bluuebunny/crossref_metadata_2025_split huggingface dataset:
Continuous Desulfurization of Dibenzothiophene with Rhodococcus rhodochrous IGTS8 (ATCC 53968)
Authors: Schilling Bernhard M. , Alvarez Luis M. , Wang Daniel I. C. , Cooney Charles L. 
Published: December 2002

AbstractDesulfurization of a model fuel system consisting of hexadecane and dibenzothiophene (DBT) by Rhodococcus rhodochrousIGTS8 was demonstrated in a 2‐L continuous stirred tank reactor (CSTR). The reactor was operated in a semicontinuous and continuous mode with and without recycling of the model fuel. A constant volumetric desulfurization activity A(t), (in mg HBP L−1 h−1) was maintained in the reactor with a feeding strategy of fresh cell suspension based on a first‐order decay of the biocatalyst. Maximum desulfurization rates, as measured by specific desulfurization activity, of 1.9 mg HBP/g DCW h were attained. Rates of biocatalyst decay were on the order of 0.072 h−1. Theoretical predictions of a respiratory quotient (RQ) associated with this biotransformation reaction agree well with experimental data from off‐gas analysis. In addition, the ratio of the specific desulfurization activity a(t), (in mg HBP/g DCW h) of recycled and fresh biocatalyst was determined and evaluated.
Carbon Mass Balance Evaluation of Cellulase Production on Soluble and Insoluble Substrates
Authors: Carlos Sáez Juan , Schell Daniel J. , Tholudur Arun , Farmer Jody , Hamilton Jenny , Colucci José A. , McMillan James D. 
Published: December 2002

AbstractA methodology is described and applied for performing carbon mass balances across cellulase enzyme production processes using both soluble sugar and insoluble cellulose substrates. The fungus Trichoderma reesei was grown on either glucose, lactose, or cellulose in aerobic batch mode, and the evolution of the main carbonaceous components (cell mass, cellulose, soluble protein, adsorbed protein, sugars, and carbon dioxide) was followed. A variety of analytical techniques were utilized to measure these components, including (i) gravimetric analysis, (ii) near‐infrared spectroscopy, (iii) bicinchoninic acid based soluble protein measurement, (iv) gas mass spectrometry and flow rate, (v) CHNS/O elemental analyses, and (vi) high‐performance liquid chromatography. The combined set of measurements allowed carbon mass balances across the cellulase production process to be assessed to determine the consistency of the underlying kinetic data. Results demonstrate the capability to determine the levels and distribution of all major carbonaceous components during the cellulase production process on both soluble and insoluble substrates. Average carbon mass balance closures were near 100% during early stages (<72 h) of the cultivations using glucose, lactose, or cellulose as the substrates, but carbon mass closures trended high later in the cultivation. Analysis of carbon allocation results suggests that an error in the gas mass flow rate measurement was the primary cause for carbon mass balance closures to exceed 110% late in the process.
True and Apparent Temperature Dependence of Protein Adsorption Equilibrium in Reversed‐Phase HPLC
Authors: Szabelski Paweł , Cavazzini Alberto , Kaczmarski Krzysztof , Van Horn Jennifer , Guiochon Georges 
Published: December 2002

AbstractThe adsorption behavior of bovine insulin on a C8‐bonded silica stationary phase was investigated at different column pressures and temperatures in isocratic reversed‐phase HPLC. Changes in the molar volume of insulin (Δ Vm) upon adsorption were derived from the pressure dependence of the isothermal retention factor ( k′). The values of Δ Vm were found to be practically independent of the temperature between 25 and 50 °C at –96 mL/mol and to increase with increasing temperature, up to –108 mL/mol reached at 50 °C. This trend was confirmed by two separate series of measurements of the thermal dependence of ln( k′). In the first series the average column pressure was kept constant. The second series involved measurements of ln( k′) under constant mobile‐phase flow rate, the average column pressure varying with the temperature. In both cases, a parabolic shape relationship was observed between ln( k′) and the temperature, but the values obtained for ln k′ were higher in the first than in the second case. The relative difference in ln( k′), caused by the change in pressure drop induced by the temperature, is equivalent to a systematic error in the estimate of the Gibbs free energy of 12%. Thus, a substantial error is made in the estimates of the enthalpy and entropy of adsorption when neglecting the pressure effects associated with the change in the molar volume of insulin. This work proves that the average column pressure must be kept constant during thermodynamic measurements of protein adsorption constants, especially in RPLC and HIC. Our results show also that there is a critical temperature, Tc ≈ 53 °C, at which ln( k′) is maximum and the insulin adsorption process changes from an exothermic to an endothermic one. This temperature determines also the transition point in the molecular mechanism of insulin adsorption that involves successive unfolding of the protein chain.
Mature Experimental Constructed Wetlands Treating Urban Water Receiving High Metal Loads
Authors: Scholz Miklas , Höhn Patrick , Minall Rowland 
Published: December 2002

AbstractThe aim was to assess over 2 years the treatment efficiencies of vertical‐flow wetland filters containing macrophytes and granular media of different adsorption capacities. Different concentrations of lead and copper sulfate (constant for 1 year each) were added to urban beck inflow water in order to simulate pretreated (pH adjustment assumed) mine wastewater. After 1 year of operation, the inflow concentrations for lead and copper were increased from 1.30 to 2.98 and from 0.98 to 1.93 mg/L, respectively. However, the metal mass load rates (mg/m2/d) were increased by a factor of approximately 4.9 for lead and 4.3 for copper. No breakthrough of metals was recorded. Lead and copper accumulated in the biomass of the litter zone and rhizomes of the macrophytes. Furthermore, microbiological activity decreased during the second year of operation. Bioindicators such as ciliated protozoa and zooplankton decreased sharply in numbers but diatoms increased. In conclusion, the use of macrophytes and adsorption media did not significantly enhance the filtration of lead and copper. Particulate lead is removed by filtration processes including straining. Furthermore, some expensive and time‐consuming water quality variables can be predicted with less expensive ones such as temperature in order to reduce sampling costs.
Adsorption of Cadmium Ion and Gallium Ion to Immobilized Metallothionein Fusion Protein
Authors: Terashima Masaaki , Oka Noriyuki , Sei Takamasa , Yoshida Hiroyuki 
Published: December 2002

AbstractA fusion protein made from maltose binding protein (pmal) and human metallothionein (MT) was expressed using E. coli. The purified recombinant protein (pmal‐MT) was immobilized on Chitopearl resin, and characteristics of pmal‐MT for metal binding were evaluated. As expected from the tertiary structure of metallothionein, the pmal‐MT ligand adsorbed 12.1 cadmium molecules per one molecule of the ligand at pH 5.2. The pmal‐MT ligand also bound 26.6 gallium molecules per one molecule of the ligand at pH 6.5. Neither cadmium ion nor gallium ion bound to a control protein bovine serum albumin (BSA). Adsorption isotherms for both ions were correlated by Langmuir‐type equations. Two types of binding sites have been elucidated on the basis of HSAB (hard and soft acid and base) theory. It was suggested that gallium ion specifically binds to amino acid residues containing oxygen and nitrogen atoms, while cadmium ion binds to specific binding sites formed by multiple cysteine residues. The pmal‐MT ligand bound these metals in the concentration range of 0.2–1.0 mM, and the bound metal ions could be eluted under relatively mild conditions (pH 2.0). The pmal‐MT Chitopearl resin was stable and could be used repeatedly without loss of binding activity. Thus, this new ligand would be useful for recovery of toxic heavy metals and/or valuable metal ions from various aqueous solutions.
Enhanced Stress Tolerance in Escherichia coli and Nicotiana tabacum Expressing a Betaine Aldehyde Dehydrogenase/Choline Dehydrogenase Fusion Protein
Authors: Yilmaz Jenny Lindberg , Bülow Leif 
Published: December 2002

AbstractIn Escherichia coli the osmoprotective compound glycine betaine is produced from choline by two enzymes; choline dehydrogenase (CDH) oxidizes choline to betaine aldehyde and then further on to glycine betaine, while betaine aldehyde dehydrogenase (BADH) facilitates the conversion of betaine aldehyde to glycine betaine. To evaluate the importance of BADH, a BADH/CDH fusion enzyme was constructed and expressed in E. coli and in Nicotiana tabacum. The fusion enzyme displayed both enzyme activities, and a coupled reaction could be measured. The enzyme was characterized regarding molecular weight and the dependence of the enzyme activities on environmental factors (salt, pH, and poly(ethylene glycol) addition). At high choline concentrations, E. coli cells expressing BADH/CDH were able to grow to higher final densities and to accumulate more glycine betaine than cells expressing CDH only. The intracellular glycine betaine levels were almost 5‐fold higher for BADH/CDH when product concentration was related to CDH activity. Also, after culturing the cells at high NaCl concentrations, more glycine betaine was accumulated. On medium containing 20 mM choline, transgenic tobacco plants expressing BADH/CDH grew considerably faster than vector‐transformed control plants.
Rhamnolipid Biosurfactant Production by Strains of Pseudomonas aeruginosa Using Low‐Cost Raw Materials
Authors: Rahman K. S. M. , Rahman Thahira J. , McClean Stephen , Marchant Roger , Banat Ibrahim M. 
Published: December 2002

AbstractThis study was aimed at the development of economical methods for higher yields of biosurfactant by suggesting the use of low‐cost raw materials. Two oil‐degrading strains, Pseudomonas aeruginosa GS9–119 and DS10–129, were used to optimize a substrate for maximum rhamnolipid production. Among the two strains, the latter produced maxima of 4.31, 2.98, and 1.77 g/L rhamnolipid biosurfactant using soybean oil, safflower oil, and glycerol, respectively. The yield of biosurfactant steadily increased even after the bacterial cultures reached the stationary phase of growth. Characterization of rhamnolipids using mass spectrometry revealed the presence of dirhamnolipids (Rha‐Rha‐C10–C10). Emulsification activity of the rhamnolipid biosurfactant produced by P. aeruginosa DS10–129 was greater than 70% using all the hydrocarbons tested, including xylene, benzene, hexane, crude oil, kerosene, gasoline, and diesel. P. aeruginosa GS9–119 emulsified only hexane and kerosene to that level.
Effect of Long‐Term Cobalt Deprivation on Methanol Degradation in a Methanogenic Granular Sludge Bioreactor
Authors: Zandvoort Marcel H. , Geerts Roy , Lettinga Gatze , Lens Piet N. L. 
Published: December 2002

AbstractThe effect of the trace metal cobalt on the conversion of methanol in an upflow anaerobic sludge bed (UASB) reactor was investigated by studying the effect of cobalt deprivation from the influent on the reactor efficiency and the sludge characteristics. A UASB reactor (30 °C; pH 7) was operated for 261 days at a 12‐h hydraulic retention time (HRT). The loading rate was increased stepwise from 2.6 g chemical oxygen demand (COD)·L reactor−1·d−1 to 7.8 g COD·L reactor−1·d−1. Cobalt deprivation had a strong impact on the methanogenic activity of the sludge. In batch tests, the methanogenic activity of the sludge with methanol as the substrate increased 5.3 (day 28) and 2.1 (day 257) times by addition of 840 nM of cobalt. The sludge had an apparent Km for cobalt of 948 nM after 28 days of operation and 442 nM at the end of the run. Cobalt deprivation during 54 days of operation led to a methanol conversion efficiency of only 55%. Continuous addition of cobalt (330 nM) for 33 days improved the methanol removal efficiency to 100%. In this period of cobalt dosing, the cobalt concentration in the sludge increased 2.7 times up to 32 μg·g TSS−1. Upon omission of the cobalt addition, cobalt washed‐out at a stable rate of 0.1 μg·g VSS−1·d−1. At the end of the run, the cobalt concentration of the sludge was similar to that of the seed sludge.
Continuous Production of High‐Content Fructooligosaccharides by a Complex Cell System
Authors: Sheu Dey‐Chyi , Duan Kow‐Jen , Cheng Chih‐Yu , Bi Jia‐Lin , Chen Jer‐Yi 
Published: December 2002

AbstractA complex biocatalyst system with a bioreactor equipped with a microfiltration (MF) module was employed to produce high‐content fructooligosaccharides (FOS) in a continuous process initiated by a batch process. The system used mycelia of Aspergillus japonicus CCRC 93007 or Aureobasidium pullulans ATCC 9348 with β‐fructofuranosidase activity and Gluconobacter oxydans ATCC 23771 with glucose dehydrogenase activity. Calcium carbonate slurry was used to control pH to 5.5, and gluconic acid in the reaction mixture was precipitated as calcium gluconate. Sucrose solution with an optimum concentration of 30% (w/v) was employed as feed for the complex cell system, and high‐content FOS was discharged continuously from a MF module. The complex cell system was run at 30 °C with an aeration rate of 5 vvm and produced more than 80% FOS with the remainder being 5–7% glucose and 8–10% sucrose on a dry weight basis, plus a small amount of calcium gluconate. The system worked for a 7‐day continuous production process with a dilution rate of 0.04 h−1, and the volumetric productivity for total FOS was more than 160 g L−1 h−1.
Reversible Immobilization of Invertase on Sepabeads Coated with Polyethyleneimine: Optimization of the Biocatalyst's Stability
Authors: Torres Rodrigo , Mateo Cesar , Fuentes Manuel , Palomo Jose M. , Ortiz Claudia , Fernández‐Lafuente Roberto , Guisan Jose M. 
Published: December 2002

AbstractInvertase fromS. cerevisiaehas been immobilized by ionic adsorption on Sepabeads fully coated with PEI. The enzyme was strongly adsorbed on the support (no desorption of the invertase was found under conditions in which all of the enzyme was released from conventional anionic exchanger supports (e.g., DEAE‐agarose)). Nevertheless, the enzyme could still be desorbed after its inactivation, and new fresh enzyme could be adsorbed on the supports without detrimental effects on enzyme loading. This is a multimeric enzyme, its minimal oligomerization active state being the dimer, but under certain conditions of pH and concentration it may give larger multimers. Very interestingly, results suggested that the adsorption of the enzyme on this large and flexible polymeric bed was able to freeze some of the different oligomeric structures of the enzyme. Thus, we have found that the enzyme immobilized at certain pH values (pH 8.5) and high enzyme concentration, in which the main enzyme structure is the tetramer, was more stable than immobilized preparations produced in conditions under which oligomerization was not favorable (dimers at low enzyme concentration) or it was too high (e.g., hexamers‐octamers at low pH value). The optimal enzyme preparation remained fully active after a 15‐day incubation at 50 °C and pH 4.5 (conditions of standard industrial use) and presented an optimal temperature approximately 5 °C higher than that of soluble enzyme.
Strain Improvement to Enhance the Production of Recombinant Penicillin Acylase in High‐Cell‐Density Escherichia coli Cultures
Authors: Lin Y.‐H. , Hsiao H.‐C. , Chou C. P. 
Published: December 2002

AbstractUsing fed‐batch operation for high‐cell‐density cultivation, efforts are frequently made for optimization of culture parameters, particularly feeding strategy. The current study also emphasized the importance of selecting strains for the production of recombinant proteins in high‐cell‐density cultures. With Escherichia coli penicillin acylase (PAC) as a target protein, the host/vector system of MDΔP7 harboring pTrcKnPAC2902 and pKS12 was designed for optimization of fed‐batch cultivation for recombinant protein production. The host, MDΔP7, potentially had a high translational and periplasmic processing efficiency for pac expression. On the other hand, the vector, pTrcKnPAC2902, was genetically constructed for pac overexpression. Coexistence of the other vector, pKS12, significantly enhanced PAC production by improving cell physiology and reducing the amount of inclusion body formation upon pac overexpression. An extremely high volumetric PAC activity at 37,500 U/L was obtained with the use of the developed host/vector system under optimum fed‐batch culture conditions.
Nonlinear Dynamics of Eucaryotic Pyruvate Dehydrogenase Multienzyme Complex: Decarboxylation Rate, Oscillations, and Multiplicity
Authors: Zeng An‐Ping , Modak Jayant , Deckwer Wolf‐Dieter 
Published: December 2002

AbstractPyruvate conversion to acetyl‐CoA by the pyruvate dehydrogenase (PDH) multienzyme complex is known as a key node in affecting the metabolic fluxes of animal cell culture. However, its possible role in causing possible nonlinear dynamic behavior such as oscillations and multiplicity of animal cells has received little attention. In this work, the kinetic and dynamic behavior of PDH of eucaryotic cells has been analyzed by using both in vitro and simplified in vivo models. With the in vitro model the overall reaction rate (ν1) of PDH is shown to be a nonlinear function of pyruvate concentration, leading to oscillations under certain conditions. All enzyme components affect ν1 and the nonlinearity of PDH significantly, the protein X and the core enzyme dihydrolipoamide acyltransferase (E2) being mostly predominant. By considering the synthesis rates of pyruvate and PDH components the in vitro model is expanded to emulate in vivo conditions. Analysis using the in vivo model reveals another interesting kinetic feature of the PDH system, namely, multiple steady states. Depending on the pyruvate and enzyme levels or the operation mode, either a steady state with high pyruvate decarboxylation rate or a steady state with significantly lower decarboxylation rate can be achieved under otherwise identical conditions. In general, the more efficient steady state is associated with a lower pyruvate concentration. A possible time delay in the substrate supply and enzyme synthesis can also affect the steady state to be achieved and leads to oscillations under certain conditions. Overall, the predictions of multiplicity for the PDH system agree qualitatively well with recent experimental observations in animal cell cultures. The model analysis gives some hints for improving pyruavte metabolism in animal cell culture.
Operational Stability of High Initial Activity Protease Catalysts in Organic Solvents
Authors: Fernandes João F. Amorim , Halling Peter J. 
Published: December 2002

AbstractThe first studies on the operational stability of cross‐linked enzyme crystals (CLECs) in organic media are described. Although these catalysts display high initial specific activity, they inactivate rapidly, losing more than 50% of the initial activity within the first 4 h under continuous flow. Furthermore, the inactivation is not reversible when returned to an aqueous medium. The same rapid inactivation occurs with adsorbed protease preparations that show similar high initial specific activity (propanol‐rinsed enzyme preparations (PREPs) of subtilisin and α‐chymotrypsin).
Modulation of Phosphoenolpyruvate Synthase Expression Increases Shikimate Pathway Product Yields in E. coli
Authors: Yi Jian , Li Kai , Draths K. M. , Frost J. W. 
Published: December 2002

AbstractProduct yields in microbial synthesis are ultimately limited by the mechanism utilized for glucose transport. Altered expression of phosphoenolpyruvate synthase was examined as a method for circumventing these limits. Escherichia coli KL3/pJY1.216A was cultured under fed‐batch fermentor conditions where glucose was the only source of carbon for the formation of microbial biomass and the synthesis of product 3‐dehydroshikimic acid. Shikimate pathway byproducts 3‐deoxy‐d‐ arabino‐heptulosonic acid, 3‐dehydroquinic acid, and gallic acid were also generated. An optimal expression level of phosphoenolpyruvate synthase was identified, which did not correspond to the highest expression levels of this enzyme, where the total yield of 3‐dehydroshikimic acid and shikimate pathway byproducts synthesized from glucose was 51% (mol/mol). For comparison, the theoretical maximum yield is 43% (mol/mol) for synthesis of 3‐dehydroshikimic acid and shikimate pathway byproducts from glucose in lieu of amplified expression of phosphoenolpyruvate synthase.
Stabilization and Improvement of Catalytic Activity of a Low Molar Mass Cellobiase by Cellobiase‐Sucrase Aggregation in the Culture Filtrate of Termitomyces clypeatus
Authors: Saha Rina , Roy Sujata Burman , Sengupta S. 
Published: December 2002

AbstractThe extracellular cellobiase (EC 3.2.1.21) of Termitomyces clypeatus separated in two protein fractions when culture filtrate or ammonium sulfate precipitated proteins were chromatographed on BioGel P‐200 column. During purification of cellobiase (CBS) from the lower molar mass (LMM) protein fraction, the enzyme behaved like a low molecular weight multimeric protein. The purified enzyme gave a single 56 kDa band in SDS‐PAGE but ladderlike bands (14, 28, 42, and 56 kDa) on denaturation by reducing‐SDS and urea. The protein, however, dissociated on dilution and protomeric (14 kDa) and multimeric forms (28 and 60 kDa) were eluted separately during HPGPLC. Specific activity of CBS gradually decreased as the molar mass of the enzyme was lowered in different eluted peaks. Protein present in all CBS pool fractions had the same amino acid composition and all displayed the same, single protein peak in reverse‐phase HPLC and 56 kDa band in SDS‐PAGE. Thus, T. clypeatus CBS was a multimeric 14 kDa protein that was optimally active as a tetramer. CBS purified from the higher molar mass fraction (HMM) as a SDS‐PAGE homogeneous 110‐kDa protein did not dissociate on dilution or by SDS‐urea. The purified protein was a protein aggregate as CBS consistently contained 20 ± 5% sucrase (SUC) Units in the preparation. The aggregate resolved during reverse‐phase chromatography on a C4 column, and an additional protein peak other than CBS was detected. The aggregated CBS had a higher temperature optimum and was more stable toward thermal and chemical denaturations than SUC‐free CBS. Increase of stability and catalytic activity of CBS by aggregation with SUC was much higher than those by the multimerization of CBS itself. All of these observations for the first time suggested that the heterologous protein‐protein aggregation, observed for a long time for fungal enzymes, might have a significant role in modulating physicochemical properties of the extracellular enzyme.