Batch reactor performance for the enzymatic synthesis of cephalexin: influence of catalyst enzyme loading and particle size

A mathematical model is presented for the kinetically controlled synthesis of cephalexin that describes the heterogeneous reaction-diffusion process involved in a batch reactor with glyoxyl-agarose immobilized penicillin acylase. The model is based on equations considering reaction and diffusion components. Reaction kinetics was considered according to the mechanism proposed by Schro?n, while diffusion of the reacting species was described according to Fick's law. Intrinsic kinetic and diffusion parameters were experimentally determined in independent experiments. It was found that from the four kinetic constants, the one corresponding to the acyl-enzyme complex hydrolysis step had the greatest value, as previously reported by other authors. The effective diffusion coefficients of all substances were about 5×10(-10)m(2)/s, being 10% lower than free diffusion coefficients and therefore agreed with the highly porous structure of glyoxyl-agarose particles. Simulations made from the reaction-diffusion model equations were used to evaluate and analyze the impact of internal diffusional restrictions in function of catalyst enzyme loading and particle size. Increasing internal diffusional restrictions decreases the Cex synthesis/hydrolysis ratio, the conversion yield and the specific productivity. A nonlinear relationship between catalyst enzyme loading and specific productivity of Cex was obtained with the implication that an increase in catalyst enzyme loading will not increase the volumetric productivity by the same magnitude as it occurs with the free enzyme. Optimization of catalyst and reactor design should be done considering catalyst enzyme loading and particle size as the most important variables. The approach presented can be extended to other processes catalyzed by immobilized enzymes.     

Covalent bond between the enzyme amyloglucosidase and a porous glass carrier: The effect of shearing

The utility of an immobilized enzyme depends not only on initial loading but also on operational half-life. The loss of activity of an immobilized enzyme in a column reactor may occur in several ways. It is therefore of interest to determine whether the decay in activity is due to the rupture of any of the enzyme-carrier bonds in the composite. In order to do this, it is first necessary to establish whether the enzyme on the composite is in fact covalently bound or adsorbed. In this report we have shown that there are a number of covalent links per protein molecule. We have also shown that the bond energies are sufficient to prevent shearing of the enzyme itself from the composite under any stresses which may occur in a chromatography column or packed-bed reactor during continuous operation. The effect of the siloxane linkage on composite stability is also discussed.     

Modeling enzyme immobilization in porous solid supports

Enzymes are often immobilized on the internal surfaces of porous solid by immersing enzyme-free particles in a well mixed solution of enzyme. The ensuing impregnation process involves coupled transient mass transfer and surface attachment of enzyme. A mathematical model is employed to explore the influences of process parameters on the amount of enzyme loaded and the distribution of immobilized enzyme within the support particles. Nonuniform loading of the support occurs under some conditions. This is significant since the distribution of enzyme within the support particle influences the overall activity and stability of the immobilized enzyme catalyst. The model developed here may also be used to describe removal of reversibly immobilized enzyme during washing or utilization of the immobilized enzyme catalyst.     

The production of 6-aminopenicillanic acid by a multistage tubular reactor packed with immobilized penicillin amidase

A theoretical model equation was derived to find the correlation between the conversion and the amount of immobilized penicillin amidase in column. The theoretical values of the conversion were predicted form this correlation and compared with experimental results. It was observed in a column reactor that the pH drop along the column path was linear versus the enzyme loading and that the enzyme activity was also linearly dependent on pH up to 8.0. In order to diminish the effect of pH drop, a continuous two-stage plug-flow reactor (PFR) with pH adjustment between the two columns was used was used in the experiments, and two- and three-stage PFRs were simulated by computer. In the case of the two-stage PFR, the maximum productivity was demonstrated experimentally and theoretically as well. when an equal amount of the immobilized enzyme was packed in both columns. It was also predicted in the tree-stage PFR system that the optimal distributions of enzyme loading in three columns were found to be 1:1:1. It was demonstrated that the increased number of reactors in series could enhance the level of the maximum productivity with a given amount of enzyme loading.     

Performance of an immobilized fuculose-1-phosphate aldolase for stereoselective synthesis

A derivative of fuculose-1-phosphate aldolase, immobilized with high loading on glyoxal–agarose gels, has been characterized and evaluated as a biocatalyst for an aldol addition reaction. The reaction of the solid biocatalyst was diffusion-controlled for conversion of its natural substrate. Nevertheless, when catalyzing the synthesis of a biologically active aminopolyol, the lower reaction rate with non-natural substrates led to a process controlled by the intrinsic enzyme kinetics. The resulting biocatalyst has high synthetic specific activity and has been successfully used in batch synthesis reactions with high conversion. In addition, the immobilized aldolase has been employed in fed-batch synthesis, increasing the selectivity of the reaction and obtaining high conversion (88%).     

Non-porous magnetic micro-particles: comparison to porous enzyme carriers for a diffusion rate-controlled enzymatic conversion

In this study the kinetics of conversion of a low-soluble substrate by an immobilized enzyme was investigated with respect to the diffusion limitation within porous and non-porous carriers. Non-porous micro-magnetic beads in comparison to conventional porous supports like Eupergit and Sepharose were tested. Due to their small diameters and their magnetic properties, micro-magnetic beads are especially applicable in diffusion rate-controlled processes in biological suspensions. The enzymatic reaction studied was the conversion of emulsified dirhamnolipid by immobilized Naringinase from Penicillium decumbens to monorhamnolipid and L-rhamnose. Taking into account mass transfer phenomena, the variation of the reaction effectiveness factor with increasing enzyme loading was estimated and compared with experimental efficiencies utilizing different enzyme loaded immobilized preparations. For comparison, carrier activities were also determined with the model substrate p-nitro-phenyl-rhamnoside. Intrinsic enzyme activities were thereby evaluated for porous supports. Highest specific activities were obtained with the micro-magnetic beads. These non-porous micro-beads demonstrated to be the most suitable carrier for bioconversion of a low-soluble substrate like rhamnolipids, where mass diffusional resistances in the three-phase reaction process are completely overcome. However, the smaller particle surface available limited the specific activity obtained at high protein loadings.     

Comparison of batch and continuous multi‐column protein A capture processes by optimal design

Multi‐column capture processes show several advantages compared to batch capture. It is however not evident how many columns one should use exactly. To investigate this issue, twin‐column CaptureSMB, 3‐ and 4‐column periodic counter‐current chromatography (PCC) and single column batch capture are numerically optimized and compared in terms of process performance for capturing a monoclonal antibody using protein A chromatography. Optimization is carried out with respect to productivity and capacity utilization (amount of product loaded per cycle compared to the maximum amount possible), while keeping yield and purity constant. For a wide range of process parameters, all three multi‐column processes show similar maximum capacity utilization and performed significantly better than batch. When maximizing productivity, the CaptureSMB process shows optimal performance, except at high feed titers, where batch chromatography can reach higher productivity values than the multi‐column processes due to the complete decoupling of the loading and elution steps, albeit at a large cost in terms of capacity utilization. In terms of trade‐off, i.e. how much the capacity utilization decreases with increasing productivity, CaptureSMB is optimal for low and high feed titers, whereas the 3‐column process is optimal in an intermediate region. Using these findings, the most suitable process can be chosen for different production scenarios.     

Improved high-pressure enzymatic biodiesel batch synthesis in near-critical carbon dioxide

The enzymatic synthesis of biodiesel by a high-pressure semi-continuous process in near-critical carbon dioxide (NcCO(2)) was studied. Biodiesel synthesis was evaluated in both batch and semi-continuous systems to develop an effective process. Batch processing demonstrated the advantageous properties of NcCO(2) as an alternative reaction medium. Three immobilized lipases (Novozym 435, Lipozyme RM IM, and Lipozyme TL IM from Novozymes) were tested, with Lipozyme TL IM the most effective, showing the highest conversion. Biodiesel conversion from several edible and non-edible oil feedstocks reached >92%. Higher conversion (99.0%) was obtained in a shorter time by employing repeated batch processes with optimized conditions: 44.3 g (500 mM) canola oil, a substrate molar ratio (methanol:oil) of 3:1, an enzyme loading of 20 wt% (of the oil used), at 30 °C, 100 bar, and 300 rpm agitation. The enzyme maintained 80.2% of its initial stability after being reused eight times. These results suggest that this method produces biodiesel energy-efficiently and environment-friendly.     

Enhancing the catalytic potential of nitrilase from Pseudomonas putida for stereoselective nitrile hydrolysis

(R)-mandelic acid was produced from racemic mandelonitrile using free and immobilized cells of Pseudomonas putida MTCC 5110 harbouring a stereoselective nitrilase. In addition to the optimization of culture conditions and medium components, an inducer feeding approach is suggested to achieve enhanced enzyme production and therefore higher degree of conversion of mandelonitrile. The relationship between cell growth periodicity and enzyme accumulation was also studied, and the addition of the inducer was delayed by 6 h to achieve maximum nitrilase activity. The nitrilase expression was also authenticated by the sodium dodecyl phosphate-polyacrylamide gel electrophoresis analysis. P. putida MTCC 5110 cells were further immobilized in calcium alginate, and the immobilized biocatalyst preparation was used for the enantioselective hydrolysis of mandelonitrile. The immobilized system was characterized based on the Thiele modulus (ϕ). Efficient biocatalyst recycling was achieved as a result of immobilization with immobilized cells exhibiting 88% conversion even after 20 batch recycles. Finally, a fed batch reaction was set up on a preparative scale to produce 1.95 g of (R)-(-)-mandelic acid with an enantiomeric excess of 98.8%.     

Thermophilic esterase from the archaeon Archaeoglobus fulgidus physically immobilized on hydrophobic macroporous resin: A novel biocatalyst for polyester synthesis

This paper reviews the immobilization of a thermophilic esterase, AFEST from the archaeon Archaeoglobus fulgidus, on a hydrophobic macroporous resin and its application in polyester synthesis using the ring-opening polymerization of ?-caprolactone as a model. Using the physical adsorption technique, the AFEST loading concentration after 24 h was 152 mg AFEST per g of support. Particle size and surface morphology of the immobilized enzyme were investigated using laser scattering analysis and scanning electron microscopy. The effects of enzyme concentration, temperature, reaction time and reaction medium on monomer conversion and product molecular weight were systematically investigated. Through the optimization of reaction parameters, poly(?-caprolactone) was obtained at an almost 100% monomer conversion rate and with a low average molecular weight (< 1,100 g/mol). Finally, the immobilized enzyme exhibited good operational stability, with a monomer conversion value of more than 55% after four batch reactions.     

Economic impact of total solids loading on enzymatic hydrolysis of dilute acid pretreated corn stover

In process integration studies of the biomass-to-ethanol conversion process, it is necessary to understand how cellulose conversion yields vary as a function of solids and enzyme loading and other key operating variables. The impact of solids loading on enzymatic cellulose hydrolysis of dilute acid pretreated corn stover slurry was determined using an experimental response surface design methodology. From the experimental work, an empirical correlation was obtained that expresses monomeric glucose yield from enzymatic cellulose hydrolysis as a function of solids loading, enzyme loading, and temperature. This correlation was used in a technoeconomic model to study the impact of solids loading on ethanol production economics. The empirical correlation was used to provide a more realistic assessment of process cost by accounting for changes in cellulose conversion yields at different solids and enzyme loadings as well as enzyme cost. As long as enzymatic cellulose conversion drops off at higher total solids loading (due to end-product inhibition or other factors), there is an optimum value for the total solids loading that minimizes the ethanol production cost. The optimum total solids loading shifts to higher values as enzyme cost decreases.     

Production of Biodiesel Using Immobilized Lipase—A Critical Review

Increase in volume of biodiesel production in the world scenario proves that biodiesel is accepted as an alternative to conventional fuel. Production of biodiesel using alkaline catalyst has been commercially implemented due to its high conversion and low production time. For the product and process development of biodiesel, enzymatic transesterification has been suggested to produce a high purity product with an economic, environment friendly process at mild reaction conditions. The enzyme cost being the main hurdle can be overcome by immobilization. Immobilized enzyme, which has been successfully used in various fields over the soluble counterpart, could be employed in biodiesel production with the aim of reducing the production cost by reusing the enzyme. This review attempts to provide an updated compilation of the studies reported on biodiesel production by using lipase immobilized through various techniques and the parameters, which affect their functionality.     

Enzymatic synthesis of benzyl benzoate using different acyl donors: Comparison of solvent-free reaction techniques

In this study, benzyl benzoate was successfully synthesized via enzymatic acylation using three immobilized enzymes as biocatalysts. Different acyl donors (benzoic acid and benzoic anhydride), operation regimes (batch, fed-batch), mixing modes (conventional mechanical stirring and ultrasound), process parameters (temperature, substrate molar ratio of acyl donor to acyl acceptor), presence or absence of solvents, enzyme amount and type were evaluated. Benzoic acid is a solid that is difficult to solubilize and, thus, was not efficient as acyl donor for the synthesis of benzyl benzoate. On the other hand, benzoic anhydride was very effective for the acylation of benzyl benzoate, and the presence of an excess of benzyl alcohol was essential to ensure the solute-solvent intermolecular attractions and good substrate solubilization, allowing the ester synthesis to be performed in the absence of organic solvents. The ultrasound was effective in increasing increase the initial reaction rate and the final conversion (88 %). However, the Lipozyme TL-IM and RM-IM supports were damaged, and the reuse was unfeasible. The batch and fed-batch approaches in conventional stirring ensured high conversions of 92 and 90 %, respectively, for batch (anhydride: alcohol 1:6) and fed-batch (1:3) using the Lipozyme TL-IM as biocatalyst. The controlled addition of the anhydride (fed-batch) allowed the reduction of alcohol molar ratio but decreased the reaction rates, and the maximum conversions were reached only after 24 h, while the batch approach had 92 % of conversion after 6 h. The yield of benzyl benzoate was high at 6 wt.% of enzyme, low temperature (50 °C), and simple reactor operation (batch). Results show the feasibility of the synthesis of benzyl benzoate via acylation using a green process that may be an alternative route to the chemical synthesis.     

Enzymatic conversion of sunflower oil to biodiesel in a solvent-free system: Process optimization and the immobilized system stability

The feasibility of using the commercial immobilized lipase from Candida antarctica (Novozyme 435) to synthesize biodiesel from sunflower oil in a solvent-free system has been proved. Using methanol as an acyl acceptor and the response surface methodology as an optimization technique, the optimal conditions for the transesterification has been found to be: 45 ^oC, 3% of enzyme based on oil weight, 3:1 methanol to oil molar ratio and with no added water in the system. Under these conditions, >99% of oil conversion to fatty acid methyl ester (FAME) has been achieved after 50 h of reaction, but the activity of the immobilized lipase decreased markedly over the course of repeated runs. In order to improve the enzyme stability, several alternative acyl acceptors have been tested for biodiesel production under solvent-free conditions. The use of methyl acetate seems to be of great interest, resulting in high FAME yield (95.65%) and increasing the half-life of the immobilized lipase by about 20.1 times as compared to methanol. The reaction has also been verified in the industrially feasible reaction system including both a batch stirred tank reactor and a packed bed reactor. Although satisfactory performance in the batch stirred tank reactor has been achieved, the kinetics in a packed bed reactor system seems to have a slightly better profile (93.6 ± 3.75% FAME yield after 8–10 h), corresponding to the volumetric productivity of 48.5 g/(dm³ h). The packed bed reactor has operated for up to 72 h with almost no loss in productivity, implying that the proposed process and the immobilized system could provide a promising solution for the biodiesel synthesis at the industrial scale.     

Properties and application of immobilized beta-D-glucosidase coentrapped with Zymomonas mobilis in calcium alginate

The enzyme beta-D-glucosidase has been immobilized on concanavalin A-Sepharose to give a maximum loading of 2050 units/g dry weight of support material. The immobilized beta-D-glucosidase was also entrapped within calcium alginate gel spheres with apparently only 35% retention of activity when assayed with 10mM cellobiose. However, it was discovered that, unlike the immobilized enzyme, the entrapped immobilized enzyme was not subject to substrate inhibition up to 100mM cellobiose, suggesting that a concentration gradient of cellobiose existed between the bulk solution and the interior of the gel sphere. Thus, the activity of the entrapped immobilized enzyme was almost twice as high as that of the immobilized enzyme when assayed with 100mM cellobiose. Concanavalin A-Sepharose-immobilized beta-D-glucosidase and the bacterium Zymomonas mobilis coimmobilized in calcium alginate gel spheres converted cellobiose to ethanol in both batch and continuous-flow fermentation systems.     

Enzymatic modification of vegetable protein: Immobilization of Penicillium duponti enzyme on reconstituted collagen and the use of the immobilized-enzyme complex for solubilizing vegetable protein in a recycle reactor

Penicillium duponti enzyme was immobilized on reconstituted collagen by macromolecular complication, impregnation, and covalent crosslinking techniques. The immobilization of the enzyme on collagen has a twofold purpose: (1) providing a protein microenvironment for the proteolytic enzyme; and (2) extending the useful life the enzyme once immobilized on the collagen matrix. Two types of collagen were used, one produced by the United States Department of Agriculture and the other produced by FMC. The USDA collagen contained unhydrolyzed telepeptide linkages and required pretreatment to reduce collagenaselike activity of the enzyme. Activity analysis of the immobilized enzyme complex showed that membranes with enzyme loading less than 10 mg enzyme/g of wet membrane in the reactor were dimensionally stable. The degree of crosslinking was an important parameter. Membranes with structural opening up to three times the initial dry thickness were found to be the maximum limit for controlled release of enzyme from the collagen membrane during enzymatic reaction. Higher activities and better stability of the enzyme in collagen membrane were found for covalent crosslinking of the enzyme to treated collagen films. The hydrolysis of soybean vegetable protein with the immobilized enzyme in a recycle reactor at enzyme loading of mg/g of wet membrane at 40°C, pH 3.4, produced 56.5% of soluble protein in 10h. The production is equivalent to 1.84 h total contact time between the substrate and the immobilized enzyme. The average productivity based on a stable enzyme activity and 20g of dry membrane was 329 mg of protein/g/mg of active enzyme immobilized. The productivity of the free enzyme in a batch reactor was 62.5 mg protein/h/mg enzyme.     

Cell immobilization technique for the enhanced production of alpha-galactosidase by Streptomyces griseoloalbus

Streptomyces griseoloalbus was immobilized in calcium alginate gel and the optimal immobilization parameters (concentrations of sodium alginate and calcium chloride, initial biomass and curing time) for the enhanced production of alpha-galactosidase were determined. The immobilization was most effective with 3% sodium alginate and 0.1M calcium chloride. The optimal initial biomass for immobilization was approximately 2.2g (wet wt.). The alginate-entrapped cells were advantageous because there was a twofold increase in the enzyme yield (55 U/ml) compared to the highest yield obtained with free cells (23.6 U/ml). Moreover, with immobilized cells the maximum yield was reached after 72 h of incubation in batch fermentation under optimal conditions, whereas in the case of free cells the maximum enzyme yield was obtained only after 96 h of incubation. The alginate beads had good stability and also retained 75% ability of enzyme production even after eight cycles of repeated batch fermentation. It is significant that this is the first report on whole-cell immobilization for alpha-galactosidase production.     

Continuous production of L-phenylalanine by Rhodotorula glutinis immobilized cells using a column reactor

Studies have been conducted on L-phenylalanine (L-Phe) production and phenylalanine ammonia lyase (PAL) stabilization in the presence of several optimum effectors and reducing agents under bioconversion of transcinnamic acid (t-CA) conditions during repeated batch operations. L-Phe production was maximized and reuseability of PAL catalyst was extended to eight consecutive cycles (repeated batches) in the presence of optimum effectors (glutamic acid, polyethylene glycol and glycerol), thioglycolic acid and sparging with nitrogen gas. These best optimum bioconversion conditions desensitize the PAL catalyst to substantially elevated higher substrate t-CA concentrations and inhibit inactivation of PAL enzyme over longer reaction periods compared to the control. The fed batch mode operation of bioconversion of total t-CA (300 mM) to L-Phe was superior (65.2%, conversion), comparing with conventional batch and repeated batch (58.4%, conversion) operations after 120 h. Gamma irradiation process was employed to polymerize and crosslink polyvinyl alcohol (PVA) with N,N'-methylene-bisacrylamide (BIS) agent. The use of immobilized PAL biocatalyst containing cells in PVA-BIS copolymer gel carrier produced by radiation polymerization is obviously advantageous with regards to the yield of L-Phe which was increased in average 1.2-fold when compare to those obtained with free cells during optimum bioconversion process. When comparing the magnitudes of gamma irradiation effects on immobilized entrapped yeast cells in PVA-BIS copolymer gel carrier using scanning electron microscopy it was show that yeast cells were protected and capable to overcome these conditions and had normal shape and other features as free (unirradiated) intact yeast cells. Optimum conditions for continuous production of L-Phe by PVA-BIS copolymer carrier entrapped yeast cells in a packed bed column reactor in recycle fed-batch mode were investigated. Under these optimum conditions L-Phe accumulated to concentration 240.1 mM represts a total conversion yield of 80% (w/w) from (300 mM) t-CA after 84 h of reaction process, which was higher than that obtained after 120 h of reaction, 65.2% (w/w) from (300 mM) t-CA with free cells in fed-batch mode. The results also demonstrated that during about 4 weeks of repeated continuous recycle fed batch mode experiments (using immobilized cells in packed bed reactor), the final production of L-Phe concentrations decreased gradually in eight consecutive runs with no sign of breakage or disintegration of the carrier gel beads.     

Immobilization of glucoamylase and glucose oxidase in activated carbon: effects of particle size and immobilization conditions on enzyme activity and effectiveness

Glucoamylase and glucose oxidase have been immobilized on carbodiimide-treated activated carbon particles of various sizes. Loading data indicate nonuniform distribution of immobilized enzyme within the porous support particles. Catalysts with different enzyme loading and overall activities have been prepared by varying enzyme concentration in the immobilizing solution. Analysis of these results by a new method based entirely upon experimentally observable catalyst properties indicates that intrinsic catalytic activity is reduced by immobilization of both enzymes. Immobilized glucoamylase intrinsic activity decreases with increasing enzyme loading, and similar behavior is suggested by immobilized glucose oxidase data analysis. The overall activity data interpretation method should prove useful in other immobilized enzyme characterization research, especially in situations where the intraparticle distribution of immobilized enzyme is nonuniform and unknown.     

Reactor optimization for alpha-1,2 glucooligosaccharide synthesis by immobilized dextransucrase

The immobilization of dextransucrase in Ca-alginate beads relies on the close association between dextran polymer and dextransucrase. However, high amounts of dextran in the enzyme preparation drastically limit the specific activity of the immobilized enzyme (4 U/mL of alginate beads). Moreover, even in the absence of diffusion limitation at the batch conditions used, the enzyme behavior is modified by entrapment so that the dextran yield increases and the alpha-1,2 glucooligosaccharides (GOS) are produced with a lower yield (46.6% instead of 56.7%) and have a lower mean degree of polymerization than with the free dextransucrase. When the immobilized catalyst is used in a continuous reaction, the reactor flow rate necessary to obtain high conversion of the substrates is very low, leading to external diffusion resistance. As a result, dextran synthesis is even higher than in the batch reaction, and its accumulation within the alginate beads limits the operational stability of the catalyst and decreases glucooligosaccharide yield and productivity. This effect can be limited by using reactor columns with length to diameter ratio > or =20, and by optimizing the substrate concentrations in the feed solution: the best productivity obtained was 3.74 g. U(-1). h(-1), with an alpha-1,2 GOS yield of 36%.