Comparison of a batch, fed-batch and continuously operated stirred-tank reactor for the enzymatic synthesis of (R)-mandelonitrile by using a process model

The suitability of a batch, fed-batch and continuously operated stirred-tank reactor for the enzymatic production of (R)-mandelonitrile in an aqueous-organic biphasic system was investigated by using a process model. The considered biphasic system is 10-50% (v/v) 100 mM sodium citrate buffer of pH 5.5 dispersed in methyl tert-butyl ether. The constraints were that 750 moles of benzaldehyde per cubic meter should react towards (R)-mandelonitrile with an enantiomeric excess of 99% and a conversion of 98%. A continuously operated stirred-tank reactor could not meet the constraints, but the production in a batch or fed-batch reactor was feasible. The choice for a batch or fed-batch reactor is dependent on the influence of the costs for reactor operation and for the enzyme on the product costs. The choice for operating at a small or large aqueous-phase volume fraction is dependent on the costs and reusability of the enzyme. The volumetric productivity is maximal when operating as batch. The enzymatic productivity and turnover are maximal when operating as fed batch. In the fed-batch mode, the enzymatic productivity increased by 24-37%, the turnover increased by 50-60% and the volumetric productivity decreased by 33-71% as compared to a batch reactor. By enhancement of mass transfer both the volumetric and enzymatic productivity can be increased considerably, while the turnover is only slightly decreased.     

Continuous Hydrolysis of Soluble Starch by Free β-Amylase and Pullulanase Using an Ultrafiltration Membrane Reactor

Enzyme hydrolysis of soluble starch by free β-amylase and pullulanase for the production of maltose was done by the simultaneous use of a stirred tank reactor and an ultrafiltration membrane. Higher conversions of starch to maltose were obtained in the permeates than that in a batch reaction. Using the basic mass balance and rate equations, concentrations of maltose, maltotriose, and substrate in the retentates and permeates could be simulated effectively. More than 99.9% of enzyme was found to be rejected by the membrane. The obtained volumetric productivities were several times higher than those reported in other systems. This system was found to have high maltose productivity with a short mean residence time, being easily controlled by transmembrane pressure.     

Continuous alpha-amylase production using Bacillus amyloliquefaciens adsorbed on an ion exchange resin

Summary A method for the continuous production of extracellular alpha amylase by surface immobilized cells of Bacillus amyloliquefaciens NRC 2147 has been developed. A large-pore, macroreticular anionic exchange resin was capable of initially immobilizing an effective cell concentration of 17.5 g DW/1 (based on a total reactor volume of 160 ml). The reactor was operated continuously with a nutrient medium containing 15 g/l soluble starch, as well as yeast extract and salts. Aeration was achieved by sparging oxygen enriched air into the column inlet. Fermentor plugging by cells was avoided by periodically substituting the nutrient medium with medium lacking in both soluble starch and yeast extract. This fermentor was operated for over 200 h and obtained a steady state enzyme concentration of 18700 amylase activity units per litre (18.7 kU/l), and an enzyme volumetric productivity of 9700 amylase activity units per litre per hour (9.7 kU/l-h). Parallel fermentations were performed using a 2 l stirred vessel fermentor capable of operation in batch and continuous mode. All fermentation conditions employed were identical to those of the immobilized cell experiments in order to assess the performance of the immobilized cell reactor. Batch stirred tank operation yielded a maximum amylase activity of 150 kU/l and a volumetric productivity of 2.45 kU/l-h. The maximum cell concentration obtained was 5.85 g DW/l. Continuous stirred tank fermentation obtained a maximum effluent amylase activity of 6.9 kU/l and a maximum enzyme volumetric productivity of 2.73 kU/l-h. Both of these maximum values were observed at a dilution rate of 0.345 l/h. The immobilized cell reactor was observed to achieve larger volumetric productivities than either mode of stirred tank fermentation, but achieved an enzyme activity concentration lower than that of the batch stirred tank fermentor.     

Complete starch hydrolysis by the synergistic action of amylase and glucoamylase: impact of calcium ions

Starch hydrolysis was performed by the synergistic action of amylase and glucoamylase. For that purpose glucoamylase (Dextrozyme) and two amylases (Liquozyme and Termamyl) in different combinations were investigated. Experiments were carried out in the repetitive- and fed-batch modes at 65 °C and pH 5.5 with and without the addition of Ca²⁺ ions. 100 % conversion of starch to glucose was achieved in batch experiments. Calcium ions significantly enhanced stability of the amylase Termamyl. The intensity of synergism between amylase Termamyl and glucoamylase Dextrozyme was higher than in the experiments carried out with amylase Liquozyme and Dextrozyme. Mathematical model of the complete reaction system was developed. Using the model, a possible explanation of the synergism between the amylase and glucoamylase was provided.     

Continuous sucrose hydrolysis by yeast cells immobilized to wool

A novel immobilized biocatalyst with invertase activity was prepared by adhesion of yeast cells to wool using glutaraldehyde. Yeast cells could be immobilized onto wool by treating either the yeast cells or wool or both with glutaraldehyde. Immobilized cells were not desorbed by washing with 1 M KCl or 0.1 M buffers, pH 3.5–7.5. The biocatalyst shows a maximum enzyme activity when immobilized at pH 4.2–4.6 and 7.5–8.0. The immobilized biocatalyst was tested in a tubular fixed-bed reactor to investigate its possible application for continuous full-scale sucrose hydrolysis. The influence of temperature, sugar concentration and flow rate on the productivity of the reactor and on the specific productivity of the biocatalyst was studied. The system demonstrates a very good productivity at a temperature of 70 °C and a sugar concentration of 2.0 M. The increase of the volume of the biocatalyst layer exponentially increases the productivity. The productivity of the immobilized biocatalyst decreases no more than 50% during 60 days of continuous work at 70 °C and 2.0 M sucrose, but during the first 30 days it remains constant. The cumulative biocatalyst productivity for 60 days was 4.8 × 10³kg inverted sucrose/kg biocatalyst. The biocatalyst was proved to be fully capable of continuous sucrose hydrolysis in fixed-bed reactors. Received: 8 November 1996 / Received revision: 31 January 1997 / Accepted: 31 January 1997     

Influence of temperature on protein hydrolysis in a cyclic batch enzyme membrane reactor

A cyclic batch enzyme membrane reactor (CBEMR) incorporating a 8000-Da polyethersulphone membrane was intended for enhancing the enzyme (Protex 6L from Bacillus licheniformis, EC. 3.4.21.62) use in the production of a whey protein hydrolysate. A mechanistic mathematical model comprising zero-order kinetics for the hydrolysis and second-order deactivation for the enzyme was proposed and validated through experiments. The influence of reaction temperature was studied and process optimisation (given the production requirements) was performed in terms of number of batch reactions that minimise the total amount of enzyme used. The optimal operation of the CBEMR allowed savings of up to 44 and 32% of enzyme compared to the single batch operation mode at 50 and 60 °C, respectively. No enzyme savings were detected when temperature was fixed at 70 °C. In general, the optimal operation temperature was 60 °C, yielding lower enzyme consumption for all productivities of the reactor.     

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.     

Continuous production of inulo-oligosaccharides from chicory juice by immobilized endoinulinase

Several carrier materials were examined for endoinulinase immobilization. A polystyrene carrier material (UF93^®) gave the best immobilization capacity (217 units/g carrier) and operational stability. Carbohydrate compositions in the reaction product were quite similar irrespective of the support materials even though each carrier material has different pore structure associated with diffusional restriction. After immobilization the optimal pH for enzyme activity was shifted from 5.0 to 4.5, whereas optimal temperature (55v°C) was unaltered. Continuous production of inulo-oligosaccharides from chicory juice was carried out using the polystyrene-bound endoinulinase. The recommended operating conditions of the enzyme reactor for maximizing productivity were as follows: feed concentration, 100 g/l chicory juice; flow rate, as superficial space velocity 2.0 h^у; temperature, 55v°C. The enzyme reactor was run for 28 days at 55v°C achieving an oligosaccharide yield of 82% without any significant loss of initial enzyme activity, where the volumetric productivity was 200 g/l · h. Furthermore, there was no marked difference in operational stability between the two reactors fed with pure inulin solution and with chicory juice as a substrate even though chicory juice contains a lot of impurities.     

Production of levanase byRhodotorula sp.

Rhodotorula sp. produced a high yield of levanase (12.5 nkat/mL) in shake flasks in basal medium containing 1% maltose as the sole carbon source. Among the different carbon sources used, maltose was found to be the best for levanase production. The optimum temperature and pH for levanase production were 30°C and 6, respectively. In a batch reactor the enzyme productivity was higher (500 nkat L⁻¹ h⁻¹) than in shaken flasks (347 nkat L⁻¹ h⁻¹).     

Room and low temperature brewing with yeast immobilized on gluten pellets

A biocatalyst prepared by the immobilization of a cryotolerant strain of Saccharomyces cerevisiae on gluten pellets was used for batch and continuous fermentation at low temperatures. The immobilized yeast showed important operational stability in repeated batch fermentations without a decrease of activity even at 0 and 5°C. Repeated batch fermentations using the biocatalyst resulted in improvement of ethanol productivity in comparison with bottom brewing fermentation and free cells using the same yeast strain. At 0 and 10°C, the fermentation rate was four and seven times higher than that of free cells, respectively. For immobilized yeast, diacetyl and polyphenol contents were lower and the alcohol concentration higher at low temperatures (0–7°C) when compared to free cells. Fine clarity was also observed in the beers. Continuous brewing using gluten-supported biocatalyst had an operational stability of 3 months with relatively high productivity and without contamination. Polyphenol and bitterness contents were lower in the continuous process than those of batch fermentations, but at low temperature (5°C) they were higher. The diacetyl content was higher than in batch fermentations and beers had a fine aroma and taste.     

Continuous production of L-phenylalanine by transamination

L-Phenylalanine was produced continuously from L-as-partate and phenylpyruvate by transaminase from a newly screened Pseudomonas putida strain. The process was carried out with an isolated enzyme in homogeneous phase in an enzyme membrane reactor and with immobilized whole cells in a stirred tank reactor, respectively. Due to the difference in transport resistance, the productivity of the free enzyme in homogeneous phase (72 mmol/L h) was about 3 times higher than the productivity achieved using immobilized cells. However, a better stability of the biocatalyst was observed with immobilized cells.     

Continuous production of L-tert-leucine in series of two enzyme membrane reactors

The L-tert-leucine synthesis was performed continuously in series of two enzyme-membrane reactors by reductive amination of trimethylpyruvate with leucine dehydrogenase. The necessary “native” cofactor NADH is regenerated with the aid of a second enzyme, formate dehydrogenase. Considering detailed kinetic studies of initial reaction rates under conditions relevant to the process a kinetic model was developed. The model shows that the overall reaction rate is strongly inhibited by the reaction product. The reactor's models combine the mass balances and proposed kinetic equations. The model adequacy was verified by using it to simulate the experiments and by comparing experimental and computed conversion, space-time yield and enzyme consumption. The calculations for the three reactor's types (batch, single CSTR and a cascade of two CSTRs in series) were compared. The results showed that a single CSTR is no favourable reactor configuration due to the very strong product inhibition. Space-time yield drops from 560 g litre^?1 day^?1 in a batch reactor to 110 g litre^?1 day^?1 in a single CSTR at the highest conversion of 98%. At the conversion of 95% the difference in biocatalyst costs between batch and two CSTR in series is negligible. Therefore the use of two enzyme membrane reactors in series was proposed. The modelling in this work shows that the optimisation of the quantity of the enzyme used results in a minimisation of the biocatalyst costs.     

Column cellulose hydrolysis reactor: the effect of retention time,temperature, cellulase concentration and exogenously added cellobiase on the overall process

Summary A novel column cellulose hydrolysis reactor with constant enzyme recycling was operated under various conditions to determine the effects of retention time, temperature, cellulase concentration and exogenously added cellobiase on the concentration of the product stream and the productivities of the reactor. Short term (7 days) hydrolysis was best at 42°C while longer term (14 days) hydrolysis was better at 37°C. A retention time of 11 h and reactor cellulase concentration of 30 filter paper units per gram of cellulose gave the best compromise for efficient operation by minimizing product inhibition, maximizing product concentration and minimizing enzyme consumption. The addition of cellobiase to the reactor increased cellulose hydrolysis and raised the proportion of monomeric sugars in the hydrolysate. Continuous cellulose hydrolyses were maintained for 7 and 14 days at 42°C and 37°C, respectively, resulting in volumetric productivities of 6.82 and 4.84 g/l/h and average sugar concentrations of 7.3% and 6.0% (w/v), respectively. Greater than 95% (w/w) of the sugars produced were in the monomeric state. Average cellulase used for the two runs were 8.4 and 5.3 filter paper units per gram of sugar produced, respectively.     

Development of a stable continuous flow immobilized enzyme reactor for the hydrolysis of inulin

A 23.5-fold purified exoinulinase with a specific activity of 413 IU/mg and covalently immobilized on Duolite A568 has been used for the development of a continuous flow immobilized enzyme reactor for the hydrolysis of inulin. In a packed bed reactor containing 72 IU of exoinulinase from Kluyveromyces marxianus YS-1, inulin solution (5%, pH 5.5) with a flow rate of 4 mL/h was completely hydrolyzed at 55 degrees C. The reactor was run continuously for 75 days and its experimental half-life was 72 days under the optimized operational conditions. The volumetric productivity and fructose yield of the reactor were 44.5 g reducing sugars/L/h and 53.3 g/L, respectively. The hydrolyzed product was a mixture of fructose (95.8%) and glucose (4.2%) having an average fructose/glucose ratio of 24. An attempt has also been made to substitute pure inulin with raw Asparagus racemosus inulin to determine the operational stability of the developed reactor. The system remained operational only for 11 days, where 85.9% hydrolysis of raw inulin was achieved.     

Immobilization of glucoamylase by adsorption on carbon supports and its application for heterogeneous hydrolysis of dextrin

Glucoamylase (GA) was immobilized by adsorption on carbon support: on Sibunit, on bulk catalytic filamentous carbon (bulk CFC) and on activated carbon (AC). This was used to prepare heterogeneous biocatalysts for the hydrolysis of starch dextrin. The effect of the texture characteristics and chemical properties of the support surface on the enhancement of the thermal stability of the immobilized enzyme was studied, and the rates of the biocatalyst's thermal inactivation at 65-80 degrees C were determined. The thermal stability of glucoamylase immobilized on different carbon supports was found to increase by 2-3 orders of magnitude in comparison with the soluble enzyme, and decrease in the following order: GA on Sibunit>GA on bulk CFC>GA on AC. The presence of the substrate (dextrin) was found to have a significant stabilizing effect. The thermal stability of the immobilized enzyme was found to increase linearly when the concentration of dextrin was increased from 10 wt/vol % to 50 wt/vol %. The total stabilization effect for glucoamylase immobilized on Sibunit in concentrated dextrin solutions was about 10(5) in comparison with the enzyme in a buffer solution. The developed biocatalyst, 'Glucoamylase on Sibunit' was found to have high operational stability during the continuous hydrolysis of 30-35 wt/vol % dextrin at 60 degrees C, its inactivation half-time (t1/2) exceeding 350 h. To improve the starch saccharification productivity, an immersed vortex reactor (IVR) was designed and tested in the heterogeneous process with the biocatalyst 'Glucoamylase on Sibunit'. The dextrin hydrolysis rate, as well as the process productivity in the vortex reactor, was found to increase by a factor of 1.2-1.5 in comparison with the packed-bed reactor.     

Encapsulation of β-amylase in water-oil-water enzyme emulsion liquid membrane (EELM) bioreactor for enzymatic conversion of starch to maltose

Abstract

The β-amylase was encapsulated in emulsion liquid membrane (ELM), which acted as a reactor for conversion of starch to maltose. The membrane phase was consisted of surfactant (span 80), stabilizer (polystyrene), carrier for maltose transport (methyl cholate) and solvent (xylene). The substrate starch in feed phase entered into the internal phase by the process of diffusion and hydrolyzed to maltose by encapsulated β-amylase. Methyl cholate present in the membrane acts as a carrier for the product maltose, which helps in transport of maltose to feed phase from internal aqueous phase. The residual activity of β-amylase after the five-reaction cycle was found to decrease to ～70%, which indicated possibility to recycle the components of the emulsion and enzyme. The pH and temperature of the encapsulated enzyme were found to be optimum at 5.5 and 60?°C, respectively. The novelty of the present work lies in the development of Enzyme Emulsion Liquid Membranes (EELM) bioreactor for the hydrolysis of starch into maltose mediated by encapsulated β-amylase. The attempt has been made for the first time for the successful encapsulation of β-amylase into EELM. The best results gave the highest residual enzyme activity (94.1%) and maltose production (29.13?mg/mL).     

Simple strategy of reactivation of a partially inactivated penicillin g acylase biocatalyst in organic solvent and its impact on the synthesis of β‐lactam antibiotics

Some reactions of organic synthesis require to be performed in rather aggressive media, like organic solvents, that frequently impair enzyme operational stability to a considerable extent. We have studied the option of developing a reactivation strategy to increase biocatalyst lifespan under such conditions, under the hypothesis that organic solvent enzyme inactivation is a reversible process. Glyoxyl agarose immobilized penicillin G acylase and cross‐linked enzyme aggregates of the enzyme were considered as biocatalysts performing in dioxane medium. Reactivation strategy consisted in re‐incubation in aqueous medium of the partly inactivated biocatalysts in organic medium, best conditions of reactivation being studied with respect to dioxane concentration and level of enzyme inactivation attained prior to reactivation. Best results were obtained with glyoxyl agarose immobilized penicillin G acylase at all levels of residual activity studied, with reactivations up to 50%; for the case of a biocatalyst inactivated down to 75% of its initial activity, full recovery of enzyme activity was obtained after reactivation. The potential of this strategy was evaluated in the thermodynamically controlled synthesis of deacetoxycephalosporin G in a sequential batch reactor operation, where a 20% increase in the cumulative productivity was obtained by including an intermediate stage of reactivation after 50% inactivation. Biotechnol. Bioeng. 2009;103: 472–479. © 2009 Wiley Periodicals, Inc.     

Performance of a cutinase membrane reactor for the production of biodiesel in organic media

The enzymatic transesterification of oils with an alcohol, using recombinant cutinase of Fusarium solani pisi microencapsulated in sodium bis(2-ethylhexyl) sulfosuccinate (AOT)/isooctane reversed micelles, was performed in a membrane bioreactor (MBR). A tubular ceramic membrane with a nominal molecular weight cut off of 15,000 Da was used to retain the enzyme, and characterized in terms of rejection coefficients of the reaction components by transmission experiments. The performance of the MBR in a total recirculation-batch mode was compared with results obtained in a stirred batch tank reactor. The continuous operation of the MBR was also evaluated and the influence of the alcohol type and permeate flow rate on conversion degree and productivity (up to 500 g(product) /day/g(enzyme) was attained) were analyzed. Cutinase wild type and mutant T179C were tested for this process and the high long-term operational stability of the cutinase mutant demonstrated its potential as biocatalyst for the enzymatic continuous production of biodiesel.     

Kinetics of solka floc cellulose hydrolysis by trichoderma viride cellulase

A product inhibition model is developed to describe the hydrolysis of cellulose by the Trichoderma viride enzyme system. It is assumed that noncompetitive inhibition by cellobiose dominates the reaction kinetics. Experiments show that this is indeed a reasonable assumption for initial cellulose concentrations of up to 15 g/liter and at hydrolysis extents up to 65′. Kinetic parameters were determined for the noncompetitive inhibitionmodel in batch experiments with durations of up to 1.5 hr. These parameterswere then used in predicting reaction progress for up to 10 hr. Cellobiose was added to the reaction mixture at the onset of some runs and againreliable predictions were obtained for up to 8 hr of hydrolysis. Finally reaction was carried out in a membrane reactor whereby the product cellobiose was being continuously removed and again reasonable predictability was obtained with a higher net reaction rate.     

Production of a sterol esterase from Ophiostoma piceae in batch and fed‐batch bioprocesses using different Pichia pastoris phenotypes as cell factory

The potential biotechnological applications for the Ophiostoma piceae sterol esterase (OPE) are conditioned to the availability of high enzyme amounts at low prices. This enzyme is a versatile biocatalyst with different biotechnological applications. In this work a systematic study on its heterologous production in different Pichia pastoris strains and operational strategies is presented. The best results were obtained using an AOX1 defective yeast strain in a fed‐batch bioprocess using methanol as inducer substrate at a set point of 2.5 g L^?1 and sorbitol as cosubstrate by means of a preprogramed exponential feeding rate at a μ = 0.02 h^?1, reaching 30 U mL^?1 of enzyme and a volumetric productivity of 403.5 U L^?1 h^?1. These values are twofold higher than those obtained with a Mut⁺ phenotype using methanol a sole carbon source. OPE was the main protein secreted by the yeast, 55% for Mut^s versus 25% for Mut^+. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1012–1020, 2014