bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of a Phanerochaete chrysosporium Manganese Peroxidase Gene in the Yeast Pichia pastoris
Authors: Gu Lina , Lajoie Curtis , Kelly Christine
Published: October 2003

A gene encoding manganese peroxidase (mnp1) from Phanerochaete chrysosporium was cloned downstream of a constitutive glyceraldehyde‐3‐phosphate dehydrogenase promoter in the methylotrophic yeast Pichia pastoris. Three different expression vectors were constructed: pZBMNP contains the native P. chrysosporium fungal secretion signal, pαAMNP contains an α‐factor secretion signal derived from Saccharomyces cerevisiae, and pZBIMNP has no secretion signal and was used for intracellular expression. Both the native fungal secretion signal sequence and α‐factor secretion signal sequence directed the secretion of active recombinant manganese peroxidase (rMnP) from P. pastoris transformants. The majority of the rMnP produced by P. pastoris exhibited a molecular mass (55–100 kDa) considerably larger than that of the wild‐type manganese peroxidase (wtMnP, 46 kDa). Deletion of the native fungal secretion signal yielded a molecular mass of 39 kDa for intracellular rMnP in P. pastoris. Treatment of the secreted rMnP with endoglycosidase H (Endo H) resulted in a considerable decrease in the mass of rMnP, indicating N‐linked hyperglycosylation. Partially purified rMnP showed kinetic characteristics similar to those of wtMnP. Both enzymes also had similar pH stability profiles. Addition of exogenous MnII, CaII, and FeIII conferred additional thermal stability to both enzymes. However, rMnP was slightly less thermostable than wtMnP, which demonstrated an extended half‐life at 55 °C.
Mathematical and Experimental Analyses of Antibody Transport in Hollow‐Fiber‐Based Specific Antibody Filters
Authors: Hout Mariah S. , Federspiel William J.
Published: October 2003

We are developing hollow fiber‐based specific antibody filters (SAFs) that selectively remove antibodies of a given specificity directly from whole blood, without separation of the plasma and cellular blood components and with minimal removal of plasma proteins other than the targeted pathogenic antibodies. A principal goal of our research is to identify the primary mechanisms that control antibody transport within the SAF and to use this information to guide the choice of design and operational parameters that maximize the SAF‐based antibody removal rate. In this study, we formulated a simple mathematical model of SAF‐based antibody removal and performed in vitro antibody removal experiments to test key predictions of the model. Our model revealed three antibody transport regimes, defined by the magnitude of the Damköhler number Da (characteristic antibody‐binding rate/characteristic antibody diffusion rate): reaction‐limited (Da≤ 0.1), intermediate (0.1 < Da < 10), and diffusion‐limited (Da ≥ 10). For a given SAF geometry, blood flow rate, and antibody diffusivity, the highest antibody removal rate was predicted for diffusion‐limited antibody transport. Additionally, for diffusion‐limited antibody transport the predicted antibody removal rate was independent of the antibody‐binding rate and hence was the same for any antibody‐antigen system and for any patient within one antibody‐antigen system. Using SAF prototypes containing immobilized bovine serum albumin (BSA), we measured anti‐BSA removal rates consistent with transport in the intermediate regime (Da ∼3). We concluded that initial SAF development work should focus on achieving diffusion‐limited antibody transport by maximizing the SAF antibody‐binding capacity (hence maximizing the characteristic antibody‐binding rate). If diffusion‐limited antibody transport is achieved, the antibody removal rate may be raised further by increasing the number and length of the SAF fibers and by increasing the blood flow rate through the SAF.
Design of a Tubular Loop Bioreactor for Scale‐up and Scale‐down of Fermentation Processes
Authors: Papagianni Maria , Mattey Michael , Kristiansen Bjorn
Published: October 2003

Microorganisms traveling through circulation loops in large‐scale bioreactors experience variations in their environment such as dissolved oxygen concentration and pH gradients. The same changes are not experienced in small bioreactors, and it is suggested that herein lies one of the major reasons for the problems encountered when translating fermentation data from one scale to another. One approach to study this problem is to look at the circulation loop itself. The present work concerns an attempt to simulate the circulation loops inside stirred tank reactors, using a tubular loop reactor specially constructed for the purpose. The reactor carries a number of ports and probes along its length for the determination of concentration gradients within. The broth is circulated around the loop by the use of peristaltic pumps, and the circulation time (tc, s) is used as a measure of simulated reactor size. The reactor system has been evaluated using the citric acid fermentation by Aspergillus niger as a test process. Acid production and fungal morphology, in terms of the mean convex perimeter of mycelial clumps quantified by image analysis, were used as the parameters of evaluation for the two systems in comparison. From comparative experiments carried out in 10 and 200 L stirred tank bioreactors, it appears that the loop reactor simulates the corresponding stirred tank representing a valuable tool in scaling up and scaling down of fermentation process.
A Predictive Aggregate Transport Model for Microfiltration of Combined Macromolecular Solutions and Poly‐Disperse Suspensions: Model Development
Authors: Baruah Gautam Lal , Belfort Georges
Published: October 2003

A methodology, called the aggregate transport model, is presented that can a priori predict both the pressure‐independent permeation flux and yield of target species for the microfiltration of poly‐disperse solutions. The model captures the phenomenon of critical shear rate. Beyond the critical shear rate (expressed as a ratio of shear rate to permeation flux), the transmission of proteins drops sharply as a result of cake classification. The widely reported benefits of operating at uniform transmembrane pressure and constant wall concentration follow from this method. The methodology is general in nature and can be used predictively to obtain an optimal balance between flux and yield of target species during the microfiltration of many commercial poly‐disperse suspensions. In the accompanying paper we test this model for microfiltration of transgenic whole goat milk.
A Predictive Aggregate Transport Model for Microfiltration of Combined Macromolecular Solutions and Poly‐Disperse Suspensions: Testing Model with Transgenic Goat Milk
Authors: Baruah Gautam Lal , Couto Daniel , Belfort Georges
Published: October 2003

To meet the technical challenge of recovering human IgG fusion protein from transgenic whole goat milk at reasonable cost with high purity and yield, a predictive aggregate transport model for microfiltration has been developed (Baruah and Belfort, 2003). Here, to test the modelapos;s predictability of permeate flux and mass transport, a comprehensive series of experiments with varying wall shear rate, feed temperature, feed concentration, and module design are presented. A very good fit was obtained between the model predictions and measurements for a wide variety of experimental conditions. For microfiltration module design comparison, a linear hollow fiber module (representing current commercial technologies) gave lower permeation flux and higher yield than a helical hollow fiber module (representing the latest self‐cleaning methodology). These results are easily explained with the model that is now being used to define operating conditions for maximizing performance. The procedure described by the model is generalizable and can be used to obtain optimal filtration performance for applications other than milk.
Specific Oxygen Uptake Rate Variations during Batch Fermentation of Bacillus thuringiensis Subspecies kurstaki HD‐1
Authors: Rowe Gerald E. , Margaritis Argyrios , Wei Ning
Published: October 2003

The specific oxygen uptake rate (qO2, respiration rate) of Bacillus thuringiensis subsp. kurstaki HD‐1 was very high at inoculation and was found to decrease essentially monotonically throughout both vegetative growth phase and transition phase under different batch culture conditions. Average qO2 values decreased from 8–10 mmol/g h at 1 h after inoculation to less than 2 mmol/g h by the time growth ended. The results are shown to be consistent with the few previous reports on qO2 in B. thuringiensis in the literature but also novel in that this pattern of monotonic decline has not been described previously. Both pH control and EDTA in low concentration shortened the vegetative growth phase and reduced the 10 h biomass concentration. Using plots of qO2 versus specific growth rate, μ, biomass yield based on the oxygen used for growth, YmaxO2, was calculated for transition phase to be 0.041–0.047 g/mmol, consistent with literature values. The same plot also showed that the presence of EDTA resulted in an atypical qO2‐μ trajectory and apparently much higher biomass yield from the oxygen consumed.
Effect of Oxygen Delignification Operating Parameters on Downstream Enzymatic Hydrolysis of Softwood Substrates
Authors: Charles Nichola , Mansfield Shawn D. , Mirochnik Olga , Duff Sheldon J. B.
Published: October 2003

Enhanced oxygen delignification of softwood pulp samples (taken upstream and downstream of a commercial oxygen delignification unit) improved the initial rate of enzymatic saccharification and overall yield of monomeric sugars by 62–82% and 76–80%, respectively. Laboratory‐scale experiments were used to examine the effect of a broad range of operating parameters (temperature, time, caustic concentration, and oxygen partial pressure) on the effectiveness of oxygen delignification. Using empirical models, kappa number (residual lignin content) was found to effectively predict final conversion to monomeric sugars. Application of oxygen delignification to sulfite mill knots resulted in smaller (20–25%) reduction in lignin content. However, using a combination of oxygen delignification and particle size reduction, up to 80% of the carbohydrate in the reject knots could be converted to fermentable sugars.
Perspectives for the Industrial Enzymatic Production of Glycosides
Authors: de Roode B. Mattheus , Franssen Maurice C. R. , Padt Albert van der , Boom Remko M.
Published: October 2003

Glycosides are of commercial interest for industry in general and specifically for the pharmaceutical and food industry. Currently chemical preparation of glycosides will not meet EC food regulations, and therefore chemical preparation of glycosides is not applicable in the food industry. Thus, enzyme‐catalyzed reactions are a good alternative. However, until now the low yields obtained by enzymatic methods prevent the production of glycosides on a commercial scale. Therefore, high yields should be established by a combination of optimum reaction conditions and continuous removal of the product. Unfortunately, a bioreactor for the commercial scale production of glycosides is not available. The aim of this article is to discuss the literature with respect to enzymatic production of glycosides and the design of an industrially viable bioreactor system.
Supercritical Fluid Disruption of Ralstonia eutropha for Poly(β‐hydroxybutyrate) Recovery
Authors: Hejazi Parisa , Vasheghani‐Farahani Ebrahim , Yamini Yadollah
Published: October 2003

A new method for disruption of Gram‐negative bacterium Ralstonia eutrophaby supercritical CO2 for poly(β‐hydroxybutyrate) (PHB) recovery is proposed. The effects of different parameters such as exposure time, pressure, temperature, volume of methanol as a modifier, and culture history on cell disruption efficiency were investigated using Taguchiapos;s statistical approach to determine optimum conditions. The optimum conditions for cell disruption and PHB recovery were as follows: exposure time, 100 min; pressure, 200 atm; temperature, 40 °C; volume of methanol, 0.2 mL. The cell culture time was less significant. At optimum conditions, the maximum efficiency of PHB recovery was found to be 89%. The proposed method is comparable with other recovery methods in terms of the percentage of PHB recovery, while it is environmentally more benign.
Micropatterned Substrates for the Growth of Functional Neuronal Networks of Defined Geometry
Authors: Vogt Angela K. , Lauer Lars , Knoll Wolfgang , Offenhäusser Andreas
Published: October 2003

The in vitro assembly of neuronal networks with control over cell position and connectivity is a fascinating approach not only for topics in basic neuroscience research but also in diverse applications such as biosensors and tissue engineering. We grew rat embryonic cortical neurons on patterned substrates created by microcontact printing. Polystyrene was used as a cell repellent background, onto which a grid pattern of physiological proteins was applied. We printed laminin and a mixture of extracellular matrix proteins and additionally both systems mixed with polylysine. Attachment of cells to the pattern with high fidelity as well as the formation of chemical synapses between neighboring cells on the pattern could be observed in all four cases, but cell attachment was strongly increased on samples containing polylysine. Neurons grown on patterned substrates had a membrane capacity smaller than that of neurons on homogeneously coated controls, which we attributed to the geometrical restrictions, but did not differ either in resting membrane potential or in the quality of synapses they formed. We therefore believe that the cells attach and differentiate normally on the pattern and form functional, mature synapses following the predefined geometry.
Effects of Three‐Dimensional Culturing on Osteosarcoma Cells Grown in a Fibrous Matrix: Analyses of Cell Morphology, Cell Cycle, and Apoptosis
Authors: Chen Chunnuan , Chen Kathryn , Yang Shang‐Tian
Published: October 2003

Osteosarcoma cells were cultured in stirred tank bioreactors with either a fibrous matrix or nonporous microcarriers to study the environmental effects on cell growth, morphology, cell cycle, and apoptosis. Cell cycle and apoptosis were analyzed using flow cytometry and visualized using confocal laser scanning microscopy and fluorescence microscopy. The three‐dimensional (3‐D) fibrous culture had better cell growth and higher metabolic rates than the two‐dimensional (2‐D) microcarrier culture because cells in the fibrous matrix were protected from shear stress and had lower apoptosis and cell death even under suboptimal conditions (e.g., nutrient depletion). The polyester fibrous matrix used in this study also exhibited the capability of selectively retaining viable and nonapoptotic cells and disposing apoptotic and nonviable cells. Consequently, very few apoptotic cells were found in the fibrous matrix even in the long‐term (1 month) T‐flask culture. In the continuous culture with packed fibrous matrixes for cell support, most cells were arrested in the G1/G0 phase after 4 days. Decreasing the dissolved oxygen level from 60 to 10% air saturation did not significantly change cell cycle and apoptosis, which remained low at ∼15%. These results could explain why the fibrous bed bioreactor had good long‐term stability and was advantageous for production of non‐growth‐associated proteins by animal cell cultures.
Chimeric Gene Library Construction by a Simple and Highly Versatile Method Using Recombination‐Dependent Exponential Amplification
Authors: Ikeuchi Akinori , Kawarasaki Yasuaki , Shinbata Tomoya , Yamane Tsuneo
Published: October 2003

A simple and efficient method for the construction of chimeric gene libraries termed RDA‐PCR (recombination‐dependent exponential amplification polymerase chain reaction) was developed by modifying polymerase chain reaction. A chimeric gene library is generated from homologous parental genes with additional primer‐annealing sequences at their “heads” and “tails”. Two primers (“skew primers”) are designed to exclusively anneal to either the heads of maternal genes or the tails of paternal genes. During the RDA‐PCR, short annealing/extension periods facilitate homologous recombination. The chimeric sequences can be exponentially amplified to form the chimeric gene library, whereas parental sequences without crossovers are not amplified. As a model, we constructed a chimeric gene library of yellow and green fluorescent protein (yfp and gfp, respectively). The crossover point profile of RDA‐PCR clones was compared with those obtained by (modified) family shuffling. PCR restriction fragment polymorphism (PCR‐RFLP) analysis of the RDA‐PCR clones showed a high content of chimeric genes in the library, whereas family shuffling required the modification using skew primers for selective enrichment of chimeric sequences. PCR‐RFLP analysis also indicated that the crossover points of RDA‐PCR chimeras were distributed over the entire protein‐coding region. Moreover, as few as 2 bp of the continual identity of nucleotides were found at the crossover points at high frequency (30% of the tested clones), suggesting that RDA‐PCR resulted in a higher diversity in crossover points than family shuffling.
Strategic Selection of Hyperthermophilic Esterases for Resolution of 2‐Arylpropionic Esters
Authors: Sehgal Amitabh C. , Kelly Robert M.
Published: October 2003

Homologues to Carboxylesterase NP and Candida rugosa lipase, used for the chiral separation of racemic mixtures of 2‐arylpropionic methyl esters, were identified by BLAST searches of available genome sequences for hyperthermophilic microorganisms. Two potential candidates were identified: a putative lysophospholipase from Pyrococcus furiosus (Pfu‐LPL) and a carboxylesterase from Sulfolobus solfataricus P1 (Sso‐EST1). Although both enzymes showed hydrolytic preference toward the (S) methyl ester, only Sso‐EST1 yielded highly optically pure (S) naproxen (%eep ≥ 90) and was thus further investigated. Changes in pH or reaction time showed little improvement in %eep or E values with Sso‐EST1. However, the addition of 25% methanol resulted in a 25% increase in E. The effect of various cosolvents on the enantiomeric ratio showed no correlation with the log P or dielectric constant values of the solvent. However, an inverse relationship between E and the denaturation capacity (DC) of the water miscible cosolvents was observed. This was attributed to an increase in enzyme flexibility with increasing solvent DC values leading to a concomitant reduction in the resolving power of Sso‐EST1. The results here show that although bioinformatics tools can be used to select candidate biocatalysts for chiral resolution of 2‐arylpropionic esters, biochemical characterization is needed to definitively determine functional characteristics.
Production of Podophyllotoxin Using Cross‐Species Coculture of Linum flavum Hairy Roots and Podophyllum hexandrum Cell Suspensions
Authors: Lin Han‐wei , Kwok Kian H. , Doran Pauline M.
Published: October 2003

Novel cross‐species coculture systems using Linum flavumhairy roots and Podophyllum hexandrum cell suspensions were applied for in vitro production of podophyllotoxin. The hairy roots and suspensions were cocultured in Linsmaier and Skoog medium in dual shake flasks and dual bioreactors. In separate experiments, coniferin feeding was shown to be an effective strategy for increasing the accumulation of podophyllotoxin in P. hexandrum suspensions. Because roots of L. flavum are a natural source of coniferin, hairy roots of this species were used in coculture with P. hexandrum to provide an in situ supply of coniferin. Compared with P. hexandrumsuspensions cultured alone in shake flasks or bioreactors, podophyllotoxin concentrations in cocultured P. hexandrum cells were increased by 240% and 72% in dual shake flask and dual bioreactor systems, respectively. The availability and stability of coniferin in the medium are the most likely factors limiting podophyllotoxin synthesis in coculture. Intensification of the coculture process is required to further improve total podophyllotoxin accumulation on a volumetric basis.
Predicted Unfolding Order of the 13 α‐Helices in the Catalytic Domain of Glucoamylase from Aspergillusawamori var. X100 by Molecular Dynamics Simulations
Authors: Liu Hsuan‐Liang , Wang Wen‐Chi
Published: October 2003

The unfolding mechanism of the 13 α‐helices in the catalytic domain of Aspergillus awamori var. X100 glucoamylase was investigated by 200 ps molecular dynamics simulations in explicit water with temperature jump technique. Rather than a simultaneous event, the unfolding of these 13 α‐helices followed a random ordered mechanism as α8→α1→α11→α7→α10→α3→α12→α13→α4→α5→α9→α6→α2. No significant relationships were found between the unfolding order and the length and the hydrophobicity of the helix. α‐Helix 8 located in the inner region of the catalytic domain was predicted to be the first helix to unfold, indicating that the destruction of the secondary structure motif was initiated from the inner region of the catalytic domain. The dynamic behavior of these α‐helices induced by increased kinetic energy during the unfolding process is considered to be similar to the expansion and compression of a series of springs under the influence of mechanical stress.
Tuning the Transcription Rate of Recombinant Protein in Strong Escherichiacoli Expression Systems through Repressor Titration
Authors: Striedner Gerald , Cserjan‐Puschmann Monika , Pötschacher Florentina , Bayer Karl
Published: October 2003

The main goal of this work was to develop a strategy that enables tuning of recombinant gene expression relative to the metabolic capacity of the host cell synthesis machinery. In the past, strong expression systems have been developed in order to maximize recombinant gene expression. However, these systems exert an extremely high metabolic burden onto the host cell, which may even lead to cell death. Hence, the period of recombinant gene expression is significantly reduced, and therefore, maximal yield cannot be attained. To extend the production phase and to achieve optimal yields, adjustment of recombinant gene expression by modulation of the transcription rate is required. To control transcription, we designed a feed regime, which continuously supplies limiting amounts of inducer in a constant ratio to biomass. For the accurate determination of appropriate amounts of inducer, a time shifted exponential substrate and inducer feed strategy has been developed. The potential of this metabolic and engineering integrated approach was proven in fed‐batch cultivation experiments using E. coliHMS174(DE3)(pET11ahSOD) as model system. Furthermore, our strategy enables the use of lactose as inducer, since its consumption can be compensated by appropriate feed profiles. The attained results fully comply with all requirements of industrial large scale cultivation and improve the applicability of strong expression systems.
Polymerization of Guaiacol and a Phenolic β‐O‐4‐Substructure by Trameteshirsuta Laccase in the Presence of ABTS
Authors: Rittstieg Klaus , Suurnäkki Anna , Suortti Tapani , Kruus Kristiina , Guebitz Georg M. , Buchert Johanna
Published: October 2003

The reaction of the monomeric lignin model compound guaiacol and the β‐O‐4‐type dimer erol (1–(4‐hydroxy‐3‐methoxyphenyl)–2(2‐methoxyphenoxy)‐propane‐1,3‐diol with laccase from Trametes hirsuta was studied in the presence of the mediator ABTS (2,2′‐azino‐di[3ethylbenzothiazoline‐6‐sulfonic acid]). The product mixtures were analyzed by means of aqueous‐phase size exclusion chromatography (SEC) with 50 mM NaOH as eluent. Interestingly, in the laccase‐catalyzed reaction with both substrates, the mediator not only functioned as an electron carrier but underwent coupling reactions with the substrate to give polymeric coupling products. The molecular weight of these copolymeric products was significantly higher than the molecular weight of products obtained without ABTS. After ultrafiltration, 33% and 21% of the initially applied ABTS could be found in the polymeric product fraction for the substrates guaiacol and erol, respectively, on the basis of nitrogen analysis. When ABTS was added to substrates after full laccase‐catalyzed polymerization, the reaction proceeded toward higher molecular weights.
Study of Process Variables in Industrial Acetic Fermentation by a Continuous Pilot Fermentor and Response Surfaces
Authors: Garrido‐Vidal Diego , Pizarro Consuelo , González‐Sáiz José M.
Published: October 2003

The modeling and optimization of industrial processes requires an intensive study of the factors involved. In this work, a continuous pilot system for studying the industrial process of acetic fermentation is developed. A Doehlert design is applied to the five variables involved in the pilot process. This experimental design allows reduction of the experimental burden and the maximum amount of information to be obtained, studying the factors at different levels depending on their significance. The experimental system provides a robust measure of the specific growth rate and the rates of substrates consumption and acetic acid production, related to the flow of effluent stream evaluated in the steady state. The results demonstrate the growth‐associated kinetics of substrates and product, and the yield factors are calculated with low values of variances for the coefficients, i.e., within the range 1–11%. The specific growth rate suits the quadratic model proposed. The response surfaces generated by the model are applied to explain the behavior of the bacterial growth and, therefore, the effects of the process variables studied over the acetic acid production. Very low levels of ethanol or oxygen make the acetification rate decrease, and a saturation effect with high levels of ethanol or oxygen is also deduced. The effects of the aeration rate, agitation, and overpressure suggest a kind of inhibition of the acetic acid production caused by the oxygen that has not been practically studied before. The temperature strengthens the inhibitory effect of the ethanol and the oxygen. The conclusions of this work consolidate the structure of a hybrid model for the acetic fermentation.
EASE Vectors for Rapid Stable Expression of Recombinant Antibodies
Authors: Aldrich Teri L. , Viaje Aurora , Morris Arvia E.
Published: October 2003

Over the past 10 years, monoclonal antibodies and antibody fragments have become an increasingly important source of therapeutic molecules in the biotechnology industry. Drug development strategies rely on screening large numbers of candidate molecules in search of an optimized drug candidate. This strategy requires efficient production of ten to a few hundred milligrams of candidate molecules for screening in bioassays and animal models. Typically, this amount of recombinant protein expression involves large numbers of transient transfections or cloning of a recombinant cell line. Both of these approaches are time‐consuming and labor‐intensive. In this report, we describe the application of an EASE vector system that is capable of generating stable pools of transfected Chinese hamster ovary cells. These pooled populations of cells produce high quantities of antibody candidates without labor‐intensive cloning in a 3–5 week time frame. When an optimal drug candidate has been selected, pools generated with EASE‐containing vectors can also be used in subsequent cloning steps to make cell lines with improved expression levels. We demonstrate that EASE increases expression in nonamplified pools in addition to increasing amplification and viability of clonal cell lines generated with the EASE‐containing vectors compared with pools and cell lines generated without EASE.
Altered Glucose Transport and Shikimate Pathway Product Yields in E.coli
Authors: Yi Jian , Draths K. M. , Li Kai , Frost J. W.
Published: October 2003

Different glucose transport systems are examined for their impact on phosphoenolpyruvate availability as reflected by the yields of 3‐dehydroshikimic acid and byproducts 3‐deoxy‐d‐arabino‐heptulosonic acid, 3‐dehydroquinic acid, and gallic acid synthesized by Escherichia coli from glucose. 3‐Dehydroshikimic acid is an advanced shikimate pathway intermediate in the syntheses of a spectrum of commodity, pseudocommodity, and fine chemicals. All constructs carried plasmid aroFFBR and tktA inserts encoding, respectively, a feedback‐insensitive isozyme of 3‐deoxy‐d‐arabino‐heptulosonic acid 7‐phosphate synthase and transketolase. Reliance on the native E. coli phosphoenolpyruvate:carbohydrate phosphotransferase system for glucose transport led in 48 h to the synthesis of 3‐dehydroshikimic acid (49 g/L) and shikimate pathway byproducts in a total yield of 33% (mol/mol). Use of heterologously expressed Zymomonas mobilis glf‐encoded glucose facilitator and glk‐encoded glucokinase resulted in the synthesis in 48 h of 3‐dehydroshikimic acid (60 g/L) and shikimate pathway byproducts in a total yield of 41% (mol/mol). Recruitment of native E. coli galP‐encoded galactose permease for glucose transport required 60 h to synthesize 3‐dehydroshikimic acid (60 g/L) and shikimate pathway byproducts in a total yield of 43% (mol/mol). Direct comparison of the impact of altered glucose transport on the yields of shikimate pathway products synthesized by E. coli has been previously hampered by different experimental designs and culturing conditions. In this study, the same product and byproduct mixture synthesized by E. coli constructs derived from the same progenitor strain is used to compare strategies for increasing phosphoenolpyruvate availability. Constructs are cultured under the same set of fermentor‐controlled conditions.
Metabolic Engineering of Pentose Phosphate Pathway in Ralstoniaeutropha for Enhanced Biosynthesis of Poly‐β‐hydroxybutyrate
Authors: Lee Jin‐Nam , Shin Hyun‐Dong , Lee Yong‐Hyun
Published: October 2003

Poly‐β‐hydroxybutyrate (PHB) biosynthesis in Ralstonia eutrophafrom gluconate as a carbon source is carried out through the Entner‐Doudoroff (ED) pathway and the pentose‐phosphate (PP) pathway generating NADPH and glyceraldehyde‐3‐phosphate that flows to acetyl‐CoA, actively in the unbalanced PHB accumulation phase. The gnd gene encoding 6‐phosphogluconate dehydrogenase (6PGDH) and the tktA gene encoding the transketolase (TK) in PP pathway of E. coli were transformed into R. eutropha H16 to modify the metabolic flux of gluconate to the PHB biosynthesis. Over‐generated NADPH by the amplified gnd gene tended to depress the cell growth and PHB concentration. Meanwhile, the amplified tktA gene significantly increased both PHB biosynthesis and cell growth as a result of the effective flow of glyceraldehyde‐3‐phosphate into acetyl‐CoA along with the concomitant supplementation of NADPH. The amplified tktA gene also activated the enzyme activities directly associated with PHB biosynthesis. The transformant R. eutropha harboring tktA gene was cultivated using pH‐stat‐fed‐batch to achieve the overproduction of PHB.
Enzyme Distribution Derived from Macroscopic Particle Behavior of an Industrial Immobilized Penicillin‐G Acylase
Authors: Van Roon Jeroen L. , Joerink Michiel , Rijkers Marinus P. W. M. , Tramper Johannes , P. H. Schroën Catharina G. , Beeftink Hendrik H.
Published: October 2003

The macroscopic kinetic behavior of an industrially employed immobilized penicillin‐G acylase, called Assemblase, formed the basis for a discussion on some simple intraparticle biocatalytic model distributions. Assemblase catalyzes the synthesis of the widely used semisynthetic antibiotic cephalexin. Despite the obvious advantages of immobilization, less cephalexin and more of the unwanted byproduct d‐(–)‐phenylglycine are obtained due to diffusional limitations when the immobilized enzyme is employed. To rationally optimize Assemblase, the parameters particle size, enzyme loading, and enzyme distribution, which severely determine the macroscopic particle performance, were studied on the basis of macroscopic observations. Laser diffraction measurements showed that the particle sizes in Assemblase vary as much as 100‐fold. The relative and total enzyme loadings in Assemblase and fractions thereof of different sizes were determined by initial‐rate d‐(–)‐phenylglycine amide hydrolysis, cephalexin synthesis experiments, and active‐site titration. These experiments revealed that the loading of penicillin‐G acylase in Assemblase was inversely correlated with the particle diameter. Apart from enzyme loadings, estimates on the intraparticle enzyme distribution came from cephalexin synthesis experiments, where mass‐transport limitations were present. Although this method cannot provide the level of detail of specific labeling experiments, it is simple, fast, and cheap. Within the set of simple model predictions, a heterogeneous enzyme distribution with most biocatalyst present in the outer region of the particle (within the outer 100 μm) gave the best description of the observed behavior, although no exact correlation was established. Highly detailed determination of intraparticle enzyme distributions must come from immunolabeling.
Cell Surface Display of Organophosphorus Hydrolase in Pseudomonasputida Using an Ice‐Nucleation Protein Anchor
Authors: Shimazu Mark , Nguyen Anne , Mulchandani Ashok , Chen Wilfred
Published: October 2003

A surface anchor system derived from the ice‐nucleation protein (INP) from Pseudomonas syringe was used to localize organophosphorus hydrolase (OPH) onto the surface of Pseudomonas putida KT2440. Cells harboring the shuttle vector pPNCO33 coding for the INP‐OPH fusion were capable of targeting OPH onto the cell surface as demonstrated by whole cell ELISA. The whole cell activity of P. putida KT2440 was shown to be 10 times higher than those of previous efforts expressing the same fusion protein in Escherichia coli. The capability of expressing enzymes on the surface of a robust and environmentally benign P. putida KT2440 should open up new avenues for a wide range of applications such as in situ bioremediation.
Novel Fermentation Strategy for Enhancing Glycerol Production by Candidakrusei
Authors: Liu Hong‐Juan , Liu De‐Hua , Zhong Jian‐Jiang
Published: October 2003

During the later stage of glycerol production by fermentation of Candida krusei, glycerol consumption by the strain was observed, although there was residual sugar in the medium. To enhance the final glycerol accumulation, a new fermentation strategy was developed by maintaining high activities of glycerol synthetic enzymes (i.e., glycerol‐3‐phosphate dehydrogenase (ctGPD) and glycerol‐3‐phosphatase (GPP)) for a relatively long period while conducting oxygen limitation at a later stage to inhibit the increase of another enzyme activity related to glycerol degradation (i.e., mitochondrial glycerol‐3‐phosphate dehydrogenase (mtGPD)). With oxygen limitation performed from 88 h, when ctGPD and GPP activities were already at a low level while mtGPD activity was increasing, the glycerol dissimilation was efficiently reduced. The final glycerol concentration reached 55.6 g/L, which was about 18% (96 h) and 30% (104 h) higher than control, and its productivity increased to 0.54 g/(L h). The proposed strategy based on cell physiology was proved useful to the glycerol fermentation process.
Attachment of Marine Sponge Cells of Hymeniacidon perleve on Microcarriers
Authors: Zhao Quanyu , Jin Meifang , G. Müller Werner E. , Zhang Wei , Yu Xingju , Deng Maicun
Published: October 2003

Toward the development of an in vitro cultivation of marine sponge cells for sustainable production of bioactive metabolites, the attachment characteristics of marine sponge cells of Hymeniacidon perleve on three types of microcarriers, Hillex, Cytodex 3, and glass beads, were studied. Mixed cell population and enriched cell fractions of specific cell types by Ficoll gradient centrifugation (6%/8%/15%/20%) were also assessed. Cell attachment ratio (defined as the ratio of cells attached on microcarrier to the total number of cells in the culture) on glass beads is much higher than that on Cytodex 3 and Hillex for both mixed cell population and cell fraction at Ficoll 15–20% interface. The highest attachment ratio of 41% was obtained for the cell fraction at Ficoll 15–20% interface on glass beads, which was significantly higher than that of a mixed cell population (18%). The attachment kinetics on glass beads indicated that the attachment was completed within 1 h. Cell attachment ratio decreases with increase in cell‐to‐microcarrier ratio (3–30 cells/bead) and pH (7.6–9.0). The addition of serum and BSA (bovine serum albumin) reduced the cell attachment on glass beads.
Improved Mammalian Expression Systems by Manipulating Transcriptional Termination Regions
Authors: Kim Dongjun , Kim Jeong Do , Baek Kwanghee , Yoon Yeup , Yoon Jaeseung
Published: October 2003

Here we show that the transcriptional terminator element of human gastrin gene, which is the only element characterized to date in terms of its function in transcriptional termination, increases the transient expression levels of recombinant proteins. The expression of the β‐galactosidase gene was enhanced 3–4‐fold in HeLa cells by inserting the terminator element of human gastrin gene at the 3′‐side of the SV40 polyadenylation signal/cleavage site of the control vector (pSV‐β‐gal). This effect of the terminator element is orientation‐dependent but not cell‐specific since a similar enhancement of β‐galactosidase gene expression was detected in COS.M6 and CHO DG44 cells. The increased level of β‐galactosidase gene expression by the transcriptional terminator element of human gastrin gene turned out to arise from elevated cellular mRNA levels, suggesting that the terminator element stabilizes mRNA by enhancing proper 3′‐end processing of mRNA.
Size and Stability of Reconstituted Sesame Oil Bodies
Authors: Peng Chi‐Chung , Lin I‐Ping , Lin Ching‐Kuan , Tzen Jason T. C.
Published: October 2003

Oil bodies of sesame seeds comprise a triacylglycerol matrix, which is surrounded by a monolayer of phospholipids embedded with unique proteins, mainly structural proteins termed oleosins. Artificial oil bodies were successfully reconstituted with various compositions of triacylglycerols, phospholipids, and oil‐body proteins. The sizes of reconstituted oil bodies displayed a normal distribution with an average size proportional to the ratio of triacylglycerols to oil‐body proteins. Both thermostability and structural stability of reconstituted oil bodies decreased as their sizes increased, and vice versa. Proteinase K digestion indicated that oleosins anchored both native and reconstituted oil bodies via their central hydrophobic domains. The stability of reconstituted oil bodies, as well as the purified ones from sesame seeds, could be substantially enhanced after their surface proteins were cross‐linked by glutaraldehyde or genipin.
How do nitrogen inputs to the Changjiang basin impact the Changjiang River nitrate: A temporal analysis for 1968–1997
Authors: Yan Weijin , Zhang Shen , Sun Pu , Seitzinger Sybil P.
Published: October 2003

We present estimates of nitrogen (N) inputs to the Changjiang River basin for the period 1968–1997. The total N input is approximately 7.8 × 109 kg in 1997, which is a threefold increase over 1968 levels. N fixation was often a dominant input before 1978, providing about 2.2 × 109 kg year−1, while N fertilizer dominated N input after 1983, supplying an additional input of some 4.4 × 109 kg year−1. More than 40% of total N inputs is converted into manure N, and half of total manure N is returned to agricultural soil. We estimate that the river nitrate concentration and flux have increased about tenfold from 1968 to 1997. Our study suggests that the percent of N inputs to the basin that are exported by the river as NO3‐N has increased steadily over the 30‐year period and that about 30% of total N input is transported through the river. The integrated N input, budget, and storage have been linked to the increasing temporal trends of Changjiang River nitrate. N fertilizer application and human population density, as well as manure N production in the basin, are good predictors of the river's nitrate concentration and flux. Therefore, how N balance is kept (especially for effective application of N fertilizer) is a crucial problem to the sustainable development of the basin.
Aberrant youth: Chemical and isotopic constraints on the origin of off‐axis lavas from the East Pacific Rise, 9°–10°N
Authors: Sims K. W. W. , Blichert‐Toft J. , Fornari D. J. , Perfit M. R. , Goldstein S. J. , Johnson P. , DePaolo D. J. , Hart S. R. , Murrell M. T. , Michael P. J. , Layne G. D. , Ball L. A.
Published: October 2003

We report measurements of U‐series disequilibria, Sr, Nd, Hf, and Pb isotopic compositions and major and trace element abundances in a suite of well‐located, off‐axis MORBs that span the East Pacific Rise (EPR) ridge crest from 9°48′–52′N and across it for ∼4 km on either side. The geological context of the samples are well constrained as they were collected by submersible in an area that has been extensively imaged by remote sensing techniques. Sr, Nd, Hf and 208Pb/206Pb isotopic compositions of the off‐axis N‐MORB are identical to the axial lavas from this same region, suggesting that their sources are similar and that melting processes are the dominant influence in establishing the U‐Th‐Ra disequilibria and trace element fractionations. A majority of off‐axis samples have U‐Th and Th‐Ra disequilibria that are larger, and model ages that are younger, than would be predicted from their off‐axis distance and the time‐integrated spreading rate. There are, however, a few off‐axis samples with U‐Th ages that are consistent with their spreading rate ages. It is likely that these samples erupted within or close to the axial summit trough (AST) and aged at a rate proportional to the spreading rate. The anomalously young ages determined for most of the off‐axis lavas suggest that volcanic construction along this region is occurring over a zone that is wider (at least 4 km) than the AST (10s to 100s of meters). The combined observational, chemical and isotopic data support a model for the 9°0′N area that includes a significant component of crustal accumulation resulting from lavas that breach the AST and flow down the flanks of the EPR ridge crest. However, these data also require a minor component of off‐axis eruptions that occur on distinct pillow mounds and ridges. This suggests that MOR construction involves several volcanic and tectonic processes acting in concert to form a complex patchwork of lava ages and compositions along, and across, this fast spreading ridge crest.
The Sea of Marmara: A two‐dimensional seismic reflection profile data archive
Authors: Parke J. R. , White R. S. , McKenzie D. , Minshull T. A. , Bull J. , Kuşçu I. , Görür N. , Şengör A. M. C.
Published: October 2003

In 1997, 1500 km of high resolution two‐dimensional (2‐D) reflection seismic data were acquired in the Sea of Marmara region, western Turkey. An archive of the 40 profiles of processed seismic data, in SEGY format, together with the Differential Global Positioning System (DGPS) locations and acquisition parameters for each profile are available for download from IRIS.
Microseismicity at the seaward updip limit of the western Nankai Trough seismogenic zone
Authors: Obana Koichiro , Kodaira Shuichi , Kaneda Yoshiyuki , Mochizuki Kimihiro , Shinohara Masanao , Suyehiro Kiyoshi
Published: October 2003

The seismogenic zone at subduction zones does not normally extend to the trench axis. The shallowest part of the plate interface is considered to be seismically decoupled. The location of the seaward updip limit of the seismogenic zone and its relation to the crustal structure are important for understanding the transition process from aseismic slip to the seismic rupture at the plate interface. The Nankai Trough seismogenic zone, southwestern Japan, is one of the most well‐studied subduction seismogenic zones in the world. However, the offshore seismicity around the Nankai Trough is very low, and hypocenters are not determined accurately by the on‐land seismic network. We performed microseismicity observations using ocean bottom seismographs off Cape Muroto. Hypocenters were determined by using a three‐dimensional Vp and Vs structure based on seismic survey results. The observed microseismicity seems to be classifiable into two types. The first is earthquakes that occur around the plate interface; the second is a group of earthquakes that occur in the uppermost mantle of the subducting oceanic plate. The seismicity around the plate interface forms several clusters. The seaward limit of the clusters is characterized by earthquakes with a similar waveform. These similar waveform earthquakes are considered to occur at small asperities in the aseismic‐seismogenic transition zone of the plate interface. The seismicity in the uppermost mantle may be related to dehydration embrittlement of subducting serpentinized mantle.
Cross‐shore sediment transport and the equilibrium morphology of mudflats under tidal currents
Authors: Pritchard D. , Hogg A. J.
Published: October 2003

We describe a mathematical model of suspended sediment transport under cross‐shore tidal currents on an intertidal mudflat. We employ a Lagrangian formulation to obtain periodic solutions for the sediment transport over idealized bathymetries and use these to investigate the process of settling lag. In deep water away from the shoreline the concentration of suspended sediment tends to a constant value, and we may invert the relation between bathymetry and offshore concentration to estimate how the gradient of a flat in a state of equilibrium varies with the sediment properties and supply. These analytical estimates are compared successfully with the numerical experiments of previous studies. We discuss the robustness of our modeling framework and demonstrate its application to different descriptions of sediment transport and tidal regimes.
Cloud inhomogeneity and broadband solar fluxes
Authors: Scheirer R. , Macke A.
Published: October 2003

Simplified representations of spatially inhomogeneous (three‐dimensional (3‐D)) clouds in radiative transfer models provide systematic errors when calculating solar broadband radiative fluxes. An example is the neglect of horizontal photon transports as it is the case for the independent column approximation (ICA). The present work tries to quantify and interpret these errors on the basis of a large set of 3‐D mixed phase cloud scenarios with 3‐D varying extinction coefficients, scattering phase functions, and single‐scattering albedos. The cloud cases result from a mesoscale atmospheric circulation model with detailed cloud microphysics. Domain‐averaged cloud radiative fluxes are calculated by means of a Monte Carlo radiative transfer model. Depending on cloud type and solar zenith angle (SZA) the differences between 3‐D and ICA results range from +20 W m−2 to −30 W m−2 for the upward reflected fluxes and from +10 W m−2 to −7 W m−2 for the absorbed fluxes. The mean (averaged over all cloud realizations) errors of the ICA‐based upward fluxes vary between 5 W m−2 overestimation at 15°SZA and 6 W m−2 underestimation at 75°SZA. The ICA underestimates the absorbed flux by ∼1–2 W m−2 for most SZA except for 75°. It is found that neglecting the horizontal variability of the absorption and scattering properties of the cloud hydrometeors leads to a general underestimation of solar broadband absorption by as much as 15 W m−2 with average values between 4 W m−2 at small SZA and 1 W m−2 at large SZA.