bluuebunny/crossref_metadata_2025 huggingface dataset:
Coaggregate of trypsin and chymotrypsin
Authors: Rajput Yudhishthir S. , Gupta Munishwar N.
Published: December 2004

The method of chemical aggregation of enzymes has the advantage of yielding an immobilized enzyme preparation wherein reactor volume can be significantly reduced because of the absence of an inert carrier. A coaggregate of trypsin and chymotrypsin formed by extensive cross‐linking with glutaraldehyde is described. A significant property of this aggregate is the reduced autolysis of the trypsin component of the coaggregate.
Computer aided analysis for biosensing and screening
Authors: Li Xiang‐Ming , Liang Bernard S. , Wang Henry Y.
Published: December 2004

Biosensors with animal and microbial cells immobilized close to the tip of a membrane electrode have been developed for chemical and drug testing. Our experimental results show that biosensors can be used for drug screening and to provide useful information about various cell‐chemical interactions. A computer aided analysis (CAA) software package is being developed here using the biosensor for various screening purposes. This software package enables us to use a computer to analyze the biosensor dynamic responses. Computer simulation and parameter estimation techniques are used to select the best model and to describe the biochemical and pharmacologic effects of various chemicals and drugs on different cell lines.
Optimization of biphasic growth of Saccharomyces carlsbergensis in fed‐batch culture
Authors: Pyun Yu Ryang , Modak Jayant M. , Chang Yong K. , Lim Henry C.
Published: December 2004

The optimal glucose feeding policy for the fed‐batch culture of Saccharomyces carlsbergensis is presented. The biphasic nature of growth results in a singular feed rate policy that is unique to this organism. When the operating cost is high, the reduction in operating time forces the cells to utilize both glucose and ethanol toward the end of fermentation time and results in a decreasing rate of glucose addition, unlike the normally observed in creasing feed rate. The optimal feeding policy depends heavily on the initial conditions and is highly sensitive to changes in kinetic parameters. A semiempirical scheme for feedback optimization is suggested for the fed‐batch yeast culture.
Relation between stabilization and rigidification of the three‐dimensional structure of an enzyme
Authors: Germain P. , Slagmolen T. , Crichton R. R.
Published: December 2004

Purified cellobiase was coupled to periodate‐oxidized dextran by reductive alkylation using sodium cyanoborohydride, sodium borohydride, and dimethylaminoborane for various reaction times. The thermal stability of the different conjugates obtained was studied and correlated to the number of links introduced between the enzyme and the soluble support. We observe that resistance to heat inactivation increases as a function of the number of modified lysines. Sodium cyanoborohydride was the most effective reducing agent. After 24 h reaction, the modification of 92% of the lysines gave a cellobiase–dextran conjugate that is a most stable enzyme. We conclude that the thermal stability observed for the chemically modified enzyme results from the rigidification of the three‐dimensional structure of the protein. This rigidification increases with the number of links introduced between the enzyme and the polysaccharide. We also observe that chemical modification leads to a heterogeneous population of stabilized enzymes. Because of this heterogeneous population, it is necessary to develop a mathematical model of the kinetics of enzyme inactivation.
Reversibility and competition in the adsorption of Trichoderma reesei cellulase components
Authors: Kyriacou Andreas , Neufeld Ronald J. , MacKenzie C. Roger
Published: December 2004

Adsorption reversibility and competition between fractionated components of the Trichoderma reesei cellulase system were studied. Specific endoglucanase (EGI), nonspecific endoglucanases (EGII, EGIII), and cellobio‐hydrolase (CBHI) were previously grouped according to their hydrolytic function. At 5°C, direct evidence of exchange between adsorbed and free enzyme was obtained for each component using [3H] and [14C] radiolabeled tracers. No release of bound enzymes was detected upon dilution of the free enzyme solution. In simultaneous adsorption of enzyme pairs, CBHI was shown to predominate adsorption. Endoglucanase EGI was preferentially adsorbed over EGII and EGIII. Sequential adsorption studies have shown that interaction between enzyme components largely determines the degree of their adsorption. Evidence suggests that both common and distinct adsorption sites exist and that their occupation depends on which components are involved. Predominance in adsorption by any one of the enzyme components is decreased at 50°C. Light microscopy and monitoring of sugar production during cellulose hydrolysis provided evidence that reduction in the ionic strength decreases the adsorption predominance of CBHI and enhances the synergism between the cellulase components.
Control of mixed microbial cultures via specific cell adhesion
Authors: Roos Joseph W. , Hjortso Martin A.
Published: December 2004

A new method for manipulating the steady‐state behavior of a mixed culture is introduced. The method makes use of differences in adherence properties between competing populations to maintain a desired population ratio. The very specific nature of some ligand‐to‐cell interactions allows precise manipulation of even closely related populations. The control method is illustrated by analysis and simulations of models of a competitive mixed culture and a culture of an unstable recombinant organism. In both cases, retention of the disadvantaged population via cell adhesion results in creation of a stable coexistence steady state over a range of operating conditions.
Oscillatory behavior of population density in continuous culture of genetic‐engineered Bacillus stearothermophilus
Authors: Koizumi Jun‐ichi , Aiba Shuichi
Published: December 2004

An oscillatory behavior in population density was observed when a transformant of Bacillus stearothermophilus carrying a rocombinant plasmid pZAM26 was cultivated continuously in a well‐stirred reactor vessel at a fixed dilution rato. Among the transformant cells that were subjected to the continuous culture, the fraction of cells harboring p2AM26 was found to be as high as 0.98–1.00 despite the emergence of the oscillation. Cells whose plasmids underwent rearrangement of DMA in terms of structural change could not be found throughout. With reference to this observation, the dynamics of the genetic‐engineered bacterium was analyzed within the category of both the linearized stability principle and the bifurcation theory. It was concluded that Hopf bifurcation was most probable to account for the experimental oscillation.
Hydrocarbon recovery and biocompatibility of solvents for extraction from cultures of Botryococcus braunii
Authors: Frenz J. , Largeau C. , Casadevall E. , Kollerup F. , Daugulis A. J.
Published: December 2004

Various water‐immiscible solvents were tested for biocompatibility and hydrocarbon recovery under different contact conditions with the hydrocarbon‐rich microalga Botryococcus braunii. Eighteen solvents were first selected from a database of 1500 compounds (compiled for solvent selection for ethanol recovery from Saccharomyces cerevisiae fermentation). Nine of these candidate solvents were shown to be biocompatible with B. braunii following short contact times. This biocompatibility tends to be associated with high molecular weights and high boiling points but strongly depends on solvent chemical structure. A low polarity is essential to biocompatibility and calculated octanol–water partition coefficients, or capacity factors determined by reversed‐phase high‐performance liquid chromatography (HPLC), are suitable predictors of biocompatibility with B. braunii. High recoveries of hydrocarbons directly from the algal culture require relatively polar solvents and are, therefore, inimical with maintenance of cell viability. The inaccessibility of weakly polar solvents to the cell surface appears to protect the algae but also prevents substantial recovery of the hydrocarbons stored in B. braunii outer walls. In order to achieve a high recovery, contact with the solvent must be carried out on algae concentrated by filtration. Then, a large fraction of B. braunii hydrocarbons can be recovered, after a short contact time, without impairing cell viability. Under these conditions, the pertinent solvent property is affinity for the nonpolar hydrocarbons, and the highest recovery yield, ∼70% after contact for 30 min, is achieved with hexane.
A new graphical method for determining parameters in Michaelis–Menten‐type kinetics for enzymatic lactose hydrolysis
Authors: Yang Shang‐Tian , Okos Martin R.
Published: December 2004

A new graphical method was developed to determine the kinetic parameters in the Michaelis–Menten‐type equation. This method was then applied to studying the kinetics of lactose hydrolysis by Aspergillus niger β‐galactosidase. In this study, the reaction temperature ranged between 8 and 60°C, and the initial lactose concentration ranged between 2.5 and 20%. A kinetic model similar to the conventional Michaelis–Menten equation with competitive product inhibition by galactose was tested using this graphical method as well as a nonlinear computer regression method. The experimental data and the model fit together fairly well at 50°C. However, a relative large disparity was found for reactions at 30°C. A three‐parameter integrated model derived from the reversible reaction mechanism simulates the experimental data very well at all temperatures studied. However, this reversible reaction model does not follow the Arrhenius temperature dependence. Nevertheless, reaction rate constants for the proposed model involving the enzyme‐galactose complex (in addition to the Michaelis complex) as an intermediate in lactose hydrolysis follow the Arrhenius temperature dependence fairly well, suggesting that this model can be best used for describing the enzymatic lactose hydrolysis. The lack of fit between the model predictions and data may be largely attributed to the effects of galactose mutarotation and oligosaccharide formation during lactose hydrolysis.
Measurement of enzyme reaction rates using advanced pH control system: Application of repetitive PF system
Authors: Shimizu Hiroshi , Sada Eizo , Shioya Suteaki , Suga Ken‐ichi
Published: December 2004

The aim of this article is to develop the on‐line measurement system of enzyme reaction rates, using an advanced pH controller, which is called a repetitive PF(Programmed Controller/Feedback Compensator) System. In the esterolysis reaction of the N‐acetyltyrosine ethyl ester by α‐chymotrypsin, the enzyme reaction rate could be calculated from the amount of base required for keeping the pH constant. The proposed controller has a learning mechanism in which the knowledge is obtained from the former results of the repetition, and pH was controlled much more successfully by the proposed repetitive PF System than by the conventional on‐off controller, PI controller, and the adaptive controller. The enzyme reaction rate could be evaluated each time as accurately as possible based on the result of the controlled batch reaction. Using Lineweaver–Burk plots of the estimated reaction rates, kinetic parameters, such as the enzyme activity and Michaelis–Menten constant, could be estimated more accurately and from fewer experiments by the repetitive PF system than by initial reaction rates using on‐off pH‐stat.
pH‐Dependent enzyme deactivation models
Authors: Chitnis Ashish , Sadana Ajit
Published: December 2004

A pH‐dependent “series‐type” enzyme deactivation model using rapid protonation and deprotonation equilibria and the relatively slower inactivation rates is presented. From the enzyme activity‐time trajectories at different pH the models presented permit the evaluation of some of the protonation and inactivation rate constants as well as the specific activities of the different enzyme forms. pH dependence of enzyme deactivations may also exhibit deactivation disguised kinetics. Three different examples of pH‐dependent enzyme deactivations available in the literature are appropriately modeled to indicate the general applicability of the model. The model presented is consistent with the data and provides mechanistic insights into the pH‐dependent deactivation of different enzymes.
Analysis of the biomass quality and photosynthetic efficiency of a nitrogen‐fixing cyanobacterium grown outdoors with two agitation systems
Authors: Fontes Agustin G. , Moreno José , Vargas M. Angeles
Published: December 2004

The efficiency of two different agitation systems (airlift and paddlewheel) in the biomass photoproduction of a nitrogen‐fixing filamentous blue‐green alga was evaluated outdoors, and the elemental and molecular composition of the cells grown with each system was analyzed. With the paddlewheel system, the productivity values achieved were over 30% higher than with the airlift system, both in summer and winter. In this last season, a conversion efficiency of total solar energy into stored biomass energy of 3.3% was estimated for the paddlewheel system. Moreover, the algal cells grown with this system exhibited a higher net protein (58.9% of dry weight) and nitrogen (11.3%) content than those grown with the airlift device, with an estimated nitrogen fixation rate of more than 2 g N m−2 day−1. These advantages of the paddlewheel system make this procedure more appropriate for the large‐scale photoproduction of nitrogen‐fixing blue‐green algae outdoors.
Co‐immobilization by adhesion of β‐galactosidase in nonviable cells of Kluyveromyces lactis with Klebsiella oxytoca: Conversion of lactose into 2, 3‐butanediol
Authors: Champluvier B. , Francart B. , Rouxhet P. G.
Published: December 2004

Klebsiella oxytoca (NRRL‐B199), although able to produce 2, 3‐butanediol from glucose, converted lactose mainly into acetic acid. By addition of a preparation of lactase (β‐galactosidase, EC 3.2.1.23), the fermentation of lactose in a stirred vessel was three‐times faster and resulted in a high concentration of 2, 3‐butanediol. The lactase confined in dead cells of Kluyveromyces lactis (CBS 683) was prepared by permeabilization with solvents and fixation with glutaraldehyde. The cells were coimmobilized by adhesion to glass wool after treatment of the latter with chitosan, which ensured cell‐support electrostatic attraction. The cell loading (dry weight) was ca. 9 gL−1 for the yeast and ca. 2 gL−1 for the bacteria. In the presence of culture medium, the adhesion of both cells was stable and the bacteria tended to form biofilms. The stability of the coimmobilized cells was demonstrated by the continous conversion of lactose into 2, 3‐butanediol at 30oC during 25 days. The coimmobilization system gave output concentrations (14 gL−1) and rate of production (1 gL−1 h−1) of 2, 3‐butanediol from lactose, similar to those obtained in the literature with immobilized cells and glucose. Compared to the literature data on direct conversion of lactose using pure cultures, the present results showed higher butanediol concentrations and 10 to 100 times higher rates of production.
Two‐stage fermentation with bacteriophage λ as an expression vector in Escherachia coli
Authors: Park Tai Hyun , Seo Jin‐Ho , Lim Henry C.
Published: December 2004

The potential of bacteriophage λ as an expression vector for a large scale production of cloned‐gene proteins was evaluated in batch and continuous bioreactors using a temperature‐sensitive mutant in the cl gene, which allows a simple manipulation of temperature as a means to control the phage in the lysogenic or lytic state. A temperature switch from 32°C (or below) to 38°C (or above) forces the phage to go from the lysogenic state to the lytic state. Temperature cycling and a two‐reactor system were used for continuous cultures. For the latter the first reactor is maintained in the lysogenic state at a lower temperature to stably maintain the foreign DNA in the host cell, while the second reactor is maintained in the lytic state to force replication of the cloned‐gene and overproduction of its products. The results are promising but suggest a greater potential for a mutant which lacks the Q gene which is responsible for host cell lysis and packaging of phage particles.
Adaptive control of dissolved oxygen concentration in a bioreactor
Authors: Lee Seung Chul , Hwang Young Bo , Chang Ho Nam , Chang Yong Keun
Published: December 2004

A new adaptive DO (dissolved oxygen) concentration control algorithm considering DO electrode dynamics with response time delay has been developed. A system model with two time‐varying parameters was used to relate the DO concentration with two control variables: air flow rate and agitation speed. Parameters of this model were estimated on‐line using a regularized constant trace recursive least‐squares method. An extended Kalman filter was used to remove the effect of noises from the DO concentration measurements and thus to improve control performance. A discrete one‐step ahead control scheme was adopted to determine control actions based on the parameter estimation results. Experimental results showed that the new adaptive DO concentration control algorithm performed better than other algorithms tested, a PID controller and adaptive algorithms without the DO electrode dynamics.
The effect of pore‐size distribution on the binding of proteins to porous resin beads
Authors: Hasegawa Masanori , Kitano Hiromi
Published: December 2004

The effect of the distribution of pore radii in the resin beads on protein binding was taken into account to analyze the elution profiles of proteins from the polymer‐packed column obtained by repetitive injection method. By assuming that the distribution of pore radii in the resin beads is logarithmic Gaussian, the theoretical curves obtained agreed well with the experimental data.
A three‐dimensional solubility parameter approach to nonaqueous enzymology
Authors: Schneider Luko V.
Published: December 2004

Widespread commercial application of enzymes as catalysts for specialty or commodity chemical synthesis will require their use in nonaqueous systems. While a number of non‐aqueous enzyme applications have been demonstrated, the lack of useful rules for predicting enzyme–solvent interactions has hindered the development of this technology. Both Hildebrand and solvent hydrophobicity (octanol–water partition coefficient) parameters have been used previously to correlate and predict enzyme activity in nonaqueous systems, with some success, but any single‐parameter approach is inherently limited in its ability to reflect the spectrum of possible enzyme‐solvent interactions. Therefore, this study evaluates the three‐dimensional solubility parameter space, as proposed by Hansen, to correlate and predict enzyme activity in microaqueous, miscible, and biphasic nonaqueous systems. Preliminary results suggest that Hansen parameters may be useful for correlating nonaqueous enzyme activity, and that the dispersive and polar parameters may be disproportionately important in single‐phase microaqueous systems. The Hansen hydrogen‐bonding parameter appears to be the only parameter yet evaluated capable of correlating the water requirement for enzyme activity in microaqueous systems, suggesting that water affects protein structure through enthalpic rather than entropic processes in nonaqueous systems. Insufficient data are available for miscible and biphasic systems, but it is proposed that enzyme activity may correlate with the average solubility parameters of miscible systems and of the aqueous phase in biphasic systems.
On the estimation of average shear rate in bubble columns
Authors: Nishikawa Masabumi
Published: December 2004

Development of bioengineering using such biomaterials as plant tissues or genetically produced microorganisms makes it important to accurately estimate the prevailing shear rate in bioreactors because they are generally sensitive to shear. Various authors have discussed ways to estimate the average shear rate in a bubble column or an airlift reactor.1 This communication will explain the background consideration for the development of the expression as γav = 50 ug by the present authors, and give suggestions for further discussions.
Flocculation of cell debris from ultrasonicated Escherichia coli
Authors: Gellan Elaine , Martin G. F. , Cumming R. H.
Published: December 2004

Cell debris of Escherichia coli produced by ultrasonication was flocculated with acid. The debris settled at pH 3 and 4, but not at pH 2. Extensive protein precipitate formed in lysates at low pH. The results were compared with whole cells flocculated by acid.
Observations of binary population biofilms
Authors: Siebel Maarten A. , Characklis William G.
Published: December 2004

Biofilm research has focused on studies of undefined mixed microbial populations and, more recently, on investigations of monopopulation biofilms. In the first case, the biofilm is considered a homogeneous mass, ignoring the properties of individual species. The second case concentrates on the properties and processes of one microbial species in the biofilm. This article describes biofilm experiments conducted with monopopulations of Klebsiella pneumoniae and Pseudomonas aeruginosa and with binary populations of K. pneumoniae and P. aeruginosa. Process rates and stoichiometric coefficients were determined for the monopopulation and for the binary population biofilms and evaluated in light of the species distribution in the latter. Results indicate that neither the specific cellular product formation rate nor the glucose–oxygen stoichiometric ratio of K. pneumoniae or P. aeruginosa in the binary biofilm is affected by the presence of the other species. Consequently, species interaction was not observed. Although the specific cellular growth rate of K. pneumoniae is five times that of P. aeruginosa, the former species did not dominate the microbial population in the biofilm. Possible reasons for this unexpected behavior are discussed.
A production of monoclonal antibodies by a simple electrofusion technique induced by ac pulses
Authors: Takahashi Y. , Suzuki K. , Niimura T. , Kano T. , Takashima S.
Published: December 2004

A simple and effective technique of electrofusion of the mouse myeloma cells and lymphocytes induced by AC pulses was investigated. Instead of pearl chain formation, we used a mild centrifugation of high‐density cell suspensions to enhance the cell–cell contacts. The optimal frequency of the AC field used for cell fusion was 10 kHz. We confirmed the production of monoclonal antibodies against human serum albumin (HSA) by the hybridoma obtained by this method. Under the conditions used in the present work, the efficiency of hybridoma formation is several times higher than those obtained by the chemical method using polyethylene glycol (PEG). Hybridoma colonies were detected in as high as 95% of the wells and 33% of them proved positive against HSA.
Modeling fermentations with recombinant microorganisms: Formulation of a structured model
Authors: Nielsen Jens , Pedersen Annemarie G. , Strudsholm Kim , Villadsen John
Published: December 2004

A simple structured model is described and compared with experimental data for fermentations with recombinant. Escherichia coli. The model is a so‐called compartment, model, where the different biomass components are lumped together in a few intracellular variables. The model is able to describe, in a biologically reasonable fashion, a majority of the observations that have been made through fermentations with recombinant microorganisms. The model is especially suited for description of dynamic changes in plasmid copy number, e.g., runaway plasmid replication.
Calcium and phosphate effects on growth and alkaloid production in Coffea arabica: Experimental results and mathematical model
Authors: Bramble Joye L. , Graves David J. , Brodelius Peter
Published: December 2004

Plant, mammalian, and microbial cells are commonly immobilized in calcium alginate gels for the production of valuable secondary metabolites. However, calcium ions are known to inhibit growth in various types of cells, and calcium is an integral part of such gels. Therefore, an investigation was conducted to evaluate the effect of calcium on the growth and alkaloid production of a model cell‐line, Coffea arabica, in suspension culture before, attempting to immobilize such cells in alginate. A kinetic model was then developed from the results to describe cell growth and alkaloid production and the mechanism by which calcium influences these variables. In addition, it was observed that there was a characteristic relationship between the concentration of calcium in the external medium and the concentration of extra cellular and intracellular phosphate. The intracellular phosphate level was, in turn, related to the production of alkaloids. Using these results, a dynamic mathematical model of cell growth and alkaloid production was developed based on the proposed roles of calcium and phosphate. The model showed satisfactory agreement with three sets of experiments at different calcium concentrations. A possible linkage between the calcium and phosphate results is postulated based on the limited solubility of calcium phosphate.
Structured modeling of a microbial system: III. Growth on mixed substrates
Authors: Nikolajsen Karin , Nielsen Jens , Villadsen John
Published: December 2004

A two‐compartment model is extended to describe growth and product formation on a mixture of different carbohydrate sources. The open‐ended structure of the compartment model is utilized to include description of the basic mechanisms of catabolite repression. The model is used to simulate batch triauxic, steady‐state, and transient fermentation, and the simulations are compared with experimental results obtained with growth of the lactic acid bacteria Streptococcus cremoris on mixtures of glucose, galactose, and lactose. The model adequately describes characteristic experimental observations such as substrate preference and carbohydrate adoption.
Characterization of a pilot plant airlift tower loop reactor: III. Evaluation of local properties of the dispersed gas phase during yeast cultivation and in model media
Authors: Fröhlich S. , Lotz M. , Larson B. , Lübbert A. , Schügerl K. , Seekamp M.
Published: December 2004

The local properties of the dispersed gas phase (gasholdup, bubble diamater, and bubble velocity) were measured and evaluated at different positions in the riser and downcomer of a pilot plant reactor and, for comparison, in a laboratory reactor. These were described in Parts I and II of this series of articles during yeast cultivation and with model media. In the riser of the pilot plant reactor, the local gas holdup and bubble velocities varied only slightly in axial direction. The gas holdup increased considerably, while the bubble velocity increased only slightly with aeration rate. The bubble size diminished with increasing distance from the aerator in the riser, since the primary bubble size was larger than the equilibrium bubble size. In the downcomer, the mean bubble size was smaller than in the riser. The mean bubble size varied only slightly, the bubble velocity was accelerated, and the gas holdup decreased from top to bottom in the downcomer. In pilot plant at constant aeration rate, the properties of the dispersed phase were nearly constant during the batch cultivation, i.e., they depended only slightly on the cell concentration. In the laboratory reactor, the mean bubble sizes were much larger than in the pilot plant reactor. In the laboratory reactor, the bubble velocities in the riser and downcomer increased, and the mean gas holdup and bubble diameter in the downcomer remained constant as the aeration rate was increased.
Kinetic data for the BM‐5 insect cell line in repeated‐batch suspension cultures
Authors: Stavroulakis Dimitrios A. , Kalogerakis Nicolas , Behie Leo A. , Latrou Kostas
Published: December 2004

The kinetics and growth characteristics of the BM‐5 insect cell line of Bombyx mori (silkworm) have been experimentally investigated in order to develop optimal growth protocols when these cells are used to produce large quantities of human proteins by recombinant baculoviruses. Experiments were performed in 200‐mL spinner flasks, which were operated at 80 rpm with 0.3% methyl cellulose (MCL) added to the medium to protect the cells from shear stress. Exposure of the cells to varying nutrient and metabolite concentrations was accomplished through a repeated‐batch mode of feeding. The results indicate that glutamine is a limiting nutrient and that lactate has an inhibitory effect on cell growth. Ammonia depletion from the medium was accompanied by uric acid accumulation suggesting that ammonia is converted to this metabolic product by the “uricotelic” and “nucleicolytic” metabolic pathways. With the examined medium formulation, glucose, fructose, and sucrose remained at high concentrations throughout the cultivation period.
Enzymic hydrolysis of lignocellulosic materials: I. Models for the hydrolysis process — a theoretical study
Authors: Vallander Lars , Eriksson Karl‐Erik L.
Published: December 2004

At the end of an enzymic hydrolysis process involving a solid lignocellulosic substrate, enzymes are found both in solution and absorbed to the substrate residue. Removal of residue from the system will result in loss of some of the enzymes, the extent of which will depend on the design of the process. To minimize enzyme loss, a study has been conducted in which six process models have been formulated and an enzyme loss function derived for each model based on the total amount of enzymes lost through residue removal. Model 1 is a reference model, characterized by an uninterrupted hydrolysis throughout the entire hydrolysis period. The residue is then washed in order to recover both sugar and adsorbed enzymes before the residue is discarded. Models 2–6 are all characterized by the removal of hydrolysate three times during the process, recirculation of dissolved and adsorbed enzymes to various points in the process and selection of a stage at which the residue is removed. The following conclusions could be drawn from the derived enzyme loss functions:

Increased enzyme adsorption leads to increased enzyme loss.

The enzyme loss decreases if the solid residue is removed late in the process.

Both adsorbed and dissolved enzymes should be introduced at the starting point of the process. This is particularly important for dissolved enzymes. Three models were chosen for experimental studies, which are reported in a second, accompanying article. The experimental results obtained are compared with the theoretical study reported here.
Enzymatic hydrolysis of lignocellulosic materials: II. Experimental investigations of theoretical hydrolysis — process models for an increased enzyme recovery
Authors: Vallander Lars , Eriksson Karl‐Erik L.
Published: December 2004

Stream pretreatment of wheat straw solubilized most of the xylan present. Xylose and other sugars were recovered by washing the substrate with water but only a minor part (34%) was monomeric. Treatment of this solutions with celulases and hemicellulases improved the yield of monomeric sugars to 69%, the main product being xylose. Some xylose was also obtained during enzymatic hydrolysis of the solid substrate although the pretreatment step contributed 64% (mean value) of total xylose formed. A reference model, No. 1, and two other models, Nos. 2 and 4, described in the first part of this article series (this issue) have been studied experimentally and results confirm the theoretical conclusions. An uninterrupted hydrolysis over a given time period leads to a lower degree of saccharification than when hydrolysate is withdrawn several times. Saccharification is also favored if the residue is removed at a late stage, i.e., at the end of the 24 h hydrolysis cycle. Extended recirculation of the enzymes during a 4 × 24‐h experimental period gave the following average yields of saccharification on a 24‐h basis: 65% (Reference), 73% (Model 2), and 79% (Model 4). It is concluded that enzyme recovery with model 4 is 70% or more, while the Reference and Model 2 attain a lower level of recovery. The design of an improved hydrolysis model is also discussed.
Microcarrier culture of bowes melanoma cells in serum‐free medium with Human plasma fraction IV‐4+ V
Authors: Jo Eui‐Cheol , Gu Man Bock , Kim Dong‐II , Paik Sung Bok
Published: December 2004

Bowes melanoma cells were cultivated successfully in a serum‐free medium which was constructed by the concept of maximum retention of proteins from fractionated human plasma having growth stimulatory activities. The cells could be cultivated in the serum‐free medium without any adaptation period. The major serum‐free component of the medium was the fraction IV‐4 + V of the Cohn fractionation process of human plasma. Approximately six times increase of tissue‐type plasminogen activator (t‐PA) activity as compared with that in serum‐free medium even though the cell growth was much slower. In addition, the growth stimulatory activities of thrombin and fibronectin were investigated during the cultivation of Bowes melanoma cells in this serum‐free medium. These proteins contributed significantly to the enhanced growth of cells by reducing doubling time to 25 and 35 h as compared with 55 h in the serum‐free medium without them. Especially, fibronectin supported cells to propagate near to the maximum cell density achieved in the medium with 10% FBS.
A model of secretory protein trafficking in recombinant AtT‐20 cells
Authors: Sambanis A. , Lodish H. F. , Stephanopoulos Gregory
Published: December 2004

After their synthesis, secretory proteins in animal cells undergo a series of transport and processing steps before they are secreted. The amount and quality of protein obtained in culture medium depends on the rates of these intracellular steps. We present a model of recombinant protein trafficking in mouse pituitary AtT‐20 cells based on currently available biological knowledge, plausible hypotheses, and quantitative secretion results, and we use it to simulate the dynamics of basal and induced secretion and to predict the dynamics of intracellular trafficking events. Besides the endoplasmic reticulum and Golgi, the model recognizes a conversion compartment (CC) where final processing of protein occurs, a storage compartment from which protein is secreted only in the presence of secretion stimulus, and constitutive and pseudoregulated (PR) pathways of secretion. The model further assumes that the protein flux is split between CC and PR and that the storage compartment exerts a negative feedback on protein flux through CC. The model predictions are compared with experimental results on secretion of human growth hormone (hGH) and insulin related peptides and on accumulation of hGH upon removal of secretion stimulus. The model is in agreement with data when either of two hypotheses is implemented: (a) cells always exhibit a high sorting efficiency at the trans‐Golgi, but CC has the capacity to process only a fraction of the protein flux leaving the Golgi compartment; (b) the processing capacity of CC never becomes saturated, but significant missorting at the trans‐Golgi occurs; in the case, the protein flux toward the plasma membrane becomes split both at the trans‐Golgi cisternae and between CC and PR. The usefulness of the type of model considered in providing a quantitative description of intracellular events and in designing new experiments is discussed.
Protease‐catalyzed peptide synthesis using inverse substrates: The synthesis of Pro‐Xaa‐bonds by trypsin
Authors: Schellenberger Volker , Schellenberger Ute , Jakubke Hans‐Dieter , Zapevalova Nina P. , Mitin Yuri V.
Published: December 2004

Benzyloxycarbonyl‐L‐proline p‐guanidinophenyl ester is an “inverse substrate” for trypsin; i.e., the cationic center is included in the leaving group instead of being in the acyl moiety. This substrate can be used in trypsin‐catalyzed acyl‐transfer reactions leading to the synthesis of Pro‐Xaa peptide bonds. The reaction proceeds about 20 times slower than reaction with similar alanine‐containing substrates, but the ratio between synthesis and hydrolysis is more favorable. The investigation of a series of nucleophiles led to information about the specificity of the process. Nucleophiles differing only in the P1′‐position show an increasing acyl transfer efficiency in the order Phe < Gly < Ley < Ser < Ala < lle. C terminal elongation of the nucleophiles is of minor influence on their efficiency. The formation of an H bond between the acyl‐enzyme and the nucleophile seems to play an important role in the aminolysis of the acyl‐enzyme.
Fluorescence modeling in a multicomponent system
Authors: Wang Nam Sun , Simmons Michael B.
Published: December 2004

An extensive fluorescence database for binary tyrosinetryptophan mixtures utilizing 280 nm excitation was collected. The database spanned three orders of magnitude (10−6M–10−3M) and covered all compositions within this range. A generalized model for describing the multicomponent fluorescence signals as a function of emission wavelength, excitation wavelength, and sample composition was derived. A geometric integral that contained all the geometric factors affecting fluorescence was introduced; thus the model was applicable to various configurations, including the three used in this study: an NADH probe, a backscatter laser‐induced fluorescence setup, and a commercial spectroflurometer. A correction factor was proposed that allowed linearization of the fluorescence signals with respect to fluorophore concentrations. The effect of the water Raman on fluorescence spectra was also modeled. The model contains only two wavelength‐dependent parameters for each of the components present in a sample, one specifying absorption of the excitation energy and the other specifying the species' fluorescence tendency. These wavelength‐dependent parameters were correlated with polynomials. The average prediction error at each wavelength was 10–20%, a major portion of which was attributed to experimental uncertainties.
Effects of Carbon dioxide on the Rheological behavior and oxygen transfer in submerged penicillin fermentations
Authors: Ju Lu‐Kwang , Ho Chester S. , Shanahan John F.
Published: December 2004

Fermentations of Penicillium chrysogenum have been made with different CO2 contents in the influent gas streams. The rheological behavior of the culture broth was found to be significantly changed by exposure to high levels of CO2. This is attributed to the wide variation in the morphology of P. chrysogenum, from normal mycelia with long hyphae to roughly spherical pellets when subjected to high levels of CO2. A correlation has been developed relating volumetric O2 transfer coefficients, kLa, with the effective O2 diffusion coefficients, De, and the apparent viscosities, μapp, based on the results obtained in this study. The use of CO2 as a potent means for altering the rheological properties of culture broths and consequently improving the O2 transfer capabilities in penicillin fermentations was clearly demonstrated.
Studies of the reductive biotransformation of selected carbonyl compounds by whole cells and extracts of baker's yeast, Saccharomyces carevisiae
Authors: Young C. S. , Ward O. P.
Published: December 2004

The progress of reductive biotransformations of a variety of earbonyl compounds by whole cells of baker's yeast was monitored with time. Biotransformations rates ranged from 0.11 to 112.12 mg product formed per g dry yeast per h. While rapid biotransformations of citronellal and ethyl benzoylformate were observed, complete conversion of substrate to product did not occur. Reductive conversions of ethyl‐ and methyl‐acetoacetate went to completion in 6 and 12 h respectively. Ethyl mandelate was produced stereoselectively, favoring the (R)‐ stereoisomer and ethyl and methyl‐3‐hydroxybutyrate were produced with (S)‐enantiospecificity. Yeast crude extract and resuspended presence of NAD(P)H. Ethyl benzoylformate and methyl‐and ethyl‐acetoacetate were preferentially reduced by yeast crude extract as compared to resuspended pellet and, in the case of the former two substrates, the reaction manifested a preference for NADPH over NADH.
Growth and immobilization of tripterygium wilfordii cultured cells
Authors: Pépin Marie‐France , Chavarie Claude , Archambault Jean
Published: December 2004

The plant Tripterygium wilfordii produces di‐ and triterpenes of interest for male contraception and treatment of arthritis and skin disorders. Cell line TRP4a obtained form this plant in 1981 was reported to produce these valuable compounds at yields (∼0.04% of the biomass dry weight) higher than found in the plant (0.001%). In order to improve this production, studies were carried out to determine the feasibility of eliminating the troublesome component of coconut milk originally used to culture this cell line. A defined formulation suitable for growth ad maintenance has been developed. This medium consisted of Gamborg's PRL4 or B5 medium supplemented with 2 mg L−1 2,4‐dichlorophenoxyacetic acid and 20 g L−1 sucrose. Furthermore, monitoring of carbohydrate uptake revealed that T. wilfordii cells, contrary to many plant cell species, did not hydrolyze sucrose extra‐cellularly before uptake. Replacement of this disaccharide by glucose or fructose increased specific growth rate from 0.15 to 0.25 day−1. As tripdiolide is reported to be present in broth extract in significant amounts, plant cell immobilization technology offers a promising alternative to suspension cultures, especially in view to on line harvesting of the product. Surface immobilized T. wilfordii cell cultures were successfully carried out in 2‐L bioreactors. Their biomass production and carbohydrate uptake were comparable to those observed for shake flask grown suspension cultures. Higher nitrate and ammonium uptake were found in immobilized cultures.
Metabolic regulation in bacterial continuous cultures: I
Authors: Baloo S. , Ramkrishna D.
Published: December 2004

Dilution rate steps in continuous culture experiments with Klebsiella pneumoniae growing on single substrate feeds have brought out interesting features of metabolic regulation not observed in batch cultures. In a step‐up experiment, the adjustment of the culture to a new steady state is preceded by an undershoot in cell density. Results of a step‐down experiment indicate a corresponding overshoot phenomenon. These observations of the transient behavior of the culture growing on glucose and xylose as well as the steady‐state results are interpreted with cybernetic models. The development of the model explicitly accounts for the lumped internal resource, which is optimally allocated toward the synthesis of key enzymes catalyzing different cellular processes. The model also includes a description of the increased maintenance demand observed at low growth rates. It reduces to previous cybernetic models in situations where the cell does not experience a sudden change in its environment and, hence, retains their predictive capability.
Metabolic regulation in bacterial continuous cultures: II
Authors: Baloo S. , Ramkrishna D.
Published: December 2004

The transient behavior of a continuous culture of Klebsiella pneumoniae with mixed feed of glucose and xylose arising from step‐up and step‐down in dilution rates and from a feed‐switching experiment is presented. he organism gradually switches from simultaneous utilization of the substrates at low growth rates to preferred utilization of the faster substrate (i.e, supporting a higher growth rate) at high dilution rates. The metabolic lags following a step increase in dilution rate and a significant accumulation of the slower substrate during the transient period result from the effects of metabolic regulation. The cybernetic modeling approach that successfully described the foregoing situations with single‐substrate feeds is employed to describe mixed substrate behavior. The parameters in the mixed‐substrate (glucose and xylose) model are the same as those in the single‐substrate models with the singular exception of the rate constant for the xylose growth enzyme synthesis. The reason for this discrepancy is discussed in detail. It appears that the constitutive rate of enzyme synthesis for growth on a given substrate may be related to the past history of the organism in regard to whether or not the organism has been exposed to the particular substrate. Thus, the results further demonstrate the ability of the framework to effectively describe metabolic regulation in batch, fedbatch, and continuous microbial cultures.
The importance of proline on long‐term hepatocyte function in a collagen gel sandwich configuration: Regulation of protein secretion
Authors: Lee Jaegwan , Morgan Jeffrey R. , Tompkins Ronald G. , Yarmush Martin L.
Published: December 2004

A serum‐free culture system for primary hepatocytes which maintains stabel high‐level hepatocyte function for prolonged periods in culture has been developed. Isolated rat primary hepatocytes were cultured in serum‐free media between two layer of gelled collagen in a sandwich configuration which reinstates the cellular polarity necessary for long‐term function in vitro. Thsee serum‐free hepatocyte cultures maintained near physiological rates of albumin and transferrin secretion for a minimum of 26 days in culture. L‐Proline was shown to be critical for both the approach to steady state and maximal level of protein secretion. Analysis of does–response data gave Km values of 2.9 and 1.7 μg/mL for albumin and transferrin secretion, respectively.
A spin label study of immobilized enzyme spectral subpopulations
Authors: Song Yeqing , Means Gray E. , Wan Xiaoming , Berliner Lawrence J.
Published: December 2004

Electron spin resonance (ESR) spin label studies have been carried out to examine the active site conformation of α‐chymotrypsin before and after immobilization on two types of organic polymer supports: Amberlite XAD‐8 and XAD‐2. α‐Chymotryspin was first chemically modified by reaction with methyl‐4‐phenylbutyrimidate and then inhibited by the active site spin label 4‐(2,2,6,6‐tetramethyl‐piperdine‐1‐oxyl)‐m‐flurosulfonylbenzamide. In general, the ESR spectra of the active site lable revealed no significant changes in conformation for most of the enzyme before or after derivatization. On the other hand, two spectral subpopulations (A and B) of spin‐labeled enzyme were characterized on the basis of their ESR spectra after immobilization on Amberlite XAD‐8. Spectral subpopulation A (distinguished by a highly restrained spectrum) appeared to retain its active site structure and conformation and represented a large majority of the labeled chymotrypsin on the beads. Its presence correlated with the high activity and stability of phenylbutyramidinated chymotryspin on the Amberlite XAD‐8 beads. Spectral subpopulation B (distinguished by a very weakly constrained spectrum) appeared to reflect loosely bound or denatured enzyme which was removable upon washing with 40% (v/v) ethylene glycol. Two methods for examining solvent accessibility to the active site lable of the kinetics of ascorbate reduction suggested that both spectral subpopulations had identical accessibilities to the bulk solvent. Paramagnetic broadening of the signal by K3Fe(CN)6 revealed differences in the spin–spin broadening of the A and B components but is deemed and inappropriate indicator of solvent accessibility.