Intrinsic kinetic parameters of substrate utilization by immobilized anaerobic sludge

This article presents a method for evaluating the intrinsic kinetic parameters of the specific substrate utilization rate (r) equation and discusses the results obtained for anaerobic sludge-bed samples taken from a horizontal-flow anaerobic immobilized sludge (HAIS) reactor. This method utilizes a differential reactor filled with polyurethane foam matrices containing immobilized anaerobic sludge which is subjected to a range of feeding substrate flow rates. The range of liquid superficial velocities thus obtained are used for generating data of observed specific substrate utilization rates (r(obs)) under a diversity of external mass transfer resistance conditions. The r(obs) curves are then adjusted to permit their extrapolation for the condition of no external mass transfer resistance, and the values determined are used as a test for the condition of absence of limitation of internal mass transfer. The intrinsic parameters r(max), the maximum specific substrate utilization rate, and K(s), the half-velocity coefficient, are evaluated from the r values under no external mass transfer resistance and no internal mass transfer limitation. The application of such a method for anaerobic sludge immobilized in polyurethane foam particles treating a glucose substrate at 30 degrees C resulted in intrinsic r(max) and K(s), respectively, of 0.330 mg chemical oxygen demand (COD) . mg(-1) volatile suspended solids (VSS) . h(-1) and 72 mg COD . L(-1). In comparison with the values found in the literature, intrinsic r(max) is significantly high and intrinsic K(s) is relatively low. (c) 1997 John Wiley & Sons, Inc.     

Evaluation of true kinetic parameters for reversible immobilized enzyme reactions

For a reversible one-substrate reaction system that follows the Haldane reaction mechanism, a new and effective method has been proposed to extract true or intrinsic kinetic parameters of immobilized enzymes from diffusion limited rate data. The method utilizes the effectiveness factors correlated in terms of the general modulus defined by Aris and Bischoff, and a new modulus defined in the present study. It requires a trial-and-error calculation, but only a few data points. Furthermore, it provides a saving of materials such as substrates and enzymes, and takes less time for experiments compared to the initial rate methods. The usefulness of the method is demonstrated by determining the kinetic parameters for membrane bound fumarase which catalyzes the reaction of the conversion of fumarate to L-malate, for which the equilibrium constant is ca. 4.     

Enzyme immobilized in a packed-bed reactor: kinetic parameters and mass transfer effects.

To describe axial dispersion, particle film mass transfer, intraparticle diffusion, and the chemical reaction of the substrate for enzymes immobilized in porous particles in packed columns, we have developed mathematical models for first- and zero-order limits of Michaelis-Menten kinetics. Steady-state solutions were derived for both long and short column boundary conditions and for plug flow. Theory was compared to experiments by hydrolysis of sucrose catalyzed by invertase bound to porous glass particles. Steady-state conversions were measured for a range of flow rates. Pulse response experiments with inert packing were used to determine values of bed void fraction and particle porosity.     

Evaluation of intrinsic immobilized kinetic parameters for factor X activation in a flow reactor

Summary Development of a double exponential model for determining the intrinsic kinetic parameters for factor X activation by tissue factor-factor VIIa (TF:VIIa) complex, during the complete course of the reaction in a flow reactor is described. The model data reveal that the factor X activation rate constant K₁ gradually increased from 0.0225 to 0.0456 min^-1 as the shear rate increased from 50 to 3000 sec^-1, whereas the factor Xa inhibition rate constant K₂ increased dramatically from 0.307 to 1.09 min^-1 for a similar increase in shear rate.     

Monitoring electron transfer by photoacoustic spectroscopy in native and immobilized thylakoid membranes

Photoacoustic spectroscopy was used to monitor photo synthetic electron transfer in native and immobilized thylakoid membranes. The photoacoustic parameter phi(r)' (the percentage of absorbed energy that is stored in photo chemical intermediates) and i(50) (the half-saturation modulated light intensity) were directly correlated to electron transfer rates. As previously shown, thylakoids immobilized in an albumin-glutaraldehyde matrix were more resistant to aging. The inhibitory effects of the immobilization procedure and of aging at 4 degrees C were detected as a decrease in i(50) values. In analogy with enzyme kinetic analysis, the effect could be characterized as a competitive type of inhibition. Photoacoustic measurements are performed in conditions similar to a working bioreactor cell with regards to the sample preparation.     

Diffusional and electrostatic effects on apparent kinetic parameters of reactions catalyzed by enzyme immobilized on the external surface of a support

Diffusional and electrostatic effects on the apparent maximum reaction rate V_m^app and the apparent Michaelis constant K_m^app were investigated theoretically for a system in which an enzyme immobilized on the external surface of a solid support catalyzes a reaction according to Michaelis-Menten kinetics. In such a system, the dependence of V_m^app and K_m^app on the substrate concentration can be expressed analytically. When the support and substrate carry charges of the same sign, resulting in a repulsive force between them, both V_m^app and K_m^app decrease with increasing substrate concentration, but they never decrease below the respective intrinsic values. On the other hand, when the support and substrate carry charges of opposite sign and therefore an attractive force occurs, V_m^app decreases towards its intrinsic value, while K_m^app decreases to values below its intrinsic value in the region of high substrate concentration.     

Effect of high substrate concentration and cell feedback on kinetic behavior of heterogeneous populations in completely mixed systems

Growth kinetics of heterogeneous populations of sewage origin were studied in completely mixed reactors of the once-through type at a high concentration of incoming substrate, 3000 mg/l glucose, and in systems employing cell feedback or sludge recycle at an incoming substrate concentration of 1000 mg/1 glucose. The recycle flow rate employed was 25% of the incoming feed flow, and the concentration of cells in the recycle was maintained as closely as possible at 150% of the cell concentration in the reactor. Studies were made at various dilution rates. Throughout these studies, batch experiments using cells grown at the various dilution rates were made to determine k_s and μ_m values. As in previous studios using heterogeneous populations, the relationship between specific growth rates μ and substrate concentration S was represented better by the Monod equation than by any other which was tested. The growth “constants” μ_m, k_s, and Y were found to fall in the same general range as those determined in previous studies in once-through systems operated at 1000 mg/l glucose. It was observed that cell recycle, even at the relatively low concentration factor employed in these studies, greatly enhanced the flocculating and settling characteristics of the cells.     

A flow method for determination of the kinetic parameters for immobilized enzymes.

A flow method is described for determination of the kinetics parameters (V-m and K-m) for enzymes that are bound to particles, to membranes, and to the interior surfaces of tubes. Substrate solution is pumped through Tygon tubing to a microvolume flow cell and back into the reaction mixture, the flow rate being adjusted to be faster than the rate of formation of product. To illustrate the technique, it is applied to the determination of the parameters for electric-eel acetylcholinesterase attached to particles, to membranes, and to the inner surface of nylon tubing.     

Global stability of two species systems

Summary Sufficient conditions for the global stability of equilibria in general, nonlinear, two-species model ecosystems are derived. These results contain and extend the theorem of Goh (1976), which is specific to linear per capita growth rates.     

New approaches for verification of kinetic parameters of immobilized concanavalin A: invertase preparations investigated by flow microcalorimetry

In our preceding article, we demonstrated a procedure based upon enzymic flow microcalorimetry using an enzyme thermistor (ET) to characterize the microkinetic properties of an immobilized enzyme (IME) and its further application in the screening of IMEs. To consider the ET method (single ET unit, ET system 1) as standard, it was necessary to show that the estimated relative kinetic parameter (DeltaT(max)) calorimetrically corresponds with the absolute value for the reaction rate within the whole measurement range. This article presents three experimental verification procedures. Two procedures are based on adaptation of the flow-through ET column to a mini-differential-reactor (DR) system with substrate recirculation and post-ET-column methods for determination of the concentration change of the product (spectrophotometrically in ET system 2) or the substrate (calorimetrically in ET system 3) with the IME-catalyzed enzymic hydrolysis. The third procedure is an independently operating DR system which spectrophotometrically estimates the concentration change of the product. The results obtained exhibited good correlation (r = 0.921) between the relative kinetic parameter DeltaT(max), as determined calorimetrically by ET system 1, and the absolute value for the reaction rate (r(max)) as determined by ET systems 2 and 3. These data proved that, within the whole range of experimental conditions applied in this study, the parameter DeltaT(max) instead of the true reaction rate could be employed for the IME screening. Moreover, the generality of the detection principle and the standardized configuration of the ET favor ET systems 2 and 3 for normal screening of IMEs and as miniaturized DR systems allowing dual measurements of kinetic parameters. (c) 1996 John Wiley & Sons, Inc.     

Association between plasma zinc concentration and zinc kinetic parameters in premenopausal women

The objective of this study was to measure relationships between plasma zinc (Zn) concentrations and Zn kinetic parameters and to measure relationships of Zn status with taste acuity, food frequency, and hair Zn in humans. The subjects were 33 premenopausal women not taking oral contraceptives and dietary supplements containing iron and Zn. Main outcomes were plasma Zn concentrations, Zn kinetic parameters based on the three-compartment mammillary model using 67Zn as a tracer, electrical taste detection thresholds, and food frequencies. Lower plasma Zn was significantly (P < 0.01) associated with smaller sizes of the central and the lesser peripheral Zn pools, faster disappearance of tracer from plasma, and higher transfer rate constants from the lesser peripheral pool to the central pool and from the central pool to the greater peripheral pool. The break points in the plasma Zn-Zn kinetics relationship were found between 9.94 and 11.5 micromol/l plasma Zn. Smaller size of the lesser peripheral pool was associated with lower frequency of beef consumption and higher frequency of bran breakfast cereal consumption. Hypozincemic women with plasma Zn <10.7 micromol/l or 700 ng/ml had decreased thresholds of electrical stimulation for gustatory nerves. Our results based on Zn kinetics support the conventional cutoff value of plasma Zn (10.7 micromol/l or 700 ng/ml) between normal and low Zn status.     

Comparative analysis of the kinetic parameters and thermal stability of two matrix metalloproteinases expressed in the developing sea urchin embryo

The extracellular matrix is now recognized as a dynamic structure which influences cellular properties. Many matrix metalloproteinase activities have been identified and characterized in vertebrates and constitute important agents in controlling the composition of the extracellular matrix. We have begun a study of matrix metalloproteinase activities in the developing sea urchin embryo. Using sea urchin peristome collagen or gelatin as physiological substrates we have determined the kinetic parameters, K_m and V_max, for an 87 kDa gelatinase activity expressed in late stage sea urchin embryos. We also determined the kinetic parameters K_m, V_max and k_cat, for a 41 kDa species, expressed in the early sea urchin embryo, which possesses both collagenase and gelatinase activities. All values determined were similar to those reported in the literature for vertebrate collagenases and gelatinases and K_m values in the micromolar range suggest that both species possess physiologically relevant activities. Both activities have previously been shown to require Ca²⁺ for activity. Using an assay for quantitating the cleavage of gelatin into trichloroacetic acid soluble peptides we report here markedly different effects of Ca²⁺ on the thermal denaturation profiles of the gelatinases. This latter finding may be indicative of different modes of action for this activating cation. Collectively, these results demonstrate both similarities and differences between vertebrate and invertebrate sea urchin gelatinases.     

On the use of apparent kinetic parameters for immobilized enzyme with noncompetitive substrate inhibition

The use of a simple rate equation with apparent parameters to describe the kinetic behavior of an immobilized enzyme with noncompetitive substrate inhibition was assessed. To do so, the reaction rate was calculated as a function of the interfacial substrate concentration, and the results were used to identify the apparent kinetic parameters by nonlinear regression. This procedure was repeated for different values of the diffusional constraints and of the inhibition constant. The equation using apparent parameters can describe the global kinetic behavior, provided that the diffusional and inhibitory constraints are not too high. When the constraints are high, a Michaelis-Menten equation can be used to model the kinetics for interfacial concentrations lower than the concentration leading to the maximum reaction rate.     

Modulation of lipase properties in macro-aqueous systems by controlled enzyme immobilization: enantioselective hydrolysis of a chiral ester by immobilized Pseudomonas lipase

Lipase from Pseudomonas fluorescens (PFL) has been immobilized by using different immobilization protocols. The catalytic behavior of the different PFL derivatives in the hydrolytic resolution of fully soluble (R,S) 2-hydroxy 4-phenyl butanoic acid ethyl ester (HPBE) in aqueous medium was analyzed. The soluble enzyme showed a significant but low enantioselectivity, hydrolyzing the S isomer more rapidly than the R-isomer (E = 7). The enzyme, immobilized via a limited attachment to a long and flexible spacer arm, showed almost identical activity and specificity to the soluble enzyme. However, other derivatives, e.g. PFL adsorbed on supports covered by hydrophobic moieties (octyl, decaoctyl), exhibited significant hyperactivation on immobilization (approximately 7-fold). Simultaneously, the enantioselectivity of the PFL-immobilized enzyme was significantly improved (from E = 7 to E = 80). By using such derivatives, almost pure R ester isomer (e.e. > 99%) has been obtained after 55% hydrolysis of the racemic mixture of a solution of 10% (w/v) (R,S) HPBE. The derivatives could be used for 10 cycles without any significant decrease in the activity of the biocatalyst.     

Processing, stability, and kinetic parameters of C5a peptidase from Streptococcus pyogenes.

A recombinant streptococcal C5a peptidase was expressed in Escherichia coli and its catalytic properties and thermal stability were subjected to examination. It was shown that the NH2-terminal region of C5a peptidase (Asn32-Asp79/Lys90) forms the pro-sequence segment. Upon maturation the propeptide is hydrolyzed either via an autocatalytic intramolecular cleavage or by exogenous protease streptopain. At pH 7.4 the enzyme exhibited maximum activity in the narrow range of temperatures between 40 and 43 degrees C. The process of heat denaturation of C5a peptidase investigated by fluorescence and circular dichroism spectroscopy revealed that the protein undergoes biphasic unfolding transition with Tm of 50 and 70 degrees C suggesting melting of different parts of the molecule with different stability. Unfolding of the less stable structures was accompanied by the loss of proteolytic activity. Using synthetic peptides corresponding to the COOH-terminus of human complement C5a we demonstrated that in vitro peptidase catalyzes hydrolysis of two His67-Lys68 and Ala58-Ser59 peptide bonds. The high catalytic efficiency obtained for the SQLRANISHKDMQLGR extended peptide compared to the poor hydrolysis of its derivative Ac-SQLRANISH-pNA that lacks residues at P2'-P7' positions, suggest the importance of C5a peptidase interactions with the P' side of the substrate.     

Bacterial community dynamics in two full‐scale wastewater treatment systems with functional stability

Aims: To characterize the bacterial community dynamics over 1 year in two full‐scale wastewater treatment systems operated under constant conditions and exhibiting stable performance. Methods and Results: Functional stability was defined and quantified by the effluent concentration of biological oxygen demand, total nitrogen and ammonia. Community dynamics were investigated using specific PCR followed by terminal restriction fragment length polymorphism (T‐RFLP) of the 16S rRNA gene. The T‐RFLP results indicated that during the period of functional stability, the bacterial community structures in two full‐scale wastewater treatment systems were not stable, and the average change rates every 15 days of the two systems were 22·6 ± 6·9 and 21·6 ± 7·3%, respectively. The corresponding species with dominant T‐RFs were determined by clonal sequencing and T‐RFLP. Based on Pareto–Lorenz distribution curves, it was observed that only a small number of micro‐organisms were numerically dominant in the two systems. Conclusions: The results of this study showed that, throughout the period of the study, the bacterial community structure changed significantly in two full‐scale wastewater treatment systems despite the stable function. Significance and Impact of the Study: The findings enrich the theory involving the relation between bacterial community dynamics and functional stability in full‐scale wastewater treatment plants.     

Continuous production of ethanol in high concentration using immobilized growing yeast cells

Summary Application of an immobilized growing yeast cell system to continuous production of ethanol in high concentration (10%) was investigated using Saccharomyces cerevisiae IFO 2363. When a medium containing 25% glucose was fed, the growth of yeast cells in gel was inhibited. The inhibitory effect was found to be reduced by a stepwise increase in concentration of glucose in the feed medium. The stepwise operation resulted in constant growth of cells in the gel even in the medium containing 25% glucose. By this stepwise feeding system, continuous production of ethanol of 114 mg/ml was maintained at a retention time of 2.6 h for over 2 months and a conversion rate of glucose to ethanol of over 95% of theoretical, was achieved.     

Modulation of citrate metabolism alters aluminum tolerance in yeast and transgenic canola overexpressing a mitochondrial citrate synthase

Aluminum (Al) toxicity is a major constraint for crop production in acid soils, although crop cultivars vary in their tolerance to Al. We have investigated the potential role of citrate in mediating Al tolerance in Al-sensitive yeast (Saccharomyces cerevisiae; MMYO11) and canola (Brassica napus cv Westar). Yeast disruption mutants defective in genes encoding tricarboxylic acid cycle enzymes, both upstream (citrate synthase [CS]) and downstream (aconitase [ACO] and isocitrate dehydrogenase [IDH]) of citrate, showed altered levels of Al tolerance. A triple mutant of CS (Deltacit123) showed lower levels of citrate accumulation and reduced Al tolerance, whereas Deltaaco1- and Deltaidh12-deficient mutants showed higher accumulation of citrate and increased levels of Al tolerance. Overexpression of a mitochondrial CS (CIT1) in MMYO11 resulted in a 2- to 3-fold increase in citrate levels, and the transformants showed enhanced Al tolerance. A gene for Arabidopsis mitochondrial CS was overexpressed in canola using an Agrobacterium tumefaciens-mediated system. Increased levels of CS gene expression and enhanced CS activity were observed in transgenic lines compared with the wild type. Root growth experiments revealed that transgenic lines have enhanced levels of Al tolerance. The transgenic lines showed enhanced levels of cellular shoot citrate and a 2-fold increase in citrate exudation when exposed to 150 micro M Al. Our work with yeast and transgenic canola clearly suggest that modulation of different enzymes involved in citrate synthesis and turnover (malate dehydrogenase, CS, ACO, and IDH) could be considered as potential targets of gene manipulation to understand the role of citrate metabolism in mediating Al tolerance.     

Microbial maximal specific growth rate as a square-root function of biomass yield and two kinetic parameters

Understanding how growth rates changes under different perturbations is fundamental to many aspect of microbial physiology. In this work, we experimentally showed that maximal specific growth rate is a square-root function of the biomass yield, the substrate turnover number, and the maximum synthesis rate of the substrate transporter under that condition. We used Escherichia coli cultures in lactose minimal medium as a model system by introducing genetic modifications, in vitro evolution, and ethanol stress to the cell. Deletion of crr affected all three parameters in different directions while deletion of ptsG decreased only the biomass yield. Ethanol stress negatively impacted all three parameters, while anaerobicity decreased biomass yield and transporter synthesis rate. In addition, laboratory evolution increased the growth rate in lactose mostly through enhancing the expression rate of the lac operon. Despite all these changes, the growth rate of the perturbed strain was successfully related to the three parameters by the square-root equation. Thus, this square-root relationship provides insight into how growth rate is altered by different physiological parameters.     

Urea hydrolysis by immobilized urease in a fixed-bed reactor: analysis and kinetic parameter estimation

Urea hydrolysis by urease immobilized onto ion exchange resins in a fixed-bed reactor has been studied. A modified Michaelis-Menten rate expression is used to describe the pH-dependent, substrate- and product-inhibited kinetics. Ionic equilibria of product and buffer species are included to account for pH changes generated by reaction. An isothermal, heterogeneous plug-flow reactor model has been developed. An effectiveness factor is used to describe the reaction-diffusion process within the particle phase. The procedure for covalent immobilization of urease onto macroporous cation exchangers is described. Urea conversion data are used to estimate kinetic parameters by a simplex optimization method. The best-fitted parameters are then used to predict the outlet conversions and pH values for systems with various inlet pH values, inlet urea and ammonia concentrations, buffers, particle sizes, and spacetimes. Very good agreement is obtained between experimental data and model predictions. This immobilized urease system exhibits quite different kinetic behavior from soluble urease because the pH near the enzyme active sites is different from that of the pore fluid. This effect results in a shift of the optimal pH value of the V(max) (pH) curve from 6.6 (soluble urease) to ca. 7.6 in dialysate solution, and ca. pH 8.0 in 20mM phosphate buffer. The reactor model is especially useful for estimating intrinsic kinetic parameters of immobilized enzymes and for designing urea removal columns.