Influence of viscosity of enzymatic reactions studied with glucoamylase of Aspergillus niger.

Viscosity can be interpreted in terms of transport of momentum and, therefore, it should influence the kinetics of enzyme reactions. A theory, developed by Somogyi and Damjanovich ((1975) J. Theor. Biol. 51, 393--401), is based on this idea. Transport of momentum must always be accompanied by the transport of mass and this second influence of viscosity is a limiting factor for fast reactions in the liquid phase. A third aspect is, that the chemical potentials of the components of viscous solutions are altered. This paper reports experiments concerning the influence of the viscosigens (compounds that increase the viscosity of solvents), alginate, sucrose, and maltose on the kinetic behaviour of glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3). The observed invariance of V and the decrease of Km are explained by the increase of chemical potentials and restriction of momentum transport.

Studies on an enzyme, S-formylglutathione hydrolase, of the dissimilatory pathway of methanol in Candida boidinii.

In Candida boidinii, S-formylglutathione formed by reaction of the glutathione-dependent formaldehyde dehydrogenase is hydrolyzed to formate and glutathione by a special enzyme, S-formylglutathione hydrolase which is induced in C. boidinii along with the other enzymes of the dissimilatory pathway during growth on CH3OH. The S-formylglutathione hydrolase was purified to apparent homogeneity and a specific activity of 1390 U/mg. The molecular weight of the native enzyme was determined as 61 000 by gel filtration and 64 000 by sedimentation-diffusion equilibrium. It is composed of two nonidentical polypeptide chains of 35 000 and 25 000 daltons. The Km-value of S-formylglutathione was found to be 0.21 mM. Glutathione is a competitive inhibitor with a Ki vaue of 18.5 mM. The enzyme is very specific for S-formylglutatione, S-acetylglutathione gave 1.3%, respectively. Other glutathione derivatives of hydroxyacids tested were not split by the S-formylglutatione hydrolase.

Purification and characterization of a nicotinamide adenine dinucleotide-dependent secondary alcohol dehydrogenase from Candida boidinii.

From the yeast Candida boidinii grown on glucose a new secondary alcohol dehydrogenase was purified 426-fold by heat treatment, column chromatography on DEAE-Sephacel, affinity chromatography on Blue Sepharose Cl-6b, and gel filtration on Sephacryl S-300. The purified enzyme was homogeneous as judged by analytical polyacrylamide gel electrophoresis. The molecular weight was found to be 150000 by sedimentation equilibrium as well as by gel filtration. The enzyme appears to be composed of four identical subunits (Mr=38000) as determined by SDS-gel electrophoresis. The enzyme catalyzes the oxidation of isopropanol to acetone in the presence of NAD+ as an electron acceptor. The Km values were found to be 0.099 mM for isopropanol and 0.14 mM for NAD+. Besides isopropanol also other secondary alcohols like butan-2-ol, pentan-2-ol, pentan-3-ol, hexan-2-ol, cyclobutanol, cyclopentanol, and cyclohexanol served as a substrate and were oxidized to the corresponding ketones. Isopropanol seems to be the best substrate for this enzyme which we therefore call isopropanol dehydrogenase. Primary alcohols are not oxidized by the enzyme. The optimum pH for enzymatic activity in the oxidation reaction was found to be 9.0, the optimal temperature is 45 degrees C. The isoelectric point of the isopropanol dehydrogenase was found to be pH 4.9. The enzyme is inactivated by mercaptide-forming reagents and chelating agents, 2-mercaptoethanol is an inhibitor. Zinc ions appear necessary for enzyme production.

Influence of substrate or product inhibition on the performance of enzyme reactors.

For the design of an enzyme reactor a detailed knowledge of the kinetic parameters of the catalyst under operational conditions is essential. For technical applications high initial substrate concentrations and high degrees of conversions are desirable, in order to save reactor volume and energy in recovery processes. Most of the kinetic data available in the literature have been derived from dilute solutions under initial rate conditions. These data cannot be extrapolated with confidence for technically interesting concentrations because substrate as well as product-inhibition may occur, which would not be observed in dilute solutions and by initial rate measurements. Because of this difficulty effective and fast methods to obtain significant data for technical applications have been developed based on-line rate determinations. Such extensive treatment has proved necessary for the following enzymes: alanine dehydrogenase, formate dehydrogenase and alpha-glucosidase, indicating that we are dealing with a general phenomenon.

Technical aspects of extractive enzyme purification.

We see the advantages of extraction processes for large-scale enzyme isolation and purification in the high capacity of the method, savings in process time and energy, high activity yields and the possibilities for continuous processing at all stages. Commercially available equipment can be used for separation of aqueous two-phase systems with minor modifications. It is hoped that, through using such technology, intracellular enzymes will become available in large quantities and at lower cost.

Purification of UDP-glucose pyrophosphorylase from germinated barley (malt).

UDP-glucose pyrophosphorylase was purified from germinated barley (malt) using anion exchange and hydrophobic interaction chromatography followed by preparative gel filtration. Gel filtration and SDS-PAGE revealed a molecular mass of 51 to 54 kDa for the monomeric protein. Microsequencing of the blotted protein by Edman degradation gave 20 N-terminal amino acids. UDP-glucose pyrophosphorylase from malt could be markedly stabilized by the addition of bovine serum albumin. The enzyme preparation is free of contaminating nucleoside triphosphatases (UTPases) and can be utilized for the enzymatic synthesis of activated sugars.

Monitoring of enzymes during chromatographic separations.

An on-line enzyme assay is presented based on flow injection techniques combined with fluorimetric detection. It allows to monitor NAD-dependent oxidoreductases during the purification of microbial crude extracts or partially purified enzymes by fast protein liquid chromatography (FPLC) in a near real-time mode. The arrangement is simple and can be easily integrated in the chromatographic system avoiding dead volumes. A high measuring frequency (up to 180 samples h-1) and a short response time (10-30 s) are achieved. The method has a low limit of detection (approximately 0.01 U ml-1), and a good reproducibility (1-4%), the injected sample volume is only 2 microliters.

Purification and characterization of a novel carbonyl reductase isolated from Rhodococcus erythropolis.

During growth on n-tetradecane a novel NADH-dependent carbonyl reductase is induced in the Gram-positive bacterium Rhodococcus erythropolis (Peters, P., Zelinski, T. and Kula, M.R. (1992) Appl. Microbiol. Biotechnol. 38, 334-340). The enzyme has been purified to homogeneity using fractional pH precipitation, anion exchange chromatography and affinity chromatography. The isoelectric point of the oxidoreductase is 4.4. The apparent molecular mass of the native enzyme is 161 kDa, that of the subunits 40 kDa as determined by SDS gel electrophoresis. A tetrameric structure of the carbonyl reductase is consistent with these results. Important biochemical data concerning the application of the reductase are: a broad pH-optimum, temperature optimum at 40 degrees C and stability at room temperature for more than 5 days. The oxidoreductase accepted as substrate aliphatic and aromatic ketones, keto esters (esters of keto carboxylic acids) and halogenated carbonyl compounds and reduced them to the corresponding hydroxyl compounds with (S)-configuration with more than 98% enantiomeric excess. The NAD(+)-dependent oxidation of primary alcohols was not catalyzed by the carbonyl reductase, whereas secondary alcohols and hydroxy acid esters were oxidized to the corresponding carbonyl compounds at about 10-fold slower reaction rates compared to the reduction.

Cloning, sequencing and overexpression of the leucine dehydrogenase gene from Bacillus cereus.

The L-leucine dehydrogenase gene from Bacillus cereus (DSM 626) was cloned from a partial genomic library and sequenced. The open reading frame has 1101 bp and codes for a protein of 39.9 kDa. The deduced amino acid sequence of the LeuDH from B. cereus shares 70-80% identity with LeuDH's from the thermophilic strains B. stearothermophilus and Thermoactinomyces intermedius. The active protein was overexpressed in Escherichia coli to yield approximately 30% of the total soluble protein.

Rapid SDS-Gel capillary electrophoresis for the analysis of recombinant NADP(+)-dependent formate dehydrogenase during expression in Escherichia coli cells and its purification.

The level of expression in Escherichia coli cells and different steps of purification of the recombinant NADP(+)-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from bacterium Pseudomonas sp.101 was analyzed by rapid SDS-Gel capillary electrophoresis (SDS-Gel CE) and compared with SDS polyacrylamide gel electrophoresis (SDS PAGE). First standard proteins were separated in the short capillary and the calibration curve generated, then fractions taken during the fermentation and purification process were analysed. The main advantages of SDS-Gel CE are short analysis time, high sensitivity, the possibility to quantify proteins at different ultraviolet wavelength, and small injection volumes. The data for each step of the fermentation process and during the purification were controlled by spectrophotometric analysis of enzyme activity and protein concentration as well as standard SDS PAGE. The molecular mass of the purified FDH was determined as 44,078 Da by matrix-assisted laser desorption/ ionisation time of flight mass spectrometry.

Analysis of polymer molecular weight distributions in aqueous two-phase systems.

The partitioning of proteins and other biomaterials between two aqueous phases containing polyethyleneglycol and dextran is a strong function of the molecular weight of the two polymers. Although both polymers are polydispersed (especially Dx) most theoretical treatments refer only to the average molecular weight (number or mass) and assume that the molecular weight distribution of each polymer is the same in both phases. In this work the molecular weight distribution of each polymer is the same in both phases. In this work the molecular weight distributions of four stock solutions of PEG (4000, 6000, 10,000 and 20,000) and four stock solutions of Dx (10,000, 40,000, 110,000 and 500,000) were measured using High Performance Gel Chromatography. The measurements were repeated on the phases formed by the polymer solutions after they were mixed and allowed to equilibrate. The molecular weight distribution of the Dx differed in the top and bottom phase; both differed from that of the stock solution. Although we believe that the molecular weight distribution for PEG also differs in the top and bottom phases, we were unable to determine this within the resolution of our instruments.

Protein partitioning in detergent-based aqueous two-phase systems.

Aqueous solutions of nonionic polyoxyethylene detergents form two liquid phases upon temperature increase above the cloud point. One of these phases is detergent-enriched and called the coacervate phase, whereas the other is detergent-depleted. Protein partitioning in such detergent-based aqueous two-phase systems was studied systematically and quantitatively, employing a series of similar polyoxyethylene detergents and proteins of varying hydrophobicity. Increasing the detergent alkyl chain length, temperature or salt concentration leads to an increase of detergent separating into the coacervate phase and a concomitant increase of protein. The positive correlation between protein hydrophobicity and partitioning into the coacervate phase confirms that protein-detergent interactions in such systems are primarily hydrophobic. These detergent-based systems can be applied to membrane proteins as well as water-soluble proteins which possess hydrophobic domains.

Studies on the enzymatic reduction of N-Boc-4S-amino-3-oxo-5-phenylpentanoic acid methylester.

The enzymatic reduction of N-Boc-4S-amino-3-oxo-5-phenylpentanoic acid methylester, the key intermediate in the stereoselective synthesis of a statinanalogue, was studied with Hansenula anomala and Hansenula silvicola. Using whole cells of H. anomala gives complete conversion and a diastereomeric excess of 88% of the desired 3S, 4S statinanalogue. The strain contains two NADPH-dependent oxidoreductases, that can be separated by ion exchange chromatography or gelfiltration, yielding the 3S, 4S or 3R, 4S stereoisomers, respectively, with > 99% diastereomeric excess (DE). In the crude extract the 3S, 4S oxidoreductase is very unstable and could be purified with << 1% yield only. In contrast, H. silvicola, which gave poor conversions using whole cells, exhibited about 80-fold higher specific activity in the crude extract than H. anomala. The NADPH-dependent oxidoreductase was purified 317-fold in 12% yield. A single enzyme of 54 kDa reduces the substrate with 97.4% DE. Besides the statinanalogue a wide range of other compounds could be reduced, most notably diones and chinones such as isatin or campherchinone. It was demonstrated that the enzymes often discussed for the reduction of beta-ketoesters with yeast e.g. L-3-hydroxyacyl CoA dehydrogenase (EC 1.1.1.35), the beta-ketoreductase of the fatty acid synthase complex and also the 3-hydroxy-3-methyl glutaryl-CoA dehydrogenase (EC 1.1.1.34) are separated during the purification steps from the oxidoreductase acting on N-Boc-4S-amino-3-oxo-5-phenylpentanoic acid methylester. The physiological role of the new enzyme is still unknown.

Isolation of monoclonal antibodies from cell containing hybridoma broth using a protein A coated adsorbent in expanded beds.

A novel expanded bed adsorbent carrying a recombinant protein A ligand was used for the isolation of monoclonal antibodies from cell containing hybridoma fermentation broth. The untreated effluent from a continuous hybridoma cultivation was applied to the stable expanded adsorbent (Streamline rProteinA), which proved to have a high capacity for the MAb studied (14 mg MAb per ml of adsorbent). A clarified and highly concentrated (up to 50 fold) eluate of high purity was obtained. A scale up of the MAb purification is demonstrated from lab scale (250 mg MAb per purification cycle) to a small pilot scale (2 g MAb per cycle). Low product concentration in the broth in combination with the high capacity of the adsorbent caused long sample application cycles (10-11 h). Experimental problems arising from these long cycle times are discussed with regard to a large scale application of the method.

The influence of cell adsorbent interactions on protein adsorption in expanded beds.

Expanded bed adsorption (EBA) is a primary recovery operation allowing the adsorption of proteins directly from unclarified feedstock, e.g. culture suspensions, homogenates or crude extracts. Thus solid-liquid separation is combined with adsorptive purification in a single step. The concept of integration requires that the solid components of the feed solution are regarded as a part of the process, which influences stability, reproducibility, and overall performance. This aspect is investigated here at the example of the influence of presence and concentration of intact yeast cells (S. cerevisiae) on the adsorption of model proteins (hen egg white lysozyme and bovine serum albumin) to various stationary phases (cation and anion-exchange, hydrophobic interaction, immobilised metal affinity). The interaction of the cells with the adsorbents is determined qualitatively and quantitatively by a pulse response method as well as by a finite bath technique under different operating conditions. The consequence of these interactions for the stability of expanded beds in suspensions of varying cell concentration is measured by residence time distributions (RTDs) after tracer pulse injection (NaBr, LiCl). Analysis of the measured RTD by the PDE model allows the calculation of the fraction of perfectly fluidised bed (phi), a parameter which may be regarded as a critical quantity for the estimation of the quality of fluidisation of adsorbents in cell containing suspensions. The correlation between bed stability and performance is made by analysing the breakthrough of model proteins during adsorption from unclarified yeast culture broth. A clear relationship is found between the degree of cell/adsorbent interaction, bed stability in terms of the phi parameter, and the sorption efficiency. Only beds characterised by a phi value larger than 0.8 in the presence of cells will show a conserved performance compared to adsorption from cell free solutions. A drop in phi, which is due to interactions of the fluidised adsorbent particles with cells from the feed, will directly result in a reduced breakthrough efficiency. The data presented highlight the importance of including the potential interaction of solid feedstock components and the expanded adsorbents into the design of EBA processes, as the interrelation found here is a key factor for the overall performance of EBA as a truly integrated operation.

Aqueous two-phase systems containing urea: influence on phase separation and stabilization of protein conformation by phase components.

During recombinant Escherichia coli fermentation with high expression levels, inclusion bodies are often formed. Aqueous two-phase systems have been used in the presence of urea for the initial recovery steps. To investigate phase behavior of such systems we determined phase diagrams of poly(ethylene glycol) (PEG)/sodium sulfate/urea/water and PEG/dextran T-500 (DEX)/urea/phosphate buffer/water at different concentrations of urea and different molecular weight of PEG. PEG/Na2SO4 aqueous two-phase systems could be obtained including up to 30% w/w urea at 25 degrees C and PEG/dextran T-500 up to 35% w/w urea. The binodial was displaced toward higher concentrations with increasing urea concentrations. The partition coefficient of urea was near unity. An unstable mutant of T4-lysozyme with an amino acid replacement in the core (V149T) was used to analyze the effect of phase components on the conformation of the enzyme. We showed that partitioning of tryptophan was not dependent on the concentration of urea in the phase system.

The use of ion-selective electrodes for evaluating residence time distributions in expanded bed adsorption systems.

The suitability of ion-selective electrodes (ISE) for the determination of residence time distribution (RTD) in turbid, cell-containing fluids was examined. The electrodes were found to give reproducible signals in biomass-containing feedstock with up to 20% wet weight of solids. The enhanced feedstock compatibility of IES, when compared to other tracer sensing devices, allows the study of expanded bed system hydrodynamics under relevant operating conditions. Within the linear range of the corresponding ISE-tracer pair, both examined ISE (Li(+)- or Br(-)-selective) showed to be insensitive against the range of flow rate and pH normally employed during expanded bed adsorption (EBA) of proteins. Analyzing the RTD obtained after a perfect ion tracer pulse in terms of the PDE model (PDE, axially dispersed plug-flow exchanging mass with stagnant zones) gave a quantitative description of the underlying hydrodynamic situation during EBA processing. These data provided a powerful tool to make predictions on the adsorptive global process performance with a defined feedstock type and composition. The link between the hydrodynamic events during feedstock application and the actual process performance was shown when applying intact yeast cell suspensions at different biomass content (up to 7.5% wet weight) and buffer conductivity (5-12 mS) onto an EBA column filled with the adsorbent Streamline Q XL as fluidized phase. On the basis of our experimental results, a guideline for the successful application of the ISE/RTD method to EBA process design is presented.

Human chymotrypsinogen B production from Pichia pastoris by integrated development of fermentation and downstream processing. Part 2. Protein recovery.

The purification of human chymotrypsinogen B (hCTRB) after expression and secretion by the yeast Pichia pastoris is described based on two different approaches using integrated initial recovery. Extraction employing aqueous two-phase systems (ATPS) from poly(ethylene glycol) and sodium sulfate allows direct processing of cell containing yeast suspensions of 50% wet weight. The target protein is obtained partially purified in the top phase while cells and cell debris are partitioned to the bottom phase of the system. hCTRB is further purified by adsorption from the top phase to the cation exchanger SP Sepharose Big Beads and elution in a salt step. The single step isolation of hCTRB is possible by expanded bed adsorption (EBA) using a fluidized cation exchanger (Streamline SP XL). A design strategy is shown taking both target protein binding and stable fluidization of the stationary phase in cell containing suspensions into consideration. For the example of hCTRB isolation from cell containing P. pastoris suspensions, a successful use of this strategy is demonstrated. Both initial recovery strategies deliver a product that can be further purified and formulated by ultrafiltration/diafiltration followed by lyophilization, resulting in a homogeneous product. Scale-up to 30-90 L of culture suspension was shown for both methods, resulting in a product of similar quality. Comparing both strategies reveals that the two-step ATPS route is better suited for high cell density cultures, while the single step EBA method is preferred for cultures of moderate cell density. This is due to the fact that application of EBA is restricted to suspensions of 10-12.5% wet weight cell concentration, thus necessitating dilution of the original broth prior to sample application. The data presented show that integrated recovery operations are a valuable alternative to traditional processing for systems that are problematic during initial solid-liquid separation.

Isolation of a recombinant formate dehydrogenase by pseudo-affinity expanded bed adsorption.

Formate dehydrogenase (FDH) is an enzyme of industrial interest, which is recombinantly expressed as an intracellular protein in Escherichia coli. In order to establish an efficient and reliable purification protocol, an expanded bed adsorption (EBA) process was developed, starting from the crude bacterial homogenate. EBA process design was performed with the goal of finding operating conditions which, on one hand, allow efficient adsorption of the target protein and which, on the other hand, support the formation of a perfectly classified fluidised bed (expanded bed) in the crude feed solution. A pseudo-affinity ligand (Procion Red HE3B) was used to bind the FDH with high selectivity and reasonable capacity (maximum equilibrium capacity of 30 U/ml). Additionally, a simplified modelling approach, involving small packed beds for generation of process parameters, was employed for defining the operating conditions during sample application. In combination with extended elution studies, a process was set up, which could be scaled up to 7.5 l of adsorbent volume yielding a total amount of 100,000 U of 94% pure FDH per run. On this scale, 19 l of a benzonase-treated E. coli homogenate of 15% wet-weight (pH 7.5, 9 mS/cm conductivity) were loaded to the pseudo-affinity adsorbent (0.25 m sed. bed height, 5 x 10(-4) m/s fluid velocity). After a series of two wash steps, a particle-free eluate pool was obtained with 85% yield of FDH. This excellently demonstrates the suitability of expanded bed adsorption for efficient isolation of proteins by combining solid-liquid separation with adsorptive purification in a single unit operation.