bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractStream 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uninterrupted hydrolysis over a given time period leads to a lower degree of saccharification than when hydrolysate is withdrawn several times.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

AbstractBowes 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

AbstractAfter 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

AbstractBenzyloxycarbonyl‐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

AbstractAn 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

AbstractFermentations 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

AbstractThe 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

AbstractThe 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

AbstractDilution 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

AbstractThe 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

AbstractA 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

AbstractElectron 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.
High stability to irreversible inactivation at elevated temperatures of enzymes covalently modified by hydrophilic reagents: α‐Chymotrypsin
Authors: Mozhaev Vadim V. , Melik‐Nubarov Nickolay S. , Levitsky Vladislav Y. U. , Siksnis Virginius A. , Martinek Karel 
Published: December 2004

AbstractBased on the idea that proteins can be stabilized by a decrease in the thermodynamically unfavorable contact of the hydrophobic surface clusters with water, α‐chymotrypsin (CT) was acylated with carboxylic acid anhydrides or re‐ductively alkylated with aliphatic aldehydes. Modification of CT with hydrophilic reagents leads to 100‐1000‐fold increase in stability against the irreversible thermoinactivation. The correlation holds: the greater the hydrophilization increment brought about by the modification, the higher is the protein thermostability. After some limiting value, however, a further increase in hydrophilicity does not change thermostability.It follows from the dependence of the thermoinactivation rate constants on temperature that for hydrophilized CT there is the conformational transition at 55‐65°C into an unfolded state in which inactivation is much slower than that of the low‐temperature conformation. The thermodynamic analysis and fluorescent spectral data confirm that the slow inactivation of hydrophilized CT at high temperatures proceeds via a chemical mechanism rather than Incorrect refolding operative for both the native and low‐temperature form of the modified enzyme. Hence, the hydrophilization stabilizes the unfolded high‐temperature conformation by eliminating the incorrect refolding. © 1992 John Wiley & Sons, Inc.
Enhanced β‐galactosidase production by high cell‐density culture of recombinant Bacillus subtilis with glucose concentration control
Authors: Park Yong Soo , Kai Kenichi , Iijima Shinji , Kobayashi Takeshi 
Published: December 2004

AbstractCell concentration, recombinant protein (β‐galactosidase) level, and the specific enzyme expression level were increased from 19 to 184 g/L, 18.3 to 129 U/mL, and 3.2 to 5.7 U/mg protein, respectively, in fed‐batch culture of recombinant Bacillus subtilis when glucose concentration was controlled at 1 g/L as compared with those of conventional fed‐batch culture. Glucose concentration of the culture broth was monitored by an automatic on‐line glucose analyzer and controlled with a moving identification combined with optimal control (MICOC) strategy. When glucose concentrations were controlled at 10, 1, and 0.2 g/L, accumulated propionic acid concentrations and specific enzyme activities were 18.5, 4.4, and 0.6 g/L and 2.9, 5.7, and 7.1 U/mg protein, respectively. The addition of various concentrations of sodium propionate to the growth medium in batch cultures resulted in a drastic decrease in the growth rate with respect to propionate concentration. The propionic acid was shown to be responsible for cell growth inhibition and enzyme activity reduction in fed‐batch culture. © 1992 John Wiley & Sons, Inc.
Cultivation of Methylosinus trichosporium OB3b: III. Production of particulate methane monooxygenase in continuous culture
Authors: Shah Nilesh N. , Park Sunghoon , Taylor Robert T. , Droege Michael W. 
Published: December 2004

AbstractContinous culture experiments with the obligatory methanotroph, Methylosinus trichosporium OB3b, were conducted to study the whole‐cell methane monooxygenase (MMO) and nitrogenase activities in a nitrate minimal salts medium under oxygen‐limited conditions with methane as the carbone source. The important variables investigated were the feed medium concentrations of copper and nitrate, CO2 addition, the agitation speed, and the dilution rate. M. trichosporium OB3b required quantitative amounts of copper (2.6 × 10−4 g Cu/g dry cell Wt) for the exclusive production of particulate MMo during continous culture growth. When the feed medium nitrate concentration was varied in the range of 5‐50 mM, the whole‐cell specific pMMO activity exhibited a maximum at 40 mM. The elimination of external CO2 gassing decreased pMMO activity by more than 30%. The steady‐state cell density increased continuously over a 300‐700 rpm range of agitation speed, whereas, the pMMO activity became maximal at 400 rpm. Also, the pMMO activity increased with the dilution rate up to 0.06 h−1 and remained constant thereafter. Maximal continuous pMMO productivity was, thus, achieved in Higgin's medium containing 10 μM Cu, 80 μM Fe, and 40 mM nitrate with an agitation speed of 500 rpm and a dilution rate of 0.06 h−1. Nitrogenase activity, on the other hand, increased over a feed medium copper concentration of 2‐15 μM, falling sharply at 20 μM, and it exhibited a minimum at 20 mM when the feed medium nitrate concentration was varied. © 1992 John Wiley & Sons, Inc.
Influence of total and oxygen partial pressure on growth and metabolism of Methylomonas clara
Authors: Liefke E. , Onken U. 
Published: December 2004

AbstractThe effect of total and oxygen partial pressure on metabolic activities of Methylomonas clara has been investigated in batch and continuous cultivation experiments using a pressurizable airlift loop reactor. At Oxygen partial pressures of more than 1000 mbar growth of M. Clara is retarded and completely inhibited at 1200 mbr. However, after several hours of incubation at elevated oxygen partial pressures, biomass formation is nearly doubled in subsquent continuous operation, Cells pretreated with oxygen are characterized by a change of cytochrome content; they excret carboxylic acids into the medium. The results indicate that, by sparging an aerobic culture intermittently wich pure oxygen, formation of biomass might be enbanced. © 1992 John Wiley & Sons, Inc.
Improvement of shikonin productivity in Lithospermum erythrorhizon cell culture by alternating carbon and nitrogen feeding strategy
Authors: Srinivasan Venkatesh , Ryu Dewey D. Y. 
Published: December 2004

AbstractStationary phase cell suspension cultures of Agrobacterium tumefaciens transformed Lithospermum erythrorhizon respond to additions of sucrose‐rich (C‐rich) medium with a 2–3‐fold increase in the accumulation of shikonin derivatives and a 3–3.5‐fold increase in the accumulation of soluble phenolics while showing a modest (10–30%) increase in cell concentration. Conversely, the addition of nitrate‐rich (N‐rich) medium resulted in 25–35% increase in biomass concentration but only 2–9% increase in shikonin production and ∼ 3% increase in the yield of soluble phenolics. Repeated additions of C‐rich medium resulted in only a modest (less than 10%) improvement in shikonin production over the levels obtained after the first application. No obvious correlation could be discerned between intracellular ATP levels or protein synthesis patterns and the pattern of shikonin accumulation following the addition of C‐rich medium, suggesting that the precursor diversion mechanism is not generally applicable in our cell line. It was found that alternating feeding of N‐rich and C‐rich media could be used as an effective strategy for enhancing the productivity of plant secondary metabolite. © 1993 John Wiley & Sons, Inc.