Determination of kinetic parameters of fermentation processes by a continuous unsteady-state method: application to the alcoholic fermentation of D-xylose by Pichia stipitis

A quick technique for determination of kinetic parameters of fermentation processes is proposed and applied to the transformation of D-xylose into ethanol by Pichia stipitis. The commonly used method to evaluate these parameters is based on achieving several steady states. In the proposed procedure, mu(m) and K(s) can be determined from only one steady state, by provoking a disturbance over it, after allowing the system to return to the original conditions. The main difference between the steady and unsteady state methods is the required fermentation time; while the former method lasted 350 h, the latter required a period 25 times lower. Kinetic and stoichiometric parameters were determined with both methods under anoxic and limited oxygen concentration conditions. Results from the two methods were compared, giving only 2% and 4.5% differences in the values of K(s) and mu(m) and a little over 4% for mu(m) were the deviations under the latter ones. (c) 1993 Wiley & Sons, Inc.     

Physiological changes ofCandida utilis in transient state during continuous cultivation

In a continuous culture ofCandida utilis, the air supply was interrupted for 15 min at 1-d intervals after the steady state had been reached. Analysis of morphology and physiology of the cell showed that after this intervention the indicators of the physiological state changed their values with different time delays and needed different times to resume their steady-state values. Another consequence of the interrupted aeration was a higher degree of the culture synchronization. The possibility to bring about a transient (i. e. unsteady) state offers a tool for a directed control of mutual proportion of intracellular components.     

Phenol biodegradation by Pseudomonas putida DSM 548 in a batch reactor

Phenol biodegradation in a batch reactor using a pure culture of Pseudomonas putida DSM 548 was studied. The purpose of the experiments was to determine the kinetics of biodegradation by measuring biomass growth rates and phenol concentration as a function of time in a batch reactor. The Haldane equation μ=μ(m)S/((K(s)+S+S(2))/K(i)) adequately describes cell growth with kinetic constants μ(m)=0.436h(-1), K(s)=6.19mgl(-1), K(i)=54.1mgl(-1). These values are in the range of those published in literature for pure or mixed cultures degrading phenol.     

Low-temperature stress induces transient oscillations in sucrose metabolism in Solanum tuberosum

Exposure to low but nonfreezing temperatures induces the net breakdown of starch and the accumulation of sucrose, glucose and fructose in potato tuber tissue, a complex phenomenon known as low-temperature sweetening (LTS). When transferred to 4 degrees C storage, tissue sucrose levels in LTS-sensitive potato tubers (Solanum tuberosum cv. Norchip) did not change monotonically to a new steady state, but rather transiently oscillated about the trajectory to the new steady state. The dynamic patterns observed in sensitive tubers grown in 1993 and 1994 were qualitatively similar. Quantitatively, however, the transient oscillation had a period of 11.5 days in 1993, whereas a period of 80 days was observed in 1994. In contrast, the sucrose levels of the LTS-tolerant potato tubers (Solanum tuberosum seedling ND860-2) increased monotonically to a higher level upon exposure to low temperatures.     

Evaluation of kinetic parameters of biochemical reaction in three-phase fluidized bed biofilm reactor for wastewater treatment

This study evaluates the kinetic parameters of biochemical reaction in three-phase fluidized bed biofilm reactor from the steady state values of the response of the system to step changes in inlet concentration. It was observed from the outlet biological oxygen demand (BOD(5)) plot of the response of the system that as the inlet BOD(5) was increased, the outlet BOD(5) also increased, reached a peak value and then decreased until it leveled to a new steady state value corresponding to the new inlet concentration level. The increase in BOD(5) was attributed to the accumulation of substrate within the reactor as well as the decrease in biofilm substrate consumption rate as the microorganisms adjusted to the new environment. Using the substrate balance at steady state and assuming Monod kinetics, an equation relating the substrate consumption rate to substrate concentration (BOD(5)) and total biofilm surface area had been established. Monod kinetic parameters were found to be K=2.20g/m(2)/day, K(m)=17.41g/m(3) and K/K(m)=0.13m/day. The ratio K/K(m) can be taken as the indicator for biofilm substrate degradation effectiveness at low substrate concentrations.     

噬菌体RB69 DNA聚合酶以不正确的核苷酸为底物的聚合反应的稳态动力学研究

测定了ＲＢ６９ ＤＮＡ 聚合酶以不正确的核苷酸（ｒＮＴＰ、ｄｄＮＴＰ以及碱基不配对的ｄＮＴＰ）为底物进行聚合反应的稳态动力学常数,并与Ｋｌｅｎｏｗ 酶进行了比较．结果表明,ＲＢ６９ ＤＮＡ 聚合酶在以不正确的核苷酸为底物进行聚合反应时,其Ｋｍ 值与正确底物参入时相比有大幅度提高,而ｋｃａｔ保持不变或下降幅度较小．而Ｋｌｅｎｏｗ 酶在利用不正确的核苷酸为底物时,与正确底物参入时相比,其ｋｃａｔ大幅度下降,而Ｋｍ （或ＫＤ）基本保持不变或上升较小幅度．两种酶不同的动力学特点反映出它们不同的底物选择机制．     

Nonfixed relationship of the Michaelis constant and maximum velocity with their corresponding rate constants

The Michaelis constant (K(m)) and V(mas) (E0k(cat)) values for two mutant sets of enzymes were studied from the viewpoint of their definition in a rapid equilibrium reaction model and in a steady state reaction model. The "AMP set enzyme" had a mutation at the AMP-binding site (Y95F, V67I, and V67I/L76V), and the "ATP set enzyme" had a mutation at a possible ATP-binding region (Y32F, Y34F, and Y32A/Y34A). Reaction rate constants obtained using steady state model analysis explained discrepancies found by the rapid equilibrium model analysis. (i) The unchanged number of bound AMPs for Y95F and the wild type despite the markedly increased K(m) values for AMP of the AMP set of enzymes was explained by alteration of the rate constants of the AMP step (k(+2), k(-2)) to retain the ratio k(+2)/k(-2). (ii) A 100 times weakened selectivity of ATP for Y34F in contrast to no marked changes in K(m) values for both ATP and AMP for the ATP set of enzymes was explained by the alteration of the rate constants of the ATP steps. A similar alteration of the K(m) and k(cat) values of these enzymes resulted from distinctive alterations of their rate constants. The pattern of alteration was highly suggestive. The most interesting finding was that the rate constants that decided the K(m) and k(cat) values were replaced by the mutation, and the simple relationships between K(m), k(cat), and the rate constants of K(m)1 = k(+1)/k(-1) and k(cat) = k(f) were not valid. The nature of the K(m) and k(cat) alterations was discussed.     

Oxidation of an inhibitory substrate by washed cells (oxidation of phenol by Pseudomonas putida)

The specific uptake rate of phenol by washed cells of Pseudomonas putida grown on phenol in steady-state continuous culture at various dilution rates was studied. The Monod-Haldane-type equation was applied to fit the data and the best kinetic parameters were determined by nonlinear least-squares techniques. The values of the kinetic parameters were found to increase monotonically with the phenol concentration in the original chemostat. The relations between the values of kinetic parameters and phenol concentration in the chemostat were described by empirical equations. Then the equation governing the instant uptake of phenol by microorganisms in chemostat in the high conversion range of phenol was proposed. This equation together with the mass balance equations can be used to determine the stability range of continuous stirred tank biochemical reactors (CSTBR) utilizing phenol.     

Volume regulatory activity of the Ehrlich ascites tumor cell and its relationship to ion transport

The volume regulatory response of the Ehrlich ascites tumor was studied in KCl-depleted, Na+-enriched cells. Subsequent incubation in K+-containing NaCl medium results in the reaccumulation of K+, Cl-, water and the extrusion of Na+. The establishment of the physiological steady state is due primarily to the activity of 2 transport systems. One is the Na/K pump (KM for K+o = 3.5 mM; Jmax = 30.1 mEq/kg dry min), which in these experiments was coupled 1K+/1 Na+. The second is the Cl--dependent (Na+ + K+) cotransport system (KM for K+o = 6.8 mM; Jmax = 20.8 mEq/kg dry min) which mediates, in addition to net ion uptake in the ratio of 1K+:1Na+:2Cl-, the exchange of K+i for K+o. The net passive driving force on the cotransport system is initially inwardly directed but does not decrease to zero at the steady state. This raises the possibility of the involvement of an additional source of energy. Although cell volume increases concomitant with net ion uptake, this change does not appear to be a major factor regulating the activity of the cotransport system.     

Role of Met-542 as a guide for the conformational changes of Phe-601 that occur during the reaction of β-galactosidase (Escherichia coli)

The Met-542 residue of β-galactosidase is important for the enzyme's activity because it acts as a guide for the movement of the benzyl side chain of Phe-601 between two stable positions. This movement occurs in concert with an important conformational change (open vs. closed) of an active site loop (residues 794-803). Phe-601 and Arg-599, which interact with each other via the π electrons of Phe-601 and the guanidium cation of Arg-599, move out of their normal positions and become disordered when Met-542 is replaced by an Ala residue because of the loss of the guide. Since the backbone carbonyl of Phe-601 is a ligand for Na(+), the Na(+) also moves out of its normal position and becomes disordered; the Na(+) binds about 120 times more poorly. In turn, two other Na(+) ligands, Asn-604 and Asp-201, become disordered. A substrate analog (IPTG) restored Arg-599, Phe-601, and Na(+) to their normal open-loop positions, whereas a transition state analog d-galactonolactone) restored them to their normal closed-loop positions. These compounds also restored order to Phe-601, Asn-604, Asp-201, and Na(+). Binding energy was, however, necessary to restore structure and order. The K(s) values of oNPG and pNPG and the competitive K(i) values of substrate analogs were 90-250 times higher than with native enzyme, whereas the competitive K(i) values of transition state analogs were ~3.5-10 times higher. Because of this, the E?S energy level is raised more than the E?transition state energy level and less activation energy is needed for galactosylation. The galactosylation rates (k?) of M542A-β-galactosidase therefore increase. However, the rate of degalactosylation (k?) decreased because the E?transition state complex is less stable.     

Electrical and mechanical activity of isoproterenol-damaged frog heart

The electrical and mechanical activities of myocardial strips from Rana pipiens were studied in the steady state at various stimulation frequencies and after a period of rest. The temperature of the bath was varied between + 12 and 35 degrees C. Normal myocardium was compared with that damaged by isoproterenol (ISO). The percentage change of action potential durations (APD50) and isometric force (P) was similar in ISO-damaged and control hearts at various bath temperatures with steady state stimulation rates between 20 and 100/min. At low stimulation rates (3-6/min) the prolongation of the action potential (AP) was more pronounced and the P-decrease was less in ISO-damaged myocardium compared to controls. These differences became more apparent at low bath temperatures. After 10 min of rest, APD50-90 was significantly prolonged in ISO-damaged heart and P was increased by a factor of 2 compared to controls (stimulation rate 20/min). Steady state values, on the other hand, were nearly the same in both groups. These findings are interpreted as indicating a temperature or ISO-dependent increase of electrogenic trans-sarcolemmal Ca2+-uptake during low frequency, or post-rest stimulation, either directly by an increase of the slow inward current (Isi), or indirectly by decreased K+-permeabilities.     

The effect of K+ concentration on the energy metabolism in perfused rat heart

From experiments at various perfusion pressures in hemoglobin-free perfused rat hearts, oxygen consumption and redox shift of pyridine nucleotide were found to vary linearly with cardiac work. This relation was used for analysis of the energy metabolism associated with ion pumps. Mechanical activities such as left ventricular pressure and heart rate varied with the extracellular K+ concentration. Ion-pump dependent changes in oxygen consumption and redox state of pyridine nucleotide, estimated as the difference of the values at normal (4.7 mM) and various other extracellular K+ concentrations with corrections for the change due to mechanical work, were found to vary linearly with the K+ concentration. The slope for oxygen consumption was about 0.1 mumol/min/g X wet wt per mM K+. Lactate release changed markedly but transiently, about 1 min after changing the extracellular K+ concentration, and its amount varied linearly with the K+ concentration. In the steady state, however, lactate release was almost independent of the extracellular K+ concentration, although oxidized pyridine nucleotide increased with increasing K+ concentration. Coronary flow increased with the extracellular K+ concentration. Heart rate changed little between 1 and 12 mM K+, but decreased sharply above 12 mM K+. At 20 mM K+, heart beat was arrested and approximately 40% of myoglobin was deoxygenated. The intracellular oxygen concentration was estimated to be about 10 microM even during aerobic perfusion. Similarly, Ca2+-free arrested heart was found to be in a hypoxic state. The results showed that oxygen entry into cardiac tissue is facilitated by the cardiac cycle.     

Influencing effect of intra-granule mass transfer in expanded granular sludge-bed reactors treating an inhibitory substrate

Two expanded granular sludge-bed (EGSB) reactors (superficial velocity u s=6.0 and 9.0m/h) were used to treat an inhibitory substrate phenol. The granule diameter (dp) increased with increasing organic loading rate (OLR) and u(s). At the OLRs of 1.67-4.44 kg phenol/m3 d, the accumulation of volatile fatty acids (VFAs) was insignificant; whereas at the OLR of 5.11 kg phenol/m3 d, both the accumulation of VFAs and the washout of large hollow granules (average dp=2.90-3.12 mm) occurred. The comparative experimental and simulated results showed that the proposed kinetic model is suitable for design and predicting purposes. The calculation results of mass transfer parameters (Thiele modulus, Biot number, diffusion layer thickness, and overall effectiveness factor) and parametric sensitivity analysis results (half-saturation constant Ks and dp) showed that the intra-granule mass transfer would lead to a more influencing effect than the external mass transfer on the overall substrate removal rate in EGSB reactors.     

Kinetics of turnover of cefotaxime by the Enterobacter cloacae P99 and GCl beta-lactamases: two free enzyme forms of the P99 beta-lactamase detected by a combination of pre- and post-steady state kinetics

Third-generation cephalosporins bearing oximino side chains are resistant to hydrolysis by class C beta-lactamases such as that from Enterobacter cloacae P99. For example, steady state parameters for hydrolysis of cefotaxime by this enzyme are as follows: k(cat) = 0.41 s(-1), K(m) = 17.2 microM, and k(cat)/K(m) = 2.3 x 10(4) s(-1) M(-1). On the other hand, however, the K(i) value for cefotaxime as an inhibitor of cephalothin hydrolysis is 27 nM. The discrepancy between K(m) and K(i) indicated that a real steady state had not been achieved in at least one of these experiments. Analysis indicated that only two to three cefotaxime turnovers occurred during the K(i) determination. This suggested that the first few turnovers of cefotaxime by the P99 beta-lactamase may be different from those in the subsequent steady state. A direct pre-steady state experiment confirmed this hypothesis. The simplest reaction scheme that fitted the data involved replacement of the initial enzyme form, E, which bound cefotaxime tightly, with a second more weakly binding form, E', by partitioning of the acyl-enzyme intermediate during the first few turnovers. Steady state turnover of cefotaxime then largely involved E' as the free enzyme form. E' slowly reverted to E in the post-steady state regime. Further evidence for this scheme included quantitative analysis of the post-steady state and observation of a difference in the catalytic activity of E and E' in 2 M ammonium sulfate. The kinetics of P99 beta-lactamase-catalyzed hydrolysis of an acyclic depsipeptide substrate bearing a third-generation cephalosporin side chain showed that the side chain is necessary but not sufficient for production of resistance to beta-lactamase; a combination of the side chain and the dihydrothiazine ring of a cephalosporin is required. The beta-lactamase of E. cloacae GC1, an extended spectrum mutant of the P99 enzyme, rapidly hydrolyzes third-generation cephalosporins, without the structural transition described above. The flexibility of the extended Omega loop of the GC1 enzyme probably leads to this situation. Conformational restriction of the loop in the P99 enzyme is probably responsible for the long-lived acyl-enzyme intermediate and the transition to E' induced by cefotaxime.     

Immobilization of tyrosinase on chitosan-clay composite beads

Tyrosinase was immobilized on glutaraldehyde crosslinked chitosan-clay composite beads and used for phenol removal. Immobilization yield, loading efficiency and activity of tyrosinase immobilized beads were found as 67%, 25% and 1400 U/g beads respectively. Optimum pH of the free and immobilized enzyme was found as pH 7.0. Optimum temperature of the free and immobilized enzyme was determined as 25-30 °C and 25 °C respectively. The kinetic parameters of free and immobilized tyrosinase were calculated using l-catechol as a substrate and K(m) value for free and immobilized tyrosinase were found as 0.93 mM and 1.7 mM respectively. After seven times of repeated tests, each over 150 min, the efficiency of phenol removal using same immobilized tyrosinase beads were decreased to 43%.     

Kinetic characterisation of the light-driven protochlorophyllide oxidoreductase (POR) from Thermosynechococcus elongatus.

The light-driven enzyme NADPH:protochlorophyllide oxidoreductase (POR) catalyses the reduction of the C17-C18 double bond of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), which is a key regulatory step in the chlorophyll biosynthesis pathway. POR from the thermophilic cyanobacterium Thermosynechococcus elongatus is an attractive system for following the reaction and in the present work we have carried out a detailed steady state kinetic characterisation of this enzyme. The thermophilic POR was shown to have maximal activity at approximately 50 degrees C, which is similar to the growth temperature of the organism. The V(max) was calculated to be 0.53 microM min(-1) and the K(m) values for NADPH and Pchlide were 0.013 microM and 1.8 microM, respectively. The binding properties for both substrates as well as the NADP(+) product have been analysed by using fluorescence emission measurements, which have allowed the dissociation constants for binding to be calculated. These results represent the first steady state kinetic characterisation of a thermophilic version of POR.     

胆碱脱氢酶的动力学性质

本文对增溶胆碱脱氢酶的稳态初速度及产物抑制动力学做了。底物胆碱和ＰＭＳ的相互影响：变化一个底物的浓度,另一个底物的Ｋｍ及Ｖｍａｘ均变化。该酶的产物三甲胺 地 制表现为对底物胆碱非竞争性而地ＰＭＳ竞争性,在胆碱饱和的情况下,三甲胺乙醛对酶的抑制仍表现为对ＰＭＳ竞争性。这些结果表明增溶胆碱脱氢酶的催化机制搂双底物双产物乒乓机制。１－ＰＣ与９－ＡＣ对增溶胆碱脱氢酶均有抑制作用,且均为混和型抑制,Ｋ１分别     

Chemical and steady-state kinetic analyses of a heterologously expressed heme dependent chlorite dismutase

Chlorite dismutase carries out the heme-catalyzed decomposition of ClO2- to Cl- and O2, an unusual transformation with biotechnological and bioremediative applications. The enzyme has been successfully overexpressed for the first time in highly functional form in Escherichia coli and its steady state kinetics studied. The purified enzyme is abundant (55 mg/L cell culture), highly active (approximately 4.7 x 10(3) micromol of ClO2- min(-1) mg(-1) subunit) and nearly stoichiometric in heme; further, it shares spectroscopic and physicochemical features with chlorite dismutases previously isolated from three organisms. A careful study of the enzyme's steady state kinetics has been carried out. ClO2- consumption and O2 release rates were measured, yielding comparable values of kcat (4.5 x 10(5) min(-1)), K(m) (approximately 215 microM), and kcat/Km (3.5 x 10(7) M(-1) s(-1) via either method (4 degrees C, pH 6.8; all values referenced per heme-containing subunit). ClO2-:O2 stoichiometry exhibited a 1:1 relationship under all conditions measured. Though the value of kcat/Km indicates near diffusion control of the reaction, viscosogens had no effect on k(cat)/K(m) or V(max). The product O2 did not inhibit the reaction at saturating [O2], but Cl- is a mixed inhibitor with relatively high values of KI (225 mM for enzyme and 95.6 mM for the enzyme-substrate complex), indicating a relatively low affinity of the heme iron for halogen ions. Chlorite irreversibly inactivates the enzyme after approximately 1.7 x 10(4) turnovers (per heme) and with a half-life of 0.39 min, resulting in bleaching of the heme chromophore. The inactivation K(I) (K(inact)) of 166 microM is similar in magnitude to Km, consistent with a common Michaelis complex on the pathway to both reaction and inactivation. The one-electron peroxidase substrate guaiacol offers incomplete protection of the enzyme from inactivation. Mechanisms in keeping with the available data and the properties of other well-described heme enzymes are proposed.     

The conformational stability of the Streptomyces coelicolor histidine-phosphocarrier protein. Characterization of cold denaturation and urea-protein interactions.

Thermodynamic parameters describing the conformational stability of the histidine-containing phosphocarrier protein from Streptomyces coelicolor, scHPr, have been determined by steady-state fluorescence measurements of isothermal urea-denaturations, differential scanning calorimetry at different guanidinium hydrochloride concentrations and, independently, by far-UV circular dichroism measurements of isothermal urea-denaturations, and thermal denaturations at fixed urea concentrations. The equilibrium unfolding transitions are described adequately by the two-state model and they validate the linear free-energy extrapolation model, over the large temperature range explored, and the urea concentrations used. At moderate urea concentrations (from 2 to 3 m), scHPr undergoes both high- and low-temperature unfolding. The free-energy stability curves have been obtained for the whole temperature range and values of the thermodynamic parameters governing the heat- and cold-denaturation processes have been obtained. Cold-denaturation of the protein is the result of the combination of an unusually high heat capacity change (1.4 +/- 0.3 kcal.mol(-1).K(-1), at 0 m urea, being the average of the fluorescence, circular dichroism and differential scanning calorimetry measurements) and a fairly low enthalpy change upon unfolding at the midpoint temperature of heat-denaturation (59 +/- 4 kcal.mol(-1), the average of the fluorescence, circular dichroism and differential scanning calorimetry measurements). The changes in enthalpy (m(DeltaH(i) )), entropy (m(DeltaS(i) )) and heat capacity (m(DeltaC(pi) )), which occur upon preferential urea binding to the unfolded state vs. the folded state of the protein, have also been determined. The m(DeltaH(i) ) and the m(DeltaS(i) ) are negative at low temperatures, but as the temperature is increased, m(DeltaH(i) ) makes a less favourable contribution than m(DeltaS(i) ) to the change in free energy upon urea binding. The m(DeltaC(pi) ) is larger than those observed for other proteins; however, its contribution to the global heat capacity change upon unfolding is small.     

Disposition of methadone in the ovine maternal-fetal unit

The distribution of methadone between mother and fetus after a single dose and at steady state was determined using the chronic pregnant ewe preparation. Chronic indwelling catheters were placed in the maternal aorta and vena cava, umbilical vein and fetal aorta. Following a single i.v. dose (0.5 mg/kg) to the mother, methadone was rapidly distributed to the fetus, with peak concentration in the umbilical vein occurring within two min. An umbilical venous-arterial gradient existed for 10–15 min after drug administration, indicating uptake of methadone by fetal tissues. Methadone concentration in the fetus was 2–5 times lower than those in the mother even in the post-distribution phase. The terminal half-life of methadone in 4 animals was 57±7.6 (S.E.) min in the mother, and 58.5±10.0 (S.E.) min in the fetus. When methadone was infused at a constant rate to the mother (0.01 mg/kg/min), steady state was achieved in both mother and fetus by 4–5 hrs. In 5 animals, maternal steady state was found to be 203±18.8 (S.E.) ng/ml, and fetal steady state was found to be 29.7±2.9 (S.E.) ng/ml. These studies show that methadone is rapidly distributed to the fetus, but fetal concentration remain lower than maternal concentration at all times.