Effects of yeast-originating polymeric compounds on ethanol pervaporation.

During ethanol fermentation with in situ pervaporation, membrane fouling might occur due to polymers originating from yeast cell lysis. The aim of this study was to evaluate the influence of yeast cellular polymers on pervaporative membrane performance. Lipids were identified as the most detrimental components among these cellular polymers causing 50% and 33% flux decrease in polydimethylsiloxane (PDMS) and polyoctylmethylsiloxane (POMS) membranes, respectively. This fouling was irreversible and might be due to hydrophobic interactions between lipids and membranes resulting in high lipid adsorption on membrane surface. The relatively hydrophobic model protein BSA also contributed to flux decrease in PDMS membrane but RNA and the model polysaccharide glycogen did not. The PDMS membrane selectivity for ethanol/water remained ~4.5 in all cases. All the cellular components decreased the water flux through the POMS membrane. However, the ethanol flux through the membrane was not altered very much, resulting in increased membrane selectivity.

Influence of fermentation by-products on the purification of ethanol from water using pervaporation.

Pervaporation is claimed to be a promising separation technique for the purification of ethanol from fermentation broths during bio-ethanol production. In this study, influence of fermentation by-products on the purification of ethanol from water during hydrophobic pervaporation was investigated. Sugars and salts were found to increase the membrane performance. Reason for this was a change in vapor/liquid equilibrium. 2,3-butanediol decreased the ethanol flux and selectivity factor, while glycerol exhibited no effect. This was explained by a strong sorption of butanediol into PDMS and no sorption of glycerol. Due to the presence of carboxylic acids, hydrophobicity degree of the Pervap 4060 membrane decreased, which resulted in an irreversible increase in water flux and decrease in separation performance. These observations suggested the presence of silicalite-based fillers in the membrane. When the pH was raised to a value above the dissociation constant, no changes in hydrophobicity degree and membrane performance were found.

Liquid-liquid extraction of fermentation inhibiting compounds in lignocellulose hydrolysate.

Several compounds that are formed or released during hydrolysis of lignocellulosic biomass inhibit the fermentation of the hydrolysate. The use of a liquid extractive agent is suggested as a method for removal of these fermentation inhibitors. The method can be applied before or during the fermentation. For a series of alkanes and alcohols, partition coefficients were measured at low concentrations of the inhibiting compounds furfural, hydroxymethyl furfural, vanillin, syringaldehyde, coniferyl aldehyde, acetic acid, as well as for ethanol as the fermentation product. Carbon dioxide production was measured during fermentation in the presence of each organic solvent to indicate its biocompatibility. The feasibility of extractive fermentation of hydrolysate was investigated by ethanolic glucose fermentation in synthetic medium containing several concentrations of furfural and vanillin and in the presence of decanol, oleyl alcohol and oleic acid. Volumetric ethanol productivity with 6 g/L vanillin in the medium increased twofold with 30% volume oleyl alcohol. Decanol showed interesting extractive properties for most fermentation inhibiting compounds, but it is not suitable for in situ application due to its poor biocompatibility.

Hydrogel coated monoliths for enzymatic hydrolysis of penicillin G.

The objective of this work was to develop a hydrogel-coated monolith for the entrapment of penicillin G acylase (E. coli, PGA). After screening of different hydrogels, chitosan was chosen as the carrier material for the preparation of monolithic biocatalysts. This protocol leads to active immobilized biocatalysts for the enzymatic hydrolysis of penicillin G (PenG). The monolithic biocatalyst was tested in a monolith loop reactor (MLR) and compared with conventional reactor systems using free PGA, and a commercially available immobilized PGA. The optimal immobilization protocol was found to be 5 g l(-1) PGA, 1% chitosan, 1.1% glutaraldehyde and pH 7. Final PGA loading on glass plates was 29 mg ml(-1) gel. For 400 cpsi monoliths, the final PGA loading on functionalized monoliths was 36 mg ml(-1) gel. The observed volumetric reaction rate in the MLR was 0.79 mol s(-1) m(-3) (monolith). Apart from an initial drop in activity due to wash out of PGA at higher ionic strength, no decrease in activity was observed after five subsequent activity test runs. The storage stability of the biocatalysts is at least a month without loss of activity. Although the monolithic biocatalyst as used in the MLR is still outperformed by the current industrial catalyst (immobilized preparation of PGA, 4.5 mol s(-1) m(-3) (catalyst)), the rate per gel volume is slightly higher for monolithic catalysts. Good activity and improved mechanical strength make the monolithic bioreactor an interesting alternative that deserves further investigation for this application. Although moderate internal diffusion limitations have been observed inside the gel beads and in the gel layer on the monolith channel, this is not the main reason for the large differences in reactor performance that were observed. The pH drop over the reactor as a result of the chosen method for pH control results in a decreased performance of both the MLR and the packed bed reactor compared to the batch system. A different reactor configuration including an optimal pH profile is required to increase the reactor performance. The monolithic stirrer reactor would be an interesting alternative to improve the performance of the monolith-PGA combination.

Adsorption of fluorescein by protein crystals.

Adsorption characteristics of native and cross-linked lysozyme crystals were examined using fluorescein as model adsorbate. The adsorption isotherms exhibited Langmuir or linear behavior. The affinity constant (b1) and the adsorption capacity (Qsat) for fluorescein were found to depend on the type and concentration of co-solute present in the solution. The dynamics of adsorption isotherm transition from Langmuir to linear showed that affinity of lysozyme for solutes increases in the order 2-(cyclohexylamino)ethanesulphonic acid (CHES), 4-morpholinepropanesulphonic acid (MOPS), acetate, fluorescein. Furthermore, the crystal morphology, the degree of cross-linking of the crystals, and, in particular, solution pH were identified as factors determining fluorescein adsorption by the lysozyme crystals. These factors seem to affect crystal capacity for the solute more than affinity for the solute. Adsorption of fluorescein by cross-linked tetragonal lysozyme crystals was exponentially dependent on the lysozyme net charge calculated from the final solution pH. The 3-5-fold increase in the fluorescein adsorption as a result of cross-linking is presumably due to the increasing hydrophobicity of the lysozyme crystal.

Quantifying anisotropic solute transport in protein crystals using 3-D laser scanning confocal microscopy visualization.

The diffusion of a solute, fluorescein into lysozyme protein crystals has been studied by confocal laser scanning microscopy (CLSM). Confocal laser scanning microscopy makes it possible to non-invasively obtain high-resolution three-dimensional (3-D) images of spatial distribution of fluorescein in lysozyme crystals at various time steps. Confocal laser scanning microscopy gives the fluorescence intensity profiles across horizontal planes at several depths of the crystal representing the concentration profiles during diffusion into the crystal. These intensity profiles were fitted with an anisotropic model to determine the diffusivity tensor. Effective diffusion coefficients obtained range from 6.2 x 10(-15) to 120 x 10(-15) m2/s depending on the lysozyme crystal morphology. The diffusion process is found to be anisotropic, and the level of anisotropy depends on the crystal morphology. The packing of the protein molecules in the crystal seems to be the major factor that determines the anisotropy.

Auxiliary phase guidelines for microbial biotransformations of toxic substrate into toxic product.

When an industrial process is developed using the microbial transformation of a precursor into a desired chemical compound, high concentrations of substrate and product will be involved. These compounds may become toxic to the cells. In situ product removal (ISPR) may be carried out, using auxiliary phases such as extractants or adsorbents. Simultaneously, in situ substrate addition (ISSA) may be performed. It is shown that for uncharged substrates and products, the aqueous solubilities of substrate and product can be used to predict if ISPR might be required. When a particular auxiliary phase is selected and the distribution coefficients of substrate and product are known, it is possible to estimate a priori if this auxiliary phase might be good enough and how much of it might be needed for an efficient (fed-)batch biotransformation process. For biotransformation products of intermediate polarity (aqueous solubility of about 1-10 g/L) there seems to be a lack of extractants and adsorbents with the capacity to raise the product concentrations to commercially more interesting levels.

A 'Fine' chemical industry for life science products: green solutions to chemical challenges.

Modern biotechnology, in combination with chemistry and process technology, is crucial for the development of new clean and cost effective manufacturing concepts for fine-chemical, food specialty and pharmaceutical products. The impact of biocatalysis on the fine-chemicals industry is presented, where reduction of process development time, the number of reaction steps and the amount of waste generated per kg of end product are the main targets. Integration of biosynthesis and organic chemistry is seen as a key development. The advances in bioseparation technology need to keep pace with the rate of development of novel bio- or chemocatalytic process routes with revised demands on process technology. The need for novel integrated reactors is also presented. The necessary acceleration of process development and reduction of the time-to-market seem well possible, particularly by integrating high-speed experimental techniques and predictive modelling tools. This is crucial for the development of a more sustainable fine-chemicals industry. The evolution of novel 'green' production routes for semi-synthetic antibiotics (SSAs) that are replacing existing chemical processes serves as a recent and relevant case study of this ongoing integration of disciplines. We will also show some challenges in this specific field.

Development of (R)-4-hydroxymandelonitrile synthesis in an aqueous-organic biphasic stirred tank batch reactor.

A relatively new hydroxynitrile lyase-catalyzed reaction was optimized to be suitable for rapid and efficient development of a full-scale production process. The conversion of 4-hydroxybenzaldehyde into (R)-4-hydroxymandelonitrile, catalyzed by Prunus amygdalus hydroxynitrile lyase, was carried out in a biphasic system of aqueous buffer (pH 5.5) and methyl tert-butyl ether and is described with a process model. The process model consists of a description of the reaction kinetics, mass transfer kinetics, and mass balances for both the aqueous and the organic phase. Values are determined for the equilibrium constant, the enzyme kinetic parameters, the lumped mass transfer coefficient for benzaldehyde, and the partition coefficients. By using estimated prices of enzyme and reactor use, the optimum aqueous phase volume fraction and required enzyme concentration were calculated at a temperature of 20 degrees C for a batch-operated stirred tank reactor. According to the process model it was possible to convert 90% of the 4-hydroxybenzaldehyde into (R)-4-hydroxymandelonitrile with 95% enantiomeric excess. The price optimum for this reaction was found at an aqueous phase volume of 17% of the total volume. The required enzyme concentration to meet the targets was 28.6 g/L aqueous phase. At the predicted optimum, the synthesis was performed experimentally and the results were in accordance with the simulation regarding the extent of conversion and the enantiomeric excess.

Equilibrium modeling of extractive enzymatic hydrolysis of penicillin G with concomitant 6-aminopenicillanic acid crystallization.

In the present downstream processing of penicillin G, penicillin G is extracted from the fermentation broth with an organic solvent and purified as a potassium salt via a number of back-extraction and crystallization steps. After purification, penicillin G is hydrolyzed to 6-aminopenicillanic acid, a precursor for many semisynthetic beta-lactam antibiotics. We are studying a reduction in the number of pH shifts involved and hence a large reduction in the waste salt production. To this end, the organic penicillin G extract is directly to be added to an aqueous immobilized enzyme suspension reactor and hydrolyzed by extractive catalysis. We found that this conversion can exceed 90% because crystallization of 6-aminopenicillanic acid shifts the equilibrium to the product side. A model was developed for predicting the equilibrium conversion in batch systems containing both a water and a butyl acetate phase, with either potassium or D-p-hydroxyphenylglycine methyl ester as counter-ion of penicillin G. The model incorporates the partitioning equilibrium of the reactants, the enzymatic reaction equilibrium, and the crystallization equilibrium of 6-aminopenicillanic acid. The model predicted the equilibrium conversion of Pen G quite reasonably for different values of pH, initial penicillin G concentration and phase volume ratio. The model can be used as a tool for optimizing the enzymatic hydrolysis.

Influence of the ethanol and glucose supply rate on the rate and enantioselectivity of 3-oxo ester reduction by baker's yeast.

Baker's-yeast-mediated reductions of ketones hold great potential for the industrial production of enantiopure alcohols. In this article we describe the stoichiometry and kinetics of asymmetric ketone reduction by cell suspensions of bakers' yeast (Saccharomyces cerevisiae). A system for quantitative analysis of 3-oxo ester reduction was developed and allowed construction of full mass and redox balances as well as determination of the influence of different process parameters on aerobic ketone reduction. The nature of the electron donor (ethanol or glucose) and its specific consumption rate by the biomass (0-1 mol.kg dw(-1).h(-1)) affected the overall stoichiometry and rate of the process and the final enantiomeric excess of the product. Excess glucose as the electron donor, i.e. a very high consumption rate of glucose, resulted in a high rate of alcoholic fermentation, oxygen consumption, and biomass formation and therefore causing low efficiency of glucose utilization. Controlled supply of the electron donor at the highest rates applied prevented alcoholic fermentation but still resulted in biomass formation and a high oxygen requirement, while low rates resulted in a more efficient use of the electron donor. Low supply rates of ethanol resulted in biomass decrease while low supply rates of glucose provided the most efficient strategy for electron donor provision and yielded a high enantiomeric excess of ethyl (S)-3-hydroxybutanoate. In contrast to batchwise conversions with excess glucose as the electron donor, this strategy prevented by-product formation and biomass increase, and resulted in a low oxygen requirement.

Electrospray ionization mass spectrometry, circular dichroism and SAXS studies of the (S)-hydroxynitrile lyase from Hevea brasiliensis.

We report on experiments pertaining to solution properties of the (S)-hydroxynitrile lyase from Hevea brasiliensis (HbHNL). Small angle X-ray scattering unequivocally established the enzyme to occur in solution as a dimer, presumably of the same structure as in the crystal. The acid induced, irreversible deactivation of HbHNL was examined by electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD) and by measuring the enzyme activity. The deactivation is paralleled by an unfolding of the enzyme. ESI-MS of this 30000 Da per monomer heavy protein demonstrated that unfolding took place in several stages which are paralleled by a decrease in enzyme activity. Unfolding can also be observed by CD spectroscopy, and there is a clear correlation between enzyme activity and unfolding as detected by ESI-MS and CD.

Hydrolytic activity in baker's yeast limits the yield of asymmetric 3-oxo ester reduction.

Microbial reductions of ketones hold great potential for the production of enantiopure alcohols, as long as highly selective redox enzymes are not interfered with by competing activities. During reduction of ethyl 3-oxobutanoate by baker's yeast (Saccharomyces cerevisiae) to ethyl (S)-3-hydroxybutanoate, a high enantiomeric excess (> 99%) can be obtained. However, reported yields do not exceed 50-70%. In this article, three main causes are shown to be responsible for these low to moderate yields. These are evaporation of the substrate and product esters, absorption or adsorption of the two esters by the yeast cells and hydrolysis of the two esters by yeast enzymes. The hydrolysis products are further metabolized by the yeast. By reducing the evaporation and absorption losses, the reduction yield can easily be improved to about 85%. Improvement of the efficiency of the reduction and hence the reduction/hydrolysis ratio should lead to a further increase in yield.

Competitive lipase-catalyzed ester hydrolysis and ammoniolysis in organic solvents; equilibrium model of a solid-liquid-vapor system.

Enzymatic ester hydrolysis and ammoniolysis were performed as competitive reactions in methyl isobutyl ketone without a separate aqueous phase. The reaction system contained solid ammonium bicarbonate, which dissolved as water, ammonia, and carbon dioxide. During the reaction an organic liquid phase, a vapor phase, and at least one solid phase are present. The overall equilibrium composition of this multiphase system is a complex function of the reaction equilibria and several phase equilibria. To gain a quantitative understanding of this system a mathematical model was developed and evaluated. The model is based on the mass balances for a closed batch system and straightforward relations for the reaction equilibria and the solubility equilibria of ammonium bicarbonate, the fatty acid ammonium salt, water, ammonia, and carbon dioxide. For butyl butyrate as a model ester and Candida antarctica lipase B as the biocatalyst this equilibrium model describes the experiments satisfactorily. The model predicts that high equilibrium yields of butyric acid can be achieved only in the absence of ammoniolysis or in the presence of a separate water phase. However, high yields of butyramide should be possible if the water concentration is fixed at a low level and a more suited source of ammonia is applied.

Study of the (S)-hydroxynitrile lyase from Hevea brasiliensis: mechanistic implications.

Investigations of the (S)-selective hydroxynitrile lyase from Hevea brasiliensis were performed by electrospray mass spectroscopy, (1)H-NMR and with an enzyme activity assay. For the trans-cyanohydrin reaction (transcyanation) a two step reaction could be established. The results furthermore indicate a fast deactivation of the enzyme at low pH and a strong substrate dependence of its stability. They rule out an enzyme-HCN complex or a covalently bound carbonyl compound. Therefore the earlier postulated reaction intermediate as well as the proposed action of the catalytic triad have to be reevaluated. The calculated molecular mass could be confirmed by mass spectroscopy.

Understanding the influence of temperature change and cosolvent addition on conversion rate of enzymatic suspension reactions based on regime analysis.

It is a commonly held belief that enzymatic conversions of substrate in aqueous suspensions can be speeded up by raising the temperature or adding organic solvents to promote dissolution of the substrate. To quantify the impact of such changes, we studied the alpha-chymotrypsin-catalyzed hydrolysis of dimethyl benzylmethylmalonate as a model system. It was found that, upon addition of organic cosolvents, longer process times were actually required, even though the substrate solubility increased severalfold as expected. Upon raising the temperature from 25 degrees C to 37 degrees C, on the other hand, both the substrate solubility, the substrate dissolution rate, and the enzymatic reaction rate increased, leading to shorter process times. A dissolution-reaction model incorporating the kinetics of enzyme deactivation could be developed. A simple relation for the prediction of the overall process time was established by evaluating the time constants for the subprocesses: substrate dissolution; enzymatic conversion; and enzyme deactivation. Using regime analysis, rules of thumb for the optimization of an enzymatic suspension reaction were derived.

Characterization of the Enantioselective Properties of the Quinohemoprotein Alcohol Dehydrogenase of Acetobacter pasteurianus LMG 1635. 1. Different Enantiomeric Ratios of Whole Cells and Purified Enzyme in the Kinetic Resolution of Racemic Glycidol.

Resting cells of Acetobacter pasteurianus LMG 1635 (ATCC 12874) show appreciable enantioselectivity (E=16-18) in the oxidative kinetic resolution of racemic 2,3-epoxy-1-propanol, glycidol. Distinctly lower values (E=7-9) are observed for the ferricyanide-coupled oxidation of glycidol by the isolated quinohemoprotein alcohol dehydrogenase, QH-ADH, which is responsible for the enantiospecific oxidation step in whole cells. The accuracy of E-values from conversion experiments could be verified using complementary methods for the measurement of enantiomeric ratios. Effects of pH, detergent, the use of artificial electron acceptors, and the presence of intermediate aldehydes, could be accounted for. Measurements of E-values at successive stages of the purification showed that the drop in enantioselectivity correlates with the separation of QH-ADH from the cytoplasmic membrane. It is argued that the native arrangement of QH-ADH in the membrane-associated complex favors the higher E-values. The consequences of these findings for the use of whole cells versus purified enzymes in biocatalytic kinetic resolutions of chiral alcohols are discussed.

Simple dissolution-reaction model for enzymatic conversion of suspension of solid substrate.

Although reactions in substrate suspension are employed in industry for several bioconversion processes, there appears to be no quantitative model available in the literature to rationalize the optimization of these processes. We present a simple model that incorporates the kinetics of substrate dissolution and a simultaneous enzymatic reaction. The model was tested in the alpha-chymotrypsin-catalyzed hydrolysis of an aqueous suspension of dimethyl benzylmethylmalonate to a homogeneous solution of enantiomerically pure monoester. This reaction occurs in the bulk phase, so catalysis by enzyme absorbed at the solid-liquid interface plays no role. The value of the parameters in the model (i.e., the mass transfer coefficient of substrate dissolution (k(L)), the substrate solubility, and the rate constant for the enzymatic reaction) were determined in separate experiments. Using these parameter values, the model gave a good quantitative prediction of the rate of the overall dissolution-reaction process. When the particle size distribution is known, k(L) may also be calculated instead. The model seems to be applicable also for other poorly soluble substrates, other enzymes, and other solvents. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 433-440, 1997.

New constraints between kinetic parameters explain the (Un)identifiability of enzymatic rate constants.

For an enzymatic reaction the rate constants in the assumed mechanism (k(1), k(-1), etc.) sometimes can be calculated from the steady-state parameter values (V(max), K(m), etc.) and sometimes cannot. When identifiability problems occur, these are obscured by redundancy occurring among the steady-state parameters. This redundancy is only partly revealed by the known Haldane relations. We found the additional constraints between the parameters. These relations allow to predict in which situation rate constants are identifiable by steady-state kinetic methods. (c) 1996 John Wiley & Sons, Inc.

Mass balancing in kinetic resolution: calculating yield and enantiomeric excess using chiral balance.

When kinetic resolution is applied for the production of enantiomerically pure compounds, process options may be used which involve more than one chiral substrate and one chiral product, such as sequential or parallel enzymatic kinetic resolutions or hydrolysis of diastereomers. Although the relation between the yields (y) of the chiral compounds is straightforward in these cases, the relation between their enantiomeric excess (ee) values is not. Combining mass balances into a so-called chiral balance (Sigma y x ee(R) = 0) provides the relation between enantiomeric excess values in a useful manner. This chiral balance easily shows which nonmeasured enantiomeric excess values and yields can be calculated from measured values. The chiral balance is only valid when configurations at chiral centers are conserved.