bluuebunny/crossref_metadata_2025 huggingface dataset:
Quasielastic light scattering, electrophoresis, and fluorescence studies of lysozyme‐poly(2‐acrylamido‐methylpropylsulfate) complexes
Authors: Xia Jiulin , Dubin Paul L. , Morishima Yotaro , Sato Takeshi , Muhoberac Barry B.
Published: December 2004

Complexation between lysozyme and sodium poly(2‐acrylamido‐2‐methylpropanesulfonate) (PAMPS) was studied by quasielastic light scattering, electrophoretic light scattering, fluorescence, and turbidimetry in electrolyte solution. These techniques show that complexation occurs at pH 9.6 in an ionic strength buffer of 0.25M NaCl + 25 mM Na2B4O7. At constant lysozyme concentration (Cpro). The structure of the complex depends on the polymer concentration. At low polymer concentration (relative to Cpro), an intrapolymer complex is formed. This intrapolymer complex aggregates to an interpolymer species upon increase in polymer concentration. Complex formation was also studied by fluorescence using pyrene‐labeled PAMPS (Py‐PAMPS). Energy transfer from singlet‐excited tryptophan residues in lysozyme to the pyrene label occurs when the complex is formed. Fluorescence and turbidity data indicate that lysozyme interacts with Py‐PAMPS preferentially at pyrene sites, which leads to static quenching of tryptophan fluorescence via energy transfer to the pyrene label. © 1995 John Wiley & Sons, Inc.
Solution behavior of α‐chymotrypsin dissolved in nonpolar organic solvents via hydrophobic ion pairing
Authors: Meyer Jeffrey D. , Matsuura James E. , Kendrick Brent S. , Evans Emily S. , Evans Gabriel J. , Manning Mark C.
Published: December 2004

Dissolution of α‐chymotrypsin in nonpolar organic solvents can be achieved using hydrophobic ion pairing, whereby the polar counterions are replaced by a stoichiometric number of detergent molecules. Using Aerosol OT[AOT, sodium bis(2‐octyl)sulfosuccinate], it is possible to partition significant amounts of the enzyme into alkanes and chlorocarbons. Apparent solubility in isooctane is greater than 1 mg/mL (80 μM). Necessary conditions for achieving effective partitioning of α‐chymotrypsin into these solvents are described. Using CD spectroscopy, it can be shown that the AOT–α‐chymotrypsin (CMT) complex retains its native secondary and tertiary structure when dissolved in alkanes, and that the globular structure is stable to more than 100°C. In contrast, α‐chymotrypsin unfolds at 54°C in aqueous solution. The relative solubility of the AOT–CMT complex in a variety of alkanes and chlorocarbons is also reported. The native structure of α‐chymotrypsin is maintained in carbon tetrachloride, but not in methylene chloride or chloroform. © 1995 John Wiley & Sons, Inc.
Competitive reactions in solutions of DNA and water‐soluble interpolyelectrolyte complexes
Authors: Izumrudov V. A. , Kargov S. I. , Zhiryakova M. V. , Zezin A. B. , Kabanov V. A.
Published: December 2004

The reaction of competitive binding of two polyanions—DNA and synthetic fluorescence‐tagged poly(methacrylate) (PMA*)—with the polycation‐quencher poly(N‐ethyl‐4‐vinyl‐pyridinium) (PEVP) was studied by fluorescence quenching technique. It was found that ability of DNA to displace PMA *from the water‐soluble nonstoichiometric interpolyelectrolyte complex (NPEC) formed by PMA* and PEVP—NPEC(PMA*‐PEVP)—and to form water‐soluble NPEC(DNA‐PEVP)

can be determined by the parameter Ψ = PPMA*/PPEVP where PPMA* and PPEVP are the degrees of polymerization of PMA* and PEVP, respectively. In the case of Ψ < 1 the decrease of Ψ leads to the shift of the reaction equilibrium to the right, which can be explained by the gain of entropy due to the increase of the total number of polymeric particles in the solution. Introduction of alkali metal cations into the reaction mixture results in the shift of the reaction equilibrium, and according to their ability to shift the equilibrium to the right the cations can be arranged in the series Na+ > K+ > Li+. The substitution of native DNA by denatured DNA practically does not affect the reaction equilibrium in solutions of NaCl and KCl but considerably shifts it to the right in solutions of LiCl. The data obtained are in accordance with the differences in the selectivity of alkali cations binding with competitive polyanions. © 1995 John Wiley & Sons, Inc.
Sequence and temperature dependence of the interbase hydrogen‐bond breathing modes in B‐DNA polymers: Comparison with low‐frequency Raman peaks and their role in helix melting
Authors: Chen Y. Z. , Prohofsky E. W.
Published: December 2004

We carry out temperature‐dependent lattice dynamics calculations to determine the vibrational normal modes associated with the interbase H‐bond breathing motion in several B‐DNA copolymers at temperatures from room temperature to the melting temperatures. We take into consideration Raman selection rules and incorporate a simple empirical model of Raman susceptibility in the interbase H bonds in our calculation and compare them to Raman measurements. Our calculations are carried out using empirical force constants that are not further refined to low‐frequency spectra. Our calculations show the existence of strong interbase H‐bond breathing modes at frequencies and with relative oscillator strengths close to the observed Raman peaks in the range of 60–140 cm−1 for the DNA sequences considered except for one helix. The correlation between the calculated and observed frequencies and oscillator strengths indicates that the observed Raman peaks in the frequency range are likely interbase H‐bond breathing modes. We find that these modes exhibit sizable temperature as well as sequence dependence. We show the softening of these modes on approaching thermal denaturation that is also in agreement with the observed behavior in Raman and melting measurements. The sensitivity of the calculation on the empirical model of Raman susceptibility and the possible reasons for the discrepancy between a few calculated values and observations are discussed. © 1995 John Wiley & Sons, Inc.
Observation of unfreezable water in aqueous solution of globule protein by microwave dielectric measurement
Authors: Miura Nobuhiro , Hayashi Yoshihito , Shinyashiki Naoki , Mashimo Satoru
Published: December 2004

A freezing process analyzed by the dielectric method on aqueous solution of albumin has revealed water structure around protein molecule. A relaxation peak due to bound water attached on the protein surface around 100 MHz at room temperature was found. It could be seen commonly in globule proteins. Another peak due to a different kind of unfreezable water was found around 1 GHz at −6°C. The amount of this water is estimated as 0.36 g water/g protein and in good agreement with that obtained by differential scanning calorimetry and nmr measurements. The water molecules form a shell layer around the protein molecule. © 1995 John Wiley & Sons, Inc.
Searching for foldable protein structures using optimized energy functions
Authors: Govindarajan Sridhar , Goldstein Richard A.
Published: December 2004

During evolution, the effective interactions between residues in a protein can be adjusted through mutations to allow the protein to fold to its native structure on an adequate time scale. We seek to address the question: Are there some structures that can be better optimized than others? Using exhaustive enumeration of the compact conformations of short proteins confined to simple lattices, we find that the best structures are those that contain contacts rare in random structures, indicating the importance of nonlocal contacts for assisting the folding process. Certain structural motifs such as long β‐hairpins, Greek‐key motifs, and jelly rolls, commonly found in proteins of known structure, have a high degree of optimizability. Contrary to what might be expected, positive correlations between the various interactions reduce optimizability. The optimization procedure produces a correlated energy landscape, which might assist folding. © 1995 John Wiley & Sons, Inc.
A reexamination of the double‐helix model for agarose gels using optical rotation
Authors: Schafer Steven E. , Stevens Eugene S.
Published: December 2004

The diffuse x‐ray diffraction patterns of agarose gels are compatible with both the “chicken‐wire” gel model consisting of associated double‐helix chain conformations and a network of more extended, but unentwined helices. Preference f or the double‐helix model has largely rested on an early interpretation of optical rotation data. Applying a recent, less empirical, chiroptical model of saccharide rotation, we confirm the earlier analysis by concluding that the rotation observed in the gel can be satisfactorily accounted for in terms of wide‐diameter agarose helices, capable of intertwining. In addition, we have found that the extended helices found in dried sols do not yield the observed gel rotations. © 1995 John Wiley & Sons, Inc.
Docking of congo red to the surface of crystalline cellulose using molecular mechanics
Authors: Woodcock Steve , Henrissat Bernard , Sugiyama Junji
Published: December 2004

A method for the docking of ligands to crystalline surfaces using molecular mechanics was developed and applied to study the molecular features of the adsorption of Congo Red to cellulose crystals. The docking procedure was based on a grid search exploring the surface repeat unit of the cellulose crystals using several orientations of Congo Red at each grid point. Results suggest a preferential adsorption of Congo Red onto certain faces of the cellulose crystals and that the lower energy conformers have a similar positioning and orientation with respect to the cellulose chains at the surface repeat unit. © 1995 John Wiley & Sons, Inc.
Polyelectrolyte electrostatics: Salt dependence, entropic, and enthalpic contributions to free energy in the nonlinear Poisson–Boltzmann model
Authors: Sharp Kim A.
Published: December 2004

Using the nonlinear Poisson–Boltzmann (PB) equation, expressions for the nonspecific salt dependence of the free energy, and the entropic and enthalpic contributions to the electrostatic free energy of a polyelectrolyte are derived. The results are generalized for any number and valence of mobile ions, and now permit a more rigorous treatment of the salt dependence of polyelectrolyte–ligand binding using the PB equation. The salt dependence of the electrostatic free energy depends on an “osmotic pressure like” term identified previously [K. Sharp and B. Honig (1990), Journal of Physical Chemistry, Vol. 94, pp. 7684–7692], which in turn depends on the integrated excess/deficit of all mobile ion species around the polyelectrolyte. For the simple salt case (two ions only) this expression is equivalent to that derived previously [H. Eisenberg (1976), Biological Macromolecules and Polyelectrolytes in Solution, Clarendon. Oxford] in terms of Donnan, or preferential interaction coefficients. Moreover, since all ions enter into the PB expression for the salt dependence of the free energy in an identical way, it is equally applicable to polyelectrolytes with charge of one sign (e.g., nucleic acids), with oppositely charged groups (such as proteins), and even polyelectrolytes that have no net charge, for which the Donnan coefficient is ill‐defined. The distribution of counterions around three simple polyelectrolyte geometries—spherical, cylindrical, and planar—was examined. In each case the excess number of counterions associated with the polyelectrolyte is not constant, but increases with decreasing salt. Although a limiting value is reached at low salt for a cylinder, in agreement with the limiting law condensation models, for the spherical geometry the number of associated counterions decreases to zero, while for the planar case it increases to 100%. For the cylindrical case Donnan coefficients agree closely with those computed previously by Monte Carlo simulations [M. C. Olmstead, C. F. Anderson, and M. T. Record (1991) Biopolymers, Vol. 31, pp. 1593–1604]. An all atom B‐DNA model shows about 5% less accumulation of counterions than a cylinder, due to the helical charge arrangement and groove structure. The entropic and enthalpic contributions to the salt‐dependent free energy of a cylinder were examined in the PB model. In addition to the well known ion cratic (redistribution) entropy, there are significant contributions from electrostatic enthalpy, and from dielectric entropy (water re‐orientation due to the electrostatic field from polyelectrolyte charges and salt ions). The latter entropy contribution has not previously been taken into account in salt‐dependent polyelectrolyte phenomena, but at higher salt concentrations (> 0.01M), this can be as large as the cratic entropy contribution. © 1995 John Wiley & Sons, Inc.
Comparison of base inclination in ribo‐AU and deoxyribo‐AT polymers
Authors: Jin Xiaokui , Johnson W. Curtis
Published: December 2004

The inclination angle between the base normal and the helix axis is measured for ribo‐AU polymers by using flow linear dichroism (LD), and compared to measurements for deoxyribo‐AT polymers under dehydrating conditions. The CD of the DNA polymers under the dehydrating conditions is not the same as the corresponding RNA polymers, which are presumed to be in the A form. However, the LD indicates that poly(dAdT)‐poly(dAdT) can assume the A form in 80% 2,2,2‐trifluoroethanol, although poly(dA)‐poly(dT) retains B form structure in this dehydrating solvent. The inclination angles are similar for B form poly(dAdT)‐poly(dAdT) and poly(dA)‐poly(dT), and these parameters are also similar for A form poly(rArU) ‐poly(rArU) and poly(rA) ‐poly(rU). All the inclination axes are similar. © 1995 John Wiley & Sons, Inc.
Effect of anisotropy of the bending rigidity on the supercoiling free energy of small circular DNAs
Authors: Schurr J. Michael , Babcock Hazen P , Gebe John A
Published: December 2004

In principle, the supercoiling free energy of a small circular DNA will be enhanced by increasing the anisotropy of its bending potential at constant persistence length. The magnitude of this effect is investigated by Monte Carlo simulation using an extension of a previously proposed algorithm. The supercoiling free energy at 298 K is simulated for circular DNAs containing N = 100 bp with torsion constant α = 5.8 × 10−12 dyne cm. persistence lengths P = 500 Å and 10,000 Å and a range of anisotropies of the bending potential from p = 1.0 to 16.0. The apparent torsion constants, reckoned from these supercoiling free energies by assuming an isotropic bending potential, are found to increase by less than 3% as the input anisotropy increases from 1.0 to 16.0. When P = 500 Å, the apparent torsion constant never rises significantly above the input value over the entire range of input anisotropies. When P = 10,000 Å, the apparent torsion constant rises only about 3% above the input value for anisotropies ρ = 8.0 and 16.0. Evidently, anisotropy of the bending potential cannot account for the fact that the torsion constants reported for small circular DNAs exceed those reported for longlinear DNAs by a factor of 1.6 or more. © 1995 John Wiley & Sons, Inc.
A molecular mechanics/continuum reaction field investigation of the interactions between polar amino acid side chains in water and organic solvents
Authors: Langlet J , Gresh N , Giessner‐Prettre C
Published: December 2004

An integrated procedure that computes in a consistent way both the intermolecular interaction energies and the solvation energies is reported. It interfaces the Sum of Interactions Between Fragments Ab initio computed molecular mechanics and the Langlei–Claverie continuum reaction field procedures. These two methodologies formulate the interaction energy as a sum of separate electrostatic, polarization, dispersion, and repulsion terms; the first two are computed with the help of distributed charges, dipoles, and quadrupoles derived from the ab initio SCF or MP2 molecular wave fundctions of individual solutes. This computational procedure will be used to estimate the solvent contribution to the interaction energy between polar amino acids side chains. We will consider, on the one hand, the terminal fragments of the side chains of aspartate and glutamate—namely the acetate anion and its protonated counterpart, acetic acid—and on the other hand, the terminal fragments of the cationic residues lysine, histidinium, and arginine, as represented by methylammonium, imidazolium, and methylguanidinium cations, respectively. The deprotonated counterpart of imidazolium, imidazole, is also investigated. With water as a solvent, and for each of the three anion‐cation complexes investigated, the total energy value DE (intermolecular + solvation) of the associated ion pair is only slightly larger (≈ 5 kcal/mol out of ≈ 170) than that of the fully dissociated arrangement and has a virtually flat dependence as a function of intermolecular separation. The associated complex has an enhanced stability in DMSO, a trend accented in chloroform and carbon tetrachloride. In water, the acetic acid–imidazole complex is less than 1 kcal/mol more stable than the dissociated pair. Energy balances, taking into account the experimental values of protonation energies of acetate and imidazole, indicate that the acetate‐imidazolium complex is more stable than its nonzwitterionic counterpart, acetic acid‐imidazole in water. In carbon tetrachloride, by contrast, the two complexes are of similar stabilities in terms of internal energies. When we consider free instead of internal energies of solvation, the three organic solvents stabilize the complex between the neutral molecules. The investigation of the interaction of two methylguanidiniums in the polar solvents suggests that the solvation energy of a complex, larger than that of the two isolated entities, could be able to overcome their electrostatic repulsion. This results in a small preference in favor of the complex. Implications of the findings of this study, and future prospects of applications to molecular recognition and conformational studies of oligopeptides and possibly proteins, are discussed. © 1995 John Wiley & Sons, Inc.
Interaction of mastoparan‐B from venom of a hornet in taiwan with phospholipid bilayers and its antimicrobial activity
Authors: Park Nam Gyu , Yamato Yuhji , Lee Sannamu , Sugihara Gohsuke
Published: December 2004

Mastoparan B (MP‐B), an amphiphiphilic α‐helical peptide newly isolated from the hornet Vespa basalis, was studied in comparison with mastoparan (MP), in terms of interaction with the phospholipid bilayer and of hemolytic and antimicrobial activity. The amphiphiphilic structure of MP‐B has more hydrophilic amino acid residues in the hydrophilic surface than that of MP. Although each peptide had a considerably different effect on the interaction with lipid bilayers (e. g., their conformation in the presence of acidic and of neutral lipids and dye‐release ability from the encapsulated liposomes), on the whole the interaction mode was similar. MP‐B caused a change in the shape of erythrocytes from normal discoid to a crenated form (named echinocytes). MP exhibited strong activity against gram‐positive bacteria but not against gram‐negative ones. Contrary to this, MP‐B showed both strong activity against gram‐positive bacteria and potent activity against gram‐negative bacteria. Whereas both peptides have almost the same residues on the hydrophobic side, the difference in the hydrophilic surface area on the molecules seems to lead to the subtle change in its interaction with membranes, resulting in the alteration of biological activity. © 1995 John Wiley & Sons, Inc.
Dielectric study of the cooperative order–disorder transition in aqueous solutions of schizophyllan, a triple‐helical polysaccharide
Authors: Teramoto Akio , Gu Hong , Miyazaki Yuji , Sorai Michio , Mashimo Satoru
Published: December 2004

Schizophyllan exists in aqueous solution as a triple helix, which is intact at room temperature. Its aqueous solution forms some ordered structure at low temperatures but undergoes a sharp transition to a disordered structure as the temperature is raised. The transition temperature Tc is about 7 and 18°C for H2O and D2O solutions, respectively. This transition was followed by time‐domain reflectometry to investigate dynamic aspects of the transition. In addition to a major peak around 10 GHz, the dielectric dispersion curve of a 20 wt % schizophyllan in D2O exhibited a small peak around 100 MHz below Tc and around 10 MHz above Tc. The major peak is due to bulk water, whereas the 100 MHz peak is assigned to “bound” or “structured” water, and that around 10 MHz to side‐chain glucose residues. However, unlike usual bound water reported for biopolymer solutions, this “structured” water disappears abruptly when the temperature becomes close to Tc without accompanying a conformational transition of the main chain. The above assignment is consistent with the structure of the ordered phase derived from previous static data that it consists of side‐chain glucose residues along with nearby water molecules surrounding the helix core that are interacting with each other loosely through hydrogen bonds, and spreads radially only a layer of one or two water molecules but a long distance along the helix axis. © 1995 John Wiley & Sons, Inc.
Design and development of a vasoactive intestinal peptide analog as a novel therapeutic for bronchial asthma
Authors: Bolin David R. , Michalewsky Joseph , Wasserman Martin A. , O'Donnell Margaret
Published: December 2004

Analogs of vasoactive intestinal peptide (VIP) were synthesized and screened as bronchodilators with the ultimate goal of enhancing the potency and extending the duration of action of the native peptide. Several design approaches were applied to the problem. First, the amino acid residues required for receptor binding and activation were identified. A model of the active pharmacophore was developed. With knowledge of the secondary structure (NMR) of the peptide, various analogs were synthesized to stabilize α‐helical conformations. Having achieved a level of enhanced bronchodilator potency, our approach then concentrated on identification of the sites of proteolytic degradation and synthesis of metabolically‐stable analogs. Two primary cleavage sites on the VIP molecule were identified as the amide bonds between Ser25‐Ile26 and Thr7‐Asp8. This information was used to synthesize cyclic peptides which incorporated disulfide and lactam ring structures. Analog work combined the best multiple‐substitution sites with potent cyclic compounds which resulted in identification of a cyclic lead peptides. This compound, Ro 25‐1553, exhibited exceptionally high potency, metabolic stability, and a long duration of action and may be an effective therapeutic for the treatment of bronchospastic diseases. © 1994 John Wiley & Sons, Inc.
One‐bead–one‐structure combinatorial libraries
Authors: Lebl Michal , Krchňák Viktor , Sepetov Nikolai F. , Seligmann Bruce , Štrop Peter , Felder Stephen , Lam Kit S.
Published: December 2004

Combinatorial libraries employing the one‐bead–one‐compound technique are reviewed. Two distinguishing features characterize this technique. First, each compound is identified with a unique solid support, enabling facile segregation of active compounds. Second, the identity of a compound on a positively reacting bead is elucidated only after its biological relevance is established. Direct methods of structure identification (Edman degradation and mass spectroscopy) as well as indirect “coding” methods facilitating the synthesis and screening of nonpeptide libraries are discussed. Nonpeptide and “scaffold” libraries, together with a new approach for the discovery of a pentide binding motif using a “library of libraries,” are also discussed. In addition, the ability to use combinatorial libraries to optimize initially discovered leads is illustrated with examples using peptide libraries. © 1994 John Wiley & Sons, Inc.
Mechanisms for the modulation of membrane bilayer properties by amphipathic helical peptides
Authors: Epand Richard M. , Shai Yechiel , Segrest Jere P. , Anantharamiah G. M.
Published: December 2004

The amphipathic helix, in which hydrophobia and hydrophilic residues are grouped on opposing faces, is a structural mot if found in many peptides and proteins that bind to membranes. One of the physical properties of membranes that can be altered by the binding of amphipathic helices is membrane monolayer curvature strain. Class A amphipathic helices, which are present in exchangeable plasma lipoproteins, can stabilize membranes by reducing negative monolayer curvature strain; proline‐punctuated class A amphipathic helical segments are particularly effective in this regard. This property is suggested to be associated with some of the beneficial biological effects of this protein. On the other hand, lytic amphipathic helical peptides can act by increasing negative curvature strain or by forming pores composed of helical clusters. Thus, different amphipathic helical peptides can be membrane stabilizing or be lytic to membranes, depending on the structural motif of the helix, which in turn determines the nature of its association with membranes. Features of these peptides that are responsible for their specific properties are discussed. © 1994 John Wiley & Sons, Inc.
The measurement of transmembrane helices by the deconvolution of CD spectra of membrane proteins: A review
Authors: Fasman Gerald D.
Published: December 2004

The interpretation of the CD spectra of proteins to date requires additional secondary structural information of the proteins to be analyzed, such as x‐ray or nmr data. Therefore, these methods are inappropriate for a CD data base whose secondary structures are unknown, as in the case of the membrane proteins. The Convex Constraint Analysis algorithm [A. Perczel, M. Hollósi, G. Tusnády, and G. D. Fasman (1991) Protein Engineering, Vol. 4, 669–679], on the other hand, operates only on a collection of spectral data to extract the common spectral components with their spectral weights. The linear combinations of these derived “pure” CD curves can reconstruct the original data set with great accuracy. For a membrane protein data set, the five‐component spectra so obtained from the deconvolution consisted of two different types of α‐helices (the α‐helix in the soluble domain and the αT‐helix, for the transmembrane α‐helix), a β‐pleated sheet, a class C‐like spectrum related to β‐turns, and a spectrum correlated with the unordered conformation. The deconvoluted CD spectrum for the αT‐helix was characterized by a positive red‐shifted band in the range 195–200 nm (+95,000 deg cm2 dmol−l), with the intensity of the negative band at 208 nm being slightly less negative than that of the 222 nm band (−50,000 and −60,000 deg cm2 dmol−1, respectively) in comparison with the regular α‐helix, with a positive band at 190 nm and two negative bands at 208 and 222 nm with magnitudes of + 70,000, −30,000, and −30,000 deg cm2 dmol−1, respectively. © 1994 John Wiley & Sons, Inc.
Concept and progress in the development of RGD‐containmg peptide pharmaceuticals
Authors: Craig William S. , Cheng Soan , Mullen Daniel G. , Blevitt Jon , Pierschbacher Michael D.
Published: December 2004

The cell adhesion domain, arginine‐glycine‐aspartic acid (RGD), has been incorporated into synthetic peptides to perform either of two modes of drug action, antagonist or agonist. Short, conformationally constrained peptides have been developed as antagonists for the platelet membrane glycoprotein complex, the integrin α11b, β3, using cell‐based and integrin‐based assays. In combination with a comparative molecular modeling study, these results have helped identify common conformotional elements in the pharmacophore of this class of molecules. Peptides are presented that are highly potent, integrin specific, and that possess reduced pharmacological side effects. Also presented is the development of a peptide that modifies, noncovalently, the surfaces of a wide variety of synthetic materials used in medical implants. The agonist activity of this molecule is evident from its ability to stimulate cell attachment on these surfaces. This is shown to translate into an in vivo activity of faster and more complete tissue integration, and a reduction in foreign body response. © 1994 John Wiley & Sons, Inc.
The kinetics of cholesterol oxidase synthesis by Nocardia rhodocrous
Authors: Buckland B. C. , Lilly M. D. , Dunnill P.
Published: December 2004

The production of cholesterol oxidase by 3 liter batch cultures of Nocardia rhodocrous growing on a glycerol/yeast extract medium was investigated. Cholesterol was shown to be a good inducer of the enzyme. The optimum time for cholesterol addition and the quantity to be added were determined, resulting in a 15‐fold yield increase. Cholesterol oxidase synthesis was influenced by the dissolved oxygen tension. Maximum cholesterol oxidase production was obtained at 30–40% air saturation. The effect of growth conditions on the extraction of cholesterol oxidase by Triton X‐100 was investigated. The scale‐up of the fermentation to 800 liters in a pilot‐plant fermenter is described.
Effects of immobilization on the kinetics of enzyme‐catalyzed reactions. II. Urease in a packed‐column differential reactor system
Authors: Ramachandran K. B. , Perlmutter D. D.
Published: December 2004

Urease from Jack bean was immobilized on nonporous glass beads by covalent bonding and its kinetics were studied in a packed‐column differential reactor. To facilitate comparison, the urease was immobilized by both diazo and glutaraldehyde coupling. The kinetic properties of immobilized urease were similar to those of the soluble enzyme and different immobilization methods did not appreciably alter the kinetic properties. The affects of three different amino acid activators appear to follow predictions obtained from a relatively simple competitive model, except at very low substrate levels.
Biological treatment of waste with high ash content using a hydrolytically assisted extended aeration process
Authors: Gaudy A. F. , Manickam T. S. , Saidi H. , Reddy M. P.
Published: December 2004

In previous reports from this laboratory it has been shown that the extended aeration process for biological treatment of organically laden municipal and/or industrial waste could be successfully employed for concurrent purification and sludge disposal. Also results using a modified process in which autodigestion was aided and controlled by periodic partial hydrolysis of small portions of the recycle sludge showed that operational control was feasible. There was some question regarding the success of such a process if the original waste contained a large portion of inorganic solids. Accordingly, a 1½ year pilot plant study was made using a waste (hydrolyzed trickling filter sludge) of exceptionally high ash content (50–60%). It was found that the ash content of activated sludge grown on this substrate did not continually increase nor did the high ash content of the waste interfere in any way with the efficiency of removal of organic matter. In general it exceeded 90 percent. Also a highly nitrified effluent was produced. A variety of analyses were performed: COD, BOD, TOC, suspended solids, NH3‐N, organic‐N, NO3‐N, etc. Interrelationships between these important monitoring parameters for assessing plant performance offered useful insight into operational control for hydrolytically assisted extended aeration processes.
Yield and maintenance relations of yeast growth in the chemostat at superoptimal temperatures
Authors: Van Uden N. , Madeira‐Lopes A.
Published: December 2004

A model is proposed that accounts for the decreases in yield which occur in chemostat cultures of mesophilic yeasts at superoptimal growth temperatures. Two yield depressing effects were identified, one due to increased maintenance requirements by the viable fraction of the population, the other due to energy substrate dissipation by the nonviable fraction. The two effects are functions of the dilution rate, as is the fraction of nonviable cells. Experimental results were obtained on the yield, maintenance, and dissipation of energy substrate in a glucose‐limited chemostat culture of a respiration‐deficient mutant of Saccharomyces cerevisiae at 39°C. The rates of glucose utilization for maintenance and for dissipation constituted, respectively, 33–28% and 15–9% of the total glucose utilization rate over the range of dilution rates tested (0.038–0.064 hr−1), while the yield varied over this range from 0.066–0.085 g of biomass (dry wt) per gram of glucose.
Influence of cell concentration, temperature, and press performance on flow characteristics and disintegration in the freeze‐pressing of Saccharomyces cerevisiae with the X‐press
Authors: Magnusson K.‐E. , Edebo L.
Published: December 2004

The pressure required for initiation of flow when freeze‐pressing with the X‐press is related to the phase boundaries of water, particularly those between ice I and liquid even at temperatures around −25°C and lower. Widening the orifice of the pressure chamber to diameters larger than 2.5 mm leads to lower pressures and less extensive cell disintegration.

Pressing Saccharomyces cerevisiae slowly with the aid of a manual hydraulic jack at −25°C produces a disintegration of 60–75% irrespective of cell concentration. Pressing at −35°C shows no clear differences.

Pressing more rapidly with the aid of a motor‐driven hydraulic press produces a similar extent of disruption of diluted cell suspensions (5.4 mg/g) as slow pressing. However, freeze‐pressing a paste of baker's yeast (270 mg/g) increases the degree of disintegration. Under these conditions the disintegration is further enhanced by a lower temperature, −35°C, and by a high velocity of flow through the orifice, such that more than 95% of the S. cerevisiae is disrupted by one pressing at less than 2 × 108 Pa.

Mechanisms for flow through the X‐press are suggested and discussed in relation to the phase diagram of water.
Aroma of sherry wines
Authors: Webb A. Dinsmoor , Noble Ann C.
Published: December 2004

Over 130 volatile components have been identified in aromas of film (F), submerged‐culture flor (S), and baked (B) sherries. The composition of these volatiles, which include more than 25 alcohols, five carbonyls, ten acetals, three amides, 35 esters, and five lactones, is influenced by the variety of grape used, the nature of the primary alcoholic fermentation, type of cooperage, aging and blending system, and filmphase yeast metabolism in (F) and (S) or reactions occurring during “baking” in (B). The influence of these factors on the aroma and several volatile formation mechanisms which contribute to the characteristic flavor of sherry, particularly during the film growth and during baking, are discussed.
The mode of interaction between the substrate and cell surface of the hydrocarbon‐utilizing yeast Candida tropicalis
Authors: Kaeppeli O. , Fiechter A.
Published: December 2004

A method for the measurement of the affinity of the cell surface to hydrocarbons is described. The affinity was basically unaffected by different pH values and temperature as well as by the chain length of the substrate. The contact time required for saturation of the cell surface with substrate was 30 sec. Cells grown on glucose showed a 25% lower adsorption capacity compared to those grown on n‐alkane. The glucose grown cells showed also a more distinguished dependence of the amount of adsorbed hydrocarbon on the quality of the emulsion. The interaction between the substrate and cell surface turned out to be an adsorption that did not involve an enzymatic reaction. These results led to the conclusion that a lipopolysaccharide moiety present at the cell surface is responsible for the affinity.
Large‐scale disintegration of microorganisms by freeze‐pressing
Authors: Magnusson K.‐E. , Edebo L.
Published: December 2004

A semicontinuous press has been constructed for the disintegration of microorganisms and other biological material by freeze‐pressing, i.e., pressure extrusion of frozen material through a narrow hole. The material to be freeze‐pressed is frozen in the form of cylindrical rods, which fit into the pressure chamber and are extruded by a piston forced back and forth by means of a hydraulic pump. At a sample temperature of −35°C and a press temperature of −20°C, about 90% disruption is achieved in a single passage of undiluted baker's yeast (Saccharomyces cerevisiae, 270 mg/g) through the orifice of the pressure chamber. With this press about 10 kg of material can be freeze‐pressed per hour.
A mathematical model for microbial growth under limitation by conservative substrates
Authors: Nyholm Niels
Published: December 2004

A mathematical model is suggested for growth of microorganisms under limitation by “conservative” substrates such as inorganic ions or vitamins that are not broken down after uptake into the cells, but that wholely or partly remain available for production of biomass. The specific growth rate is expressed here as a function of the intracellular “concentration” of the limiting substrate, defined as the amount of substrate within the cells per unit of cell dry weight.

In the model, the intracellular substrate is divided into two parts. One part is a “structural” substrate not available for further growth. The other part is an “excess” or “functional” substrate that is used for biomass production and is assumed to be converted into structural substrate proportionally to growth. The rate of growth is believed to be controlled by the intracellular concentration of excess substrate.
Temperature‐influenced species competition in mass cultures of marine phytoplankton
Authors: Goldman Joel C. , Ryther John H.
Published: December 2004

Five marine phytoplankton species (Phaeodactylum tricornutum, Thalassiosira pseudonana, Skeletonema costatum, Monochrysis lutheri, and Dunaliella tertiolecta) were grown in enriched laboratory continuous cultures and natural populations were mass cultured outdoors for 16 months. Competition among the species was shown to be highly dependent on temperature, although the actual production of plant organic matter at the low growth rates used was relatively independent of this variable. Control of marine species in mass cultures does not appear economically feasible, but this drawback may be overcome by selecting herbivorous shellfish that are capable of assimilating those temperature‐dependent phytoplankton species dominating in a particular locale.
A fermentation process for producing both ethanol and lysine‐enriched yeast
Authors: Tanner Robert D. , Souki Nabil T. , Russell Richard M.
Published: December 2004

In 18 batch‐fermentation experiments, baker's yeast was grown in an enriched mineral medium, containing 10% by weight glucose, at various pH and temperature levels. The pH and temperature are just two representative engineering variables which and be easily varied at negligible cost. The commercial yeast inoculum, .20% by weight or about .16% viable cells, was selected to represent industrial (nonsterile) conditions. Free L‐lysine, ethanol, and cell growth were followed in time for each batch run held at a fixed pH and temperature. The maximum free lysine level reached at either 10½ or 24 hr occurred at a pH of 5 and 32°C. At 24 hr, the peak free lysine level, 120 mg/liter, is three times as great as the uncontrolled pH counterpart. In terms of total L‐lysine, (free plus protein‐bound) the peak represents a 25% improvement over the uncontrolled case, based on an average 3.5% lysine level per cell weight. The greatest measured cell level, .9% by weight in the fermentation broth, or a 5½‐fold increase over the inoculum, was reached during the 36°C and pH 3 run, while the largest measured ethanol value (3%, or 30% conversion by weight from glucose) was achieved during the 28°C and pH 6 experiment. The optimal lysine run produced, however, no less than 15% of the maximum cell and 30% of the maximum ethanol levels.
Effects of quantitative shock loadings on the constant recycle sludge concentration activated‐sludge process
Authors: Wong Y. K. , Yang P. Y.
Published: December 2004

The reliability of the process of Ramanathan and Gaudy (Biotechnol Bioeng., 13, 125 (1971)) for the completely mixed activated‐sludge process holding the recycle cell concentration, XR, as a system constant with respect to step changes in hydraulic retention time was investigated. The experiments were run at initial dilution rates of ⅛, ⅙, ¼, and ½ hr−1 treating a soft drink bottling wastewater. The influent substrate concentration was maintained at 1000 mg/liter chemical oxygen demand and the hydraulic recycle ratio at 0.3. The recycle sludge concentration was maintained at about 7000 mg/liter.

It was found that the system could accommodate hydraulic shock loads up to 200% positive changes and down to 50%negative changes without disruption of the effluent quality. Shorter retention time of the range studied, from 2 to 8 hr, has the advantage of shorter response time with respect to the response of the concentration of biological solids in the reactor.
Computer‐aided material balancing for prediction of fermentation parameters
Authors: Cooney Charles L. , Wang Henry Y. , Wang Daniel I. C.
Published: December 2004

Despite the importance of biomass as a parameter in fermentation processes, there are no commercially available sensors suitable for its measurement. An indirect approach for the assessment of biomass concentration can be based on material balances and on the direct monitoring of fermentation parameters for which there are established sensors (e.g., gaseous oxygen and carbon dioxide). As a consequence, this method requires no assumption of cellular yield coefficients or rate constants. This approach is also readily adaptable to general use since it requires only some knowledge of the compositions of the substrate, cells, and noncellular products.
Nonporous magnetic materials as enzyme supports: Studies with immobilized chymotrypsin
Authors: Munro P. A. , Dunnill P. , Lilly M. D.
Published: December 2004

Chymotrypsin has been immobilized to several nonporous magnetic materials. Nickel particles were considered to be most suitable as immobilized enzyme supports. Chymotrypsin immobilized to nonporous magnetic supports was not fouled significantly by6 either whole milk or clarified yeast homogenate. AE‐cellulose‐chymotrypsin was rapidly fouled by both these materials and chymotrypsin immobilized to acrylic‐based ion exchangers was slowly fouled. Immobilized enzyme activity was found to be inverted proportional to particle diameter for nonporous rock magnetite particles. Immobilization by adsorption and then glutaraldehyde crosslinking was used to produce controlled amounts of chymotrypsin on the particles. Esterolytic activity increased with enzyme loading but caseinolytic activity did not increase. Chymotrypsin is inhibited by metal ions from the magnetic supports. It is partially protected by use of a preliminary protein coating and may be reactivated by incubation with EDTA or BSA.
Properties of intracellular ribonuclease utilized for RNA reduction in disintegrated cells of Saccharomyces cerevisiae
Authors: Lindblom Marianne
Published: December 2004

The properties of intracellular RNase in disintegrated cell suspensions of Saccharomyces cerevisiae have been studied. The influence of salt addition and/or incubation of the suspension on the activity of RNase and on the degradation of endogenous RNA was determined. No significant change in the RNase activity in the disintegrated suspensions was obtained by addition of 3% NaCl or by incubation at 50°C with 3% NaCl. During the incubation with NaCl the active RNase was able to degrade endogenous RNA. By incubation without salt the RNase was inactivated. Inactivation also occurred after extraction at alkaline pH. The RNase had an optima at pH 5–6 and temperatures between 50–60°C. The main part of the RNase in the unincubated suspension was soluble at pH 5.6 but not at pH 4.0. After incubation with NaCl the RNase was soluble at pH 4.0. No serious protein degradation occurred during the short time incubation needed for RNA reduction. 70% of the protein in the suspensions was recovered in the precipitate at pH 4.0 after 20 min of incubation. The corresponding protein recovery from unincubated suspensions was 77%.
Continuous fermentation in slant tubes
Authors: Wick Emil , Popper Karel
Published: December 2004

Continuous, steady‐state grape‐juice fermentation, carried out in experimental slant‐tube fermentors, was characterized by complete fermentation, even at fast pumping rates, because efficient cell retention, as well as rapid yeast growth, quickly built cell density up to the limit permitted by the juice supply rate. Essentially complete fermentation was easily achieved with a juice residence time of 3.1 hr, compared to about 120 hr in normal batch fermentation. Successful continuous fermentation required constancy of gas and liquid flow through the tubes, placing some restrictions on choice of tube configurations. The validity of design equations, developed previously from a settling model, was tested, and some practical aspects of their application to fermentor design and operation were demonstrated.
Aminoacylase pellets
Authors: Hirano Ken‐Ichi , Karube Isao , Suzuki Shuichi
Published: December 2004

Aminoacylase was immobilized on the mycelium pellets of Aspergillus ochraceus by using albumin and glutaraldehyde. No difference in the optimum pH was observed between native aminoacylase and aminoacylase pellets. The amino‐acylase pellets were stable in pH 4–8 but they were unstable in alkaline conditions. The aminoacylase pellets were more stable against heavy metal ions and inhibitors than native aminoacylase. However, the degree of the activation of aminoacylase with cobalt ion decreased with the immobilization. It was suggested that most of aminoacylase was covalently coupled to the mycelium with glutaraldehyde.
The growth of L‐cells and Vero cells on an autoclavable MEM–peptone medium
Authors: Keay Leonard
Published: December 2004

The growth of L‐60TM cells (a suspension culture adapted L‐cell) on media composed of MEM (minimum essential medium (Eagle)) and bactopeptone autoclaved together or separately under a variety of conditions has been determined. It has been found that MEM autoclaved with 0.5% bactopeptone at 15 psi for 20 min, cooled and then neutralized with NaHCO3, consistently supported good cell growth of L‐60TM and L‐929 cells. Similar results were obtained when the MEM and bactopeptone were autoclaved separately. The cells grew initially as a monolayer, subsequently becoming a stationary suspension. Some experiments were carried out with agitated suspension culture of L‐60TM cells in the autoclaved MEM–bactopeptone combination with and without added methylcellulose and results were obtained which indicate that large scale suspension culture is possible in this system. Other peptones were also found to support cell growth.

The autoclaved MEM–bactopeptone combination also supported the growth of Chang liver and Vero cells. The Chang liver cells rapidly dissociated from the plastic surface but the Vero cells remained sufficiently securely attached so that it was possible to grow them near to confluency in roller bottles.