Studies on the Polymorphism of L-Glutamic Acid

The growth rate of the α-crystal of L-glutamic acid was measured under various degrees of supersaturation and temperatures. The rate constants and the activation energies for (0 0 1) and (1 1 1) faces were measured and the latter values were 6.7 and 11.5 kcal/mol, respectively. The controlling process of the α-crystal growth was investigated by comparison of Sherwood numbers of dissolution and crystallization, and the crystallization process was found to be controlled by the surface reaction.     

Staining kinetics in single cells. Part I. Influence of convective diffusion on the staining rate

The staining kinetics of single cells have been investigated using a perfusion cuvette in combination with a computer controlled microscope spectrometer. The physicochemical hydrodynamics of staining are characterized. Using a steady-state laminar flow parallel to the cell surface a hydrodynamic and a diffusional boundary layer are observed which are determined by the flow rate. The thickness of the diffusional boundary layer revealed by experimental data is in agreement with theoretically calculated values. At certain well-defined hydrodynamic conditions convective diffusion has no further effect on the staining rate.     

Growth rate control in fed-batch cultures of recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg)

Summary A recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg) exhibited growth-assciated product formation. By controlling the medium feed rate, based on the calculated amount of medium required for 1 h, a constant specific growth rate was obtained in the range of 1.12-0.18 h^–1. In order to prolong the exponential growth phase, the medium feed rate was increased exponentially. A fedbatch cultivation method based on the production kinetics of batch culture enhanced HBsAg production ten times more than in batch culture. The reason for the increase can be explained by the fact that the production of HBsAg is expressed as an exponential function of time when the specific growth rate is controlled to a constant value in growth-associated product fromation kinetics. In the scale-up of this culture to 91, the specific growth rate could also be maintained constant and the HBsAg production trend was similar to that in a 1-l culture. However, ethanol accumulation occurred at a late stage in fed-bach culture. Ethanol produced was not reutilized and inhibited further cell growth. Offprint requests to: M. B. Gu     

Growth rate control in fed-batch cultures of recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg).

A recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg) exhibited growth-associated product formation. By controlling the medium feed rate, based on the calculated amount of medium required for 1 h, a constant specific growth rate was obtained in the range of 0.12-0.18 h-1. In order to prolong the exponential growth phase, the medium feed rate was increased exponentially. A fed-batch cultivation method based on the production kinetics of batch culture enhanced HBsAg production ten times more than in batch culture. The reason for the increase can be explained by the fact that the production of HBsAg is expressed as an exponential function of time when the specific growth rate is controlled to a constant value in growth-associated product formation kinetics. In the scale-up of this culture to 91, the specific growth rate could also be maintained constant and the HBsAg production trend was similar to that in a 1-1 culture. However, ethanol accumulation occurred at a late stage in fed-bach culture. Ethanol produced was not reutilized and inhibited further cell growth.     

Kinetics of Growth and Substrate Uptake in a Biological Film System

The rates of growth and substrate uptake in a biological film continuous-flow reactor were studied. The experiments were performed with high fluid velocities to bring the reactor operation to the reaction-controlled regime, thus avoiding external diffusional resistances. The glucose uptake experiments were performed with small film thicknesses so that full substrate penetration within the entire film thickness could be obtained. In this way, the catalyst effectiveness factor was 1.0 and the observed rate was the true, or intrinsic, rate. The results of the experiments indicate that both the intrinsic rate of substrate uptake and the rate of film growth are independent of the substrate concentration remaining in the reactor (zero-order reactions). However, the value of the initial substrate concentration when the film is in the early stages of growth defines the magnitude of both the rate of uptake and growth. This effect of the initial substrate concentration follows a saturation-function pattern.     

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.     

Mathematical simulation of a biofilm process

A mathematical model has been developed for a fixed-film biological process (film flow over a flat plate) that describes bulk liquid transport, diffusional transport of oxygen and organics across a stagnant film, diffusional transport of oxygen and organics into the biofilm, biochemical reactions by the individual cells within the biofilm, biofilm growth, and cell density changes within the biofilm due to cellular decay. Simulation studies are presented to show how contact time and diffusion layer thickness affect process performance.     

Production of HBsAg by growth rate control with recombinantSaccharomyces cerevisiae in fed-batch

Summary To maintain a constant specific growth rate for a recombinantS.cerevisiae in fed-batch, the medium feeding rate has been controlled with respect to the hourly calculated glucose uptake rate. The recombinant yeast producing HBsAg showed the exponential production trend in proportion to the exponential cell growth. Total cell yield in fed-batch was about 0.402 g cells/g glucose, and HBsAg was produced about ten times more than in batch. Decrease of growth rate by HBsAg produced was not shown.     

Effect of initial substrate concentration on the rate of nitrification in a batch experiment

The intrinsic rate of nitrification was observed in a batch reactor by eliminating external and internal diffusional resistances. The former were minimized by means of intense agitation, and the latter by mechanical rupture of the activated sludge flocs using high mixer rotational speeds. The optimum temperature and pH for the intrinsic nitrification rate were found to be 30–35°C and 8.0, respectively. Initial ammonium concentration was found to have a strong effect on the value of the kinetic parameters of the Michaelis–Menten rate expression at low ammonium levels. However, at high initial concentrations both parameters attained a constant maximum value that is independent of the initial substrate level.     

Kinetic behavior of immobilized Penicillin acylase

Penicillin acylase has been immobilized to carboxymethylcellulose and to the resin Amberlite XAD7. The reaction kinetics of the enzyme were affected by both intrinsic (molecular) and microenvironmental effects. The Michaelis constant for the enzyme increased after immobilization as a result of an intrinsic effect of the reagent, glutaraldehyde, used for enzyme immobilization. Microenvironmental effects were of two types: diffusional limitation of access of substrate and a reaction-generated pH depression in the support particles. This depression of internal pH was observed in all the preparations and could be reduced by addition of pH buffering salts to reactor. An adsorbed pH-indicating dyc was used to determine the surface and internal pH of particles of XAD7–penicillin acylase under various reaction conditions. The extent of diffusional rate limitation in XAD7–penicillin acylase was related to the penetration depth of protein into the porous support particles. The penetration depth of protein and thus the diffusional limitation of the reaction rate could be controlled by the conditions of preparation of the immobilized enzyme. A staining technique was used to observe the location of the protein.     

Temperature effects on the presteady-state and transport-associated currents of GABA cotransporter rGAT1

The effects of temperature on the gamma-aminobutyric acid (GABA) uptake and on the presteady-state and transport-associated currents of the GABA cotransporter, rat gamma-aminobutyric acid transporter 1 (rGAT1), have been studied using heterologous oocyte expression and voltage-clamp. Increasing temperature from 15 to 30 degrees C increased GABA uptake, diminished the maximal value of the relaxation time constant of the presteady-state currents and increased the amplitude of the current associated with the transport of GABA. The curve of the presteady-state charge versus voltage was shifted toward negative potentials by increasing the temperature, while the maximal amount of charge (Q(max)) remained constant; the tau versus V curve was also negatively shifted by increasing temperatures. Analysis of the outward (alpha) and inward (beta) rate constants as functions of temperature showed that they are affected differently, with a Q(10)=3.4 for alpha and Q(10)=1.5 for beta. The different temperature coefficients of the rate constants account for the observed shifts. These observations are consistent with a charge moving mechanism based on a conformational change of the protein; the weaker temperature sensitivity of the inward rate constant suggests a rate-limiting diffusional component on this process.     

Evaluation of a model for seeded isothermal batch protein crystallization

Bulk protein crystallization, unlike small molecule crystallization, has found very limited use in biopharmaceutical manufacture. Most work in this area targets obtaining single large crystals for molecular structure determination by crystallography. Design and optimization of bulk crystallization for protein recovery and purification is much less common, and requires a mathematical model for analysis of laboratory data suitable for scale-up purposes. Traditionally, the crystal size distribution and method of moments is used to characterize the crystallization process. A simpler method is presented in this paper that utilizes the desupersaturation curve. The method uses an approach that does not require expensive instrumentation or characterization of the seed crystal size distribution. The method is extended to allow determination of both the mass deposition rate constant and the growth rate order from a single desuperaturation curve. Experimental data for the bulk crystallization of ovalbumin are used to validate the method. The rate constants and rate order obtained using the new method compare well with literature values. Scale-up is illustrated by prediction of the impact of changes in seed mass on protein crystallization. This new method offers a straightforward and low-cost alternative to traditional methods for the analysis and scale-up of protein crystallization data.     

Theory of friction-limited DNA unwinding

The theory of friction-limited DNA unwinding is developed explicitly for moderate tind large perturbations. This extension of the earlier theory of the relaxation kinetics is necessary because of the complex nature of the rate limitation for small perturbations. The assumption of the theory that is violated under relaxation conditions is that base pairing reactions occurring at a constant local degree of twist of the strands are fast compared to the net unwinding of the molecule. However, these reactions that are slow for small perturbations have a large activation energy, and become faster than friction-limited un winding for large enough temperature jumps and sufficiently large DXA molecules. Thus only the rate for moderate and large perturbations is clearly limited by frictional resistance to turning the molecule in solution. The model used is a diffusional unwinding of the two strands, driven by the accompanying decrease in free energy. For large perturbations a numerical solution of the diffusion equation is required, since the diffusion coefficient is not constant. Two new parameters must be introduced into the equilibrium statistical theory to describe friction-limited unwinding kinetics. These are the force constant b, for winding up coil regions and the frictional coefficient per base pair β_cfor rotating coil regions in solution. We find by fitting the theory to experiment that b = 1.8 × 10^?13 ergs/ rad²- and β_c = 3.5 × 10^?21 erg see/base pair, both for DNA melted in alkali at 0.4.M Na ⁺ and ～30 °C. The latter value is in agreement with predictions based on the viscosity of single stranded DNA in alkali. The quoted value of bcan be interpreted to mean that the number of conformational states of a nucleolide is reduced by an average factor of 1.55 when it is wound around another strand to the degree of twist in a double helix, but without forming a base pair.     

水/乙醇混合溶剂中的阿奇霉素结晶动力学

利用粒数密度和粒度之间的关系判别晶体生长模型;采用间歇动态法,以粒数衡算方程、溶质质量守恒和McCabe定律为基础,利用Beer-Lambert定律,借助光学关联的方法,建立了包含透光率变量的伴有成核和晶体生长的动力学模型;通过在线测量溶液密度与透光率数据,采用非线性最小二乘法拟合得到了晶体成核和生长动力学经验方程,并以实时浓度为目标验证了动力学参数的准确性以及模型表达式的正确性。     

A collagen-alkaline phosphatase conjugate membrane: enzymatic kinetics in-vitro stability

Collagen-alkaline phosphatase membranes have been prepared, and their enzymatic kinetics and in-vitro stability analyzed. Collagen-alkaline phosphatase dispersions were prepared by complexation in aqueous alkaline solution and cast into membranes by controlled dehydration. These membranes were then crosslinked in glutaraldehyde solution, washed thoroughly, and dried. Crosslinking in glutaraldehyde confers increased stability of catalytic activity to these collagen-enzyme membranes, especially when compared to uncrosslinked collagen-alkaline phosphatase membranes assayed in a similar fashion. Crosslinking in glutaraldehyde also appears to inhibit gross leaching of the soluble enzyme from the carrier matrix. Apparent intrinsic kinetic properties of the collagen-alkaline phosphatase conjugate were analyzed in membranes of various thickness in order to determine the effect of internal diffusion resistances on the kinetics of the immobilized enzyme. The apparent Michaelis constant of the immobilized enzyme decreased as a function of decreasing membrane thickness, reaching an observed apparent Michaelis constant of 1.6mM at a membrane thickness of 0.2 mm. Extrapolation of the apparent Michaelis constant to zero membrane thickness, using a linear plot of the natural logarithm of the apparent Michaelis constant versus membrane thickness, allowed estimation of the true Michaelis constant of the immobilized enzyme. The estimated value for the true Michaelis constant of the collagen-alkaline phosphatase complex was 0.7mM. This value agrees closely with reported values for several purified mammalian alkaline phosphatase. The apparent Michaelis constant for the 0.2mm collagen-enzyme membrane agrees closely with the Michaelis constant reported for an alkaline phosphate purified from chondrocyte matrix vesicles. The intrinsic maximum reaction velocity (V(m)) of the collagen-enzyme complex was estimated b plotting the observed reaction rate as a function of decreasing membrane thickness and extrapolating such plots, at various substrate concentrations, to the limiting case of zero membrane thickness. The maximum reaction velocity was obtained by the common intercept of these plots as they approached zero membrane thickness.     

β-Mannanase production and kinetic modeling from carob extract by using recombinant Aspergillus sojae

The main objectives of this study were to optimize β-mannanase fermentation conditions by using Response Surface Methodology (RSM) and to model kinetically using the kinetic models. Based on the results, the optimum fermentation conditions were found to be initial sugar concentration of 10°Bx, whey concentration of 0.75% [w/v], and inoculum size of 8% (v/v). Under optimized conditions, β-mannanase activity (P), sugar consumed (ΔS), maximum β-mannanase production rate (Q_P), and sugar utilization yield (SUY) were 687.89 U/mL, 47.38 g/L, 118.54 U mL^–1 day^–1, and 69.73%, respectively. Kinetic models were employed to describe the optimum β-mannanase fermentation process. The kinetic analysis of β-mannanase fermentation showed that β-mannanase fermentation is growth associated because the α value (U/mgX) is approximately 330-fold higher than the β value (U/mgX·hr). Nevertheless, maintenance value (Z) was lower than γ value, thus showing that Aspergillus niger mainly utilizes the sugars for β-mannanase production and fungal growth. Consequently, carob extract and whey powder could be used to be cost-effective carbon and organic nitrogen sources, respectively. It was clearly indicated that the suggested kinetic models can successfully describe the fungal growth, β-mannanase production, and substrate consumption.     

Effects of Light Intensity on Tracheid Dimensions in Picea sitchensis

In seedlings of Picea sitchensis grown in constant conditions,or within older trees in the field, light intensity had no neteffect on the wall thickness of tracheids produced at the samepoint in time. This appears to be due to a balanced regulatorysystem, effects of light intensity on rate of accumulation ofwall volume per leaf being offset by differences in rate ofxylem increment, and differences in wall material per tracheidbeing nullified in their effects on wall thickness by effectson tracheid diameter. Mean tracheid wall thickness across the growth ring increasedwith light intensity, due to increase in proportion of late-woodassociated with the longer duration of cambial activity at higherlight intensity, duration of wall thickening increasing duringthe season. Duration of wall thickening did not vary with lightintensity. The rate of increase in wall volume was limited by light intensity(and hence possibly by substrate availability) at all lightintensities in the field, but in seedlings in controlled conditionsthe rate of wall production was no greater at 20 000 lx thanat 6700 lx.     

An experimental study of acid production rate controlled operations of a continuous fermentor

The efficacy of acid production rate (APR) controlled operations of a continuous fermentor supporting the growth of a methylotroph, L3, was experimentally examined. Direct digital control of pH at a constant value allowed for on-line estimation of APR during the fermentation. Two types of APR controlled operations were studied. In the first type of operation, the APR was controlled at a constant value according to a predetermined program by manipulating the feed flow rate to the fermentor. Such an operation effectively stabilized the cell mass productivity of a continuous fermentor subjected to disturbances in the feed nutrient concentration. It resulted in a near complete conversion of methanol to yield a cell mass product with very low amounts of unutilized methanol at both steady state and transient fermentation situations. In the second type of operation, the feed flow rate was manipulated to optimize the steady state value of APR during the fermentation. This method shows promise for on-line steady state optimization of cell mass productivity in a continuous fermentor.     

Molecular dynamics study of the isothermal crystallization mechanism of polyethylene chain: the combined effects of chain length and temperature

A molecular level understanding of the polyethylene (PE) crystallization process was elucidated by molecular dynamics simulation of three states, with varying chain length and temperature. The process can be classified into the following three states: (1) nucleation controlled state, (2) competitive state of crystal growth process and new nuclei formation, and (3) crystal growth controlled state, which could be quantified by the evolution of nuclei number. With increasing chain length, two phenomena occur: the single crystallization mechanism changes from state (1) to (3), and the crystal size increases while the b/a axial ratio in the lateral surface decreases. These changes can be explained from a thermodynamic point of view, in that the van der Waals (vdW) interaction per CH₂ unit is strengthened and more nucleation sites are generated for longer chain. Size effect (meaning different surface fractions when the chain collapses into a globule) was an important factor determining vdW energy per unit and the crystallization states of a single PE chain. On the other hand, the crystallization states were independent of chain length for short chains systems with the same size effect. In both conditions, a long chain generates multi-crystal domains, and a short chain prefers a single crystal domain. Our results not only provide molecular level evidence for crystallization states but also clarify the influence of chain length on the crystallization process.