Ceramic membrane microfilter as an immobilized enzyme reactor.

This study investigated the use of a ceramic microfilter as an immobilized enzyme reactor. In this type of reactor, the substrate solution permeates the ceramic membrane and reacts with an enzyme that has been immobilized within its porous interior. The objective of this study was to examine the effect of permeation rate on the observed kinetic parameters for the immobilized enzyme in order to assess possible mass transfer influences or shear effects. Kinetic parameters were found to be independent of flow rate for immobilized penicillinase and lactate dehydrogenase. Therefore, neither mass transfer nor shear effects were observed for enzymes immobilized within the ceramic membrane. Both the residence time and the conversion in the microfilter reactor could be controlled simply by regulating the transmembrane pressure drop. This study suggests that a ceramic microfilter reactor can be a desirable alternative to a packed bed of porous particles, especially when an immobilized enzyme has high activity and a low Michaelis constant.     

Thermal stability of lactate dehydrogenase and alcohol dehydrogenase incorporated into highly concentrated gels]

The rate constants for inactivation of lactate dehydrogenase and alcohol dehydrogenase in solution at 65 degrees C (pH 7,5) are 0,72 and 0,013 min-1, respectively. The enzyme incorporation into acrylamide gels results in immobilized enzymes, whose residual activity is 18--25% of the original one. In 6,7% gels the rate of thermal inactivation for lactate dehydrogenase is decreased nearly 10-fold, whereas the inactivation rate for alcohol dehydrogenase is increased 4,6-fold as compared to the soluble enzymes. In 14% and 40% gels the inactivation constants for lactate dehydrogenase are 6,3.10(-3) and 5,9.10(-4) min-1, respectively. In 60% gels the thermal inactivation of lactate dehydrogenase is decelerated 3600-fold as compared to the native enzyme. The enthalpy and enthropy for the inactivation of the native enzyme are equal to 62,8 kcal/mole and 116,9 cal/(mole.grad.) for the native enzyme and those of gel-incorporated (6,7%) enzyme -- 38,7 kcal/mole and 42 cal/(mole.grad.), respectively. The thermal stability of alcohol dehydrogenase in 60% gels is increased 12-fold. To prevent gel swelling, methacrylic acid and allylamine were added to the matrix, with subsequent treatment by dicyclohexylcarbodiimide. The enzyme activity of the modified gels is 2,7--3% of that for the 6,7% gels. The stability of lactate dehydrogenase in such gels is significantly increased. A mechanism of stabilization of the subunit enzymes in highly concentrated gels is discussed.     

Experimental measurement of dynamic fluid shear stress on the aortic surface of the aortic valve leaflet

Aortic valve (AV) calcification is a highly prevalent disease with serious impact on mortality and morbidity. Although exact causes and mechanisms of AV calcification are unclear, previous studies suggest that mechanical forces play a role. Since calcium deposits occur almost exclusively on the aortic surfaces of AV leaflets, it has been hypothesized that adverse patterns of fluid shear stress on the aortic surface of AV leaflets promote calcification. The current study characterizes AV leaflet aortic surface fluid shear stresses using Laser Doppler velocimetry and an in vitro pulsatile flow loop. The valve model used was a native porcine valve mounted on a suturing ring and preserved using 0.15% glutaraldehyde solution. This valve model was inserted in a mounting chamber with sinus geometries, which is made of clear acrylic to provide optical access for measurements. To understand the effects of hemodynamics on fluid shear stress, shear stress was measured across a range of conditions: varying stroke volumes at the same heart rate and varying heart rates at the same stroke volume. Systolic shear stress magnitude was found to be much higher than diastolic shear stress magnitude due to the stronger flow in the sinuses during systole, reaching up to 20 dyn/cm² at mid-systole. Upon increasing stroke volume, fluid shear stresses increased due to stronger sinus fluid motion. Upon increasing heart rate, fluid shear stresses decreased due to reduced systolic duration that restricted the formation of strong sinus flow. Significant changes in the shear stress waveform were observed at 90 beats/min, most likely due to altered leaflet dynamics at this higher heart rate. Overall, this study represents the most well-resolved shear stress measurements to date across a range of conditions on the aortic side of the AV. The data presented can be used for further investigation to understand AV biological response to shear stresses.     

Immobilization of lactate dehydrogenase on polyacrylamide beads

Pig muscle lactate dehydrogenase (L-lactate:NAD oxidoreductase, EC 1.1.1.27) was covalently immobilized on polyacrylamide beads containing carboxylic functional groups activated by water-soluble carbodiimide. The effects of immobilization on the catalytic properties and stability of the lactate dehydrogenase were studied. There was no shift in the pH optimum of the immobilized enzyme compared to that of the soluble one. The apparent optimum temperature of the soluble enzyme was 65 degrees C, while that of the immobilized enzyme was between 50 and 65 degrees C. The apparent Km values of the immobilized enzyme with pyruvate and NADH substrates were higher than those of the soluble enzyme. As a result of immobilization, enhanced stabilities were found against heat treatment, changes in pH, and urea denaturation.     

Affinity chromatography of lactate dehydrogenase on immobilized nucleotides

The interaction of two isoenzymes of lactate dehydrogenase from pig heart muscle (H(4)) and rabbit skeletal muscle (M(4)), with immobilized nucleotides was examined: the effects of pH and temperature on the binding of lactate dehydrogenase were studied with immobilized NAD(+) matrices. The influence of substrate, product and sulphite on the binding of heart muscle lactate dehydrogenase to immobilized NAD(+) was investigated. The interaction of both lactate dehydrogenase isoenzymes with immobilized pyridine and adenine nucleotides and their derivatives were measured. The effects of these parameters on the interaction of lactate dehydrogenase with immobilized nucleotides were correlated with the known kinetic and molecular properties of the enzymes in free solution.     

Improved oxygen transfer and increased l-lactic acid production by morphology control of Rhizopus oryzae in a static bed bioreactor

Rhizopus oryzae was immobilized on a cotton matrix in a static bed bioreactor. Compared with free cells in a stirred tank bioreactor, immobilized R. oryzae in this bioreactor gave higher lactic acid production but lower ethanol production. The highest lactic acid production rate (2.09 g/L h) with the final concentration of 37.83 g/L from 70 g/L glucose was achieved when operating the bioreactor at 700 rpm and 0.5 vvm air. To better understand the relationship between shear effects (agitation and aeration) and R. oryzae morphology and metabolism, oxygen transfer rate, fermentation kinetics, and lactate dehydrogenase activity were determined. In immobilized cell culture, higher oxygen transfer rate and lactic acid production were achieved but lower lactate dehydrogenase activity was found as compared with those in free cell culture operated at the same conditions. These results clearly imply that mass transport was the rate controlling step in lactic acid fermentation by R. oryzae.     

Flow kinetics of lactate dehydrogenase chemically attached to nylon tubing.

Rabbit muscle lactate dehydrogenase (EC 1.1.1.27) was attached covalently to the inner surface of nylon tubing; a modified technique, involving benzidine and glutaraldehyde, was used, and the resulting immobilized enzyme showed no loss of activity over a period of several months. An experimental study was made of the flow kinetics for the reaction between pyruvate and reduced nicotinamide adenine dinucleotide in two limiting cases, one substrate in excess and the concentration of the other one varied. A range of flow rates and temperatures was covered. The results were analyzed in various ways on the basis of the Kobayashi--Laidler treatment of flow systems. It was concluded that the kinetics are largely diffusion-controlled, especially at the lower substrate concentrations and flow rates. The values of the apparent Michaelis constants vary with flow rate vf, being linear in vf-1/3, and the values extrapolated to infinite flow rate (vf-1/3 = 0) approach the values for the enzyme in free solution. Analysis of the rates led to activation energies for the diffusion of the two substrates.     

Physical degradation of proteins in well-defined fluid flows studied within a four-roll apparatus

In most applications of biotechnology and downstream processing proteins are exposed to fluid stresses in various flow configurations which often lead to the formation of unwanted protein aggregates. In this paper we present physical degradation experiments for proteins under well-defined flow conditions in a four-roll apparatus. The flow field was characterized numerically by computational fluid dynamics (CFD) and experimentally by particle image velocimetry (PIV). The local shear strain rate as well as the local shear and elongation rate was used to characterize the hydrodynamic stress environment acting on the proteins. Lysozyme was used as a model protein and subjected to well-defined fluid stresses in high and low stress environment. By using in situ turbidity measurements during stressing the aggregate formation was monitored directly in the fluid flow. An increase in absorbance at 350 nm was attributed to a higher content of visible particles (>1 μm). In addition to lysozyme, the formation of aggregates was confirmed for two larger proteins (bovine serum albumin and alcohol dehydrogenase). Thus, the presented experimental setup is a helpful tool to monitor flow-induced protein aggregation with high reproducibility. For instance, screening experiments for formulation development of biopharmaceuticals for fill and finish operations can be performed in the lab-scale in a short time-period if the stress distributions in the application are transferred and applied in the four-roll mill.     

Kinetcs and decay of fumarase activity of immobilized Brevibacterium ammoniagenes cells for continuous production of L-malic acid

The kinetics of the reversible fumarase reaction of immobilized Brevibacterium ammoniagenes cells and the decay behavior of enzyme activity were investigated in a plug flow system. The time course of the reaction in the immobilized cell column was well explained by the time-conversion equation including the apparent kinetic constants of the immobilized cell enzyme. The decay rate of fumarase activity was faster in the upper sections of the column (inlet side of the substrate solution) compared with the lower sections when 1M sodium fumarate (pH 7.0) was continuously passed through the column at 37°C. It was shown that the decay rate of the fumarase activity in the immobilized cell column depends on the flow rate of the substrate solution. The effect of flow rate on the decay rate of enzyme activity was considered to be related to the rate of contamination of enzyme with poisonous substances derived from the substrate solution or to the rate of leakage of enzyme stabilizers and/or enzyme itself from the immobilized cells.     

Affinity precipitation and site-specific immobilization of proteins carrying polyhistidine tails

Proteins carrying genetically attached polyhistidine tails have been purified using affinity precipitation with metal chelates. DNA fragments encoding four or five histidine residues have been genetically fused to the oligomeric enzymes lactate dehydrogenase (Bacillus stearothermophilus), beta-glucoronidase (Escherichia coli), and galactose dehydrogenase (Pseudomonas fluorescens) as well as to the monomeric protein A (Staphylococcus aureus). The chimeric genes were subsequently expressed in E. coli. The engineered enzymes were successfully purified from crude protein solutions using ethylene glycolbis (beta-aminoethyl) tetraacetic acid (EGTA) charged with Zn(2+) as precipitant, whereas protein A, carrying only one attached histidine tail, did not precipitate. However, all of the engineered proteins could be purified on immobilized metal affinity chromatography (IMAC) columns loaded with Zn(2+). The potential of using the same histidine tails for site-specific immobilization of proteins was also investigated. The enzymes were all catalytically active when immobilized on IMAC gels. For instance, immobilized lactate dehydrogenase, carrying tails composed of four histidine residues, displaced 83% of the soluble enzyme activity. (c) 1996 John Wiley & Sons, Inc.     

A technique for determining the shear sensitivity of mammalian cells in suspension culture

Summary A murine hybridoma was subjected to constant shear rates within a couette viscometer for periods of 15 hours. Shear effects on cells were determined by live cell counts, cell viability and by the release of the cytoplasmic enzyme lactate dehydrogenase into the culture medium. Cell damage was observed at a shear rate of 870s^-1 but not at 420s^-1.     

Rheo-NMR Studies of an Enzymatic Reaction: Evidence of a Shear-Stable Macromolecular System

Understanding the effects of shear forces on biopolymers is key to understanding how biological systems function. Although currently there is good agreement between theoretical predictions and experimental measurements of the behavior of DNA and large multimeric proteins under shear flow, applying the same arguments to globular proteins leads to the prediction that they should only exhibit shear-induced conformational changes at extremely large shear rates. Nevertheless, contradictory experimental evidence continues to appear, and the effect of shear on these biopolymers remains contentious. Here, a custom-built rheo-NMR cell was used to investigate whether shear flow modifies enzyme action compared with that observed quiescently. Specifically, ¹H NMR was used to follow the kinetics of the liberation of methanol from the methylesterified polysaccharide pectin by pectinmethylesterase enzymes. Two different demethylesterifying enzymes, known to have different action patterns, were used. In all experiments performed, Couette flows with shear rates of up to 1570 s⁻¹ did not generate detectable differences in the rate of methanol liberation compared to unsheared samples. This study provides evidence for a shear-stable macromolecular system consisting of a largely β-sheet protein and a polysaccharide, in line with current theoretical predictions, but in contrast to some other experimental work on other proteins.     

Coimmobilization of lactate dehydrogenase and low molecular weight thiols on sepharose

Lactate dehydrogenase (EC 1.1.1.27) and dithiothreitol (DTT) were coimmobilized on Sepharose activated with cyanogen bromide. It was demonstrated that addition of 10 mM DTT (but not 2-mercaptoethanol) during immobilization increased the enzyme specific activity 1.5-5-fold, depending on the initial extent of Sepharose activation by cyanogen bromide. The total activity increased two- to threefold. The lactate dehydrogenase preparations were rich in matrix-immobilized sulfhydryl groups (1.8-13.0 nmol per ml gel). The presence of DTT increased the stability of immobilized lactate dehydrogenase.     

Immobilization of glyceraldehyde-3-phosphate dehydrogenase by non-covalent binding to specific antibodies and Fab-fragments coupled to Sepharose]

Rabbit antibodies to rat skeletal muscle glyceraldehyde-3-phosphate dehydrogenase, as well as monovalent Fab fragments of these antibodies were coupled to CNBr-activated Sepharose 4B. Rat skeletal muscle glyceraldehyde-3-phosphate dehydrogenase was then immobilized on a matrix by non-covalent binding to specific antibodies. Immobilized enzyme retains approximately 90% catalytic activity of the soluble dehydrogenase; pH optimum of activity and the Km value observed are changed as compared to the enzyme in solution. Glyceraldehyde-3-phosphate dehydrogenase immobilized on specific antibodies is shown to undergo adenine nucleotide-induced dissociation into dimers. The immobilized dimeric form of the enzyme thus obtained is catalytically active and capable of reassociating with the dimers of apoglyceraldehyde-3-phosphate dehydrogenase added in solution to the suspension of Sepharose.     

Immobilization of glucose oxidase and lactate dehydrogenase onto magnetic nanoparticles for bioprocess monitoring system

Glucose oxidase (GOD) and lactate dehydrogenase (LDH) were immobilized onto magnetic nanoparticles, viz. Fe₃O₄, via carbodiimide and glutaraldehyde. The immobilization efficiency was largely dependent upon the immobilization time and concentration of glutaraldehyde. The magnetic nanoparticles had a mean diameter of 9.3 nm and were superparamagnetic. The immobilization of GOD and LDH on the nanoparticles slightly decreased their saturation magnetization. However, the FT-IR spectra showed that GOD and LDH were immobilized onto the nanoparticles by different binding mechanisms, the reason for which was not well explained. The optimum pH values of the immobilized GOD and LDH were changed to 8 and 10, respectively. The free and immobilized enzyme kinetic parameters (K_m and V_max) were determined by Michaelis-Menten enzyme kinetics. The Km values for free and immobilized GOD were 0.168 and 0.324 mM, respectively, while those for free and immobilized LDH were 0.19 and 0.163 mM for NAD, and 2.976 and 4.785 mM for lactate, respectively. High operational stability was observed, with more than 80% of the initial enzyme activity being retained for the immobilized GOD up to 12 h and for the immobilized LDH up to 24 h. The immobilized GOD was applied to a sequential injection analysis system for the application of bioprocess monitoring.     

Protein fractions of the ova, embryos and skeletal muscles of the loach affecting the stability and activity of lactate dehydrogenase

When lactate dehydrogenase obtained from Misgurnus muscles and purified to the homogeneous state is incubated for 16 hat 38 degrees C, its activity lowers down to 10% of the initial value. Extracts of egg cells, embryos or skeletal muscles of the mentioned fish species added to the enzyme solution decrease considerably its inactivation. Proteins stabilizing the activity of lactate dehydrogenase are revealed in the supernatant liquid obtained after salting out of the above extracts with 60% sulphate ammonium saturation. These proteins are in fractions with the molecular weight below 45 kDa. Among proteins with the molecular weight 10 kDa there are polypeptides which exert an activation effect on lactate dehydrogenase. This effect is intensified with the presence of insulin.     

Determinations of lactate and lactate dehydrogenase activity in serum with the flow injection analysis system involving immobilized enzyme column and chemiluminescence

The methods for the highly sensitive flow injection analysis of lactate and lactate dehydrogenase (LDH) activity in serum using immobilized enzymes in column form and chemiluminescence detection which does not require a blank correction are described. The methods were based on the determination of chemiluminescence formed by the reaction of a luminol-ferricyanide mixture with hydrogen peroxide. This hydrogen peroxide was produced by the lactate oxidase (LOD) reaction from lactate, which was in serum or was produced by the action of LDH in serum. The action of LDH in a flow injection analysis system was performed for 2 min in an incubation coil placed parallel to the substrate-buffer line between the LOD column and the LOD/catalase column. Endogenous lactate in serum was removed by an immobilized LOD/catalase column prior to the action of LDH. The present method gave perfect linearity of the data up to 5.6 mmol/liter for lactate and 1840 IU/liter for LDH activity with satisfactory precision, reproducibility, and accurate reaction recoveries. The results from the lactate and LDH activity correlated satisfactorily with those obtained by other well-established methods.     

Kinetics and stability of immobilized chicken liver xanthine dehydrogenase.

Xanthine dehydrogenase (EC 1.2.1.37) was isolated from chicken livers and immobilized by adsorption to a Sepharose derivative, prepared by reaction of n-octylamine with CNBr-activated Sepharose 4B. Using a crude preparation of enzyme for immobilization it was observed that relatively more activity was adsorbed than protein, but the yield of immobilized activity increased as a purer enzyme preparation was used. As more activity and protein were bound, relatively less immobilized activity was recovered. This effect was probably due to blocking of active xanthine dehydrogenase by protein impurities. The kinetics of free and immobilized xanthine dehydrogenase were studied in the pH range 7.5-9.1. The Km and V values estimated for free xanthine dehydrogenase increase as the pH increase; the K'm and V values for the immobilized enzyme go through a minimum at pH 8.1. By varying the amount of enzyme activity bound per unit volume of gel, it was shown that K'm is larger than Km are result of substrate diffusion limitation in the pores of the support material. Both free and immobilized xanthine dehydrogenase showed substrate activation at low concentrations (up to 2 microM xanthine). Immobilized xanthine dehydrogenase was more stable than the free enzyme during storage in the temperature range of 4-50 degrees C. The operational stability of immobilized xanthine dehydrogenase at 30 degrees C was two orders of magnitude smaller than the storage stability, t 1/2 was 9 and 800 hr, respectively. The operational stability was, however, better than than of immobilized milk xanthine oxidase (t 1/2 = 1 hr). In addition, the amount of product formed per unit initial activity in one half-life, was higher for immobilized xanthine dehydrogenase than for immobilized xanthine oxidase. Unless immobilized milk xanthine oxidase can be considerable stabilized, immobilized chicken liver xanthine dehydrogenase is more promising for application in organic synthesis.     

Coimmobilization of Lactate Dehydrogenase and Low-Molecular-Weight Thiols on Sepharose

Lactate dehydrogenase (EC 1.1.1.27) and dithiothreitol (DTT) were coimmobilized on Sepharose activated with cyanogen bromide. It was demonstrated that the addition of 10 mM DTT (but not 2-mercaptoethanol) during immobilization increased the enzyme specific activity 1.5–5-fold depending on the initial extent of Sepharose activation by cyanogen bromide. The total activity increased two- to threefold. The lactate dehydrogenase preparations were rich in matrix-immobilized sulfhydryl groups (1.8–13.0 nmol per ml gel). The presence of DTT increased the stability of immobilized lactate dehydrogenase.     

NAD recycling in the collagen membrane.

NAD recycling in the collagen membrane was investigated as follows: (1) Alcohol dehydrogenase and lactate dehydrogenase were co-immobilized in the collagen membrane and the rate of lactate production by immobilized enzymes was compared with that of free enzymes by using free NAD. An increased rate was observed in the case of immobilized enzyme. (2) The soluble high molecular weight derivatives of NAD (dextran-NAD) were immobilized in the collagen membrane with the two dehydrogenases and recycling of dextran-NAD in the membrane was examined. Lactate was produced by the membrane without adding free NAD. The interaction between the high molecular weight NAD derivatives and enzymes are also discussed.