bluuebunny/crossref_metadata_2025_split huggingface dataset:
Physiological and Genetic Strategies for Enhanced Subtilisin Production by Bacillus subtilis
Authors: Pierce Jeffrey A. , Robertson Channing R. , Leighton Terrance J. 
Published: March 2005

AbstractDefined minimal media conditions were used to assess and subsequently enhance the production of subtilisin by genetically characterized Bacillus subtilis strains. Subtilisin production was initiated by the exhaustion or limitation of ammonium in batch and fed‐batch cultures. Expression of the subtilisin gene (aprE) was monitored with a chromosomal aprE::lacZ gene fusion. The β‐galactosidase production driven by this fusion reflected subtilisin accumulation in the culture medium. Subtilisin gene expression was temporally extended in sporulation‐deficient strains (spoIIG), relative to co‐genic sporogenous strains, resulting in enhanced subtilisin production. Ammonium exhaustion not only triggered subtilisin production in asporogenous spoIIG mutants but also shifted carbon metabolism from acetate production to acetate uptake and resulted in the formation of multiple septa in a significant fraction of the cell population. Fed‐batch culture techniques, employing the spoIIG strain, were investigated as a means to further extend subtilisin production. The constant provision of ammonium resulted in linear growth, with doubling times of 11 and 36 h in each of two independent experiments. At the lower growth rate, the responses elicited (subtilisin production, glucose metabolism, and morphological changes) during the feeding regime closely approximated the ammonium starvation response, while at the higher growth rate a partial starvation response was observed.
Measurement of Heat Generated by Mixing during the Batch Production of Single Cell Protein from Cheese Whey
Authors: Ghaly A. E. , Ben‐Hassan R. M. , Ben‐Abdallah N. 
Published: March 2005

AbstractA procedure for measuring the rate of heat production from fermentation systems has been developed. The heat generated by mixing and lactose metabolism during the batch production of single cell protein from cheese whey lactose was calculated using a heat balance equation. The technique presented accounts for all the heat produced in and lost from the fermentation unit. Most of the heat generated by mixing in the cell‐free system (98.93 %) was lost with exhaust gas while a very small amount (1.07 %) was lost through the fermenter lid, wall, and bottom. The heat generated by mixing was 26 % of the total heat generated in the fermentation system with an active yeast population present. The heat produced by lactose metabolism depended on the yeast cell number. A maximum yeast population size of 820 million cells/mL and a lactose removal efficiency of 97 % were observed. About 93 % of the lactose consumed was used for growth. A yield of 0.78 g of cells/g of lactose was achieved.
Enzymatic Esterification of Fatty Acid Mixtures from Milk Fat and Anhydrous Milk Fat with Canola Oil in Supercritical Carbon Dioxide
Authors: Yu Zer‐Ran , Rizvi Syed S. H. , Zollweg John A. 
Published: March 2005

AbstractThe ester ethyl oleate was synthesized from oleic acid and ethanol by using the lipase, Candida cylindracea, immobilized on Celite 545 and in supercritical carbon dioxide (SC‐CO2) as the solvent. The enzymatic esterification reaction is much faster in SC‐CO2 than in organic solvents. This technique was then applied to the hydrolysis products of milk fat to determine the feasibility of using it as a process to enhance flavor. Gas chromatographic (GC) analysis of the ethyl esters showed that short‐chain fatty acids (C4‐C8) esterify more completely than long‐chain fatty acids (C16‐C18). The technique was then applied to interesterify a mixture of triglycerides from canola oil (CNO) and anhydrous milk fat (AMF). The starting mixture contained predominantly C52‐C56 triglycerides from the CNO and C32‐C38 triglycerides from the AMF. The product exhibited predominantly the C42‐C50 and C54 triglycerides.
Applications of Ultrafast HPLC to Process Development of Recombinant DNA‐Derived Proteins
Authors: Olson Kenneth C. , Gehant Richard L. 
Published: March 2005

AbstractThe application of ultrafast HPLC to the development of recovery processes for proteins produced by recombinant DNA technology has been explored using wide‐pore HPLC resins and instrumentation designed for rapid analysis. High‐resolution analysis of complex samples was achieved with a total analysis time of less than 5 min from injection to injection. Fractions collected during preparative chromatography were analyzed by SDS gel electrophoresis and fast HPLC. Specific proteins in the fractions were detected and quantitated by fast HPLC providing real‐time analysis for pooling. The technique was also applied to the formidable task of detecting and quantitating protein variants during the development of recovery processes. Several examples of post‐translational variant detection are shown. Ultrafast HPLC is a new analytical tool that can be applied to the development of robust manufacturing processes producing therapeutic proteins essentially free of known impurities and variants.
Mathematical Modeling of the Production of Cyclosporin A by Tolypocladium inflation: Effect of L‐Valine
Authors: Agathos Spiros N. , Lee Jaegwan 
Published: March 2005

AbstractA mathematical model was developed to describe the kinetics of submerged fungal growth, consumption of nutrients, and production of the immunosuppressant drug, cyclosporin A (CyA). Special emphasis was placed on the experimentally observed stimulatory effect of L‐valine on CyA production. The proposed model was based on kinetic information and emerging mechanistic data on CyA biosynthesis. It was assumed that L‐valine acts as both precursor and inducer of the CyA‐synthesizing multienzyme (CyA synthetase), and an unmethylated intermediate of CyA was postulated in the biosynthetic process. The model consisted of two parts: one describing cell growth and substrate consumption and the other addressing the kinetics of internal properties such as endogenous valine, biosynthetic enzyme, CyA intermediate, and CyA. The success of this extensive model was confirmed by its ability to simulate adequately the kinetic profiles of both external and internal variables. For instance, the model correctly predicted the time course of intracellular L‐valine accumulation. In addition, both the optimal level and timing of exogenous L‐valine addition could be predicted for maximum drug production. This study suggests rational new ways of improving the fungal production process of this important immunosuppressant.
Optimal Design of an Enzymatic Reactor for Flow Injection Analysis
Authors: Poch M. , Montesinos J. L. , Valle M. Del , Alonso J. , Araujo A. , Lima J. L. F. C. 
Published: March 2005

AbstractA simulation procedure for the optimization of enzymatic reactors used in sandwich flow injection systems is evaluated. The system is modeled as a plug‐flow reactor with axial dispersion. To calibrate it, dispersion coefficients can be evaluated using residence time distribution techniques; meanwhile, enzymatic kinetics must be determined for the system considered, according to the values of the substrate conversion attained. The model has been linked to an optimization routine based on the Powell algorithm. The proposed approach has been evaluated in a system performing simultaneous determinations of glucose and glycerol, considered the common carbon sources in a fermentation process.
Dehalogenation by Cytochrome P‐450cam: Effect of Oxygen Level on the Decomposition of l,2‐Dibromo‐3‐chloropropane
Authors: Koe Gary S. , Vilker Vincent L. 
Published: March 2005

AbstractA P‐450cam monooxygenase enzyme system, obtained through separate purification of the three protein subunits, was used as a biocatalyst in the dehalogenation of 1,2‐dibromo‐3‐chloropropane (DBCP). Incubations in a solution containing the reconstituted enzyme system were performed in two oxygen environments, oxygen‐rich (oxygen‐saturated solution) and oxygen‐lean (oxygen concentration reduced to 1% of saturation), and analyzed for hydrocarbon and bromide ion (Br‐) product formation and the kinetics of DBCP decomposition. l‐Bromo‐3‐chloroacetone (BCA) was identified by gas chromatography‐mass spectrometry as a major product of oxygen‐rich incubations, while 3‐chloro‐l‐propene (allyl chloride) was verified as an oxygen‐lean product. The identification of these two products suggests that P‐450cam activity is occurring in two separate mechanisms, oxidative and reductive, which is governed by the availability of oxygen. This suggestion is further supported by differences that were observed in the DBCP decomposition rate, which is an order of magnitude slower under oxygen‐lean conditions, and differences in bromide ion formation, where approximately one Br‐stoichiometric equivalent is released per mole of DBCP lost under oxygen‐rich conditions versus approximately two Br‐ stoichiometric equivalents for oxygen‐lean conditions.
Concentrating Cellulases Using a Temperature‐Sensitive Hydrogel: Effect of Gel Particle Size and Geometry
Authors: Park Chang‐Ho , Orozco‐Avila Ignacio 
Published: March 2005

AbstractA temperature‐sensitive poly (N‐isopropylacrylamide) hydrogel was used to concentrate cellulases from Trichoderma reesei. The effect of gel size and geometry on separation efficiency and weight change rate was studied. Gel weight change was reproducible over six cycles of short swelling and collapsing (2 h each) experiments, but the gel weight change capacity of small and large cubes was 90% and 62% of small extruded gel, respectively. The gel weight change capacity of large extruded gel and chopped gel was comparable to that of small extruded gel. Cellulase separation efficiency increased up to 64% for large cubes, but it was lower than 80–97% for comparably sized solutes reported in the literature. Small extruded gel reached its equilibrium weight in 2–3 h, but it took more than 200 times longer for gel disks to reach equilibrium weights. Initial solution flux (surface‐specific water release/sorption rate) during swelling and collapsing was 1065–2450 g/m2·h, but flux decreased rapidly with time. Skin formation was partially responsible for the slower weight change rate of larger gels, but chopped gel also formed skin at collapse temperatures higher than 37 °C. Skin formation was not observed for small extruded gel up to 50 °C. During gel disk collapsing experiments, transparent bubbles trapping pressurized water initially were observed all over the gel surface, and some of the bubbles on the disk edge did not collapse until the gel approached an equilibrium weight.
Thermal Time Distributions in Tubular Heat Exchangers during Aseptic Processing of Fluid Foods
Authors: Bhamidipati S. , Singh R. K. 
Published: March 2005

AbstractThe objectives of this study were to develop a mathematical model for thermal time distributions (TTD) in tubular heat exchangers and to perform a computer simulation to study the role of TTD in food process design. A computer simulation of heat transfer to a pseudoplastic fluid with variable thermophysical properties heated in a tubular heat exchanger was carried out. A power law model, with the flow behavior consistency indices modeled as temperature‐dependent properties, is used to described the flow behavior of the fluid. The coupled continuity, momentum, and energy equations with corresponding boundary conditions for constant wall temperature and heat flux are solved numerically by a Dufort‐Frankel finite difference technique. Thermal processing models are then applied to obtain TTD by tracking fluid elements along the flow path in a heating section. TTD and F curves are used to evaluate process effectiveness. Two tube radii (0.0254 and 0.0382 m) and two wall temperatures (125 and 130 °C) were studied. Increasing the wall temperature from 125 to 130 °C reduced the length of the heat exchanger by 11.3%. Increasing the tube radius by 150% caused an 84.2 % increase in the length of the heating section.
A Kinetic Model for Substrate and Energy Consumption of Microbial Growth under Substrate‐Sufficient Conditions
Authors: Zeng A.‐P. , Deckwer W.‐D. 
Published: March 2005

AbstractThe growth of heterotrophic microorganisms can be classified into substrate‐limited and substrate‐sufficient growth according to the relative availability of the substrate (carbon and energy source) and other nutrients. It is generally observed that the consumption rates of substrate and energy (ATP) are higher under substrate‐sufficient conditions than under conditions of substrate limitation. The excess substrate and ATP consumption is often influenced by the residual concentration of substrate in a. relatively wide range. To account for these effects, a kinetic model is proposed to describe substrate and ATP consumption rates of microbial growth under substrate‐sufficient conditions. According to the model, the specific substrate consumption rate of a substrate‐sufficient culture can be expressed as the sum of the substrate consumption rate under substrate‐limited conditions at the corresponding specific growth rate and an additional consumption rate due to excess substrate. The same kinetic form also applies to the specific ATP consumption rate and to the specific oxygen consumption rate of an aerobic culture, respectively. The linear equations for substrate and ATP consumption rates of Pirt and of Stouthamer and Bettenhausen can be used for substrate‐limited growth. The excess of substrate and ATP consumption rates at carbon surplus can be described in a form similar to that of Michaelis&&beta;;Menten kinetics. The proposed kinetic model has been verified with experimental data from three continuous cultures representing both anaerobic and aerobic microbial growth on substrates with low and high degrees of reductance. Using this model, the parameters maximum growth yield and maintenance requirement (both in terms of substrate and ATP) of a culture under different growth limitations can be better defined and quantified. The range of residual substrate concentrations in which the specific rates of substrate and ATP consumption are affected can also be assessed. This information should be helpful in designing medium and reactor operating conditions and in interpreting experimental results obtained under different limiting conditions.
Fractionation of Citrus Oils Using a Membrane‐Based Extraction Process
Authors: Brose Daniel J. , Chidlaw Mark B. , Friesen Dwayne T. , Lachapelle Ed D. , Van Eikeren Paul 
Published: March 2005

AbstractA membrane‐based extraction process is described for the fractionation of citrus oils to produce an oil stream enriched in oxygenated components. The process relies on the high selectivity of cyclodextrins (CDs) to preferentially bind the desirable oxygenates. In the process, low‐temperature citrus oil flows on one side of a nonporous membrane, and an aqueous CD solution flows on the other side of the membrane. Oxygenates diffuse through the water‐swollen hydrophilic membrane and preferentially partition into the aqueous CD solution. The aqueous CD solution is then heated and circulated past a second membrane, where the CD/oxygenate complex dissociates and the liberated oxygenates diffuse into a citrus oil solution. Disassociated CD is recycled to the first membrane. Data are presented on the effect of temperature and CD type on the partitioning of oxygenates from orange oil into aqueous CD solutions. Operation of a pilot‐scale test loop that capitalizes on the high selectivity of CDs and the temperature dependence of the CD binding is described for the production of oxygenate‐enriched orange oil.
Biosorption of Heavy Metals
Authors: Volesky B. , Holan Z. R. 
Published: March 2005

AbstractOnly within the past decade has the potential of metal biosorption by biomass materials been well established. For economic reasons, of particular interest are abundant biomass types generated as a waste byproduct of large‐scale industrial fermentations or certain metal‐binding algae found in large quantities in the sea. These biomass types serve as a basis for newly developed metal biosorption processes foreseen particularly as a very competitive means for the detoxification of metal‐bearing industrial effluents. The assessment of the metal‐binding capacity of some new biosorbents is discussed. Lead and cadmium, for instance, have been effectively removed from very dilute solutions by the dried biomass of some ubiquitous species of brown marine algae such as Ascophyllum and Sargassum, which accumulate more than 30% of biomass dry weight in the metal. Mycelia of the industrial steroid‐transforming fungi Rhizopus and Absidia are excellent biosorbents for lead, cadmium, copper, zinc, and uranium and also bind other heavy metals up to 25% of the biomass dry weight. Biosorption isotherm curves, derived from equilibrium batch sorption experiments, are used in the evaluation of metal uptake by different biosorbents. Further studies are focusing on the assessment of biosorbent performance in dynamic continuous‐flow sorption systems. In the course of this work, new methodologies are being developed that are aimed at mathematical modeling of biosorption systems and their effective optimization. Elucidation of mechanisms active in metal biosorption is essential for successful exploitation of the phenomenon and for regeneration of biosorbent materials in multiple reuse cycles. The complex nature of biosorbent materials makes this task particularly challenging. Discussion focuses on the composition of marine algae polysaccharide structures, which seem instrumental in metal uptake and binding. The state of the art in the field of biosorption is reviewed in this article, with many references to recent reviews and key individual contributions.
Fnr, a Global Transcriptional Regulator of Escherichia coli, Activates the Vitreoscilla Hemoglobin (VHb) Promoter and Intracellular VHb Expression Increases Cytochrome d Promoter Activity
Authors: Tsai Philip S. , Kallio Pauli T. , Bailey James E. 
Published: March 2005

AbstractThe oxygen‐regulated promoter (Pvhb) of the Vitreoscilla hemoglobin gene has been applied to direct high‐level expression of several cloned proteins, including Vitreoscilla hemoglobin (VHb), which improves productivity of many aerobic processes. In an effort to gain a better understanding of the regulation of Pvhb, and to guide further optimization of this technology, we investigated whether the Escherichia coli global regulatory molecules Fnr and the Arc system (ArcA and ArcB), which control the expression of various genes under either aerobic or anaerobic conditions, also regulate Pvhb activity in E. coli. The activity of Pvhb and the expression of VHb in E. coli were activated by Fnr, but were relatively unaffected by the Arc system under microaerobic conditions (DO less than 2% air saturation). We also examined the possibility of VHb affecting cytochrome d promoter activity during microaerobiosis. The presence of VHb increased the activity of β‐galactosidase from a cytochrome d promoter‐lacZ fusion by 1.5‐fold. This indicates that VHb affects oxygen‐regulated transcription of E. coli genes and may contribute to the modified physiology observed in VHb‐expressing E. coli.
Noncompetitive Immunoassays Using Bifunmctional Unilamellar Vesicles or Liposomes
Authors: Singh Anup K. , Kilpatrick Peter K. , Carbonell Ruben G. 
Published: March 2005

AbstractSmall unilamellar vesicles (SUVs) functionalized with an enzyme label and with specific ligands for biological molecules are useful as signal enhancement vehicles in the development of enzyme‐linked immunoadsorbent assays and other biosensor applications. Bifunctional vesicles were prepared by covalently attaching horseradish peroxidase (HRP) and an antibody to the outside of the lipid bilayer of an SUV. The reaction conditions were optimized to obtain 7–12 antibody molecules and 100–200 HRP molecules per vesicle. The enzyme retained 70–80% of its specific activity after immobilization, and the presence of immobilized proteins on the vesicle surface apparently increased the vesicle stability. To minimize the background signal and maximize the specific signal, the immunoassay protocol was optimized with respect to (1) the type and concentration of blocking agent, (2) the diluents for HRP‐antibodyvesicles and sample, (3) the incubation period, and (4) the incubation temperature. The bifunctional vesicles were used in a noncompetitive immunoassay to detect d‐dimer, a fibrin dimer formed at the early stages of thrombosis. A second conjugate, HRP‐antibody, was prepared, characterized, and used as a control against which to compare the assay using vesicles. The assay results using vesicles led to a detection limit for d‐dimer in human plasma 9 times lower than what was achieved using the conventional enzyme‐antibody conjugate assay.
Studies on Affinity Constants of Hapten‐Specific Monoclonal Antibodies Using an Antibody‐Immobilized ELISA
Authors: Chu Kent S. , Jin Shan , Guo Jingrong , Jue Marina , Huang John J. 
Published: March 2005

AbstractAn enzyme‐linked immunosorbent assay to measure affinity constants of hapten‐specific monoclonal antibodies has been developed. In this method an antibody‐immobilizing format is used. Antibody affinity toward hapten—enzyme tracer is first calculated by using Scatchard's method. Using this affinity, antibody‐hapten affinity is then calculated by following the principles of chemical thermodynamics and mass conservation. The affinity from this method agrees with the IC50 values in ELISA. This method is useful in early determination of affinity during monoclonal antibody development. Compared with the antigen‐coated format, this method avoids the problem of distinguishing binding of unliganded antibody and singly liganded antibody to antigens on solid phase.
Purification and Characterization of an Amyloglucosidase from Termitomyces clypeatus That Liberates Glucose From Xylan
Authors: Ghosh A. K. , Naskar A. K. , Jana M. L. , Khowala S. , Sengupta S. 
Published: March 2005

AbstractAn amyloglucosidase was purified to homogeneity from the culture filtrate of Termitomyces clypeatus, using the following steps: ammonium sulfate fractionation, DEAE‐Sephadex chromatography, and HP‐GPLC on an Ulstropac TSK‐G3000 SWG column. The enzyme was a glycoprotein with a minimum molecular weight of 56 000. It had appreciable activity on glycogen and amylopectin, moderate activity on maltose, and little activity on panose. The enzyme, unlike fungal amyloglucosidase (Aspergillus niger), could liberate glucose from xylans. The enzyme had Km = 1.81 mg/mL and Vm = 82.1 μmol/min/mg for starch hydrolysis and Km = 4.36 mg/mL and Vm = 57.7 μmol/min/mg for the hydrolysis of larch wood xylan. Among the different inhibitors, NBS and CDTA were the most potent. Previously the enzyme was shown [Khowala, S.; et al. Appl. Microbiol. Technol. 1992, 37, 287–292] to have synergistic activity on xylan hydrolysis similar to other xylanolytic enzymes: α‐arabinofuranosidase or α‐glucuronidase. Since the amyloglucosidase was not active on cellulose, arabinogalactan, or β‐glucans, which may be present as contaminants in xylan, the probable liberation of glucose directly from xylan by the enzyme was indicated.