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Biomedical subjects

C L Cooney

Publications and source records attributed to C L Cooney.

At least 37 records · Page 2Linked to original sources
PubMed1993

Kinetics of immobilized heparinase in human blood.

Immobilized enzyme reactors can form the basis of useful blood detoxification systems. One such reactor was developed for heparin neutralization by immobilized heparinase. In this article, reactor kinetics were studied under clinically relevant conditions. Heparin neutralization was assessed in vitro in whole human blood using (a) a well-mixed batch reactor, and (b) an oscillating, continuous-flow reactor. The kinetics of heparin neutralization in human blood were first order over the entire range of heparin and enzyme concentrations and particle fractions tested. The kinetic rate was not sensitive to physiological variations in the concentration of antithrombin, a heparin binding protein in blood. Enzyme activity did not decrease significantly over the 2 hour test period. Kinetic control of the system with minimal intraparticle diffusional limitations was suggested by the Thiele moduli (0.11-0.67) and effectiveness factors (0.98 +/- 0.01). The ratio kcat/Km obtained in batch studies was 0.0028 +/- 0.0008 cm3/microgram-min. A continuous-flow oscillating reactor within a closed recirculation loop performed as a single well mixed batch reactor; there was a short mixing time of recirculating blood when compared to reaction time. A model based on this mixing pattern and the kinetics obtained in independent batch studies accurately predicted heparin neutralization profiles observed in the continuous-flow system.

Antithrombins↗
PubMed1992

Fermentation monitoring.

Fermentation monitoring continues to be the focus of much research. Over the last year, important strides were made in improving bioprocess monitoring using NADH fluorescence, viscosity, affinity techniques, enzyme and microbial sensors, calorimetry, flow injection analysis and bioluminescence. Better fermentation monitoring is important for improving understanding, operation, development and control of the process. We expect progress in these areas of research to continue. In addition, we highlight some non-conventional approaches.

Biosensing Techniques↗
PubMed1991

The release of heparinase from the periplasmic space of Flavobacterium heparinum by three-step osmotic shock.

Heparinase was released from the periplasmic space of Flavobacterium heparinum by three-step osmotic shock procedure. The procedure involves resuspending exponentially growing cells consecutively into (1) 40% sucrose, (2) 10 mM sodium phosphate, 2 mM magnesium chloride, pH 7, and (3) 10 mM sodium phosphate, 300 mM sodium chloride, 2 mM magnesium chloride, pH 7. Typically, 50-75% of the total heparinase activity is recovered by this procedure with an observed 7-15-fold increase in purity. The majority of heparinase activity is released in the final step of the procedure allowing for resolution from cytoplasmic and nonspecific periplasmic material. F. heparinum cells can be stored in 40% sucrose at 4 degrees C for up to one week without significant losses in recovery yields.

Biotechnology↗
PubMed1990

Intracellular lytic enzyme systems and their use for disruption of Escherichia coli.

This article focusses on lytic enzyme systems available in E. coli and their potential use for cellular disruption. In the systems described here the genetic information for lysis would be carried within the microbial host, either integrated or naturally occurring on chromosomal DNA, or on extrachromosomal elements such as plasmids. Each microbe would carry complete information for endogenous enzymatic lysis, and lysis would occur in a controlled manner after being triggered by an external factor such as temperature or inducer addition. The lytic systems explored in this review include the autolytic enzymes, colicin lytic enzymes, and bacteriophage lytic enzymes from phage phiX174, T4, lambda, MS2 and Q beta. Many of the colicin lytic enzymes and all of the bacteriophage lytic enzymes described here have been cloned, and in some instances examined as cellular disruption methods. None of the E. coli autolytic enzymes have been cloned, but information pertinent for use as a disruption method is described.

Autolysis↗
PubMed1990

An overview of continuous protein purification processes.

As the sphere of influence of recombinant technology moves away from the laboratory bench, towards product commercialization, development of manufacturing and large scale process technology is becoming a major challenge and determinant for commercial success. The challenge is particularly acute for protein purification process development where protein purification costs tend to dominate overall process economics. The primary objective for process scale purification is to minimize cost for a purified product which meets specifications. Continuous processes may be used to facilitate achievement of the overall objectives. This review critically examines the use of continuous processing for protein purification and recovery operations. The processes have been divided into three general areas: adsorptive and chromatographic, electrophoretic, and extractive. Consideration is given to the operational advantages and limitations of the reviewed processes.

Journal Article↗
PubMed1990

Immobilized mammalian cell cultivation in hollow fiber bioreactors.

Cultivation of animal cells for the production of recombinant proteins is an important method for manufacturing complex proteins requiring posttranslational processing. One of the often considered methods for cultivation is by immobilization of the cells in hollow fiber bioreactors (HFBRs). These systems allow the cells to grow to high densities in a shear protected environment; furthermore the product can be accumulated in high concentration in the case of ultrafiltration HFBRs. Operation and scale-up are constrained by nutrient and product transport with oxygen transfer to growing cells being the most critical parameter. Mathematical models describing HFBRs have proved to be useful in quantitating and understanding the constraints and guiding the scale-up of this approach to animal cell cultivation.

Journal Article↗
PubMed1990

Specific plate assay for bacterial heparinase.

A procedure was developed for detecting heparinase activity on heparin agar plates. The method is based on the differential precipitation of heparin and heparinase-generated heparin fragments by protamine sulfate. Heparinase activity is detected by the presence of clear zones against a white background. This method can be used to screen for the expression of recombinant heparinase and to identify Flavobacterium heparinum mutants expressing heparinase constitutively.

Agar↗
PubMed1990

Optimization and simulation of continuous affinity-recycle extraction (care).

Simulation and optimization of continuous affinity recycle extraction (CARE), a protein purification unit operation based on protein adsorption to solid phase adsorbents, is described in this paper. Rather than packing conventional adsorbent particles in a fixed bed (column), solid/liquid contact is carried out in well-mixed reactors. Continuous operation is achieved by recirculation of the adsorbent particles between two or more contactors. The feasibility of this purification scheme was established with the recovery and isolation of the enzyme beta-galactosidase from E.coli, using the affinity support PABTG/Agarose. A mathematical model describing system performance was developed. The mathematical model was used to optimize several facets of the system design and operation. The base two-stage contractor design was modified by the addition of an intermediate wash stage as well as the incorporation of multiple adsorption stages. These design modifications serve to increase purification, concentration and recovery while utilizing the same amount of adsorbent. The methodology for defining and optimizing objective functions was developed and experimentally validated. Finally, optimum system start-up protocols, minimizing the time required to reach steady-state operation, were developed and experimentally validated. The impact of early introduction of adsorptive purification in a downstream processing sequence, with CARE, was evaluated and is described. Through the early introduction of a highly specific adsorptive step, significant purification is achieved simultaneously with clarification and concentration. In addition, purification performance in CARE was contrasted with that achievable in conventional column chromatography.

Chromatography, Affinity↗
PubMed1986

Removal of the anticoagulant activities of the low molecular weight heparin fractions and fragments with flavobacterial heparinase.

Recently, the development of low molecular weight heparin fractions and fragments (LMHF) as potential antithrombotic agents has gained increased attention. However, the lack of antagonists to neutralize the anticoagulant effects of these drugs may seriously exclude them from possible uses in extracorporeal therapy. This is mainly because of the concern that the high dosage of the drugs employed in extracorporeal therapy could lead to serious bleeding risks. Our earlier work has demonstrated that immobilized heparinase can remove polydisperse heparin both in vitro and in vivo. To examine whether such a system may be used as a novel approach to neutralize the anticoagulant effects of LMHF, different LMHF were tested using heparinase. In vitro data showed that both the APTT and anti-FXa activities of the LMHF including Kabi 2165, PK 10169, Cy 216 and CY 222 were nearly completely eliminated by heparinase in less than 20 min. This study suggests that an immobilized heparinase system may be an useful element for the acceptance of the LMHF for their use in extracorporeal therapy.

Anticoagulants↗
PubMed1986

Polysaccharide lyases.

Polysaccharide lyases (or eliminases) are a class of enzymes (EC 4.2.2.-) that act to cleave certain activated glycosidic linkages present in acidic polysaccharides. These enzymes act through an eliminase mechanism, rather than through hydrolysis, resulting in unsaturated oligosaccharide products. Acidic polysaccharides are ubiquitous and so are the lyases that degrade them. This review article examines lyases that act on acidic polysaccharides of plant, animal, and microbial origin. These lyases are predominantly of microbial origin and come from a wide variety of both pathogenic and nonpathogenic bacteria and fungi. The lyases discussed include alginate lyase (EC 4.2.2.3), pectin lyase (EC 4.2.2.10), pectate lyase (EC 4.2.2.2), oligogalacturonide lyase (EC 4.2.2.6), exopolygalacturonate lyase (EC 4.2.2.9), chondroitin lyases (EC 4.2.2.4 and EC 4.2.2.5), hyaluronate lyase (EC 4.2.2.1), heparin lyase (EC 4.2.2.7), heparan lyase (EC 4.2.2.8), and other unclassified lyases. This review examines the sources, regulation, purification, and properties of these polysaccharide lyases.

Animals↗
PubMed1986

Oxygen limitation on L-serine production in a hollow-fiber bioreactor.

Pseudomonas AM1 utilizes glycine and methanol to produce L-serine aerobically. The consumption of methanol in this bioconversion is stoichiometrically in excess of L-serine production. Consequently, the oxygen requirement associated with L-serine production is higher than expected for the conversion from glycine. One method of L-serine production investigated was a technique utilizing a hollow-fiber ultrafiltration cartridge as a bioreactor. Oxygen diffusion limitations appear to impede the consumption of methanol and, consequently, the production of L-serine in such a reactor. Methanol consumption data agree with predictions based on a hollow-fiber diffusion model.

Glycine↗
PubMed1986

Sulfur regulation of heparinase and sulfatases in Flavobacterium heparinum.

Sulfur regulation of heparinase synthesis and sulfatase synthesis was studied in Flavobacterium heparinum. Heparinase synthesis was strongly repressed by sulfate and L-cysteine, while the activity of this enzyme showed little or no inhibition by these compounds. Heparinase was synthesized in the absence of heparin when L-methionine was used as the sole sulfur source. The sulfatases produced by F. heparinum, which include the sulfatases involved in heparin catabolism, were also studied. At least some of the sulfatase activity was regulated by sulfur compounds in a manner similar to heparinase regulation. L-Cysteic acid and taurine were not suitable sulfur sources to support the growth of F. heparinum.

Ammonium Sulfate↗
PubMed1985

Purification and characterization of heparinase from Flavobacterium heparinum.

Heparinase (EC 4.2.2.7) isolated from Flavobacterium heparinum was purified to homogeneity by a combination of hydroxylapatite chromatography, repeated gel filtration chromatography, and chromatofocusing. Homogeneity was established by the presence of a single band on both sodium dodecyl sulfate and acid-urea gel electrophoretic systems. Amino acid analysis shows that the enzyme contains relatively high amounts of lysine residues (9%) consistent with its cationic nature (pI 8.5) but contains only 4 cysteine residues/polypeptide. The molecular weight of heparinase was estimated to be 42,900 +/- 1,000 daltons by gel filtration and 42,700 +/- 1,200 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is very specific, acting only on heparin and heparan monosulfate out of 12 similar polysaccharide substrates tested. It has an activity maximum at pH 6.5 and 0.1 M NaCl and a stability maximum at pH 7.0 and 0.15 M NaCl. The Arrhenius activation energy was found to be 6.3 kcal/mol. However, the enzyme is very sensitive to thermal denaturation and loses activity very rapidly at temperatures over 40 degrees C. Kinetic studies of the heparinase reaction at 37 degrees C gave a Km of 8.04 X 10(-6) M and a Vm of 9.85 X 10(-5) M/min at a protein concentration of 0.5 microgram/ml. By adapting batch procedures of hydroxylapatite and QAE (quaternary aminoethyl)-Sephadex chromatography, gram quantities of heparinase that is nearly free of catalytic enzyme contaminants can be purified in 4-5 h.

Amino Acids↗
PubMed1984

An immobilized microbial heparinase for blood deheparinization.

A new medical application of an immobilized microbial enzyme is described. Extracorporeal devices require systemic heparin administration to prevent thrombus formation; however, the use of heparin often leads to serious hemorrhagic complications. Heparinase isolated from Flavobacterium has been immobilized and used in a fluidized bed reactor to eliminate heparin from blood passing through an extracorporeal circuit both in vitro and in vivo. This paper discusses the stepwise development of this heparinase reactor including: (1) improvements in the fermentation resulting in an inexpensive large-scale source of heparinase without the addition of the previously required inducer, heparin; (2) the use of batch processes to adapt previous purification schemes to large-scale heparinase production and the subsequent purification of heparinase to a single SDS-PAGE banding protein; (3) the immobilization of heparinase with a 91% activity recovery and good stability, (4) the design and successful testing of a fluidized bed reactor containing immobilized heparinase in the removal of clinically used quantities of heparin from both human blood in vitro and canine blood in vivo; and (5) the initiation of animal studies focusing on the toxicology of heparinase-derived heparin degradation products and the short and long term effects of exposure to these products and to heparinase.

Enzymes, Immobilized↗
PubMed1983

Microbial fermentative preparation of L-[15N2]lysine and its tracer: application to serum amino acid kinetic studies.

The microorganism Brevibacterium flavum 21129 has been used to produce multigram batches of L-[15N2]lysine of high purity and isotopic enrichment by supplementation of the growth medium with (15NH4)2SO4 of 98.0 atom% excess. The doubly 15N-labeled lysine can be detected at dilutions 10 times greater than singly labeled lysine when isotope dilution curves are analyzed by gas chromatography-mass spectrometry. This enhanced sensitivity permits kinetic measurements of plasma free-lysine isotope content over a 300-fold dilution during 6 h following a single oral bolus of 5 mg/kg body wt. This inexpensive preparation method lends itself to the production of highly useful biochemical compounds for kinetic studies of human nutrition.

Absorption↗