Performance of an external loop air-lift bioreactor for the production of monoclonal antibodies by immobilized hybridoma cells

Summary The performance of an external loop air-lift bioreactor was investigated by assessing the inter-relationships between various hydrodynamic properties and mass transfer. The feasibility of using this bioreactor for the production of monoclonal antibodies by mouse hybridoma cells immobilized in calcium alginate gel beads and alginate/poly-l-lysine microcapsules was also examined. When the superficial gas velocity, V _g , in the 300 ml reactor was varied from 2 to 36 cm/min, the average liquid velocity increased from 3 to 14 cm/sec, the gas hold-up rose from 0.2 to 3.0%, and the oxygen mass transfer coefficient, k _L a, increased from 2.5 to 18.1 h^-1. A minimum liquid velocity of 4 cm/s was required to maintain alginate gel beads (1000 m diameter, occupying 3% of reactor volume) in suspension. Batch culture of hybridoma cells immobilized in alginate beads followed logarithmic growth, reaching a concentration of 4×10⁷ cells/ml beads after 11 days. Significant antibody production did not occur until day 9 into the culture, reaching a value of 100 g/ml of medium at day 11. On the other hand, bioreactor studies with encapsulated hybridoma cells gave monoclonal antibody concentrations of up to 800 g/ml capsules (the antibody being retained within the semipermeable capsule) and maximum cell densities of 2×10⁸ cells/ml capsule at day 11. The volumetric productivities of the alginate gel immobilized cell system and the encapsulated cell system were 9 and 3 g antibody per ml of reactor volume per day, respectively. The main advantage of the bioreactor system is its simple design, since no mechanical input is required to vary the hydrodynamic properties.     

Binding of chloride and a disulfonic stilbene transport inhibitor to red cell band 3

Summary The effect of chloride on 4,4-dibenzamido-2,2-disulfonic stilbene (DBDS) binding to band 3 in unsealed red cell ghost membranes was studied in buffer [NaCl (0 to 500mm) + Na citrate] at constant ionic strength (160 or 600mm). pH 7.4, 25°C. In the presence of chloride, DBDS binds to a single class of sites on band 3. At 160mm ionic strength, the dissociation constant of DBDS increases linearly with chloride concentration in the range [Cl]=450mm. The observed rate of DBDS binding to ghost membranes, as measured by fluorescence stopped-flow kinetic experiments, increases with chloride concentration at both 160 and 600mm ionic strength. The equilibrium and kinetic results have been incorporated into the following model of the DBDS-band 3 interaction: The equilibrium and rate constants of the model at 600mm ionic strength areK ₁=0.67±0.16 m,k ₂=1.6±0.7 sec^–1,k _–2=0.17±0.09 sec^–1,K ₁=6.3±1.7 m,k ₂=9±4 sec^–1 andk _–2=7±3 sec^–1. The apparent dissociation constants of chloride from band 3,K _Cl, are 40±4mm (160mm ionic strength) and 11±3mm (600mm ionic strength). Our results indicate that chloride and DBDS have distinct, interacting binding sites on band 3.     

Continuous production of 5′-ribonucleotides from yeast RNA by hydrolysis with immobilized 5′-phosphodiesterase and 5′-adenylate deaminase

The production of 5-IMP and 5-GMP by enzymatic conversion from RNA using a continuous two packed-bed reactor was investigated. 5-Phosphodiesterase (5PD) and 5-adenylate deaminase (5AD) were immobilized in an acrylic resin to produce derivatives with about 15 U/g of support. The kinetic properties of the enzymes were described by Michaelis-Menten models: no significant differences were found in the K _m value of the free and immobilized 5AD (60 and 20 m, respectively), whereas for 5PD the K _m value was one order of magnitude higher for the immobilized enzyme (4.85 mg RNA/ml), probably due to diffusional limitations. Both enzymes remained stable after 8 h of use in a continuous packed-bed reactor whereas the half lives of the free enzymes were 193 min and 240 min at 40°C and 70°C for 5AD and 5PD, respectively. A procedure is proposed for the design of a continuous two packed-bed column process.F. Olmedo and F. Iturbe are with the Depto. de Alimentos y Biotecnologia, Facultad de Química, UNAM, México 04510, D.F., Mexico. J. Gomez-Hernández is with the Depto. de Biotecnología, UAM-1, Apdo. Postal 55-535, México 09340, D.F., Mexico. A. López-Munguía is with the Instituto de Biotecnología, Apartado Postal 510-3, Cuernavaca, Mor. 62271, Mexico     

Ethanol production at 45°C by Kluyveromyces marxianus IMB3 immobilized in magnetically responsive alginate matrices

Summary The thermotolerant yeast, Kluyveromyces marxianus IMB3 produced 11g ethanol/l during growth at 45°C on media containing 4% (w/v) lactose when immobilized in alginate beads whereas the free cells produced 5g ethanol/l. A magnetically responsive biocatalyst, prepared by incorporating Fe₃O₄ into the alginate matrix increased ethanol production to 12g/l in batch-fed reactors. Ethanol concentrations were further increased to a maximum of 18g/l by immobilization of the endogenous K. marxianus -galactosidase to the Fe₃O₄ particles prior to inclusion into the alginate matrix. Maximum ethanol productivity by the system was 87% of the maximum theoretical yield.     

Identification of a sheath-associated protein involved in phosphate transport in Sphaerotilus natans

Summary Sphaerotilus natans was shown to have a fourfold lower K _mof phosphate transport when grown in medium containing 0.1 mm phosphate, compared to cells grown in 10.0 mm phosphate. Analysis of sheath proteins from cells grown at these two phosphate levels revealed a protein of 53 kDa present in the sheath of cells grown at a phosphate concentration of 0.1 mm. This sheath-associated, phosphate-regulated protein, designated SapP, was gel purified and used to raise a polyclonal antibody. Enzyme-linked immunosorbent assay was used to localize this protein to the surface of the sheathed cells. Phosphate uptake assays done in the presence of the antibody also showed a rise in the K _mof phosphate transport in cells grown in 0.1 mm phosphate, indicating that this protein is involved in high-affinity phosphate transport.Offprint requests to: C. F. Kulpa Jr     

Der Einfluß von Redoxindicatoren auf die Säurebildung von Streptococcus lactis

Zusammenfassung Der Einfluß von acht Redoxindicatoren mit abgestuften Normalpotentialen zwischen E ₀=-340 mV und E ₀=+115 mV ist in einem Flüssigsubstrat auf die Säurebildung von Streptococcus lactis untersucht worden.Durch Farbstoffe mit Normalpotentialen zwischen-122 mV und +115 mV (Nilblau, Methylenblau, Brillantkresylblau, Toluylenblau) konnte die Milchsäurebildung, besonders bei niederen Farbstoffkonzentrationen (4·10^-5 und 10^-4 m/l), signifikant gefördert werden. Farbstoffe mit Normalpotentialen zwischen-289 mV und-122 mV (Safranin T, Phenosafranin, Janusgrün, Nilblau) hemmten dagegen die Produktion von Milchsäure in allen oder in der Mehrzahl der angewendeten Konzentrationen. Niblau, das am übergang der beiden Potentialbereiche liegt, förderte die Milchsäurebildung sehr stark bei niederen und hemmte ebenso stark bei höheren Konzentrationen.Die Bildung von flüchtigen Säuren wurde durch keinen der Farbstoffe gefördert. Eine Hemmung trat durch Farbstoffe mit Normalpotentialen zwischen-289 mV und +47 mV (Safranin T, Phenosafranin, Janusgrün, Nilblau, Methylenblau, Brillantkresylblau) ein. Janusgrün hemmte die Bildung flüchtiger Säuren in allen untersuchten Konzentrationen zwischen 4·10^-5 und 4·10^-4 m/l. Je weiter das Normalpotential des hemmenden Farbstoffes von dem Normalpotential des Janusgrüns abwich, desto geringer wurde die Hemmung in der geringsten Konzentrationsstufe von 4·10^-5 m/l. Diese Unterbindung der Hemmwirkung wirkte sich in Richtung auf negativere Normalpotentiale mehr aus als in Richtung auf positivere Normalpotentiale.Durch die beiden Farbstoffe mit den extremsten Normalpotentialen (Neutralrot E ₀=-340 mV, Toluylenblau E ₀=+115 mV) wurde weder die Produktion von Milchsäure noch die von flüchtigen Säuren merklich gehemmt. Eine Förderung der Milchsäurebildung konnte von beiden Farbstoffen nur durch Toluylenblan in der geringsten Konzentration (4×10^-5 m/l) erzielt werden.

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The influence of redox indicators on the acid formation in Streptococcus lactis

Summary The influence of 8 redox indicators with graded standard redox potentials between E ₀=-340 mV and E ₀=+115 mV was tested for the acidification by Streptococcus lactis in a liquid medium.By redox indicators with standard redox potentials between-122 mV and +115 mV (Nile blue, methylene blue, brillant cresyl blue, and toluylene blue) the formation of lactic acid could be significantly increased, especially with low dye concentrations (4×10^-5 and 10^-4 m/l). Dyes with standard redox potentials between-289 mV and-122 mV (safranine T, phenosafranine, Janus green, Nile blue) on the other hand retarded the production of lactic acid by Strept. lactis in all or most of the dye concentrations used. Nile blue-representing the transition point between the two sections of redox indicators-increased the production of lactic acid very much in low concentrations and inhibited it as much in high concentrations.The production of volatile acids was not increased by any dyestuff. It was delayed by dyes with standard redox potentials between-289 mV and +47 mV (safranine T, phenosafranine, Janus green, Nile blue, methylene blue, brillant cresyl blue). Janus green retarded the production of volatile acids in all analysed concentrations between 4×10^-5 and 4×10^-4 m/l. The greater the difference between the standard redox potential of the inhibiting dye and the standard redox potential of Janus green the smaller was the retarding effect in the lowest degree of concentration of 4×10^-5 m/l. This stopping of the retarding effect was more effective towards standard redox potentials more negative then towards more positive ones.The two dyes with the most extreme standard redox potentials (neutral red E ₀=-340 mV, toluylene blue E ₀=+115 mV) didn't obviously delay any acid formation, neither the production of lactic acid nor that of volatile acids. Of the two dyes only toluylene blue in the lowest concentration used (4×10^-5 m/l) caused an increased production of lactic acid.

     

Immobilization of the methanogenic bacterium Methanosarcina barkeri

Whole cells of the methanogen Methanosarcina barkeri were immobilized in an alginate network which was crosslinked with Ca²⁺ ions. The rates of methanol conversion to methane of entrapped cells were found to be in the same range as the corresponding rates of free cells. Furthermore, immobilized cells were active for a longer period than free cells. The particle size of the spherical alginate beads (1.2 mm-3.7 mm ?) and thus diffusion had no obvious influence on the turnover of methanol. The half-value period for methanol conversion activity determined in a buffer medium was approximately 4 days at 37°C for entrapped cells. The apparent K_m value K for such cells was nearly 140mM and the V_max value was about 1.2 μmol methanol/min/mg entrapped protein. Therefore the high rates of methanol degradation measured, e.g., 0.5 μmol methanol/min/mg entrapped protein, indicated that the immobilization technique preserved the cellular functions of this methanogenic bacterium.     

Interaction among anion,cation and glucose transport proteins in the human red cell

Summary The time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to band 3 can be measured by the stopped-flow method. We have previously used the reaction time constant, _DBDS, to obtain the kinetic constants for binding and, thus, to report on the conformational state of the band 3 binding site. To validate the method, we have now shown that the ID₅₀ (0.3±0.1 m) for H₂-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of _DBDS is virtually the same as the ID₅₀ (0.47±0.04 m) for H₂-DIDS inhibition of red cell Cl^– flux, thus relating _DBDS directly to band 3 anion exchange. The specific glucose transport inhibitor, cytochalasin B, causes significant changes in _DBDS, which can be reversed with intracellular, but not extracellular,d-glucose. ID₅₀ for cytochalasin B modulation of _DBDS is 0.1±0.2 m in good agreement withK _D =0.06±0.005 m for cytochalasin B binding to the glucose transport protein. These experiments suggest that the glucose transport protein is either adjacent to band 3, or linked to it through a mechanism, which can transmit conformational information. Ouabain (0.1 m), the specific inhibitor of red cell Na⁺,K⁺-ATPase, increases red cell Cl^– exchange flux in red cells by a factor of about two. This interaction indicates that the Na⁺,K⁺-ATPase, like the glucose transport protein, is either in contact with, or closely linked to, band 3. These results would be consistent with a transport proteincomplex, centered on band 3, and responsible for the entire transport process, not only the provision of metabolic energy, but also the actual carriage of the cations and anions themselves.     

Endopolygalacturonase immobilized on epoxide-containing supports

Summary Endopolygalacturonase was immobilized onto 3-(2,3-epoxypropoxypropyl)-silica and oxirane-acrylic beads. Optimum conditions of immobilization and catalytic properties of the immobilized enzyme preparations are described.     

A new method for immobilization of acetylcholinesterase

A new method for immobilization of acetylcholinesterase (AChE) to alginate gel beads by activating the carbonyl groups of alginate using carbodiimide coupling agent has been successfully developed. Maximum reaction rate (V _max) and Michaelis–Menten constant (K _m) were determined for the free and binary immobilized enzyme. The effects of pH, temperature, storage stability, reuse number and thermal stability on the free and immobilized AChE were also investigated. For the free and binary immobilized enzyme on the Ca–alginate gel beads, optimum pH values were found to be 7 and 8, respectively. Optimum temperatures for the free and immobilized enzyme were observed to be 30 and 35 °C, respectively. Upon 60 days of storage the preserved activity of free and immobilized enzyme were found as 4 and 68%, respectively. In addition, reuse number, and thermal stability of the free AChE were increased by as a result of binary immobilization.     

Adenosine 5′-phosphosulfate sulfotransferase from the marine macroalgaPorphyra yezoensis Ueda (Rhodophyta): stabilization,purification, and properties

Adenosine 5-phosphosulfate sulfotransferase (APSSTase) was purified over 2700-fold to homogeneity from the thalli of the marine macroalgaPorphyra yezoensis Ueda (Rhodophyta), using a combination of ammonium sulfate precipitation, hydrophobic chromatography, anion-exchange chromatography and gel-filtration. The native M_r measured by gel-filtration was 350 000. The subunit M_r was estimated to be 43 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In addition, APSSTase had a relatively broad pH optimum of pH 9.0–9.8 with a peak at pH 9.5. The apparentK _m value for adenosine 5-phosphosulfate (APS) was 2.1 M, when dithiothreitol was acceptor substrate. 3-Phosphoadenosine 5-phosphosulfate and inosine 5-phosphosulfate could not substitute for APS as a sulfate donor. The enzyme utilized several organic thiols as acceptor substrates (artificial substrates): dithiothreitol (apparentK _m = 1.5 mM) and dithioerythritol (apparentK _m = 1.5 mM) gave the highest activity, and appreciable activity was also obtained usingl-glutathione (reduced form) which exhibited slight substrate inhibition (apparentK _m = 0.6 mM; the initial velocity was maximal at 3.0–4.0 mM). While APSSTase was markedly unstable in vitro: the half-life for activity loss at 25°C and pH 9.5 was about 8 min, the instability was decreased in the presence of a relatively high concentration of Na₂SO₄ or (NH₄)₂SO₄, and in the presence of APS or its analogs (AMP and -methylene-APS). Most of the thiols, with the sole exception of glutathione, were found to inactivate APSSTase irreversibly. The thiol-mediated inactivation was completely inhibited by the high concentration of Na₂SO₄, and by the analogs of APS.Abbreviations APS adenosine 5-phosphosulfate - APSSTase adenosine 5-phosphosulfate sulfotransferase - -m-APS -methylene-adenosine 5-phosphosulfate - DTT dithiothreitol - IPS inosine 5-phosphosulfate - PAPS 3-phosphoadenosine 5-phosphosulfate We wish to thank Mr. I. Kashiwase, Mr. Y. Endo and Mr. Y. Mimura, School of Fisheries Sciences, Kitasato University, for their technical assistance in this study. The research described in this paper was partly supported by the Kitasato Research Grant (H5-9 and H6-13 to N.K.).     

Purification and properties of cAMP independent glycogen synthase kinase and phosvitin kinase from human leukocytes

Summary cAMP independent glycogen synthase kinase and phosvitin kinase activity was purified from the 180 000 × g supernatant of human polymorphonuclear leukocytes by ammonium sulphate precipitation and phosphocellulose chromatography. The cAMP independent glycogen synthase kinase eluted from the phosphocellulose at 0.54 m NaCl (peak A) separate from the major phosvitin kinase eluting at 0.68 m NaCl (peak B). The kinase activity of both peaks tended to form aggregates, but in the presence of 0.6 m NaCl, the peak B enzyme had M_r 250 000, 7.2S and the peak A enzyme M_r 38 000, 3.8S. The ratio between synthase kinase and phosvitin kinase activity in peak A was 1:3.2 and in peak B 1:31.4. In addition the kinase activities differed with respect to sensitivity to temperature, ionic strength and CaCl₂. It is suggested that the peak A enzyme represents the cAMP independent glycogen synthase kinase of leukocytes, whereas the peak B enzyme is a phosvitin kinase, which is insignificantly contaminated with some synthase kinase (peak A) and contains a separate, second synthase kinase.Synthase kinase had K _m ^app 4.2 m for muscle glycogen synthease I and K _m ^app 45 m for ATP. GTP was a poor substrate. The activity was not influenced by cyclic nucleotides, Ca²⁺, or glucose-6-P. Synthase I from muscle and leukocytes was phosphorylated to a ratio of independence of less than 0.05.Abbreviations cAMP adenosine cyclic 3:5-monophosphate - DTT dithiothreitol - EGTA ethylene glycol-bis-(-amino-ethylether)-N,N-tetraacetic acid - PMSF phenylmethylsulfonylfluoride - PKI protein kinase inhibitor - RI ratio of independence for glycogen synthase - SDS sodium dodecyl sulphate     

Isolation of mutants with improved production of cAMP from Microbacterium sp. no. 205 (ATCC21376)

Summary Mutants were isolated from Microbacterium sp. no. 205 (ATCC 21376) producing 13–30mm cyclic adenosine 3,5-monophosphate (cAMP) by salvage biosynthesis, through sequential improvements of the bacterium for the purpose of improving cAMP production. The mutants produced 50–75 mm cAMP on 100 mm inosine 5-monophosphate as a precursor. Mutants resistant to the inhibition of growth by cAMP at high concentrations were isolated; the resistance was one of four characteristics effective for improved production of cAMP.     

Evaluation of some gelling agents for immobilization of aerobic microbial cells in alginate and carrageenan gel beads

Summary Different gelling agents were used to immobilized viable cells in either alginate or -carrageenan gel beads. Based on cell leakage from the gel beads, oxygen and glucose diffusion coefficients and toxicity of the gelling agents, SrCl₂ was found to be the best for immobilization of aerobic microbial cells in, not only alginate but also carrageenan gel beads.     

IP3-and cAMP-induced responses in isolated olfactory receptor neurons from the channel catfish

Summary Olfactory receptor neurons enzymatically dissociated from channel catfish olfactory epithelium were depolarized transiently following dialysis of IP₃ or cAMP (added to the patch pipette) into the cytoplasm. Voltage and current responses to IP₃ were blocked by ruthenium red, a blocker of an IP₃-gated Ca²⁺-release channel in sarcoplasmic reticulum. In contrast, the responses to cAMP were not blocked by extracellularly applied ruthenium red, nor by l-cis-diltiazem or amiloride and two of its derivatives. The current elicited by cytoplasmic IP₃ in neurons under voltage clamp displayed a voltage dependence different from that of the cAMP response which showed marked outward rectification. A sustained depolarization was caused by increased cytoplasmic IP₃ or cAMP when the buffering capacity for Ca²⁺ of the pipette solution was increased, when extracellular Ca²⁺ was removed or after addition of 20–200 nm charibdotoxin to the bathing solution, indicating that the repolarization was caused by an increase in [Ca _i ] that opened Ca²⁺-activated K⁺ channels. The results suggest that different conductances modulated by either IP₃ or cAMP are involved in mediating olfactory transduction in catfish olfactory receptor neurons and that Ca²⁺-activated K⁺ channels contribute to the termination of the IP₃ and cAMP responses.Abbreviations ATP adenosine 5-triphosphate - BAPTA (bis-(o-aminophenoxy)-ethane-N-N-N-N)-tetraacetic acid - cAMP adenosine cyclic 3,5-monophosphate - cGMP guanosine cyclic 3,5-monophosphate - CTX charybdotoxin - DCB 3,4-dichlorobenzamil - EDTA ethylenediaminetetraacetic acid - EGTA ethylenglycol-bis-(b-aminoethyl)-N-N-N-N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - IP₃ inositol-1,4,5-triphosphate - NMDG N-methyl-d-glucamine We would like to thank the Tanabe Seiyaku Co., Ltd., for their gift of l-cis-diltiazem. This work was supported by National Institutes of Health grants DC00566 and BRSG S07RR05825.     

Apical membrane Cl-butyrate exchange: Mechanism of short chain fatty acid stimulation of active chloride absorption in rat distal colon

The cellular model of short chain fatty acid stimulation of electroneutral Na-Cl absorption in large intestine proposes that SCFA, following its uptake across the apical membrane, recycles and is coupled to functional Na-H and Cl-short chain fatty acid exchanges. To establish the presence of a Cl-butyrate exchange (used as a model short chain fatty acid), studies of ³⁶Cl and ¹⁴C-butyrate uptake across apical membrane vesicles of rat distal colon were performed. An outward butyrate-gradient stimulated transient accumulation of ³⁶Cl uptake that was not inhibited by pH clamping with valinomycin (a K ionophore) and FCCP (a proton ionophore). Outward butyrate-gradient-stimulated ³⁶Cl uptake was inhibited by 4,4-diisothiocyanatostilbene2,2-disulfonic acid (DIDS) with a half-maximal inhibitory concentration (IC₅₀) of 68.4 m, and was saturated by both increasing extravesicular Cl concentration (K _m for Cl of 26.8 ±3.4 mm and a V _max of 12.4±0.6 nmol/mg protein·9 sec) and increasing intravesicular butyrate concentration (K _m for butyrate of 5.9 mm and a V _max for Cl of 5.9 nmol/mg protein · 9 sec). ³⁶Cl uptake was also stimulated by outward gradients of other short chain fatty acids (e.g., propionate, acetate and formate). In contrast, an outward Cl gradient failed to enhance ¹⁴C-butyrate uptake. Extravesicular Cl more than extravesicular butyrate enhanced ³⁶Cl efflux from apical membrane vesicles. These studies provide compelling evidence for the presence of an electroneutral, pH-activated, Cl-butyrate exchange which in concert with Na-H exchange is the mechanism by which butyrate stimulates electroneutral Na-Cl absorption.Abbreviations used AMV apical membrane vesicles - BLMV basolateral membrane vesicles - DIDS 4,4-diisothiocyanatostilbene 2,2-disulfonic acid - FCCP carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone - MES 1-[N-morpholino]ethanesulfonic acid - NMG N-memyl-d-glucamine - SCF Ashort chain fatty acid This study was supported in part by a Public Health Service research Grant (DK 14669) provided by the National Institute of Diabetes, Digestive and Kidney Diseases. Ms. Mary Guidone provided excellent secretarial assistance.     

Kinetics of DIDS inhibition of swelling-activated K-Cl contrasport in low K sheep erythrocytes

Summary The inhibitory effect of various stilbene disulfonates was examined on the swelling-activated Cl-dependent K transport (K-Cl cotransport) in low K sheep erythrocytes. Both diisothiocy-anatostilbenes H₂DIDS and DIDS were found to be potent inhibitors. The DIDS concentration yielding 50% inhibition (IC₅₀) of KCl cotransport was 60 m in the absence of external K and 3 m at physiological K concentration. Other stilbene derivatives, such as SITS (4-acetamido-4 isothiocyanatostilbene-2,2-disulfonic acid), were only effective in the presence of external K, whereas DNDS (4,4-dinitrostilbene-2,2-disulfonic acid) and ISA (4-sulfophenyl isothiocyanate) had only slight effects at a concentration of 1 mm. The augmenting effect of external K is due to a second K site, distinguishable from the K transport site by its much higher affinity. No inhibition occurred in the absence of external Cl, whether or not external Rb(K) was present. Additionally, DIDS inhibited K-Cl cotransport activated by thiol alkylation with N-ethylmaleimide (NEM) as well as by Mg depletion in the presence of A23187 and a chelator. We conclude that allosteric sites affect the stilbene binding. When these sites are saturated, changes in external K or Cl concentration do not affect the affinity for DIDS (noncompetitive inhibition).This work was supported by grants in aid from the American Heart Association.     

Enzymatic splitting of penicillin V for the production of 6-APA using immobilized penicillin V acylase

Penicillin V acylase from Fusarium sp. SKF 235 was immobilized on several cation-exchange resins, of which Amberlite CG-50 was preferred. Maximum activity of the immobilized penicillin V acylase was 250 to 280 IU/g dry beads. The pH and temperature optima of the enzyme shifted from 6.5 to 6.8 and 55°C to 60°C, respectively, as a result of immobilization. However, the K _m for penicillin V remained at 10mm. Parameters for producing 6-aminopenicillanic acid were investigated and the immobilized penicillin V acylase was used for 68 cycles in a stirred tank reactor.     

Amino acid metabolism in the thermophilic phototroph,Chloroflexus aurantiacus: properties and metabolic role of two l-threonine (l-serine) dehydratases

Two l-threonine (l-serine) dehydratases (EC 4.2.1.16) of the thermophilic phototrophic bacterium Chloroflexus aurantiacus Ok-70-fl were purified to electrophoretic homogeneity by procedures involving anion exchange and hydrophobic interaction chromatography. Only one of the two enzymes was sensitive to inhibition by l-isoleucine (K _i=2 M) and activation by l-valine. The isoleucine-insensitive dehydratase was active with l-threonine (K _m=20 mM) as well as with l-serine (K _m=10 mM) whereas the other enzyme, which displayed much higher affinity to l-threonine (K _m=1.3 mM), was inactivated when acting on l-serine. Both dehydratases contained pyridoxal-5-phosphate as cofactor. When assayed by gel filtration techniques at 20 to 25° C, the molecular weights of both enzymes were found to be 106,000±6,000. In sodium dodecylsulfate-polyacrylamide gel electrophoresis, the two dehydratases yielded only one type of subunit with a molecular weight of 55,000±3,000. The isoleucine-insensitive enzyme was subject to a glucose-mediated catabolite repression.Abbreviations A absorbance - ile isoleucine - PLP pyridoxal-5-phosphate - SDS sodium dodecyl sulfate - TDH threonine dehydratase - U unit     

Production of citric acid with immobilized Yarrowia lipolytica

Summary Citric acid was produced with immobilized Yarrowia lipolytica yeast in repeated batch-shake-flask and air-lift fermentations. In active and passive immobilization methods calcium alginate, -carrageenan, polyurethane gel, nylon web and polyurethane foams were tested as carriers in repeated-batch fermentations. The highest citric acid productivity of 155 mg l^–1 h^–1 was reached with alginate-bead-immobilized cells in the first batch. A decrease in bead diameter from 5–6 mm to 2–3 mm increased the volumetric citric acid productivity threefold. In an air-lift bioreactor the highest citric acid productivity of 120 mg l^–1 h^–1 with a product concentration of 16.4 g l^–1 was obtained with cells immobilized in -carrageenan beads. Offprint requests to: H. Kautola