilized alpha-galactosidase tested at different flow rates, resulting in a 90% reduction of raffinose-family oligosaccharides in the soymilk at a flow rate 40 ml/h. Therefore, the present study demonstrated that immobilized alpha-galactosidase in a continuous mode is efficient for reducing the oligosaccharides present in soymilk, which may be of considerable interest for industrial application.     

Cyclodextrin glycosyltransferase from Bacillus circulans DF 9R: Activity and kinetic studies

Activity characteristics and kinetic aspects of a cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans DF 9R were studied. A mixture of α-, β- and γ-cyclodextrins (CDs), glucose, maltose and negligible amounts of longer linear dextrins were produced from gelatinized amylose, amylopectin and starch from different sources. In the coupling reaction, CDs were the substrates in the presence of acceptors such as maltose and/or longer oligosaccharides. From oligosaccharides formed by three or more glucose units, this enzyme produced linear chains of several lengths which were then cyclized. CGTase catalytic efficiency was compared employing an analytical grade starch and cassava starch for food use. Since the results obtained were similar for both starches, the use of an economic starch is an advantage. CGTase was inhibited by the substrate and its own products. Starch concentrations over 20 mg/mL inhibited the cyclizing activity. CDs behaved as competitive inhibitors and maltose as an uncompetitive inhibitor while maltotriose showed a mixed inhibition pattern. Limit dextrins showed a scarce inhibitory effect on enzyme activity. CD production could be improved with an ultrafiltration membrane reactor for continuous removal of the products; the starch concentration should be maintained below an inhibitory concentration and limit dextrins would remain in the reactor without affecting enzyme activity.     

Unique mode of acetylation of oligosaccharides in aqueous two-phase system by Trichoderma reesei acetyl esterase

Trichoderma reesei RUT C-30 acetyl esterase, known to catalyze transacetylation reactions in water/vinyl acetate two-phase mixtures, was studied with respect to regioselectivity of acetylation of oligosaccharides in aqueous environment. Using series of oligosaccharides and their methyl glycosides, it was found that the enzyme catalyzes an efficient acetylation at O-3 position of the non-reducing terminal units of gluco-, xylo- and manno-oligosaccharides and a less efficient acetylation of O-2 position of the reducing end units of gluco- and xylo-oligosaccharides. The axial hydroxyl group at O-2 position of the reducing end mannose in mannooligosaccharides was not recognized by the enzyme and its acetylation was not observed. The structure of isolated transacetylation products was established by NMR, ESI-MS analysis and on the basis on their resistance towards action of glycosidases acting from the non-reducing end of oligosaccharides. The position of acetylation allowed deduce on some of the structural requirements of the enzyme for the acetyl group acceptors. T. reesei RUT C-30 acetyl esterase was also found to be capable of liberation of acetyl groups from terminal units of oligosaccharides, which speaks for its classification as an exo-acting acetyl esterase.     

Membrane reactor for enzymatic hydrolysis of cellobiose

A pressurized, stirred vessel attached with an ultrafiltration membrane was used as a membrane reactor, Cellobiose hydrolysis by cellobiase was carried out and theoretically analyzed in terms of steady-state conversion and flow rate through the membrane. When the flow rate exceeds a critical value, a significant fraction of the enzyme inside the reactor is localized in the concentration polarization layer where shear from stirring is high. Consequently, enzyme deactivation inside the concentration polarization layer is accelerated and the conversion decreases due to an exchange of active enzyme in bulk with deactivated enzyme in the polarization layer via convection and back diffusion. Successful operation can be obtained at flow rates lower than the critical point to avoid the polarization and thus the deactivation. It is shown that 6.5 L of 2 mg/mL of cellobiose solution is hydrolyzed to glucose with a conversion of 91% in 20 h with 1.617 mg of cellobiase enzyme, in a reactor attached with a PM 10 membrane of an effective surface area of 39.2 cm².     

Alcoholysis and reverse hydrolysis reactions in organic one-phase system with a hyperthermophilic beta-glycosidase

Alcoholysis and reverse hydrolysis reactions were performed enzymatically in one-phase water-saturated 1-heptanol systems. Lactose or glucose was used as substrate to produce heptyl-beta-galactoside and/or heptyl-beta-glucoside, respectively. When alcoholysis of lactose was performed at 37 degrees C with beta-galactosidase from Escherichia coli, the initial rate was 14 nmol/mL min, and the limiting factors were the poor solubility of the substrate in 1-heptanol and low thermal stability of the enzyme. When a hyperthermophilic beta-glycosidase was used at 90 degrees C, the rate was 3.14-fold higher; in this case a higher concentration of soluble lactose in the water-saturated heptanol was available to the enzyme due to the higher temperature. The hyperthermophilic beta-glycosidase was also able to use glucose and galactose as substrates to achieve the reverse hydrolysis reaction. As a consequence, when lactose was used as substrate, heptyl-beta-galactoside was formed by alcoholysis, while the released glucose moiety was used in a secondary reverse hydrolysis reaction to produce heptyl-beta-glucoside. Both reactions followed Michaelis-Menten kinetics behavior. Neither lactose nor heptyl glycosides were hydrolyzed by this enzyme in water-saturated heptanol. However, the conversion was limited by a strong product inhibition and the formation of oligosaccharides, especially at high substrate concentrations, reducing the final glycoside yield.     

Rapid and sensitive screening of N-glycans as 9-fluorenylmethyl derivatives by high-performance liquid chromatography: a method which can recover free oligosaccharides after analysis

There are a large number of labeling methods for asparagine-type oligosaccharides with fluorogenic and chromophoric reagents. We have to choose the most appropriate labeling method based on the purposes such as mass spectrometry, high-performance liquid chromatography and capillary electrophoresis. Asparagine-type glycans are released from core proteins as N-glycosylamine at the initial step of the releasing reaction when glycoamidase F is employed as the enzyme. The N-glycosylamine-type oligosaccharides thus released by the enzyme are subjected to hydrolysis or mutarotation to form free-form oligosaccharides. In the detailed studies on the enzyme reaction, we found a condition in which the released N-glycosylamine-type oligosaccharides were exclusively present at least during the course of enzyme reaction, and developed a method for in situ derivatization of the glycosylamine-type oligosaccharides with 9-fluorenylmethyl chloroformate (Fmoc-Cl). The Fmoc labeled sialo- and asialo- (or high-mannose and hybrid) oligosaccharides were successfully analyzed on an amine-bonded polymer column and amide-silica column, respectively. The present method showed approximately 5 times higher sensitivities than that using 2-aminobenzoic acid (2-AA). The separation profile was similar to that observed using 2-AA method as examined by the analyses of carbohydrate chains derived from several glycoproteins including complex-type, high-mannose type and hybrid type of N-linked oligosaccharides. The labeled oligosaccharides were stable at least for several months when stored at -20 degrees C. Furthermore, it should be emphasized that the Fmoc-derivatized oligosaccharides could be easily recovered as free reducing oligosaccharides simply by incubation with morpholine in dimethylformamide solution. We obtained a pure triantennary oligosaccharide with 3 sialic acid residues as a free reducing form from fetuin in good yield after isolation of the corresponding Fmoc oligosaccharide followed by removing reaction of the Fmoc group. The proposed method will be useful for preparation of free oligosaccharides as standard samples at pmol-nmol scale from commercially available glycoproteins.     

Immobilization of beta-galactosidase on fibrous matrix by polyethyleneimine for production of galacto-oligosaccharides from lactose

The production of galacto-oligosaccharides (GOS) from lactose by Aspergillus oryzae beta-galactosidase immobilized on cotton cloth was studied. A novel method of enzyme immobilization involving PEI-enzyme aggregate formation and growth of aggregates on individual fibrils of cotton cloth leading to multilayer immobilization of the enzyme was developed. A large amount of enzyme was immobilized (250 mg/g support) with about 90-95% efficiency. A maximum GOS production of 25-26% (w/w) was achieved at near 50% lactose conversion from 400 g/L of lactose at pH 4.5 and 40 degrees C. Tri- and tetrasaccharides were the major types of GOS formed, accounting for about 70% and 25% of the total GOS produced in the reactions, respectively. Temperature and pH affected not only the reaction rate but also GOS yield to some extend. A reaction pH of 6.0 increased GOS yield by as much as 10% compared with that of pH 4.5 while decreased the reaction rate of immobilized enzyme. The cotton cloth as the support matrix for enzyme immobilization did not affect the GOS formation characteristics of the enzyme under the same reaction conditions, suggesting diffusion limitation was negligible in the packed bed reactor and the enzyme carrier. Increase in the thermal stability of PEI-immobilized enzyme was also observed. The half-life for the immobilized enzyme on cotton cloth was close to 1 year at 40 degrees C and 21 days at 50 degrees C. Stable, continuous operation in a plug-flow reactor was demonstrated for about 3 days without any apparent problem. A maximum GOS production of 26% (w/w) of total sugars was attained at 50% lactose conversion with a feed containing 400 g/L of lactose at pH 4.5 and 40 degrees C. The corresponding reactor productivity was 6 kg/L/h, which is several-hundred-fold higher than those previously reported.     

Properties and action pattern of the recombinant mannuronan C-5-epimerase AlgE2

The mannuronan C-5-epimerase AlgE2 is one of a family of Ca²⁺-dependent epimerases secreted by Azotobacter vinelandii. These enzymes catalyze the conversion of β- -mannuronic acid residues (M) to - -guluronic acid residues (G) in alginate. AlgE2 has been produced by fermentation with a recombinant strain of Escherichia coli, isolated and partially purified. Epimerization with AlgE2 increased the content of G-residues in different alginates from starting values of 0–45% up to approximately 70%. The new G-residues were mainly present in short blocks. Although G-residues may be introduced next to pre-existing G-residues, AlgE2 was not able to epimerize strictly alternating MG-structures. The epimerization with AlgE2 was greatly affected by the concentration of Ca²⁺. The type of alginate used as substrate affected the reaction rate and the reaction pattern especially at low Ca²⁺ concentration. AlgE2 appears to act by a preferred attack mechanism where the enzyme associates with different sequences in the alginate depending on the concentration of Ca²⁺. During epimerization, AlgE2 occasionally causes cleavage of the alginate chain. The observed frequency corresponds to 1–3 breaks per 1,000 M-units epimerized.     

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.