bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of galP and glk in a Escherichia coli PTS mutant restores glucose transport and increases glycolytic flux to fermentation products
Authors: Hernández‐Montalvo Verónica , Martínez Alfredo , Hernández‐Chavez Georgina , Bolivar Francisco , Valle Fernando , Gosset Guillermo 
Published: July 2003

AbstractIn Escherichia coli, the uptake and phosphorylation of glucose is carried out mainly by the phosphotransferase system (PTS). Despite the efficiency of glucose transport by PTS, the required consumption of 1 mol of phosphoenolpyruvate (PEP) for each mol of internalized glucose represents a drawback for some biotechnological applications where PEP is a precursor of the desired product. For this reason, there is considerable interest in the generation of strains that can transport glucose efficiently by a non‐PTS mechanism. The purpose of this work was to study the effect of different gene expression levels, of galactose permease (GalP) and glucokinase (Glk), on glucose internalization and phosphorylation in a E. coli PTS− strain. The W3110 PTS−, designated VH32, showed limited growth on glucose with a specific growth rate (μ) of 0.03 h−1. A low copy plasmid family was constructed containing E. coli galP and glk genes, individually or combined, under the control of a trc‐derived promoter set. This plasmid family was used to transform the VH32 strain, each plasmid having different levels of expression of galP and glk. Experiments in minimal medium with glucose showed that expression of only galP under the control of a wild‐type trc promoter resulted in a μ of 0.55 h−1, corresponding to 89% of the μ measured for W3110 (0.62 h−1). In contrast, no increase in specific growth rate (μ) was observed in VH32 with a plasmid expressing only glk from the same promoter. Strains transformed with part of the plasmid family, containing both galP and glk genes, showed a μ value similar to that of W3110. Fermentor experiments with the VH32 strain harboring plasmids pv1Glk1GalP, pv4Glk5GalP, and pv5Glk5GalP showed that specific acetate productivity was twofold higher than in W3110. Introduction of plasmid pLOI1594, coding for pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis, to strain VH32 carrying one of the plasmids with galP and glk caused a twofold increase in ethanol productivity over strain W3110, also containing pLOI1594. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 687–694, 2003.
Effects of β‐cyclodextrin–dextran polymer on stability properties of trypsin
Authors: Fernández Michael , Villalonga Maria L. , Caballero Julio , Fragoso Alex , Cao Roberto , Villalonga Reynaldo 
Published: July 2003

AbstractDextran modified with the mono‐6‐pentylene‐diamino‐6‐deoxy‐β‐cyclodextrin derivative was evaluated as a thermoprotectant additive for trypsin. The optimum temperature for trypsin activity was increased by 7°C in the presence of this polymer. The enzyme thermostability was increased from 48.5 to 64°C over 10 min of incubation, and the activation free energy of thermoinactivation at 50°C was increased by 4.1 kJ/mol in the presence of the additive. Trypsin was 6‐fold more resistant to autolytic inactivation at alkaline pH in the presence of the polymer. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 743–747, 2003.
Simultaneous nitrification and denitrification using stored substrate (phb) as the electron donor in an SBR
Authors: Third Katie A. , Burnett Natalie , Cord‐Ruwisch Ralf 
Published: July 2003

AbstractThe potential for PHB (poly‐β‐hydroxybutyrate) to serve as the electron donor for effective simultaneous nitrification and denitrification (SND) was investigated in a 2‐L sequencing batch reactor (SBR) using a mixed culture and acetate as the organic substrate. During the feast period (i.e., acetate present), heterotrophic respiration activity was high and nitrification was prevented due to the inability of nitrifying bacteria to compete with heterotrophs for oxygen. Once acetate was depleted the oxidation rate of PHB was up to 6 times slower than that of soluble acetate and nitrification could proceed due to the decreased competition for oxygen. The slow nature of PHB degradation meant that it was an effective substrate for SND, as it was oxidised at a similar rate to ammonium and was therefore available for SND throughout the entire aerobic period. The percentage of nitrogen removed via SND increased at lower DO concentrations during the famine period, with up to 78% SND achieved at a DO concentration of 0.5 mg L−1. However, the increased percentage of SND at a low DO concentration was compromised by a 2‐times slower rate of nitrogen removal. A moderate DO concentration of 1 mg L−1 was optimal for both SND efficiency (61%) and rate (4.4 mmol N. Cmol X−1 · h−1). Electron flux analysis showed that the period of highest SND activity occurred during the first hour of the aerobic famine period, when the specific oxygen uptake rate (SOUR) was highest. It is postulated that a high SOUR due to NH and PHB oxidation decreases oxygen penetration into the floc, creating larger zones for anoxic denitrification. The accumulation of nitrate towards the end of the SND period showed that SND was finally limited by the rate of denitrification. As PHB degradation was found to follow first‐order kinetics (dfPHB/dt = −0.19 · fPHB), higher PHB concentrations would be expected to drive SND faster by increasing the availability rate of reducing power and reducing penetration of oxygen into the floc, due to the corresponding increased SOUR. Process control techniques to accumulate higher internal PHB concentrations to improve PHB‐driven SND are discussed. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 706–720, 2003.
Detection and identification ofEscherichia coli, Shigella,andSalmonellaby microarrays using thegyrBgene
Authors: Kakinuma Kenichi , Fukushima Masao , Kawaguchi Ryuji 
Published: July 2003

AbstractCommonly, 16S ribosome RNA (16S rRNA) sequence analysis has been used for identifying enteric bacteria. However, it may not always be applicable for distinguishing closely related bacteria. Therefore, we selectedgyrBgenes that encode the subunit B protein of DNA gyrase (a topoisomerase type II protein) as target genes. The molecular evolution rate ofgyrBgenes is higher than that of 16S rRNA, andgyrBgenes are distributed universally among bacterial species. Microarray technology includes the methods of arraying cDNA or oligonucleotides on substrates such as glass slides while acquiring a lot of information simultaneously. Thus, it is possible to identify the enteric bacteria easily using microarray technology. We devised a simple method of rapidly identifying bacterial species through the combined use ofgyrBgenes and microarrays. Closely related bacteria were not identified at the species level using 16S rRNA sequence analysis, whereas they were identified at the species level based on the reaction patterns of oligonucleotides on our microarrays usinggyrBgenes. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 721–728, 2003.
Scale‐up of Artemisia annua L. hairy root cultures produces complex patterns of terpenoid gene expression
Authors: Souret Frédéric F. , Kim Yoojeong , Wyslouzil Barbara E. , Wobbe Kristin K. , Weathers Pamela J. 
Published: July 2003

AbstractHairy roots grow quickly, reach high densities, and can produce significant amounts of secondary metabolites, yet their scale‐up to bioreactors remains challenging. Artemisia annua produces a rich array of terpenoids, including the sesquiterpene, artemisinin, and transformed roots of this species provide a good model for studying terpenoid production. These cultures were examined in shake flasks and compared with cultures grown in two types of bioreactors, a mist reactor and a bubble column reactor, which provide very different environments for the growing roots. Mist reactors have been shown previously to result in cultures that produce significantly more artemisinin per gram fresh weight of culture, while bubble column reactors have produced greater biomass. We have compared expression levels of four key terpenoid biosynthetic genes: 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase (HMGR), 1‐deoxy‐D‐xylulose‐5‐phosphate synthase (DXS), 1‐deoxy‐D‐xylulose‐5‐phosphate reductoisomerase (DXR), and farnesyl diphosphate synthase (FPS) in the three culture conditions. In shake flasks we found that although all four genes showed temporal regulation, only FPS expression correlated with artemisinin production. Light also affected the transcription of all four genes. Although expression in reactors was equivalent to or greater than that of roots grown in shake flasks, no correlation was found between expression level within six different zones of each reactor and their respective oxygen levels, light, and root‐packing density. Surprisingly, transcriptional regulation of HMGR, DXS, DXR, and FPS was greatly affected by the position of the roots in each reactor. Thus, relying on a single reactor sample to characterize the gene activity in a whole reactor can be misleading, especially if the goal is to examine the difference between reactor types or operating parameters, steps essential in scaling up cultures for production. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 653–667, 2003.
Chitosan scaffolds for biomolecular assembly: Coupling nucleic acid probes for detecting hybridization
Authors: Yi Hyunmin , Wu Li‐Qun , Sumner James J. , Gillespie James B. , Payne Gregory F. , Bentley William E. 
Published: July 2003

AbstractChitosan, a naturally occurring biopolymer, was used as a scaffold for the covalent binding of single‐stranded DNA oligonucleotide probes in a fluorescence‐based nucleic acid hybridization assay. Chitosan's pH dependent chemical and electrostatic properties enable its deposition on electrodes and metal surfaces, as well as on the bottom of microtiter plates. A combinatorial 96‐well microtiter plate format was used to optimize chemistries and reaction conditions leading to hybridization experiments. We found the coupling of oligonucleotides using relatively common glutaraldehyde chemistry was quite robust. Our hybridization results for complementary ssDNA oligonucleotides (E. coli dnaK sequences) demonstrated linear fluorescence intensity with concentration of E. coli dnaK‐specific oligonucleotide from 0.73 μM to 6.6 μM. Moreover, hybridization assays were specific as there was minimal fluorescence associated with noncomplementary groEL oligonucleotide. Finally, these results demonstrate the portability of a DNA hybridization assay based on covalent coupling to chitosan, which, in turn, can be deposited onto various surfaces. More arduous surface preparation techniques involving silanizing agents and hazardous washing reagents are eliminated using this technique. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 646–652, 2003.
The effects of metabolites from the indigenous Acidithiobacillus thiooxidans and temperature on the bioleaching of cadmium from soil
Authors: Liu Hsuan‐Liang , Chiu Chi‐Wei , Cheng Yang‐Chu 
Published: July 2003

AbstractThe effect of metabolites from the indigenous Acidithiobacillus thiooxidans and temperature on the bioleaching of cadmium from soil was investigated in the present study. Bioleaching was found to be more effective than chemical leaching of cadmium. The metabolite, mainly sulfuric acid, which was shown to be growth‐associated in the exponential phase, plays a major role in bioleaching. The maximum amount of cadmium leached was obtained after 8 days of precultivation when cells were directly involved in the leaching process. It indicates that cells in the exponential growth phase exhibit higher activity toward bioleaching. In contrast, the maximum amount of cadmium leached and the maximum initial rate for bioleaching were reached after 16 days of precultivation when only metabolites were involved in the bioleaching process. It implies that higher sulfuric acid concentration results in higher leaching efficiency. In addition, higher temperature leads to higher leaching efficiency. The optimal operation condition for bioleaching was determined to be a two‐stage process: The first stage involves the precultivation of the indigenous A. thiooxidans at 30°C for 8 days followed by 20 minutes of centrifugation to discard cells. The second stage involves the bioleaching with the subsequent supernatant at 50°C. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 638–645, 2003.
Global incorporation of norleucine in place of methionine in cytochrome P450 BM‐3 heme domain increases peroxygenase activity
Authors: Cirino Patrick C. , Tang Yi , Takahashi Katsuyuki , Tirrell David A. , Arnold Frances H. 
Published: July 2003

AbstractIn this study we have replaced all 13 methionine residues in the cytochrome P450 BM‐3 heme domain (463 amino acids) with the isosteric methionine analog norleucine. This experiment has provided a means of testing the functional limits of globally incorporating into an enzyme an unnatural amino acid in place of its natural analog, and also an efficient way to test whether inactivation during peroxide‐driven P450 catalysis involves methionine oxidation. Although there was no increase in the stability of the P450 under standard reaction conditions (in 10 mM hydrogen peroxide), complete substitution with norleucine resulted in nearly two‐fold‐increased peroxygenase activity. Thermostability was significantly reduced. The fact that the enzyme can tolerate such extensive amino acid replacement suggests that we can engineer enzymes with unique chemical properties via incorporation of unnatural amino acids while retaining or improving catalytic properties. This system also provides a platform for directing enzyme evolution using an extended set of protein building blocks. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 729–734, 2003.
A novel method of simulating oxygen mass transfer in two‐phase partitioning bioreactors
Authors: Nielsen David R. , Daugulis Andrew J. , Mclellan P. James 
Published: July 2003

AbstractAn empirical correlation, based on conventional forms, has been developed to represent the oxygen mass transfer coefficient as a function of operating conditions and organic fraction in two‐phase, aqueous–organic dispersions. Such dispersions are characteristic of two‐phase partitioning bioreactors, which have found increasing application for the biodegradation of toxic substrates. In this work, a critical distinction is made between the oxygen mass transfer coefficient, kLa, and the oxygen mass transfer rate. With an increasing organic fraction, the mass transfer coefficient decreases, whereas the oxygen transfer rate is predicted to increase to an optimal value. Use of the correlation assumes that the two‐phase dispersion behaves as a single homogeneous phase with physical properties equivalent to the weighted volume‐averaged values of the phases. The addition of a second, immiscible liquid phase with a high solubility of oxygen to an aqueous medium increases the oxygen solubility of the system. It is the increase in oxygen solubility that provides the potential for oxygen mass transfer rate enhancement. For the case studied in which n‐hexadecane is selected as the second liquid phase, additions of up to 33% organic volume lead to significant increases in oxygen mass transfer rate, with an optimal increase of 58.5% predicted using a 27% organic phase volume. For this system, the predicted oxygen mass transfer enhancements due to organic‐phase addition are found to be insensitive to the other operating variables, suggesting that organic‐phase addition is always a viable option for oxygen mass transfer rate enhancement. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 735–742, 2003.
Resistance of Lactobacillus casei in plastic‐composite‐support biofilm reactors during liquid membrane extraction and optimization of the lactic acid extraction system
Authors: Demirci Ali , Cotton Julie C. , Pometto Anthony L. , Harkins Kristi R. , Hinz Paul N. 
Published: July 2003

AbstractLactic acid fermentations were performed with plastic‐composite‐support (PCS) disks in solvent‐saturated media with Lactobacillus casei subsp. rhamnosus (ATCC 11443). The PCS disks contained 50% (w/w) polypropylene, 35% (w/w) ground soybean hulls, 5% (w/w) yeast extract, 5% (w/w) soybean flour, and 5% (w/w) bovine albumin. Bioassays were performed by growing L. casei in solvent‐saturated media after soaking the PCS disks. Eighteen different solvent and carrier combinations were evaluated. Overall, L. casei biofilm fermentation demonstrated the same lactic acid production in solvent‐saturated medium as suspended cells in medium without solvents (control). To evaluate PCS solvent‐detoxifying properties, two bioassays were developed. When solvent‐saturated medium in consecutive equal volumes (10 mL then 10 mL) was exposed to PCS, both media demonstrated lactic acid fermentation equal to the control. However, when solvent‐saturated medium with two consecutive unequal volumes (10 mL then 90 mL) was exposed to PCS, some degree of toxicity was observed. Furthermore, iso‐octane, tributylphosphate (TBP), and Span 80 were optimized for recovery as 91%, 5%, and 4% (v/v), respectively, with a 1:1 ratio of 1.2 M Na2CO3 stripping solution. Also, recovery by emulsion liquid extraction in the hollow‐fiber contactor was minimal due to low recovery at pH 5.0 and incompatibility of the solvent and hollow‐fiber material. These results suggest that PCS biofilm reactors can benefit lactic acid fermentation by eliminating the toxic effect from solvent leakage into the fermentation medium from liquid–liquid extractive integrated fermentations. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 749–759, 2003.
Three‐dimensional numerical approach to investigate the substrate transport and conversion in an immobilized enzyme reactor
Authors: Esterl Stefan , Özmutlu Özlem , Hartmann Christoph , Delgado Antonio 
Published: July 2003

AbstractThis numerical study evaluates the momentum and mass transfer in an immobilized enzyme reactor. The simulation is based on the solution of the three‐dimensional Navier‐Stokes equation and a scalar transport equation with a sink term for the transport and the conversion of substrate to product. The reactor consists of a container filled with 20 spherical enzyme carriers. Each of these carriers is covered with an active enzyme layer where the conversion takes place. To account for the biochemical activity, the sink term in the scalar transport equation is represented by a standard Michaelis‐Menten approach. The simulation gives detailed information of the local substrate and product concentrations with respect to external and internal transport limitations. A major focus is set on the influence of the substrate transport velocity on the catalytic process. For reactor performance analysis the overall and the local transport processes are described by a complete set of dimensionless variables. The interaction between substrate concentration, velocity, and efficiency of the process can be studied with the help of these variables. The effect of different substrate inflow concentrations on the process can be seen in relation to velocity variations. The flow field characterization of the system makes it possible to understand fluid mechanical properties and its importance to transport processes. The distribution of fluid motion through the void volume has different properties in different parts of the reactor. This phenomenon has strong effects on the arrangement of significantly different mass transport areas as well as on process effectiveness. With the given data it is also possible to detect zones of high, low, and latent enzymatic activity and to determine whether the conversion is limited due to mass transfer or reaction resistances. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 780–789, 2003.
Modeling chromate reduction in Shewanella oneidensis MR‐1: Development of a novel dual‐enzyme kinetic model
Authors: Viamajala Sridhar , Peyton Brent M. , Petersen James N. 
Published: July 2003

AbstractChromate (Cr(VI)) reduction tests were performed with nitrate‐ and fumarate‐grown stationary phase cultures of Shewanella oneidensis MR‐1 (henceforth referred to as MR‐1) and disappearance of Cr(VI) was monitored over time. A rapid initial decrease in Cr(VI) concentration was observed, which was followed by a slower, steady decrease. These observations appear to be consistent with our previous results indicating that Cr(VI) reduction in MR‐1 involves at least two mechanisms (Viamajala et al., 2002b). Modeling of metal reduction kinetics is often based on single‐enzyme Michaelis‐Menten equations. However, these models are often developed using initial rates and do not always match actual reduction profiles. Based on the hypothesis that multiple Cr(VI) reduction mechanisms exist in MR‐1, a model was developed to describe the kinetics of Cr(VI) reduction by two parallel mechanisms: (1) a rapid Cr(VI) reduction mechanism that was deactivated (or depleted) quickly, and (2) a slower mechanism that had a constant activity and was sustainable for a longer duration. Kinetic parameters were estimated by fitting experimental data, and model fits were found to correspond very closely to quantitative observations of Cr(VI) reduction by MR‐1. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 790–797, 2003.
Cometabolism of Cr(VI) by Shewanella oneidensis MR‐1 produces cell‐associated reduced chromium and inhibits growth
Authors: Middleton Sarah S. , Latmani Rizlan Bencheikh , Mackey Mason R. , Ellisman Mark H. , Tebo Bradley M. , Criddle Craig S. 
Published: July 2003

AbstractMicrobial reduction is a promising strategy for chromium remediation, but the effects of competing electron acceptors are still poorly understood. We investigated chromate (Cr(VI)) reduction in batch cultures of Shewanella oneidensis MR‐1 under aerobic and denitrifying conditions and in the absence of an additional electron acceptor. Growth and Cr(VI) removal patterns suggested a cometabolic reduction; in the absence of nitrate or oxygen, MR‐1 reduced Cr(VI), but without any increase in viable cell counts and rates gradually decreased when cells were respiked. Only a small fraction (1.6%) of the electrons from lactate were transferred to Cr(VI). The 48‐h transformation capacity (Tc) was 0.78 mg (15 μmoles) Cr(VI) reduced · [mg protein]−1 for high levels of Cr(VI) added as a single spike. For low levels of Cr(VI) added sequentially, Tc increased to 3.33 mg (64 μmoles) Cr(VI) reduced · [mg protein]−1, indicating that it is limited by toxicity at higher concentrations. During denitrification and aerobic growth, MR‐1 reduced Cr(VI), with much faster rates under denitrifying conditions. Cr(VI) had no effect on nitrate reduction at 6 μM, was strongly inhibitory at 45 μM, and stopped nitrate reduction above 200 μM. Cr(VI) had no effect on aerobic growth at 60 μM, but severely inhibited growth above 150 μM. A factor that likely plays a role in Cr(VI) toxicity is intracellular reduced chromium. Transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS) of denitrifying cells exposed to Cr(VI) showed reduced chromium precipitates both extracellularly on the cell surface and, for the first time, as electron‐dense round globules inside cells. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 627–637, 2003.