Quantitative analysis of Penicillium chrysogenum Wis54-1255 transformants overexpressing the penicillin biosynthetic genes

The low penicillin-producing, single gene copy strain Wis54-1255 was used to study the effect of overexpressing the penicillin biosynthetic genes in Penicillium chrysogenum. Transformants of Wis54-1255 were obtained with the amdS expression-cassette using the four combinations: pcbAB, pcbC, pcbC-penDE, and pcbAB-pcbC-penDE of the three penicillin biosynthetic genes. Transformants showing an increased penicillin production were investigated during steady-state continuous cultivations with glucose as the growth-limiting substrate. The transformants were characterized with respect to specific penicillin productivity, the activity of the two pathway enzymes delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and isopenicillin N synthetase (IPNS) and the intracellular concentration of the metabolites: delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (bisACV), isopenicillin N (IPN), glutathione (GSH), and glutathione disulphide (GSSG). Transformants with the whole gene cluster amplified showed the largest increase in specific penicillin productivity (r(p))-124% and 176%, respectively, whereas transformation with the pcbC-penDE gene fragment resulted in a decrease in r(p) of 9% relative to Wis54-1255. A marked increase in r(p) is clearly correlated with a balanced amplification of both the ACVS and IPNS activity or a large amplification of either enzyme activity. The increased capacity of a single enzyme occurs surprisingly only in the transformants where all the three biosynthetic genes are overexpressed but is not found within the group of pcbAB or pcbC transformants. The indication of the pcbAB and pcbC genes being closely regulated in fungi might explain why high-yielding strains of P. chrysogenum have been found to contain amplifications of a large region including the whole penicillin gene cluster and not single gene amplifications. Measurements of the total ACV concentration showed a large span of variability, which reflected the individual status of enzyme overexpression and activity found in each strain. The ratio ACV:bisACV remained constant, also at high ACV concentrations, indicating no limitation in the capacity of the thioredoxin-thioredoxin reductase (TR) system, which is assumed to keep the pathway intermediate LLD-ACV in its reduced state. The total GSH pool was at a constant level of approx. 5.7 mM in all cultivations.     

The RAS-adenylate cyclase pathway and cell cycle control inSaccharomyces cerevisiae

The cell cycle ofSaccharomyces cerevisiae contains a decision point in G₁ called start, which is composed of two specific sites. Nutrient-starved cells arrest at the first site while pheromone-treated cells arrest at the second site. Functioning of the RAS-adenylate cyclase pathway is required for progression over the nutrient-starvation site while overactivation of the pathway renders the cells unable to arrest at this site. However, progression of cycling cells over the nutrient-starvation site does not appear to be triggered by the RAS-adenylate cyclase pathway in response to a specific stimulus, such as an exogenous nutrient. The essential function of the pathway appears to be limited to provision of a basal level of cAMP. cAMP-dependent protein kinase rather than cAMP might be the universal integrator of nutrient availability in yeast. On the other hand stimulation of the pathway in glucose-derepressed yeast cells by rapidly-fermented sugars, such as glucose, is well documented and might play a role in the control of the transition from gluconeogenic growth to fermentative growth. The initial trigger of this signalling pathway is proposed to reside in a glucose sensing complex which has both a function in controlling the influx of glucose into the cell and in activating in addition to the RAS-adenylate cyclase pathway all other glucose-induced regulatory pathways in yeast. Two crucial problems remaining to be solved with respect to cell cycle control are the nature of the connection between the RAS-adenylate cyclase pathway and nitrogen-source induced progression over the nutrient-starvation site of start and second the nature of the downstream processes linking the RAS-adenylate cyclase pathway to Cyclin/CDC28 controlled progression over the pheromone site of start.Abbreviations cAMP-PK cAMP-dependent protein kinase     

External anions relieve DIDS inhibition of SO4 efflux from placental membrane vesicles

Sulphate efflux from human placental microvillus membrane vesicles was inhibited by external DIDS (K_I10 .6 M). This inhibition was partially reversed on addition of the translocated substrates sulphate or selenate to the external medium: selenite which is not translocated does not protect against DIDS inhibition. These findings show that the mechanism responsible for sulphate efflux can be modified by substrate in the external medium.Abbreviations DIDS 4-acetamido-4-isothiocyanostilbene-2-2-disulphonate     

Analysis of the regulation of penicillin biosynthesis in Aspergillus nidulans by targeted disruption of the acvA gene

To analyse the regulation of the biosynthesis of the secondary metabolite penicillin in Aspergillus nidulans, a strain with an inactivated acvA gene produced by targeted disruption was used. acvA encodes -(l--aminoadipyl)-l-cysteinyl-d-valine synthetase (ACVS), which catalyses the first step in the penicillin biosynthetic pathway. To study the effect of the inactivated acvA gene on the expression of acvA and the second gene, ipnA, which encodes isopenicillin N synthase (IPNS), A. nidulans strain XEPD, with the acvA disruption, was crossed with strain AXB4A carrying acvA-uidA and ipnA-lacZ fusion genes. Ascospores with the predicted non-penicillin producing phenotype and a hybridization pattern indicating the presence of the disrupted acvA gene, and the fusion genes integrated in single copy at the chromosomal argB locus were identified. Both fusion genes were expressed at the same level as in the non-disrupted strain. Western blot analysis (immunoblotting) revealed that similar amounts of IPNS enzyme were present in both strains from 24 to 68 h of a fermentation run. In the acvA disrupted strain, IPNS and acyl-CoA: 6-aminopenicillanic acid acyltransferase (ACT) specific activities were detected, excluding a sequential induction mechanism of regulation of the penicillin biosynthesis gene ipnA and the third gene aat.     

Cephamycin production and isopenicillin N synthetase activity in cultures of Streptomyces clavuligerus

Summary The relationships between growth, cephamycin production and isopenicillin N synthetase (IPNS) activity in cultures of Streptomyces clavuligerus were examined to establish conditions that optimize the yield and specific activity of the enzyme. Unexpectedly for a secondary metabolic pathway component, IPNS was synthesized preferentially during rapid growth and reached its maximum specific activity in cultures supplied with readily assimilated sources of nitrogen. The activity decreased sharply as cultures entered stationary phase. On the other hand, comparisons of growth and antibiotic production on a range of carbon and nitrogen sources as well as measurements of IPNS activity in chemostat cultures implicated catabolite repression, a mechanism usually associated with separation of trophophase and idiophase activities, as an important factor in controlling expression of the secondary metabolic pathway. An explanation for the timing of IPNS biosynthesis is suggested.Dedicated to Professor H. J. Rehm on the occasion of his 60th birthday     

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.     

Absence of the glutamine-synthetase-linked methylammonium (ammonium)-transport system in the cyanobiont of Cycas-cyanobacterial symbiosis

Using the ammonium analogue ¹⁴CH₃NH ₃ ⁺ , ammonium transport was studied in the cyanobiont cells freshly isolated from the root nodules of Cycas revoluta. An L-methionine-dl-sulphoximine (MSX)-insensitive ammonium-transport system, which was dependent on membrane potential (), was found in the cyanobiont. However, the cyanobiont was incapable of metabolizing exogenous ¹⁴CH₃NH ₃ ⁺ or NH ₄ ⁺ because of the absence of another ammonium-transport system responsible for the uptake of ammonium for assimilation via glutamine synthetase (EC 6.3.1.2). Such a modification seems to be the result of symbiosis because the free-living cultured isolate, Anabaena cycadeae, has been shown to possess both the ammonium-transport systems.Abbreviations and symbol ATS/ATSs ammonium transport system/systems - Chl chlorophyll - GS glutamine synthetase - MSX L-methionine-dl-sulphoximine - membrane potential     

Kinetic characterization of carboxypeptidase-Y-catalyzed peptide semisynthesis Prediction of yields

Summary Carboxypeptidase-Y-catalyzed peptide semisynthesis has been characterized at pH 7.5, 25°C from initial rate steady state kinetic and progress reaction studies of hydrolysis and aminolysis of-N-benzoyl-L-tyrosine 4-nitro-anilide using the natural L-amino acids and their amides as nucleophiles. The reaction mechanism previously shown to account for carboxypeptidase-Y-catalyzed aminolysis reactions (Christensen et al., 1992) was found also to account for all of the reactions studied here. It involves in addition to the classical serine proteinase mechanism: i) complex formation between the free enzyme and the nucleophile, an interaction characterized by the competitive inhibition constant,K _i, and ii) reaction of the nucleophile with the acylated enzyme forming a complex of enzyme and aminolysis product, characterized by the aminolysis kinetic parameter,K _N.A competitive inhibitory effect showing binding to the free enzyme is seen mainly with large hydrophobic amino acids and their amides i.e. the same residues as those preferred on either side of the scissile bond in carboxypeptidase-Y substrates. The stoichiometry of the inhibition is 1 : 1 and the actual binding position most likely is that of the leaving group of substrates,S ₁.Aminolysis effects are obtained with a wide range of amino acids and amino acid amides, exceptions are Pro and, probably due to their low solubility, Tyr, Trp, Asp and Glu. TheK _N-values show relatively little dependence on the chemical nature of the side groups, but a marked difference between the amino acid and its amide. The amides interact more strongly. The kinetic parameter,k _c/K_m, of the hydrolysis of the aminolysis products is another important factor in peptide semisynthesis. Thek _c/K_m-values obtained of the amidated aminolysis products are much less than those of the products formed with free amino acids. All in all this leads to rather efficient aminolysis with the L-amino acid amides and poor aminolysis with the L-amino acids.Abbreviations BzTyrNHPhNO₂ -N-benzoyl-L-tyrosinyl 4-nitro-aniline - Xaa L-amino acids - Xaaa L-amino acid amides - Z-Phe Carbobenzoxy-L-phenylalanine - Z-Met Carbobenzoxy-L-methionine - BzTyr -N-benzoyl-L-tyrosine - AlaVal L-alanyl-L-valine - ValAla L-valyl-L-alanine     

Osmoregulation and the control of phloem-sap composition in Ricinus communis L.

Phloem-sap composition was studied in plants of Ricinus communis L. grown on a waterculture medium. The sap possessed a relatively high K⁺:Na⁺ ratio and low levels of Ca²⁺ and free H⁺. Sucrose and K⁺ (together with its associated anions) accounted for 75% of the phloem-sap solute potential (_s). In plants kept in continuous darkness, a decrease in phloem-sap sucrose levels over 24h was accompanied by an increase in K⁺ levels. Measurements of phloem-sap _s and xylem water potential () indicated that this resulted in a partial maintenance of phloem turgor pressure _p. In darkness there was also a marked decrease in the malate content of the leaf tissue, and it is possible that organic carbon from this source was mobilized for export in the phloem. The results support the concept of the phloem sap as a symplastic phase. We interpret the increase in K⁺ levels in the phloem in darkness as an osmoregulatory response to conditions of restricted solute availability. This reponse can be explained on the basis of the sucrose-H⁺ co-transport mechanism of phloem loading.Abbreviations water potential - _s solute potential - _p pressure potential     

Formation of the imidazolides of dinucleotides under potentially prebiotic conditions

Summary Imidazolides of dinucleotides such as ImpApA can be formed from the corresponding dinucleotides in a two-stage process, which gives up to 15% yields under potentially prebiotic conditions. First a solution of the dinucleotide and sodium trimetaphosphate is dried out at constant temperature and humidity. This produces polyphosphates such as pnApA in excellent yield (80%). The products are dissolved in water, imidazole is added, and the solution is dried out again. This yields the 5-phosphorimidazolides.Abbreviations P₃! trimetaphosphate - A adenosine - U uridine - EDTA ethylenediaminetetraacetic acid - Ap adenosine 2(3)-phosphate - Ap! adenosine cyclic 2:3-phosphate - pA adenosine 5-phosphate - pA²p adenosine 2, 5-diphosphate - pA³p adenosine 3, 5-diphosphate - pAp! 5-phospho-adenosine cyclic 2:3-phosphate - ATP adenosine 5-triphosphate - ImpA adenosine 5-phosphorimidazolide - A²pA adenylyl-[25]-adenosine - A³pA adenylyl-[35]-adenosine - A²pU adenylyl-[25]-uridine - A³pU adenylyl-[35]-uridine - pA²pA 5-phosphoadenylyl-[25]-adenosine - pA³pA 5-phospho-adenylyl-[35]-adenosine - pA²pU 5-phospho-adenylyl-[25]-uridine - pA³pU 5-phospho-adenylyl-[35]-uridine - pApN (N= A, U) 5-phosphate of a dinucleoside phosphate - p_nApN (N = A, U; n = 2, 3, 4.) 5-polyphosphate of a dinucleoside phosphate - ImpA²pA imidazolide of pA²pA - ImpA³pA imidazolide of pA³pA - ImpA²pU imidazolide of pA²pU - ImpA³pU imidazolide of pA³pU - ImpApN imidazolide of pApN     

Chitin synthetase mutants of Phycomyces blakesleeanus

Mutants resistant to nikkomycin, an inhibitor of chitin biosynthesis, were isolated after exposure of wild-type spores of the fungus Phycomyces blakesleeanus to N-methyl-N-nitro-N-nitrosoguanidine. Genetic analysis revealed that nikkomycin resistance was due to mutations in a single gene, chsA. Mutants and wild type grew equally well in the absence of nikkomycin. In contrast to the wild type, whose spore germination and mycelial growth were inhibited by 5 M nikkomycin, chsA mutants grew reasonably well in the presence of 50 M nikkomycin. Chitin synthesis in vivo was much less affected by the drug in the mutants than in the wild type. Resistance was not due to impaired uptake or detoxification of the drug. Analysis of the kinetics of chitin synthesis in vitro showed that the mutants had a decreased K_a for the allosteric activator, N-acetylglucosamine, and gross alterations in nikkomycin inhibition kinetics. These results indicate that chsA is the structural gene for chitin synthetase, or at least for the polypeptide that bears the catalytic and allosteric sites.     

The Cytochrome cbo from the Obligate Methylotroph Methylobacillus flagellatus KT Is a Cytochrome c Oxidase

The cbo-type oxidase of Methylobacillus flagellatus KT was purified to homogeneity by preparative native gel electrophoresis, and the kinetic properties and substrate specificity of the enzyme were studied. Ascorbate and ascorbate/N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) were oxidized by cytochrome cbo with a pH optimum of 8.3. With TMPD as an electron donor for the cbo-type oxidase, the optimal pH (7.0 to 7.6) was determined from the difference between respiration rates in the presence of ascorbate/TMPD and only ascorbate. The kinetic constants determined at pH 7.0 were as follows: oxidation by the enzyme of reduced TMPD was characterized by K _M = 0.86 mM and V _max = 1.1 mol O₂/(min mg protein), and oxidation of reduced horse heart cytochrome c was characterized by K _M = 0.09 mM and V _max = 0.9 mol O₂/(min mg protein). Cyanide inhibited ascorbate/TMPD–oxidase activity (K _i = 4.5–5.0 M). The soluble cytochrome c _H (12 kDa), partially purified from M. flagellatus KT, was found to serve as a natural electron donor for the cbo-type oxidase.     

The kinetics and feedback inhibition of cytidine 5′-triphosphate synthetase in wild-type and mutant Chinese hamster cells

The kinetics and cytidine 5-triphosphate (CTP) feedback inhibition of CTP synthetase in wild-type and four mutants of Chinese hamster V79 cells have been studied. The enzymes of the wild type and three of the four mutants exhibited positive cooperativity with the substrate uridine 5-triphosphate (UTP). Three of the mutants had K _{m app} and S ₅₀ valuves distinctly greater than those of the wild type, while the fourth mutant had values similar to those of the wild type. all four mutants exhibited resistance to CTP feedback inhibition, while the wild type was sensitive to such inhibition. It is postulated that a single mutational event in each mutant had caused a concomitant change of the enzyme in its binding both to the substrate UTP and to the end-product CTP.This work was supported by Grant GM 20608 from the U.S. Public Health Service.     

Characterization of a Na∶K∶2Cl cotransport system in the apical membrane of a renal epithelial cell line (LLC-PK1)

Summary ⁸⁶Rb uptake into LLC-PK₁ cells (an established renal epithelial cell line) was found to be comprised of an active ouabain-sensitive component, a loop diuretic-sensitive component which was passive and strictly dependent upon the presence of extracellular Na⁺ and Cl^– for activity, and a leak component. The diuretic-sensitive component of influx was investigated further in apical membrane vesicles derived from these cells. A large fraction of⁸⁶Rb,²²Na and³⁶Cl flux into these vesicles was sensitive to inhibition by furosemide and dependent upon the presence of the other two co-ions, in keeping with the presence of a loop diuretic-sensitive Na⁺K⁺Cl^– cotransport system. The kinetic parameters for Na⁺ and K⁺ interaction have been analyzed under initial linear zerotrans conditions. The following values were obtained:K _mNa+=0.42±0.05 mmol/liter,V _max=303±24 pmol/mg/6 sec;K _mK+=11.9±1.0 mmol/liter,V _maxK+=307±27 pmol/mg/6 sec. For Cl^– interaction evidence for two cooperative binding sites with different affinities and different specificities were obtained. Thus, a stoichiometry of 1Na⁺1K⁺2Cl^– can be calculated. It is concluded that the apical membrane of LLC-PK₁ cells contains a Na⁺K⁺2Cl^– cotransport system with properties similar to those described for the thick ascending limb of the loop of Henle.     

Ion Regulation of the Kinetics of Potential-Dependent Potassium Channels

We apply a theoretical approach developed earlier. The interaction ofions that permeate a channel with slowly relaxing charged channel-forminggroups (ion-conformational interaction – ICI) is addressed by thisapproach. One can describe the ion concentration influence (ion regulation)on channel functioning in this manner. A patch-clamp method in awhole-cell configuration is used to study the ICI. For this purpose theinfluence of an external concentration of potassium ions on thepotential-dependent potassium current (I_A) in the externalmembrane of GH₃ cells was studied. The increase of[K⁺ _out] from 5 mM to 100 mM causes anon-monotonous shift of current-voltage dependencies. The dependence of bothan activation time constant tgr_n and a steady-state activation(n) on [K⁺]_out have a minimum andmaximum respectively. The analysis of the results suggests that the observedeffects are caused by ICI. A physical model is developed to describe thedependence of the potassium channel kinetics on the external concentrationof the ions and the membrane potential. The deformation of the closedstate of the gate and the corresponding energy shifts cause the observednon-monotonous dependencies due to ICI. Thus, the general theoreticalapproach has an experimental confirmation and is applied to concreteexamples. Formulas for concentrational dependencies of the channel kineticsare given for practical uses.     

Cyclic AMP regulation of glucose transport in germinating Pilobolus longipes spores

Pilobolus longipes spores were activated by either glucose or 6-deoxyglucose. Glucose-induced spore activation was previously shown to follow an increase in intracellular cyclic AMP. Concurrent with glucose-induced spore activation, were shifts in 6-deoxyglucose transport kinetics towards higher V _max and K _m values. Cyclic AMP derivatives also caused spore activation and similar changes in the kinetic parameters of 6-deoxyglucose transport. The time course of activation was paralleled by changes in transport activity. Inhibition of phosphodiesterase alone did not cause activation or induce changes in transport activity, but in combination with sub-optimal levels of either 6-deoxyglucose or cAMP derivatives, it amplified the germination signals to produce large increases in both spore activation and 6-deoxyglucose transport activity. These results support the conclusion that glucose transport in germinating spores is regulated by cAMP.Abbreviations IBMX 3-isobutyl-1-methylxanthine; monobutyryl cyclic AMP - N⁶ monobutyryladenosine 3:5-cyclic monophosphate - 8-bromo cyclic AMP 8-bromoadenosine 3:5-cyclic monophosphate     

Electrical characteristics of stomatal guard cells: The ionic basis of the membrane potential and the consequence of potassium chlorides leakage from microelectrodes

The membrane electrical characteristics of stomatal guard cells in epidermal strips from Vicia faba L. and Commelina communis L. were explored using conventional electrophysiological methods, but with double-barrelled microelectrodes containing dilute electrolyte solutions. When electrodes were filled with the customary 1–3 M KCl solutions, membrane potentials and resistances were low, typically decaying over 2–5 min to near-30 mV and <0.2 k·cm² in cells bathed in 0.1 mM KCl and 1 mM Ca²⁺, pH 7.4. By contrast, cells impaled with electrodes containing 50 or 200 mM K⁺-acetate gave values of-182±7 mV and 16±2 k·cm² (input resistances 0.8–3.1 G, n=54). Potentials as high as (-) 282 mV (inside negative) were recorded, and impalement were held for up to 2 h without appreciable decline in either membrane parameter. Comparison of results obtained with several electrolytes indicated that Cl^- leakage from the microelectrode was primarily responsible for the decline in potential and resistance recorded with the molar KCl electrolytes. Guard cells loaded with salt from the electrodes also acquired marked potential and conductance responses to external Ca²⁺, which are tentatively ascribed to a K⁺ conductance (channel) at the guard cell plasma membrane.Measurements using dilute K⁺-acetate-filled electrodes revealed, in the guard cells, electrical properties common to plant and fungal cell membranes. The cells showed a high selectivity for K⁺ over Na⁺ (permeability ratio P_Na/P_K=0.006) and a near-Nernstian potential response to external pH over the range 4.5–7.4 (apparent P_H/P_K=500–600). Little response to external Ca²⁺ was observed, and the cells were virtually insensitive to CO₂. These results are discussed in the context of primary, charge-carrying transport at the guard cell plasma membrane, and with reference to possible mechanisms for K⁺ transport during stomatal movements. They discount previous notions of Ca²⁺-and CO₂-mediated transport control. It is argued, also, that passive (diffusional) mechanisms are unlikely to contribute to K⁺ uptake during stomatal opening, despite membrane potentials which, under certain, well-defined conditions, lie negative of the potassium equilibrium potential likely prevailing.Abbreviations and symbols EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - Mes 2-(N-morpholino) propanesulfornic acid - E equilibrium potential - G_m membrane conductance - R_in input resistance - V_m membrane potential     

Fed-batch culture of hybridoma cells: comparison of optimal control approach and closed loop strategies

Control of fed-batch culture of hybridoma cells was investigated based on two approaches optimal control theory and feedback control. Experiments were conducted for both approaches-with a feed enriched in glutamine. The optimal feed trajectory, a decreasing one, yielded a final monoclonal antibody (MAb) concentration of 170 mg/l, a three-fold increase compared to a typical batch operation.The feedback strategy relied on the on-line estimation of the net specific growth rate of cells from the measurement of the CO₂ production rate with a mass-spectrometer. A PI controller was then used to maintain the growth rate at a desired value by adjusting the dilution rate to the reactor. For the chosen set-point (0.1 d^–1), the final MAb concentration achieved was about 100 mg/1. It was found that there was a delay in the assimilation of the glutamine that should be included in the model to explain the lower MAb production in feedback mode. A higher production can be expected also for a lower set-point in feedback operation.List of Symbols Amm mM ammonia concentration - CPR l/(ld) carbon dioxide production rate - D _t l/d dilution rate - e _t l/d control error - F L/d feed flow rate - Glc mM glucose concentration - Gln mM glutamine concentration - Lac mM lactate concentration - I mg performance index - k _d l/d specific death rate - K _damm l/(mM · d) kinetic parameter for death rate - K _dgln mM kinetic parameter for death rate - K _dlac l/(mM·d) kinetic parameter for death rate - K _c l controller gain - K _glc mM kinetic parameter for growth rate - K _gln mM kinetic parameter for growth rate - K _tr L/(cell·d) transport coefficient - K l/d kinetic parameter for Mab production - m _glc mM/(cell·d) maintenance coefficient - M _Ab mg/l monoclonal antibody concentration - P _t covariance matrix - q _glc l/(l·cell·d) specific CO₂ production rate - q _glc mM/(cell·d) specific glucose uptake rate - q _gln mM/(cell·d) specific glutamine uptake rate - q _Mab mg/(l·cell·d) specific monoclonal antibody production - t _f d final culture time - T d sampling rate - u control input - V l reactor volume - X cell/l total cells concentration - X _v cell/l viable cells concentration - Y yield coefficient Greek mg/cell variable yield coefficient - ₀ mg/(cell·d) growth-associated kinetic parameter - mg/(cell·d) non growth-associated kinetic parameter - _t+1 defined by Eq. (19) - forgetting factor - l/d specific growth rate - _max l/d specific growth rate - _i d controller integral time constant     

Integral kinetics of the cleavage of maltose catalysed by α-glucosidase fromSaccharomyces carlsbergensis

Summary A new, sensitive and continuous assay for -glucosidase is described exploiting the different angles of rotation for the substrate maltose and the product glucose. Kinetic experiments revealed a very pronounced product inhibition of -glucosidase fromSaccharomyces carlsbergensis with a K_i of 4.85·10^–3 M for glucose.The K_M of maltose was found to be 37.8·10^–3 M. Taking these values, an integral kinetic curve for the enzymatic hydrolysis of maltose was calculated, which is shown to fit the experimental data.Symbols used k₁ (min^–1) pseudo first-order rate constant (for enzymatic cleavage) - k₂ (min^–1) rate constant (for mutarotation reaction) - I, P (mol/1) inhibitor (product) concentration - k_i (mmol/1) inhibitor constant - K_M (mmol/l) Michaelis constant - [M] ₅₈₉ ³⁰ (degree/m · l/mol) molecular rotation at 30°C and 589 nm - s (mmol/l) substrate concentration - R (mmol/mg · min) reaction rate - V_max (mmol/mg · min) maximal rate - U (mol/min) activity unit (here at 30°C and pH=6.8) Indices O initial value - max maximal value