bluuebunny/crossref_metadata_2025 huggingface dataset:
The preparation and characterization of a twin‐tailed, lipid‐linked polysialic acid
Authors: Matthews Ian , Petrak Karel
Published: December 2004

Reactions between water‐insoluble and water‐soluble components are often slow, giving low yields. In order to prepare glycolipids composed of polysialic acid and N‐(4‐(p‐maleimidylphenyl)butyryl)dipalmitoyl‐L‐α‐phosphatidylethanol amine triethylammonium salt, we used liposomes to enhance the interfacial reaction between the reagents. Thus, thiolreactive liposomes were reacted with polysialic acid cysteamine. A 70% yield was achieved with this addition reaction, and the product was isolated by dialysis and centrifugation © 1993 John Wiley & Sons, Inc.
Looking into the grooves of DNA
Authors: Boutonnet Nathalie , Hui Xiaowen , Zakrzewska Krystyna
Published: December 2004

The present study is aimed at understanding the effects of DNA sequence, local conformation, and curvature on groove geometry. Energy‐optimized structures are obtained by Jumna methodology; groove geometry is analyzed by a recently developed technique that allows an accurate and continuous measurement of width and depth. The mechanics of groove deformations is also studied and analyzed in terms of helicoidal parameters. © 1993 John Wiley & Sons, Inc.
Helix promotion in polypeptides by polyols
Authors: Bello Jake
Published: December 2004

The helix content of [L‐Lys(Me3)]n · ClO4, and [L‐Lys(Me3)50, L‐Ala50]n · ClO4 in water is markedly increased by the presence of sucrose and glycerol. For [L‐Lys(Me3)]n · ClO4 the ellipticity at 222 nm changes from +2 × 103 deg cm2 dmole−1 in water to −44 × 103 in 50% glycerol. Sucrose does not promote helix formation in melittin at pH 7.2, but glycerol does. At pH 5.5 sucrose and, more so, glycerol, induce helix in melittin. Glycerol induces some helix in methylated melittin, but less than in melittin. The results are discussed in relation to excluded volume effects, ΔG of transfer of peptide and hydrophobic groups from water to mixed solvents, electrostatic effects, and preferential hydration. © 1993 John Wiley & Sons, Inc.
β‐Alanine containing peptides: γ‐Turns in cyclotetrapeptides
Authors: Di Blasio B. , Lombardi A. , D'Auria G. , Saviano M. , Isernia C. , Maglio O. , Paolillo L. , Pedone C. , Pavone V.
Published: December 2004

In the present paper we describe the synthesis, purification, single‐crystal x‐ray analysis, solution conformational characterization, and conformational energy calculations of the cyclic tetrapeptide cyclo‐ (β‐Ala‐L‐Pro‐β‐Ala‐L‐Val). The peptide was synthesized by classical solution methods and the cyclization of the free tetrapeptide was accomplished in good yields in diluted methylene chloride solution using N,N‐dicyclohexyl‐carbodiimide. The compound crystallizes in the monoclinic space group P21 from ethanol with two independent molecules in the unit cell. All peptide bonds are trans. The nmr molecular conformation in the acetonitrile solution as well as that derived from the molecular dynamic simulation in vacuo is quite different from those observed in the solid state and is very similar to that previously observed for the parent compound cyclo‐(β‐Ala‐L‐Pro‐β‐Ala‐L‐Pro). © 1993 John Wiley & Sons, Inc.
Nmr and computer‐aided studies of the three interchanging stereoisomers of the cyclic hexapeptide cyclo[‐Pro1‐Gly2‐Glu3(OBzl)‐Pro4‐Phe5‐Leu6‐]: Unexpected observation of a cis isomer of a secondary amide peptide bond in the presence of two trans proline peptide bonds
Authors: Ewen Ian Mc
Published: December 2004

The cyclic hexapeptide cyclo[‐Pro1‐Gly2‐Glu3(OBzl)‐Pro4‐Phe5‐Leu6‐] (1; OBzl: benzyl ester) was modeled and synthesized to be used as a chiral site for the separation of enantiomers. Total correlation spectroscopy and nuclear Ovehauser effect spectroscopy spectra of the peptide in CDCl3 showed the presence of three stereoisomers. The two dominant stereoisomers 1a and 1b exchanged chemically with each other, while the minor stereoisomer 1c exchanged exclusively with the stereoisomer 1b. Stereoisomer 1a had two cis proline peptide bonds while stereoisomer 1b had all‐trans peptide bonds. The stereoisomer 1c had, for nonstrained peptides, an unusual cis phenylalanine peptide bond while both proline peptide bonds were trans. © 1993 John Wiley & Sons, Inc.
Solid state and solution structure of Boc‐L‐Ala‐ΔPhe‐ΔPhe‐NHMe: A dehydropeptide showing propensity for 310‐helices of both screw senses
Authors: Tuzi Angela , Ciajolo M. Rosaria , Guarino Gennaro , Temussi Pier Andrea , Fissi Adriano , Pieroni Osvaldo
Published: December 2004

The crystal and molecular structure of the peptide Boc‐L‐Ala‐Δphe‐Δphe‐NHMe, containing two consecutive dehydro‐phenylalanine (Δphe) residues, has been solved by x‐ray diffraction. Two independent molecules, X and Y, are present in the crystallographic unit. Their conformation corresponds approximately to an incipient 310‐helix stabilized by two intramolecular hydrogen bonds. The (ϕ, ψ) torsion angles, however, have negative and positive signs in the two molecules X and Y, respectively. Therefore, in spite of the presence of an amino acid residue of the L configuration, the two helical molecules have opposite screw senses, even though the right‐handed helix is less distorted than the left‐handed one in correspondence of the L‐Ala residue.

The CD spectra in various solvents exhibit exciton bands originating from dipole–dipole interaction between the Δphe side chains. Addition of DMSO to the chloroform solution produces, as a first step, a strong increasing of the CD bands, which are then progressively canceled by increasing DMSO concentration. The nmr data parallel the behavior observed in the CD spectra. In CDCl3 solution, the temperature coefficients of the NH resonances are consistent with the involvement of the last two amide protons of the sequence in intramolecular hydrogen bonds, but only negligibly small nuclear Overhauser effects (NOE) are observed. Addition of 5% DMSO‐d6 allows the observation of diagnostic NOEs. CD and nmr data indicate that the solid state structure is retained in solution, and are consistent with the presence of right‐handed and left‐handed conformers, with a prevalence of the more stable right‐handed one. © 1993 John Wiley & Sons, Inc.
DNA orientation in shear flow
Authors: Simonson Tomas , Kubista Mikael
Published: December 2004

The linear dichroism (LD) has been measured for DNA molecules 239–164,000 base pairs long oriented in shear flow over a large range of velocity gradients (30–3,000 s −1) and ionic strengths (2–250 mM). At very low gradients, the degree of DNA orientation increases quadratically with the applied shear as predicted by the Zimm theory [J. Zimm, (1956) Chemical Physics, Vol. 24, p. 269]. At higher gradients, the orientation of fragments ≥ 7 kilobase pairs (kbp) increases linearly with increasing shear, whereas the orientation of fragments ≥ 15 kbp shows a more complicated dependence. In general, the orientation decreases with increasing ionic strength throughout the studied ionic strength interval, owing to a decrease in the persistence length of the DNA. The effect is most dramatic at ionic strengths below 10 mM, and is more pronounced for longer DNA fragments. For fragments ≥ 15 kbp and velocity gradients ≥ 100 s−1, the orientation can be adequately described by the empirical relation: LDr= –(k1‐G)/(k2 + G), where k1is a linear function of the square root of the ionic strength and k2 depends on the DNA contour length. Since the DNA persistence length can be represented as a linear function of the reciprocal square root of the ionic strength [D. Porschke, (1991) Biophysical Chemistry, Vol. 40, p. 169], extrapolation of the empirical relation provides information about the stiffness of the DNA fibers. © 1993 John Wiley & Sons, Inc.
Proton magnetic resonance studies of bradykinin antagonists
Authors: Liu Xiaohong , Stewart John M. , Gera Lajos , Kotovych George
Published: December 2004

Bradykinin (BK) is a peptide hormone with sequence Arg1‐Pro2‐Pro3‐Gly4‐Phe5‐Ser6‐Pro7‐Phe8‐Arg9 and has been implicated in a multitude of pathophysiological processes such as the ability to lower systemic blood pressure and stimulate pain. BK analogues having bulky, β‐branched D‐aliphatic residues at position 7 combined with bulky L‐aliphatic residues at position 8 have now been observed to be strong antagonists. Conformational studies based on two‐dimensional nmr experiments in methanol/water (80/20 v/v) were carried out on several such active antagonists in a polar solvent. Included in this study were the very active antagonists, [D‐Arg0, Hyp3, Thi5, D‐Cpg7, Cpg8]‐BK [Cpg: α‐cyclo‐pentyl‐glycine; Hyp: trans‐4‐hydroxy‐L‐proline; Thi: β‐(2‐thienyl)‐L‐alanine] (I), [D‐Arg0, Hyp3, D‐Cpg7, Cpg8] ‐BK (II), as well as its variant with D‐Cpg7 replaced by Cpg7, namely [D‐Arg0, Hyp3, Cpg7, Cpg8]‐BK (III). A turn‐like structure, which coexists with the extended conformation, was observed between residues 2 and 5 for the most active antagonists I and II, in direct correlation with the peptide activities. No turn‐like structure was found for residues 6–9. In peptide III, a turn‐like structure was not identified. The existence of a turn at the C‐terminal end of bradykinin and its analogues has been predicted by empirical calculations and supported by nmr measurements. But the present nmr study on the most active antagonists (I, II) does not support this hypothesis. Instead, the data suggest that a turn‐like structure between residues 2 and 5 could be important for antagonist activity. Finally, one weak inhibitor [D‐Cpg7]‐BK (IV) showed no defined secondary structure. © 1993 John Wiley & Sons, Inc.
Computer simulations of the flexibility of a series of synthetic cyclic peptide analogues
Authors: Thomas Aline , Roux Benoit , Smith Jeremy C.
Published: December 2004

The flexibility of a series of cyclic peptides derived from the epitope of a snake toxin is investigated using computer simulation techniques. Molecular dynamics (MD) simulations and vibrational analyses are performed on chemically constrained peptides modeled on the parent protein loop. In the 50 ps MD simulations, large variations in the atomic fluctuations are seen between the peptides, and can be related to the nature of the chemical constraints present in the molecules. Normal mode analyses are performed on energy‐minimized configurations derived from the dynamics trajectories. The atomic fluctuations calculated from the normal modes are about 30% of those of the molecular dynamics for the more flexible peptides and 70% for the more constrained molecules. The calculated differences in flexibility between the molecules are much less significant in the harmonic approximation. © 1993 John Wiley & Sons, Inc.
On the interaction of polypeptides with bile salts or bilirubin‐IXα
Authors: D'Alagni Maria , D'Archivio Angelo A. , Giglio Edoardo
Published: December 2004

Aqueous solutions formed by polypeptides, simple models of proteins, and bile salts (sodium cholate and deoxycholate, NaC and NaDC, respectively) or bilirubin‐IXα (BR) have been studied by CD measurements. They could mimic more complicated biliary systems, thus supplying a possible interpretation of the behavior of some amino acid residues in the biliary proteins. The aggregation of NaDC and NaC in water can be monitored by CD measurements. Bile salts, in submicellar and micellar form, stabilize poly (L‐Lys) (PLL) in α‐helical conformation. The α‐helix content increases with increasing bile salt concentration and ionic strength. NaDC seems to be a slightly better stabilizing agent of the α‐helix conformation than NaC. Models characterized by hydrogen bonds between bile salts and PLL are proposed, also resorting to previous data available on the systems formed by NaDC and poly(L‐Leu‐L‐Leu‐L‐Lys) (PLLL) or poly(L‐Leu‐L‐Leu‐L‐Asp) (PLLA). Binding of BR to PLL, poly(D‐Lys), poly(L‐Glu), PLLL, and PLLA in water has been investigated by CD spectra in order to clarify the nature of the association complexes and the mechanism of the BR enantioselective complexation. Potential energy calculations provide binding models capable of explaining the enantioselective ability of the PLL and PLLL α‐helices toward the left‐ and right‐handed enantiomer of BR, respectively. BR is bound to ‐NH2 groups of PLL and PLLL lying on a right‐ and left‐handed spiral, respectively. These results, together with those formerly obtained for some bile salts–BR systems, indicate that the selectivity originates from a binding that involves large regions of the BR molecule and gives rise, very probably, to moderate conformational changes from the “ridge tile” structure observed in the crystals. In some cases van der Waals forces can play a crucial role in the chiral recognition of bilirubin. Moreover, possible interaction models of BR with human serum albumin are proposed on the basis of a recent x‐ray crystal structure of the protein. © 1993 John Wiley & Sons, Inc.
Single‐residue substitution in homopolypeptides: Perturbative helix–coil theory at a single site
Authors: Qian Hong
Published: December 2004

Based on Lifson–Roig's helix–coil transition theory, substitution of a single heteroresidue into a homopolymer host is studied. This study models recent experiments that substitute a single amino acid into a small peptide in water [A. Chakrabartty, J. A. Schellman, and R. L. Baldwin (1991), Nature, Vol. 351, pp. 586–688]. Our formalism, which is based on a perturbation method, differs from the existing theory for sequenced polymers and is naturally analogous, hence likely to be useful, to substitution experiments in the laboratory. It is shown that the intrinsic helix propensity w is directly proportional to the equilibrium constant for the helix–coil equilibrium of a single residue in a host peptide. This intuitive new result will simplify experimental data interpretations for measurements of the helical conformation on the single amino acid level. It is also shown that substitution affects the total helicity of the host peptide according to two considerations: the helicity of the replaced residue prior to the substitution, and the sensitivity of the site, a measure of neighboring interactions. The relationship between substitution stability and thermal stability is explored. © 1993 John Wiley & Sons, Inc.
Identification of the adenine binding site in the ricin toxin A‐chain by fluorescence, CD, and electron spin resonance spectroscopy
Authors: Ramalingam T. S. , Das Puspendu K. , Podder Sunil K.
Published: December 2004

CD, electron spin resonance, and fluorescence spectroscopy have been utilized to study the adenine binding site of ricin and its toxic A‐subunit. At acidic (4.5) and physiological (7.3) pH, adenine or a spin‐labeled analogue of adenine, N6‐(2,2,6,6‐tetramethyl‐1‐oxypiperidin‐4‐yl) adenine, alters the near uv CD spectra of the ricin A‐chain as well as intact ricin, whereas the far uv CD spectra of all proteins remain unchanged. Electron spin resonance data show that the adenine spin‐labeled analogue interacts strongly with the A‐chain both at pH 4.5 and 7.3, but no or very weak binding is observed for the intact ricin or the isolated B‐chain. The adenine spin label gets highly immobilized (2AII = 65.5G) by the A‐chain. The apparent dissociation constant Kd for the toxic A‐chain ligand complex is 1.55 × 10−4 M and 5.6 × 10−5 M at pH 7.3 and 4.5, respectively. Fluorescence intensity of ricin A‐chain bound 1,8‐anilinonaphthalenesulfonic acid (ANS) decreases by ∼55% at pH 4.5 with the addition of the spin‐labeled analogue of adenine, implying that both the ANS and adenine spin label (ADSL) bind to the hydrophobic domain of the A‐chain. Fluorescence of the only intrinsic tryptophan probe of the A‐chain is also efficiently quenched by ADSL, indicating that the tryptophan residue and the hydrophobic adenine binding site are closely located. All spectroscopic measurements indicate that adenine or its spin‐labeled analogue has a single binding site adjacent to the TRP211 residue in the A‐chain. Expansion of the A‐chain globule and subsequent exposure of the hydrophobic binding site seem to be responsible for the increased binding of adenine at pH 4.5. © 1993 John Wiley & Sons, Inc.
Dynamics and structures of DNA: Long‐range effects of a 16 base‐pair (CG)8 sequence on secondary structure
Authors: Kim Ug‐Sung , Fujimoto Bryant S. , Furlong Clement E. , Sundstrom Joseph A. , Humbert Richard , Teller David C. , Schurr J. Michael
Published: December 2004

The effects of inserting 16 base pair (bp) of alternating CG[(CG)8] near the middle of a much longer restriction fragment (1097 bp) are investigated by measuring various properties that are sensitive to secondary and tertiary structure. Results for this fragment are compared with those for a control fragment (1089 bp) with the identical sequence except at the insert. Another fragment (1382 bp), which contains a 296‐bp extension at the 5′‐end of the 1089‐bp control fragment, is also used as a secondary control in some experiments. When the 1097‐bp (CG)8 insert fragment is compared with the control fragments in 0.1M NaCl buffer, the (CG)8 insert is found to induce disproportionately large relative changes in the molar ellipticity at 273 nm ([θ)273], the torsion constant (α) measured by fluorescence polarization anisotropy, the optical melting profile, and the susceptibility to S1 nuclease. Estimates of the minimum distance over which the (CG)8 insert alters the secondary structure range from 330 to 550 bp. With increasing NaCl concentration, the 1097‐bp insert fragment undergoes a structural transition between 2.0 and 2.5M that is manifested in the apparent diffusion coefficient (Dplat) from dynamic light scattering at large scattering vector. This transition, which is not exhibited by the control DNAs, is presumed to involve formation of Z‐helix at the insert. However, the observed decrease in (Dplat) is attributed to an increase in bending rigidity, which perforce must be globally distributed far beyond the (CG)8 insert per se. In 4.25M NaCl (but not in 0.1M NaCl), the addition of 1 ethidium dye per 300 bp induces an extensive structural transition in the 1097 bp (CG)8 insert fragment. This transition, which also is not exhibited by the control DNAs, significantly decreases the bending rigidity, doubles [θ]273, and takes place on a time scale of a few days. Removal of ethidium and salt by dialysis vs 0.1M NaCl buffer restores the original properties of the 1097‐bp (CG)8 insert fragment. The present results are consistent with a (fluctuating, long‐range) description of the secondary structure in which a given short sequence transiently fluctuates among two or more distinct secondary structures that extend over much larger domains of variable position and size, and whose relative stabilities depend on distant as well as close‐lying base pairs. © 1993 John Wiley & Sons, Inc.
A conformational study of the dehydroalanine: Dipeptide and homopolypeptide
Authors: Alemán Carlos , Perez Juan J.
Published: December 2004

A molecular mechanics study of polydehydroalanine [poly‐(Δ Ala)] is presented. For this purpose the AMBER 3a program has been used to perform the calculations. With exception of the point charges, the parameters for the terminal groups were taken from AMBER 3a libraries, whereas those for the Δ Ala residue from Alagona et al. [J. Comp. Chem. (1991) Vol. 12, pp. 934–942]. Charges for the residue and terminal groups have been fitted from the MNDO electrostatic potential and scaled to achieve an ab initio 6‐31G* quality. Calculations have been carried out using the continuous solvent approximation with three different dielectrics ε = 1, 1r, and 4r. The results show that, despite the preferred structure for the isolated residue is an extended conformation, a 310‐helix is the preferred conformation in the solid state (ε = 1 and 1r), whereas a peculiar structure with ψ = 0° is preferred with ε = 4r. © 1993 John Wiley & Sons, Inc.
Molecular dynamics in sodium poly (L‐glutamate) aqueous solutions analyzed by means of the stretched exponential decay of the williams–watts function
Authors: Bordi F. , Cametti C. , Paradossi G.
Published: December 2004

The high‐frequency dielectric dispersions of the sodium salt of poly(L‐glutamic acid) aqueous solutions in the frequency range from 1 to 1000 MHz have been analyzed by means of the Cole–Davidson relaxation function in the frequency domain and by means of the Williams‐Watts relaxation function in the time domain. The analogies between the two descriptions are discussed on the basis of the analysis carried out by Lindsey and Patterson and the connections between the dipole–dipole correlation function based on the kinetic model for polyion dynamics proposed by Skinner and the Williams–Watts nonexponential decay function are briefly discussed. © 1995 John Wiley & Sons, Inc.
Design and synthesis of nonpeptide peptidomimetic inhibitors of renin
Authors: Smith Amos B. , Akaishi Ryouichi , Jones David R. , Keenam Terence P. , Guzman Mark C. , Holcomb Ryan C. , Sprengeler Paul A. , Wood John L. , Hirschmann Ralph , Holloway M. Katharine
Published: December 2004

The desire to replace the amide backbone of renin inhibitors with a new scaffold led us to explore vinylogous amides (enaminones). An initial attempt proved unsuccessful, a result explained after the fact via docking experiments. Based on this lesson, we designed a different vinylogous amide scaffold which incorportated one or more pyrrolinone rings into the backbone. Three of the four compounds gave IC50s in the 0.6 to 18 μM range. These compounds did not inhibit HIV‐1 protease. Taken together, the results reported herein provide insights into the role of hydrogen bonding and steric interactions for binding to renin. © 1994 John Wiley & Sons, Inc.
Submerged culture of hamster kidney cells in a stainless steel vessel
Authors: Telling R. C. , Elsworth R.
Published: December 2004

This paper describes the design and operation of an industrial type culture system for the submerged growth of BHK 21 cells at a volume of 30 l. Using a modified Eagle's medium and with the culture controlled at pH 7.4 ± 0.05 and at a temperature of 35 ± 0.25°C., the cells grew from 0.5 × 106 to about 2.5 × 106 viable cells/ml. in 50 hr. at stirrer speeds of 330 and 460 rpm. The significance of certain aspects of design and operation of a culture system of this type is discussed.
Optimal control of an enzyme reaction subject to enzyme deactivation. I. Batch process
Authors: Ho Lewis Y. , Humphrey Arthur E.
Published: December 2004

This paper is concerned with the study of an enzymatic system in a repeated batch process where the enzyme is subject to deactivation. The particular system studied was the enzymatic hydrolysis of Penicillin G to 6‐aminopenicillanic acid. Utilizing standard optimization techniques, pH and temperature control policies were determined that would maximize the product yield.
A method for large reduction of the nucleic acid content of yeast
Authors: Canepa A. , Pieber M. , Romero C. , Toha J. C.
Published: December 2004

A method for large reduction of nucleic acid content of Saccharomyces cercvisae is described. This method is based on yeast suspension in phosphate solution (50mM), heat shock following the Maul et al. (Nature 228, 181 (1970)) procedure, and dialysis or washing with phosphate solution of alkaline pH. When the extraction process is carried out at pH 12, sodium phosphate can be replaced by sodium chloride with the same results. Under these conditions the protein mg/nucleic acid mg ratio attains values of 50–60, (10 times the initial ratio). At this alkaline pH, washing can be done with distilled water with equally good results.
Kinetic behavior of mixed populations of activated sludge
Authors: Chiu S. Y. , Fan L. T. , Kao I. C. , Erickson L. E.
Published: December 2004

The kinetic behavior of heterogeneous microbial populations of sewage origin was studied in a single‐stage isothermal continuous flow completely mixed aeration tank. A series of experiments were carried out at various dilution rates using glucose as the growth limiting substrate. The steady‐state behavior of the system was observed at each dilution rate and the results were found to fit fairly well with the steady‐state equation bayed on the Monod model with an endogenous respiration term included, i.e., μ = μmS/(Ks + S) − Kd. The growth kinetics of cells harvested at steady state for each dilution rate were studied using batch experiments. The multiple response data of the system as functions of time were used to estimate the parameter values in the above kinetic model. It was found that values of the growth parameters changed significantly and systematically with cell population. For example, values of μm were high at high dilution rates and low at low dilution rates. It was also found that only those batch growth parameters from cells obtained at fairly high dilution rates are comparable with those estimated by the results of steady‐state operations. The results of this investigation suggest that (1) different cell populations pre dominated at different steady‐state dilution rates, with high dilution rates resulting in predominantly fast‐growing organisms and low dilution rates resulting in predominantly slow‐growing cells, and (2) risk exists in any randomly picked batch experiment to predict the steady‐state behavior of the system when heterogeneous microbial populations must be used.
Biotechnology report: Solid state fermentations
Authors: Hesseltine C. W.
Published: December 2004

A unique method is described by which large yields of secondary metabolites arc produced on solid substrates. The process involves the use of moist substrates which are continuously agitated in appropriate fermentation equipment. The amount of agitation, aeration, and moisture can be varied. Extremely high yields of secondary metabolites such as ochratoxin and aflatoxin were obtained using Aspergillus and Penicillium species. The process prevents sporulation of the fungus and because of the nature of the solid substrate makes recovery of the product easier than in conventional liquid media. The substrates include rice, corn, wheat, and other cereals.
Release of protein from Bakers' yeast (Saccharomyces cerevisiae) by disruption in an industrial agitator mill
Authors: Currie J. A. , Dunnill P. , Lilly M. D.
Published: December 2004

Release of protein from a suspension of bakers' yeast (Saccharomyces cerevisiae) by disruption in an industrial agitator mill has been studied. Protein release on disruption in the mill is a first‐order rate process. The rate constant is dependent on at least six parameters. Increased disruption efficiency was obtained at higher agitator speeds, greater loading of bead attritive elements and lower rates of upward recycle of yeast suspension through the mill. An increase in temperature from 5 to 42°C was accompanied by a reduction in disruption efficiency of approximately 20%. With optimal values of the parameters examined the throughput of the mill is 5.32 kg/hr of soluble protein for 90% disruption.
The effect of carbon dioxide and particle surface area on the microbiological leaching of a zinc sulfide concentrate
Authors: Torma A. E. , Walden C. C. , Duncan D. W. , Branion R. M. R.
Published: December 2004

The effects of carbon dioxide‐enriched air on the rate of zinc extraction during the microbiological leaching of a, high‐grade zinc sulfide concentrate by Thiobacillns ferrooxidans have been studied. Under normal air‐aeration conditions, the leach rate is limited initially by the availability of solid substrate surface area per unit volume of leach liquor, then by availability of carbon dioxide. If carbon dioxide‐enriched air is supplied, along with excess substrate, the zinc extraction rate increases as the carbon dioxide content increases until some other, as yet unknown, factor becomes limiting.
Model for the growth of aerobic microorganisms under oxygen limiting conditions
Authors: Ryder D. N. , Sinclair C. G.
Published: December 2004

A simple dynamic model is proposed which will allow fermenters to be run at throughputs which fully utilize the mass transfer capabilities of the fermenters while not decreasing the yield from the substrate. The model is compared with one previously proposed, which was originally formulated for double substrate limitation when both substrates were supplied in the feed. Computer solutions of the model are given which show the effects of the parameters used. Experimental results from growing Candida utilis on a high concentration of glucose were found to be similar to those predicted by the model.
Continuous production of L‐aspartic acid by immobilized aspartase
Authors: Tosa Tetsuya , Sato Tadashi , Mori Takao , Matuo Yuhsi , Chibata Ichiro
Published: December 2004

Various methods were tried for the immobilization of aspartase, and the preparation having the highest activity was obtained when partially purified aspartase from Escherichia coli was entrapped into polyacrylamide gel Iattice. Enzymatic properties of the immobilized aspartase were investigated and compared with those of the native aspartase. With regard to optimum pH, temperature, concentration of Mn++, kinetic constants and heat stability, no marked difference was observed between the native and immobilized aspartases.

By employing an enzyme column packed with the immobilized aspartase, conditions for continuous production of L‐aspartic acid from ammonium fumarate were investigated. When a solution of 1M ammonium fumarate (pH 8.5, containing 1mM MnCl2) was passed through the aspartase column at the flow rate of SV = 0.08 at 37°C, the highest rate of reaction was attained. From the column effluents, L‐aspartic acid was obtained in a good yield.
Some methods for processing of single‐cell protein
Authors: Hedenskog Gudmund , Mogren Hårkan
Published: December 2004

Methods for the production of protein concentrates, with a low content of nucleic acid, in kilogram quantities from yeast have been studied with the aid of equipment designed for operation on pilot‐plant scale. The influence of drum drying and mechanical disintegration on the nutritive value of the yeast was also investigated. Drum drying and mechanical disintegration improved the nutritive value of the yeast but high extractability of protein and nucleic acid was only obtained after mechanical disintegration.

Protein concentrates without and with cell walls were produced from mechanically disintegrated yeast. The different fractions which were obtained when separating cell walls and precipitating protein by heating at alkaline pH, were analyzed. After protein precipitation, about 90% of the RNA could be precipitated from the supernatant by addition of acid, giving a product containing 50% RNA of the dry weight. The protein precipitate obtained after cell wall separation had an RNA content of less than 2% and contained 70–l75% of the amino acids in the starting yeast material. Protein concentrates containing cell walls were produced by precipitating protein by heating at alkaline pH directly after mechanical disintegration. The content of RNA was about 2% and the yield of amino acids was 70–80%. It was found that the nutritive value of the protein concentrate was higher than that of the starting yeast material. To produce such a protein concentrate on a large scale, the process described can probably be employed.
Immobilized glucoamylase on porous glass
Authors: Marsh David R. , Lee Yoon Y. , Tsao George T.
Published: December 2004

A study was made to determine the controlling mass transfer resistance in the overall reaction rate for conversion of maltose to glucose, catalyzed by glucoamylase immobilized onto porous glass. For normal operation of a packed column and air‐stirred batch reactor, the rate controlling step was found to be the internal resistance of simultaneous pore diffusion and chemical reaction. Experimental effectiveness factors were determined and are compared with those derived from a theoretical diffusion model based on Michaelis‐Menten kinetics. Also given are temperature and pH relationships for the free and immobilized glucoamylase.
Penicillin amidase production by bacillus megaterium
Authors: Acevedo F. , Cooney C. L.
Published: December 2004

Penicillin amidase, an enzyme which hydrolyzes benzylpenicillin to 6‐aminopenicillanic acid and phenylacetic acid, is produced by Bacillus megaterium ATCC 14945 as an extracellular enzyme. We used this system as a model to examine the effects of nitrogen, sulfur, and phosphorous limitation on enzyme production in continuous culture. For these studies, we developed a minimal medium for B. megaterium which contained histidine as the sole nitrogen source. Batch experiments showed that this enzyme is produced as a growth‐associated metabolite. Enzyme production was shown to be a function of the growth‐limiting conditions and the concentration of the inducer, phenylacetic acid. Sulfur limitation in continuous culture yielded enzyme activities approximately three to five times those observed in nitrogen‐ and phosphorous‐limited chemostats. These results are discussed in terms of the environment's influence on enzyme production in continuous culture.
Hydrocarbon fermentations: Oxidation mechanism and nonionic‐surfactant effects in a culture of Candida lipolytica
Authors: Whitworth D. A. , Moo‐Young M. , Viswanatha T.
Published: December 2004

Oxidation rates of several alkane substrates by C. lipolytica ATCC 8661 grown on n‐dodecane were determined using a Warburg Respirometer. Substrates were emulsified using Span 20, Span 80, Tween 20, Tween 80, and Triton X‐100 surfactants and the effects of these surfactants on oxidation and growth were determined. The oxidation rates of a number of intermediates, including lauric acid and lauryl alcohol, were also assessed. Responses of dodecane‐grown C. lipolytica to select substrates were compared to the corresponding behavior with glucose‐grown yeast and with baker's yeast. The role of surfactants in hydrocarbon fermentations is discussed in the light of the present and previously published data.
Cofactor recycling in an enzyme reactor. A comparison Using free and immobilized dehydrogenases with free and immobilized NAD
Authors: Wykes J. R. , Dunnill P. , Lilly M. D.
Published: December 2004

The requirements for enzymic cofactor recycling have been investigated in a system employing alcohol and lactate dehydrogenases. The interactions of various combinations of free dehydrogenases or dehydrogenases immobilized either to the same or separate supports, with free NAD, a soluble highmolecular weight derivative of NAD or an insoluble derivative of NAD have been examined.
Enzymes immobilized on alumina and stainless steel supports
Authors: Charles M. , Coughlin R. W. , Paruchuri E. K. , Allen B. R. , Hasselberger F. X.
Published: December 2004

Enzymes can be sorbed on inorganic carriers followed by crosslinking with glutaraldehyde. In some cases superior results are obtained when the support is precoated with a porous layer of titanium oxide. Immobilized lactase prepared in this way retains essentially all its activity when stored under water at 23°C for long periods of time and loses activity only slowly while treating cheese whey at 55°C over the course of several weeks contact time. Furthermore, catalyst activity is unaffected by frequent sanitization. Optimum pH for these immobilized‐lactase catalysts (enzyme produced from A. niger) is about 3.0 and optimum temperature is about 60°C. Amyloglucosidase catalase, L‐asparaginase, and trypsin have also been immobilized by these techniques.
Enzyme immobilization on macroreticular polystyrene
Authors: Baum George
Published: December 2004

Macroreticular polystyrene beads may be converted into suitable supports for covalent binding of enzymes. In many respects the supports are physically similar to controlled pore glass (CPG). Our results for immobilized glucoamylase were very similar to published results using CPG as a carrier. The characteristics of immobilized papain were less satisfactory. The product exhibited a Z‐shaped activity‐time profile suggestive of the involvement of multiple mechanisms.
Production of a plasmin inhibitory substance by Scopolia japonica suspension cultures
Authors: Misawa Masanaru , Tanaka Hozumi , Chiyo Osamu , Mukai Noboru
Published: December 2004

A potent inhibitory agent against human plasmin, fibrinolytic proteinase, has been found in the extracts of callus tissue of Scopolia japonica. Effects of cultural conditions on cell growth and production of the plasmin inhibitory substance by this cell line in suspension cultures were examined in MurashigeSkoog's medium. More than l.5 mg of the inhibitor, as t‐amino cyclohexane carboxylic acid, a synthetic plasmin inhibitor, were observed to accumulate per ml of medium containing 0.83 g of NH4NO3 and 7.6 g of KNO3 per liter as well as suitable levels of growth hormones.

Addiction of antibiotics and deformers were examined in preliminary tests for large scale cultivation. Semicontinuous culture on a small scale in a glass cylinder, was also tested and growth rate of 1.29 g/liter/day (by dry wt) was obtained. Plasmin inhibitory activities in the extracts of the results intact plant and in cultured cells of S. japonica were compared and the results indicated that cell suspension culture was superior to extraction the natural plant for inhibitor production.
Effects of internal diffusion on the lineweaver‐Burk plots for immobilized enzymes
Authors: Gondo Shinichiro , Isayama Shogo , Kusunoki Koichiro
Published: December 2004

The effect of internal diffusion on the slope and the intercept of the LineweaverBurk plots for the immobilized enzyme was considered theoretically and it was found that the slope and the intercept are influenced not only by the dimensionless term M but also by the range of the dimensionless bulk substrate concentration ζb. The dependencies of the slope and the intercept on M and on the rate of ζb are shown graphically. Accurate estimations of M and the maximum velocity of the immobilized enzyme give the true, not apparent, Michaelis constant. It is shown that the linear correlations in the Lineweaver‐Burk plots do not always coincide with the correlations for the estimation of M and the maximum velocity. It also is shown that large values of M may induce a serious error in the estimation of M with large values of ζb and in an estimation of the maximum velocity with small values of ζb.
Transient kinetics of yeast growth in batch and continous culture with an inhibitory carbon and energy source
Authors: Ko R. Y. C. , Edwards V. H.
Published: December 2004

Growth and acetate metabolism by Candida utilis (ATCC 9226) is reported for both acetate‐ and zinc‐limited cultures in defined media. Acetate concentrations were varied from suboptimal to inhibitory levels in both types of media in differential shake flask culture and in batch and continuous cultures in stirred fermentors. Transient responses of steady‐state cultures to small or large additions of concentrated sodium acetate, or to shifts in dilution rate or inlet acetate concentration are compared with one another and with simple mathematical models of growth and acetate metabolism. Exponential growth was observed during unrestricted growth (differential shake flask and batch cultures) with both types of media. Addition of acetate during unrestricted growth always caused lags and for larger pulses, lower specific growth rates were observed after exponential growth resumed. Inhibition by high acetate concentrations was much greater in acetate–limited than in zinc–limited cultures. During restricted growth (steady‐state, continuous cultures), high acetate concentrations again consistently caused growth lags but stimulated, inhibited, or temporarily stopped acetage uptake. Qualitative agreement between the predictions of a simple mathematical model of acetate inhibition fitted to differential shake flask data and the observed transient data was surprisingly good.
Protease covalently coupled to porous glass: Preparation and characterization
Authors: Mason R. D. , Detar C. C. , Weetall H. H.
Published: December 2004

A microbial protease was immobilized by covalent attachment, to porous glass. This material was characterized for pH optimum, thermal stability, and operational half–life using casein as substrate.

The immobilized enzyme was used for preparation of soya hydrolysates, low in free amino acids with high solubility.
Hydrolysis of particulate tributyrin in a fluidized lipase reactor
Authors: Lieberman R. B. , Ollis D. F.
Published: December 2004

Pancreatic lipase has been immobilized onto stainless steel beads by adsorption followed by crosslinking, and onto polyacrylamide by covalent bonding. The activities of the two types of immobilized enzyme toward the particulate substrate, tributyrin emulsion droplets, were determined experimentally, and rate constants based on Michaelis‐Menten kinetics were calculated. The activity of the stainless steel–lipase was determined for various flow conditions and for various support sizes by the use of a differential fluidized bed recycle reactor. The rate constants calculated indicate that the experimental reaction rate is free from mass transfer influences, since the observed Michaelis constant does not vary with the fluidization velocity or with the support particle size. In addition, the Michaelis constant of the stainless steel–lipase was found to be equal to that of the free enzyme, suggesting that adsorption and subsequent crosslinking does not alter the enzyme–substrate affinity. The emulsion substrate mass transfer rates, calculated from the filtration theory, indicate that each substrate particle which contacts the immobilized enzyme is hydrolyzed to a significant extent. Theexperimentally determined kinetic rate constants may be used directly to predict the size of integral fluidized bed reactors.
Analysis of double‐substrate limited growth
Authors: Bader F. G.
Published: December 2004

Mathematical models which relate the growth rate of a microorganism to a single limiting substrate concentration have long been established. In recent years, it has become apparent that, under certain conditions, the growth rate of an organism may be simultaneously limited by two or more substrates. Mathematical models of double‐substrate limitation fall into two categories: interactive and no interactive models. A discussion of both types of models is presented in both conceptual and mathematical terms. An analogous case of an enzyme which requires two different substrates to produce a single product is presented. This enzyme analog indicates that both types of double‐substrate limitation models appear to be feasible under certain conditions. Based upon stoichiometry and specific growth rate‐substrate concentration contour plots, a method for determining the operational conditions which will lead to double‐substrate limitation is presented.
Preparation of immobilized soybean β‐amylase on porous cellulose beads and continuous maltose production
Authors: Maeda Hidekatsu , Tsao George T. , Chen Li Fu
Published: December 2004

Immobilized soybean β‐amylase was prepared by using porous cellulose beads. The expressed activity of the β‐amylase–cellulose beads conjugated below 35 mesh was 59–69% of the initial activity and the protein content was 10–13%. General properties of the conjugate were almost identical with those of the native enzyme except for the Km value. The Km value of the conjugate was 40mM and the Km value of the native enzyme was 0.6mM. This large difference was probably caused by pore structure, i.e., a pore diffusion problem. The film diffusion problem occurred at the flow rate below a linear velocity of 3 cm/min. Maximum maltose contents of the hydrolyzates prepared by the conjugate and the native enzyme were 69 and 71%, respectively. After a continuous column operation at 50°C for 17 days, the activity of the column was 60% of the activity. The half‐life of the column at 40°C was 40 days.
Enzymatic properties and application of L‐asparaginase–collagen membrane
Authors: Morikawa Yasushi , Karube Isao , Suzuki Shuichi , Nakano Yosuke , Taguchi Tetsuo
Published: December 2004

The activity of the L‐asparaginase–collagen membrane was 3.4 U/mg membrane (5.8 U/cm2 membrane) and the activity yield was 29%. The apparent Michaelis constant of the asparaginase–collagen membrane was 5.9 × 10−3M. The operational half‐life of the immobilized asparaginase column was 35–40 days. The L‐asparaginase–collagen membrane retained 90% of its original activity after ethylene oxide gas sterilization. The dried membrane stored at room temperature retained its original activity for five months, and the membrane stored in 0.05M phosphate buffer (pH 8.0) retained its original activity for one week at 37°C. The L‐asparaginase–collagen membrane tanned with 1% glutaraldehyde was stable against proteolytic enzymes. Complete degradation of L‐asparagine by the L‐asparaginase–collagen membrane occurred at a low concentration. The L‐asparagine in dog blood plasma was completely degraded within 20 min by the extracorporal shunt using the L‐asparaginase–collagen membrane.
Biomethanation: Anaerobic fermentation of CO2, H2 and CO to methane
Authors: Wise D. L. , Cooney C. L. , Augenstein D. C.
Published: December 2004

Studies to examine the microbial fermentation of coal gasification products (CO2, H2 and CO) to methane have been done with a mixed culture of anaerobic bacteria selected from an anaerobic sewage digestor. The specific rate of methane production at 37°C reached 25 mmol/g cell hr. The stoichiometry for methane production was 4 mmol H2/mol CO2. Cell recycle was used to increase the cell concentration from 2.5 to 8.3 g/liter; the volumetric rate of methane production ran from 1.3 to 4 liter/liter hr. The biogasification was also examined at elevated pressure (450 psi) and temperature to facilitate interfacing with a coal gasifier. At 60°C, the specific rate of methane production reached 50 mmol/g cell hr. Carbon monoxide utilization by the mixed culture of anaerobes and by a Rhodopseudomonas species was examined. Both cultures are able to carry out the shift conversion of CO and water to CO2 and hydrogen.
Preparation of succinylated yeast protein: Composition and solubility
Authors: Vananuvat P. , Kinsella John E.
Published: December 2004

A method for the succinylation of yeast Proteins during their isolation is described. Some factor affecting the extent of succinylation are described, e.g., heating of protein (80°C for 5 min) prior to dervatization reduced maximum succinylation of available ε‐amino groups from 88 to 54%. Succinylation of amino groups progressively increased as the concentration of succinic anhydride was increased and there was a concomitant decrease in nucleic acid content of the precipitated protein concentrate from 12.5 to 5.3 for 0 and 88% succinylation, respectively, succinylation enhanced the solubility of the protein concentrates particularly below pH 7 and it decreased the isoelectric point pH. 4.5 to 4.0.
Process design and economic studies of alternative fermentation methods for the production of ethanol
Authors: Cysewski Gerald R. , Wilke Charles R.
Published: December 2004

Cell recycle and vacuum fermentation processes are described for the continuous production of ethanol. Preliminary process design studies are employed to make an economic comparison of these alternative fermentation schemes with continuous and batch fermentation technologies. Designs are based on a production capacity of 78,000 gal 95% ethanol (EtOH)/day employing molasses as the fermentation substrate. The studies indicate that a 57% reduction in fixed capital investment is realized by continuous rather than batch operation. Further decreases in required capital investment of 68 and 71% over batch fermentation were obtained for cell recycle and vacuum operation, respectively. However, ethanol production costs were dominated by the cost of molasses, representing over 75% of the total manufacturing cost. But, when a reasonable yeast by‐product credit was assumed, the net production cost for 95% ethanol was estimated at 82.3 and 80.6 cent/gal, for the cell recycle and vacuum processes, respectively.