Regional heparinization via simultaneous separation and reaction in a novel Taylor-Couette flow device.

The development of a safe and efficient bioreactor design has remained a challenge for the clinical application of immobilized enzymes. Specifically, the use of immobilized heparinase I has been the target of many studies to make heparin anticoagulation therapy safer for the critically ill patient with kidney failure or heart disease. We have investigated the use of Taylor-Couette flow for a novel type of bioreactor. In a previous study, we showed that the fluidization of agarose immobilized heparinase within Taylor vortices in whole blood can lead to extensive blood damage in the form of cell depletion and hemolysis. Based on these findings, we designed and developed a reactor, referred to as vortex-flow plasmapheretic reactor (VFPR), that incorporated plasmapheresis and fluidization of the agarose in the reactive compartment, separate from the whole-blood path. In the present study, immobilized heparinase I was tested as a means of achieving regional heparinization of a closed circuit. This is a method in which heparin is infused into the extracorporeal circuit predialyzer and neutralized postdialyzer. Saline studies were performed with an immobilized heparinase I-packed bed and with the VFPR. An in vitro feasibility study was performed with the VFPR using human blood. The VFPR achieved heparin conversions of 44 +/- 0.5% and 34 +/- 2% in saline and blood, respectively. In addition, the VFPR caused no blood damage. We report a novel method to achieve fluidization which depended on secondary, circumferencial flow, and was independent of the primary flow through the device.     

Immobilized heparinase: In vitro reactor model

Heparinase immobilized to agarose has previously been shown to be useful in degrading heparin and thereby preventing thromboembolytic complications when this anticoagulant has been used in extracorporeal perfusions. The current study examined the kinetics of this immobilized enzyme. When heparinase is covalently bound to 8% agarose, the partition coefficient of heparin in the catalytic particle is 0.36 +/- 0.048 (N = 10). The immobilized enzyme has a K(m) of 0.15 +/- 0.03 mg/mL and an activation energy of 10.3 +/- 0.57 kcal/gmol (N = 5). These values are statistically indistinguishable from the values for the free enzyme. The immobilized enzyme showed a pH activity optimum between 7.0 and 7.4, compared to the optimum pH of 6.5 for the soluble enzyme. The activity optimum of immobilized heparinase with respect to salt concentration was between 0 and 0.1M. A reactor containing immobilized heparinase recirculating internally at 1300 mL/min behaved as a continuously stirred tank reactor (CSTR) when solutions at a flow rate of 120 mL/min were passed through the device. The residence time distribution was determined using blue dextran (molecular weight 2 x 10(6) daltons), which is sterically excluded from the agarose catalyst. A model of the heparinase reactor based on ideal CSTR behavior and the immobilized enzyme kinetic parameters was developed. It accurately predicted experimental conversions over a range of catalyst volumes, enzyme loadings, and substrate concentrations to within 7% in most cases and with a maximum deviation of 13%.     

Distribution of heparinase covalently immobilized to agarose: Experimental and theoretical studies

An immobilized enzyme reactor has been developed to remove heparin, the anticoagulant that is required in all extracorporeal devices for patients undergoing open-heart surgery or kidney dialysis. The device uses the enzyme heparinase (EC 4.2.2.7), which is covalently linked to agarose with cyanogen bromide. A critical parameter in the development of a model for the degradation of heparin catalyzed by immobilized heparinase is the radial concentration profile of the enzyme within the agarose matrix. Experimental determinations of bound enzyme con centrations have been conducted previously for several enzyme systems using radioactive or fluorescent labels. For the development of the heparinase reactor it is necessary to use catalytically but not electrophoretically pure enzyme, and thus it is not possible to use the labeling techniques. To obtain information about the bound enzyme distribution, an experimental study of the intrinsic binding kinetics of heparinase to cyanogen bromide-activated agarose was conducted. The binding reaction was studied as a function of both the concentration of heparinase and the gel-reactive group. At conditions of functional group excess, the binding kinetics were pseudo first order in heparinase concentration with a rate constant equal to 0.12 C(c[triple chemical bond]n) (h(-1)), where C(c[triple chemical bond]n) is the gel-reactive group concentration. The reactive group concentration remained constant within the 2-4-h experiments. Competitive binding between heparinase and the protein contaminants was unimportant. A model was formulated for the immobilization procedure based on the diffusion of heparinase within the porous network and the binding kinetics as determined above. The model predicted the immobilization of heparinase to be kinetically controlled and the enzyme to distribute uniformly within the agarose matrix. These experimental techniques could be applied to predict the immobilized enzyme distribution for different enzyme systems that are not electrophoretically pure.     

Immobilization of recombinant heparinase I fused to cellulose-binding domain.

Immobilization of biologically active proteins is of great importance to research and industry. Cellulose is an attractive matrix and cellulose-binding domain (CBD) an excellent affinity tag protein for the purification and immobilization of many of these proteins. We constructed two vectors to enable the cloning and expression of proteins fused to the N- or C-terminus of CBD. Their usefulness was demonstrated by fusing the heparin-degrading protein heparinase I to CBD (CBD-HepI and HepI-CBD). The fusion proteins were over-expressed in Escherichia coli under the control of a T7 promoter and found to accumulate in inclusion bodies. The inclusion bodies were recovered by centrifugation, the proteins were refolded and recovered on a cellulose column. The bifunctional fusion protein retained its abilities to bind to cellulose and degrade heparin. C-terminal fusion of heparinase I to CBD was somewhat superior to N-terminal fusion: Although specific activities in solution were comparable, the latter exhibited impaired binding capacity to cellulose. CBD-HepI-cellulose bioreactor was operated continuously and degraded heparin for over 40 h without any significant loss of activity. By varying the flow rate, the mean molecular weight of the heparin oligosaccharide produced could be controlled. The molecular weight distribution profiles, obtained from heparin depolymerization by free heparinase I, free CBD-HepI, and cellulose-immobilized CBD-HepI, were compared. The profiles obtained by free heparinase I and CBD-HepI were indistinguishable, however, immobilized CBD-HepI produced much lower molecular weight fragments at the same percentage of depolymerization. Thus, CBD can be used for the efficient production of bioreactors, combining purification and immobilization into essentially a single step.     

Immobilized enzyme cellulose hollow fibers: II. Kinetic analysis

Immobilized enzyme hollow fibers may be useful in the purification or treatment of whole blood under clinical conditions. In this study, catalytically pure heparinase was immobilized to cellulose to analyze the feasibility for the removal of heparin's anticoagulant activity from whole blood. The kinetics of catalytically pure heparinase immobilized to regenerated cellulose hollow fibers were quantified with respect to mass transfer coefficient and enzyme loading. The kinetic analysis showed that increases in the mass transfer coefficient of heparin in the fiber lumen decreased the apparent Michaelis constant while increases in enzyme activity immobilized to the fiber lumen increased the apparent Michaelis constant. The apparent Michaelis constant was an order of magnitude greater than the intrinsic K(m) value for the system. The intrinsic K(m) value for heparinase-cellulose is 0.4 +/- 0.3 mg/mL (N = 6) and it is the same order of magnitude as the K(m) value for soluble heparinase.     

Detoxification of organophosphate pesticides using an immobilized phosphotriesterase from Pseudomonas diminuta

A purified phosphotriesterase was successfully immobilized onto trityl agarose in a fixed bed reactor. A total of up to 9200 units of enzyme activity was immobilized onto 2.0 mL of trityl agarose (65 mumol trityl groups/mL agarose), where one unit is the amount of enzyme required to catalyze the hydrolysis of one micromole of paraoxon in one min. The immobilized enzyme was shown to behave chemically and kinetically similar to the free enzyme when paraoxon was utilized as a substrate. Several organophosphate pesticides, methyl parathion, ethyl parathion, diazinon, and coumaphos were also hydrolyzed by the immobilized phosphotriesterase. However, all substrates exhibited an affinity for the trityl agarose matrix. For increased solubility and reduction in the affinity of these pesticides for the trityl agarose matrix, methanol/water mixtures were utilized. The effect of methanol was not deleterious when concentrations of less than 20% were present. However, higher concentrations resulted in elution of enzyme from the reactor. With a 10-unit reactor, a 1.0 mM paraoxon solution was hydrolyzed completely at a flow rate of 45 mL/h. Kinetic parameters were measured with a 0.1-unit reactor with paraoxon as a substrate at a flow rate of 22 mL/h. The apparent K(m) for the immobilized enzyme was 3-4 times greater than the K(m) (0.1 mM) for the soluble enzyme. Immobilization limited the maximum rate of substrate hydrolysis to 40% of the value observed for the soluble enzyme. The pH-rate profiles of the soluble and immobilized enzymes were very similar. The immobilization of phosphotriesterase onto trityl agarose provides an effective method esterase onto trityl agarose provides an effective method for hydrolyzing and thus detoxifyuing organophosphate pesticides and mammalian acetylcholinesterase inhinbitors.     

Performance of rotating packed disk reactor with immobilized glucose oxidase

Summary Reactor performance was studied to investigate whether a rotating packed disk reactor (RPDR) can be used for the enzymatic oxidation of biochemicals. The disks were packed with calcium alginate beads with immobilized glucose oxidase and catalase, which catalyze the reaction of glucose and oxygen. The production rate of gluconic acid increased with the speed of rotation and the bulk flow rate. An optimum submergence for maximum productivity existed.     

Design of a biomedical reactor for plasma low-density lipoprotein removal

The purpose of this study was to design a biomedical reactor that reduces plasma cholesterol when incorporated in an in vivo extracorporeal system. Phospholipase A(2), immobilized onto Agarose beads and housed inside the bioreactor, modifies plasma low density lipoprotein (LDL) into a form that is rapidly removed from circulation. In a packed bed reactor, the enzymatic conversion of LDL to the modified form (with plasma taken from hypercholesterolemic New Zealand white rabbits) was relatively low, 25% +/- 6 for a single pass of plasma through the reactor. An extended bed reactor, a hybrid of fluidized and packed bed reactors, was then developed to increase the conversion. This reactor displays a single pass conversion of 60% +/- 5 under optimal flow conditions. An evaluation of the flow rate through the reactor indicates that the system is limited by external mass transfer when employed under in vivo conditions. In addition, this system requires blood separation before the enzyme modification, which complicates the circuit control. Therefore, a new system was designed for in vivo use with rabbits. The resulting design, called the plasma separator reactor (PSR), combines plasma separation and enzymatic conversion in a single chamber. The PSR has three advantages over other studied systems: improved external mass transfer conditions, easy controlability, and simple set-up procedures. Single pass conversion reached 52% +/- 12 in suboptimal flow under simulated in vivo conditions. This reactor was also tested in vivo with hypercholesterolemic New Zealand white rabbits. A continuous conversion of up to 80% +/- 6 of rabbit plasma phospholipids was observed during 90 min of blood circulation (5 mL/min). The decrease in total plasma cholesterol reached a level of 60% of the initial value and was observed to be a function of the bioreactor enzyme activity. (c) 1993 John Wiley & Sons, Inc.     

Quantitative bead assay for hyaluronidase and heparinase I

A novel, simple, and sensitive assay was developed to monitor, quantitatively, the hyaluronidase and heparinase I-catalyzed cleavage of fluoresceinamine-labeled hyaluronic acid and heparin, respectively. The fluoresceinamine-labeled substrates were hydrophobically absorbed onto 4-microm polystyrene beads. In the presence of enzyme, the change in fluorescence output of the substrate-absorbed beads was monitored in a noncontinuous manner using a flow cytometer. Our results show that hyaluronidase and heparinase I can cleave their respective substrates on the beads in a concentration- and time-dependent manner. The assay is suitable for detecting the presence of these glycosaminoglycan-degrading enzymes in cell lysates, extracts, or purified fractions, for quantifying their amounts, and for investigating the activity of potential inhibitors.     

The influence of bond chemistry on immobilized enzyme systems for ex vivo use

The ideal derivatized support for the clinical use of an immobilized enzyme system should irreversibly bind active enzyme. We have investigated the behavior of heparinase and bilirubin oxidase immobilized via cyanogen bromide, tresyl chloride, epoxide, or carbodiimidazole activated natural and synthetic matrices. The protein bound to each activated support was 90% for cyanogen bromide (CNBr) activated agarose, 50-80% for tresyl chloride activated agarose, and 50% for oxirane activated acrylic (Eupergit C). The activity retention of immobilized heparinase was greatest (50%) with CNBr activated agarose while for the immobilization of bilirubin oxidase, the activity retention was greatest (25-30%) with tresyl chloride activated agarose and oxirane activated acrylic.The stability of the different covalent bonds was studied in vitro with radioiodinated enzymes. The leaching profiles showed the same trends for each support and chemistry. A plateau in portein leaching was reached after a few hours of incubatttion and the transient leaching period was well represented byu a logarithimic function of time. The amount of enzyme released from the least stable support (CNBr activated agarose) in 24 h was injected intravenously in New Zealand white rabbits. Using an indirect enzyme-linked immunnosorbant assay (ELISA), no immune responce was detected. The transient leaching profile was shortenend by washingthe enzyme-support conjugate with 1M hydroxylamine, pH8.5 intermolecular cross-linking with glutaraldehyde also improves the enzyme-support stability. Tresyl chloride and oxirane activated supports produce bonds with improved stability without adversely affecting enzymatic activity.     

Colorimetric heparinase assay for alternative anti-metastatic activity

Heparanase has been previously associated with the metastatic potential, inflammation, and angiogenesis of tumor cells. Heparanase activity has been detected by means of UV absorption, radiolabeled substrates, electrophoretic migration, and heparan sulfate affinity assays. However, those methods have proven to be somewhat problematic with regards to application to actual biological samples, the accessibility of the immobilized substrates, experimental sensitivity, and the separation of degraded products. Rather than focusing on heparanase activity, then, we have developed a rapid, alternative colorimetric heparinase assay, on the basis of the recent finding that sulfated disaccharides generated from heparin by bacterial heparinase exhibit biological properties comparable to those from heparan sulfate by mammalian heparanase. In this study, the concentrations of porcine heparin and bacterial heparinase I were determined using a Sigma Diagnostics Kit. Morus alba was selected as a candidate through this assay system, and an inhibitor, resveratrol, was purified from its methanol extract. Its anti-metastatic effects on the pulmonary metastasis of murine B16 melanoma cells were also evaluated. Our findings suggest that this assay may prove useful as a diagnostic tool for heparinase inhibition, as an alternative anti-metastatic target.     

Hexasaccharides from the histamine-modified depolymerization of porcine intestinal mucosal heparin

Specific sequences in heparin are responsible for its modulation of the biological activity of proteins. As part of a program to characterize heparin-peptide and heparin-protein binding, we are studying the interaction of chemically discrete heparin-derived oligosaccharides with peptides and proteins. We report here the isolation and characterization, by one- and two-dimensional 1H NMR spectroscopies, of ten hexasaccharides, one pentasaccharide, and one octasaccharide serine that were isolated from depolymerized porcine intestinal mucosal heparin. Hexasaccharides were chosen for study because they fall within the size range, typically tetra- to decasaccharide in length, of heparin sequences that modulate the activity of proteins. The depolymerization reaction was catalyzed by heparinase I (EC 4.2.2.7) in the presence of histamine, which binds site specifically to heparin. Histamine increases both the rate and extent of heparinase I-catalyzed depolymerization of heparin. It is proposed that oligosaccharides produced by heparinase I-catalyzed depolymerization can inhibit the enzyme by binding to the imidazolium group of histidine-203, which together with cysteine-135 forms the catalytic domain of heparinase I. The increased rate and extent of depolymerization are attributed to competitive binding of the oligosaccharides by histamine.     

Horizontal rotary bioreactor: effect of rotation and bed characteristics on ethanol fermentation with immobilized yeast cells

The performance of a horizontal rotary bioreactor (HRBR) with immobilized cells for glucose to ethanol conversion could be significantly enhanced by rotation. Optimum speed of rotation depends on the diameter of the bead, substrate flow rate and loading ratio. The use of smaller beads is ideally suitable for application in HRBR with negligible problems due to CO₂ gas venting.     

An immobilized enzyme reactor for the detoxification of bilirubin

An immobilized enzyme reactor has been developed for the degradation of bilirubin as a potential treatment for neonatal jaundice. It utilizes the enzyme bilirubin oxidase from Myrothecium verrucaria, which in the presence of molecular oxygen converts bilirubin to biliverdin and other products that are much less toxic than bilirubin. Bilirubin oxidase was covalently attached to agarose beads using cyano transfer activation. Forty percent of the specific activity of bilirubin oxidase was retained after immmobilization, and preparations with 20 units of enzymatic activity per gram of drained wet weight of gel were obtained. The stability of bilirubin oxidase at pH 7.4 and 37 degrees C was improved fivefold by immobilization. A 15-mL column containing immobilized bilirubin oxidase, through which a 37 degrees C solution of 332muM bilirubin and 450muM human serum albumin in 0.05M phosphate buffer (pH 7.4) was passed at 1 mL/min, converted more than 60 percent of the bilirubin per pass. The substrate specificity of the enzyme and the small volume of the reactor are important characteristics for this clinical application where it is desirable to remove only one compound from the blood and to minimize the volume of blood in the extracorporeal circuit. This reactor, by detoxifying the jaundiced infant's blood of bilirubin, would eliminate the risks associated with the use of donor blood as is done currently in treating severe neonatal jaundice.     

Continuous production of gluconic acid and sorbitol from Jerusalem artichoke and glucose using an oxidoreductase of Zymomonas mobilis and inulinase

Gluconic acid and sorbitol were simultaneously produced from glucose and Jerusalem artichoke using a glucose-fructose oxidoreductase of Zymomonas mobilis and inulinase. Inulinase was immobilized on chitin by cross-linking with glutaraldehyde. Cells of Z. mobilis permeabilized with toluene were coimmobilized with chitin-immobilized inulinase in alginate beads. The optimum amounts of both chitin-immobilized inulinase and permeabilized cells for coimmobilization were determined, and operational conditions were optimized. In a continuous stirred tank reactor operation, the maximum productivities for gluconic acid and sorbitol were about 19.2 and 21.3 g/L/h, respectively, at the dilution rate of 0.23 h(-1) and the substrate concentration of 20%, but operational stability was low because of the abrasion of the beads. As an approach to increase the operational stability, a recycle packed-bed reactor (RPBR) was employed. In RPBR operation, the maximum productivities for gluconic acid and sorbitol were found to be 23.4 and 26.0 g/L/h, respectively, at the dilution rate of 0.35 h(-1) and the substrate concentration of 20% when the recirculation rate was fixed at 900 mL/h. Coimmobilized enzymes were stable for 250 h in a recycle packed-bed reactor without any loss of activity, while half-life in a continuous stirred tank reactor (CSTR) was observed to be about 150 h.     

Inhibitory effect of heparan sulfate-like glycosaminoglycans on the infectivity of Chlamydia pneumoniae in HL cells varies between strains

Glycosaminoglycans are known to participate in the attachment of several chlamydial strains. We studied the effect of heparin, enoxaparin, low-molecular-weight heparin, chondroitin sulfate A, and heparinase I on the infectivity of Chlamydia pneumoniae strain CWL029 and two Finnish isolates, Kajaani 7 and Parola, in an HL cell line which is epithelial in origin. Two Chlamydia trachomatis strains, L2 and E, were used for comparison. The infectivity of all C. pneumoniae strains and C. trachomatis serovar E was inhibited not only by heparin derivatives but also by chondroitin sulfate A and heparinase treatment. Treatment of host cells with heparin derivatives and heparinase was also inhibitory. Different chlamydial strains and species seem, however, to vary in their ability to use heparin in their attachment to host cells.     

Immobilization of heparin with trichloro-s-triazine: Purification of mouse antithrombin

A method for covalently attaching heparin to agarose in which the bound heparin is resistant to detachment is described. A controlled amount of 4-aminophenethylamine is linked to trichloro-s-triazine-activated agarose via the primary amino group. The resulting immobilized arylamine is then diazo coupled to an appropriately modified heparin. The immobilized heparin was used in a two-step procedure for the purification of mouse antithrombin. Some of the properties of the mouse antithrombin are described.     

Passage of classical swine fever virus in cultured swine kidney cells selects virus variants that bind to heparan sulfate due to a single amino acid change in envelope protein E(rns)

Infection of cells with Classical swine fever virus (CSFV) is mediated by the interaction of envelope glycoprotein E(rns) and E2 with the cell surface. In this report we studied the role of the cell surface glycoaminoglycans (GAGs), chondroitin sulfates A, B, and C (CS-A, -B, and -C), and heparan sulfate (HS) in the initial binding of CSFV strain Brescia to cells. Removal of HS from the surface of swine kidney cells (SK6) by heparinase I treatment almost completely abolished infection of these cells with virus that was extensively passaged in swine kidney cells before it was cloned (clone C1.1.1). Infection with C1.1.1 was inhibited completely by heparin (a GAG chemically related to HS but sulfated to a higher extent) and by dextran sulfate (an artificial highly sulfated polysaccharide), whereas HS and CS-A, -B, and -C were unable to inhibit infection. Bound C1.1.1 virus particles were released from the cell surface by treatment with heparin. Furthermore, C1.1.1 virus particles and CSFV E(rns) purified from insect cells bound to immobilized heparin, whereas purified CSFV E2 did not. These results indicate that initial binding of this virus clone is accomplished by the interaction of E(rns) with cell surface HS. In contrast, infection of SK6 cells with virus clones isolated from the blood of an infected pig and minimally passaged in SK6 cells was not affected by heparinase I treatment of cells and the addition of heparin to the medium. However, after one additional round of amplification in SK6 cells, infection with these virus clones was affected by heparinase I treatment and heparin. Sequence analysis of the E(rns) genes of these virus clones before and after amplification in SK6 cells showed that passage in SK6 cells resulted in a change of an Ser residue to an Arg residue in the C terminus of E(rns) (amino acid 476 in the polyprotein of CSFV). Replacement of the E(rns) gene of an infectious DNA copy of C1.1.1 with the E(rns) genes of these virus variants proved that acquisition of this Arg was sufficient to alter an HS-independent virus to a virus that uses HS as an E(rns) receptor.     

An approach for the stable immobilization of proteins

An approach is presented for the stable covalent immobilization of proteins with a high retention of biological activity. First, chemical modification studies were used to establish enzyme structural and functional properties relevant to the covalent immobilization of an enzyme to agarose based supports. Heparinase was used as a model enzyme in this set of studies. Amine modifications result in 75-100% activity loss, but the effect is moderated by a reduction in the degree of derivatization. N-hydroxysuccinimide, 1,1,1-trifluoroethanesulfonic acid, and epoxide activated agarose were utilized to determine the effect of amine reactive supports on immobilized enzyme activity retention. Cysteine modifications resulted in 25-50% loss in activity, but free cysteines were inaccessible to either immobilized bromoacetyl or p-chloromercuribenzoyl groups. Amine reactive coupling chemistries were therefore utilized for the covalent immobilization of heparinase. Second, to ensure maximal stability of the immobile protein-support linkage, the identification and subsequent elimination of the principal sources of protein detachment were systematically investigated. By using high-performance liquid chromatography (HPLC), electrophoresis, and radiolabeling techniques, the relative contributions of four potential detachment mechanisms-support degradation, proteolytic degradation, desorption of noncovalently bound protein, and bond solvolysis-were quantified. The mechanisms of lysozyme, bovine serum albumin, and heparinase leakage from N-hydroxysuccinimide or 1,1,1-trifluoroethanesulfonic acid activated agarose were elucidated. By use of stringent postimmobilization support wash procedures, noncovalently bound protein loss. An effective postimmobilization washing procedure is presented for the removal of adsorbed protein and the complete elimination of immobilized protein loss.     

Removing human immunodeficiency virus (HIV) from human blood using immobilized heparin

Heparin covalently attached to a water-insoluble resin suspended in HIV-infected aqueous buffer or whole blood captures the virus; subsequent physical separation of the immobilized heparin reduced the viral titers by over 80 and 50%, respectively. The detoxification concept has been validated by both circulating an HIV-1 solution through a column packed with the heparin–sepharose beads and successively mixing an HIV-1 solution with fresh beads.