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  • Biochemistry and Biotechnology  (16,218)
  • 1
    ISSN: 0887-3585
    Keywords: folding and binding ; kinetics ; pepstatin A ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The prediction of binding affinities from structure is a necessary requirement in the development of structure-based molecular design strategies. In this paper, a structural parameterization of the energetics previously developed in this laboratory has been incorporated into a molecular design algorithm aimed at identifying peptide conformations that minimize the Gibbs energy. This approach has been employed in the design of mutants of the aspartic protease inhibitor pepstatin A. The simplest design strategy involves mutation and/or chain length modification of the wild-type peptide inhibitor. The structural parameterization allows evaluation of the contribution of different amino acids to the Gibbs energy in the wild-type structure, and therefore the identification of potential targets for mutation in the original peptide. The structure of the wild-type complex is used as a template to generate families of conformational structures in which specific residues have been mutated. The most probable conformations of the mutated peptides are identified by systematically rotating around the side-chain and backbone torsional angles and calculating the Gibbs potential function of each conformation according to the structural parametrization. The accuracy of this approach has been tested by chemically synthesizing two different mutants of pepstatin A. In one mutant, the alanine at position five has been replaced by a phenylalanine, and in the second one a glutamate has been added at the carboxy terminus of pepstatin A. The thermodynamics of association of pepstatin A and the two mutants have been measured experimentally and the results compared with the predictions. The difference between experimental and predicted Gibbs energies for pepstatin A and the two mutants is 0.23 ± 0.06 kcal/mol. The excellent agreement between experimental and predicted values demonstrates that this approach can be used in the optimization of peptide ligands. Proteins 30:74-85, 1998. © 1998 Wiley-Liss, Inc.
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  • 2
    ISSN: 0887-3585
    Keywords: xenon ; krypton ; hydrophobic cavity ; protein-ligand binding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: X-ray diffraction is used to study the binding of xenon and krypton to a variety of crystallised proteins: porcine pancreatic elastase; subtilisin Carlsberg from Bacillus licheniformis; cutinase from Fusarium solani; collagenase from Hypoderma lineatum; hen egg lysozyme, the lipoamide dehydrogenase domain from the outer membrane protein P64k from Neisseria meningitidis; urate-oxidase from Aspergillus flavus, mosquitocidal δ-endotoxin CytB from Bacillus thuringiensis and the ligand-binding domain of the human nuclear retinoid-X receptor RXR-α. Under gas pressures ranging from 8 to 20 bar, xenon is able to bind to discrete sites in hydrophobic cavities, ligand and substrate binding pockets, and into the pore of channel-like structures. These xenon complexes can be used to map hydrophobic sites in proteins, or as heavy-atom derivatives in the isomorphous replacement method of structure determination. Proteins 30:61-73, 1998. © 1998 Wiley-Liss, Inc.
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  • 3
    ISSN: 0887-3585
    Keywords: membrane ; protein ; structure ; prediction ; hydrophobicity ; computer ; magainin ; melittin ; 18A ; M2δ ; PGLa ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The lipid bilayer is crucial for the folding of integral membrane proteins. This article presents an empirical method to account for water-lipid interfaces in the insertion of molecules interacting with bilayers. The interactions between the molecule and the bilayer are described by restraint functions designed to mimic the membrane effect. These functions are calculated for each atom and are proportional to the accessible surface of the latter. The membrane is described as a continuous medium whose properties are varying along the axis perpendicular to the bilayer plane. The insertion is analyzed by a Monte Carlo procedure applied to the restraint functions. The method was successfully applied to small α peptides of known configurations. It provides insights of the behaviors of the peptide dynamics that cannot be obtained with statistical approaches (e.g., hydropathy analysis). Proteins 30:357-371, 1998. © 1998 Wiley-Liss, Inc.
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  • 4
    ISSN: 0887-3585
    Keywords: protein modeling ; crystal structure ; conformation change ; prediction ; mechanism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The occurrence of large domain motions associated with the mechanism of action of many proteins is well established. We present a general method of predicting domain closure applicable to proteins containing domains separated by an apparent hinge. The method attempts to allow for natural directional bias within the closing protein by repeatedly applying a weak pulling force over a short distance between pairs of atoms chosen at random in the two domains in question. Appropriate parameters governing the pulling function were determined empirically. The method was applied to the bi-lobal protein PGK and a closed-form activated ternary complex generated for Bacillus stearothermophilus PGK. This model was compared with the recently determined crystal structure of closed-form Trypanosoma brucei PGK. The model predicts the correct hinge regions, although the magnitude of movement at one hinge point was overestimated, and provides a reasonable representation of the closed-form ternary complex. Proteins 30:372-380, 1998. © 1998 Wiley-Liss, Inc.
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  • 5
    ISSN: 0887-3585
    Keywords: serum amyloid A ; fluorescence ; circular dichroism ; acute phase ; denaturation ; nuclease ; amyloidosis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We developed a recombinant DNA system to overexpress a fusion protein between the small, minimally soluble acute phase serum protein, serum amyloid A (SAA), and the bacterial enzyme staphylococcal nuclease (SN). This fusion protein is very soluble and is immunoreactive to polyclonal anti-SAA antibodies. Tryptophan fluorescence shows smooth denaturation curves for the fusion protein in guanidinium HCl or potassium thiocyanate. Fluorescence also indicates that only a single tryptophan residue (of the four present) is accessible to iodide quenching and, presumably, is exposed on the surface of the fusion protein. Circular dichroism (CD) shows a significant signal indicating α-helix, which can be attributed to the SAA portion of the molecule; these are the first CD spectral data available for SAA. pH titration shows persistence of helix domains for the fusion protein at pH 3.0, in contrast to the denaturation of SN under the same conditions. (The entire fusion protein shows a random coil pattern below pH 3.0.) By exploiting the structural and solubility properties of SN, this fusion protein has provided the first structural data about SAA - the precursor of the amyloid deposits in secondary amyloidosis. This fusion protein should be useful for further physical and physiologic studies of SAA. Proteins 30:381-387, 1998. © 1998 Wiley-Liss, Inc.
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  • 6
    ISSN: 0887-3585
    Keywords: binding free energy ; electrostatics ; group contributions ; thermodynamic cycle ; solvation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The challenge of evaluating absolute binding free energies of protein-protein complexes is addressed using the scaled Protein Dipoles Langevin Dipoles (PDLD/S) model in combination with the Linear Response Approximation (LRA). This is done by taking the complex between Rap1A (Rap) and the p21ras binding domain of c-Raf (Raf-RBD) (Nassar et al., Nature 375:554-560, 1995) as a model system. Several formulations and different thermodynamic cycles are explored taking advantage of the LRA method and considering the protein reorganization during complex formation. The performance of different approximations is examined by comparing the calculated and observed absolute binding energies for the native complex and some of its mutants. The evaluation of the contributions of individual residues to the binding free energy, which is referred to here as group contributions is also examined. Special attention is paid to the role of the “dielectric constant,” εin which is in fact a scaling factor that represents the contributions that are treated implicitly. It is found that explicit consideration of protein relaxation is crucial for obtaining reasonable results with small values of εin, but it is also found that such a treatment of protein-protein interactions is very challenging and does not always give stable results. This indicates that more advanced explicit calculations should be based on experimentally determined structures of both the complex and the isolated proteins. Nevertheless, it is demonstrated that the qualitative trend of the effect of mutations can be reproduced by considering the effect of protein reorganization implicitly, using εin ˜25 for ionized residues and εin ˜4 for polar residues. Thus, it is concluded that an explicit treatment of solvent relaxation (which is common to current continuum models) does not provide sufficient compensation for turning off the charges of ionized residues on the interaction surface of the Raf-RBD/Rap complex. Representing the missing contribution by large εin can, of course, reproduce the observed effect of ionized residues, but now the contribution of uncharged residues will be largely underestimated. Regardless of these conceptual problems, it is established that a very simple nonrelaxed approach, where the relaxation of both the protein and the solvent are considered implicitly, can provide an effective qualitative way for evaluating group contributions, using large and small values for εin of ionized and neutral residues, respectively. As much as the actual system studied is concerned we find that more residues than generally assumed play a role in Raf-RBD/Rap interaction. This includes residues that are not located at the protein-protein interaction surface. These residues contribute to the binding energy through direct charge-charge interaction without leading to drastic structural changes. The overall contribution of the surface residues is quite significant since Raf and Rap are positively and negatively charged, respectively, and their charges are distributed along the interaction site between the two proteins. Proteins 30:407-423, 1998. © 1998 Wiley-Liss, Inc.
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  • 7
    ISSN: 0887-3585
    Keywords: protease II ; intrinsic fluorescence ; ionic strength ; heat denaturation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Oligopeptidase B is a member of a new serine peptidase family, unrelated to the trypsin and subtilisin families. It is a potential processing enzyme of prokaryotes, being very specific for the basic amino acid pairs of polypeptides. An understanding of the kinetics of the enzyme requires the examination of its conformational stability under a variety of conditions. To this end, the enzyme was cloned from Escherichia coli HB101 by the PCR method, expressed with high yield in E. coli XL1-Blue, and purified essentially in two chromatographic steps. The denatured enzyme failed to refold, which precluded the calculation of free energy of stability, ΔG0. Therefore, the unfolding rates were measured to probe the stability against urea, pH, and heat. Denaturation processes were monitored by intrinsic fluorescence, circular dichroism, and activity measurements. A static method, intrinsic fluorescence vs. pH, was indicative of significant changes in the tertiary structure of the enzyme pH < 6 and pH > 8.5. The more sensitive dynamic methods, unfolding rates in urea and inactivation rates at high temperature, revealed increased flexibility in the protein structure between pH 6 and pH 7, where the static method did not show significant changes. Inactivation of the enzyme in the acidic pH range correlated with the results obtained with the static rather than with the dynamic method. Acid denaturation at pH 3 was markedly retarded by 1 M NaCl. Against heat inactivation the enzyme was also considerably protected in the presence of salt, and the higher enthalpy and entropy of activation suggested the importance of hydration in the stabilization. The kinetics of unfolding followed single-exponential decay under strongly denaturing conditions (high urea concentration or high temperature), but deviated from the apparently two-state mechanism at low urea concentrations and at slightly acidic pH. The results indicate that under harsher denaturing conditions there is a single rate-limiting step in unfolding, whereas under milder conditions partly unfolded intermediates are populated. Proteins 30:424-434, 1998. © 1998 Wiley-Liss, Inc.
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  • 8
    ISSN: 0887-3585
    Keywords: molecular dynamics ; free energy perturbation ; thermodynamics integration ; spherical solvent boundary potential ; cell multipole method ; Nosé-Hoover equation ; component analysis ; chymotrypsin inhibitor 2 ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We developed a software package for improved free energy calculation, in which spherical solvent boundary potential, cell multipole method, and Nosé-Hoover equation are employed. The performance of the developed software package is demonstrated in the case of valine to alanine mutation of the 57th residue in chymotrypsin inhibitor 2. By using this package, we obtained results quantitatively comparable to experimental results. By the free energy component analysis, it is shown that leucine 51, arginine 65, arginine 67, and phenylalanine 69 residues contribute significantly to the total free energy shift, ΔΔG. Among them, contribution from the hydrophilic arginine 67 residue, which is in close contact with the mutation site, is the largest. Structure around the mutation site is largely changed by the mutation. The structure change is caused mainly by two effects, hydrophobic interaction and short-range interaction along the sequence. Effects of Nosé-Hoover algorithm and Kirkwood reaction field are also discussed. Proteins 30:388-400, 1998. © 1998 Wiley-Liss, Inc.
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  • 9
    ISSN: 0887-3585
    Keywords: protein stability ; cold shock domain ; nucleic acid binding ; hydrophobic effect ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: In the cold-shock protein CspB from Bacillus subtilis three exposed Phe residues (F15, F17, and F27) are essential for its function in binding to single-stranded nucleic acids. Usually, the hydrophobic Phe side chains are buried in folded proteins. We asked here whether the exposition of the essential Phe residues could be a cause for the very low conformational stability of CspB. Urea-induced and heat-induced equilibrium unfolding transitions were measured for three mutants of CspB, where Phe 15, Phe 17, and Phe 27 were individually replaced by alanine. Unexpectedly, all three mutations strongly destabilized CspB. The aromatic side chains of Phe 15, Phe 17, and Phe 27 in the active site are thus important for both binding to nucleic acids and conformational stability. There is no compromise between function and stability in the active site. Model calculations indicate that, although they are partially exposed to solvent, all three Phe residues nevertheless lose accessible surface upon folding, and this should favor the native state. A different result is obtained with the F38A variant. Phe 38 is hyperexposed in native CspB, and its substitution by Ala is in fact stabilizing. Proteins 30:401-406, 1998. © 1998 Wiley-Liss, Inc.
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  • 10
    ISSN: 0887-3585
    Keywords: cytochrome c ; thermal unfolding ; proteolysis ; proteinase K ; thermolysin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Recent hydrogen exchange experiments on native cytochrome c implicate a sequential unfolding pathway in contrast to a simple two-state process. We have studied the heat-induced unfolding of this protein by using spectroscopic measurements to detect changes in conformation and proteolytic enzyme digestion to identify regions of the protein that are labile. Several spectroscopic profiles were monitored: CD at 222 nm, a measurement of secondary structure change in the protein, the absorbance at 280 nm, involving the local environment of Trp 59, and absorbance at 420 nm, the Soret band of the heme. The apparent Tm values for these probes differ, consistent with an unfolding pathway containing intermediates. The limited digestion by proteinase K is consistent with population of an intermediate state in unfolding. We find a single strong region of cleavage at low temperature with retention of structure in each fragment. Proteins 30:435-441, 1998. © 1998 Wiley-Liss, Inc.
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  • 11
    ISSN: 0887-3585
    Keywords: pleckstrin homology domain ; DNA sequence homology ; DNA sequence patterns ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Pleckstrin homology (PH) domains have been proven to bind phosphoinositides (PI) and inositolphosphates (IP). On the other hand, a binding of PH domains to proteins is still a matter of debate. The goal of this work was to identify potential PH domain protein target sites and to build a model for PH domain-protein binding. A candidate sequence, called HIKE, was identified by sequence homology analysis of the proteins that are considered the strongest PH binding candidates, i.e., Gβ, PKC, and Akt. HIKE contains a PI binding sequence and fulfills several criteria for a potential PH-binding site, i.e., it is present in other PH-binding candidates, lies in regulatory regions independently predicted to bind PH domains, and is conserved in 3-D structure among different molecules. These findings and the similarities with the mode of binding of PTB and PDZ domains suggest a β strand-β strand coordination model for PH-protein binding. The HIKE model predicts that membrane anchoring of PH domains and their targets could be a critical step in their interaction, which would consistently explain why PH-protein binding has only been detected in the presence of PI. Proteins 31:1-9, 1998. © 1998 Wiley-Liss, Inc.
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  • 12
    ISSN: 0887-3585
    Keywords: protein design ; lattice model ; protein stability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A structure-based, sequence-design procedure is proposed in which one considers a set of decoy structures that compete significantly with the target structure in being low energy conformations. The decoy structures are chosen to have strong overlaps in contacts with the putative native state. The procedure allows the design of sequences with large and small stability gaps in a random-bond heteropolymer model in both two and three dimensions by an appropriate assignment of the contact energies to both the native and nonnative contacts. The design procedure is also successfully applied to the two-dimensional HP model. Proteins 31:10-20, 1998. © 1998 Wiley-Liss, Inc.
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  • 13
    ISSN: 0887-3585
    Keywords: subtilisin BPN′ ; proenzyme ; protein folding ; protein crystallography ; thermal stability ; calcium binding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The three-dimensional structure of a subtilisin BPN′ construct that was produced and folded without its prodomain shows the tertiary structure is nearly identical to the wild-type enzyme and not a folding intermediate. The subtilisin BPN′ variant, Sbt70, was cloned and expressed in Escherichia coli without the prodomain, the 77-residue N-terminal domain that catalyzes the folding of the enzyme into its native tertiary structure. Sbt70 has the high-affinity calcium-binding loop, residues 75 to 83, deleted. Such calcium-independent forms of subtilisin BPN′ refold independently while retaining high levels of activity [Bryan et al., Biochemistry, 31:4937-4945, 1992]. Sbt70 has, in addition, seven stabilizing mutations, K43N, M50F, A73L, Q206V, Y217K, N218S, Q271E, and the active site serine has been replaced with alanine to prevent autolysis. The purified Sbt70 folded spontaneously without the prodomain and crystallized at room temperature. Crystals of Sbt70 belong to space group P212121 with unit cell parameters a = 53.5 Å, b = 60.3 Å, and c = 83.4 Å. Comparison of the refined structure with other high-resolution structures of subtilisin BPN′ establishes that the conformation of Sbt70 is essentially the same as that previously determined for other calcium-independent forms and that of other wild-type subtilisin BPN′ structures, all folded in the presence of the prodomain. These findings confirm the results of previous solution studies that showed subtilisin BPN′ can be refolded into a native conformation without the presence of the prodomain [Bryan et al., Biochemistry 31:4937-4945, 1992]. The structure analysis also provides the first descriptions of four stabilizing mutations, K43N, A73L, Q206V, and Q271E, and provides details of the interaction between the enzyme and the Ala-Leu-Ala-Leu tetrapeptide found in the active-site cleft. Proteins 31:21-32, 1998. Published 1998 Wiley-Liss, Inc.This article is a US Government work and, as such, is in the public domain in the United States of America.
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  • 14
    ISSN: 0887-3585
    Keywords: quantum chemistry ; molecular mechanics ; inhibitor ; metalloenzyme complexes ; selectivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We investigated the binding properties of the metalloprotease inhibitors hydroxamate, methanethiolate, and methylphosphoramidate to a model coordination site occurring in several Zn2+ metalloproteases, including thermolysin. This was carried out using both the SIBFA (sum of interactions between fragments ab initio-computed) molecular mechanics and the SCF/MP2 procedures for the purpose of evaluating SIBFA as a metalloenzyme modeling tool. The energy-minimized structures were closely similar to the X-ray crystallographic structures of related thermolysin-inhibitor complexes. We found that selectivity between alternative geometries and between inhibitors usually stemmed from multiple interaction components included in SIBFA. The binding strength sequence is hydroxamate > methanethiolate ≥ methylphosphoramidate from multiple interaction components included in SIBFA. The trends in interaction energy components, rankings, and preferences for mono- or bidentate binding were consistent in both computational procedures. We also compared the Zn2+ vs. Mg2+ selectivities in several other polycoordinated sites having various “hard” and “soft” qualities. This included a hexahydrate, a model representing Mg2+/Ca2+ binding sites, a chlorophyll-like structure, and a zinc finger model. The latter three favor Zn2+ over Mg2+ by a greater degree than the hydrated state, but the selectivity varies widely according to the ligand “softness.” SIBFA was able to match the ab initio binding energies by <2%, with the SIBFA terms representing dispersion and charge-transfer contributing the most to Zn2+/Mg2+ selectivity. These results showed this procedure to be a very capable modeling tool for metalloenzyme problems, in this case giving valuable information about details and limitations of “hard” and “soft” selectivity trends. Proteins 31:42-60, 1998. © 1998 Wiley-Liss, Inc.
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  • 15
    ISSN: 0887-3585
    Keywords: ricin structure ; inhibitor design ; energy minimization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Ricin A-chain is an N-glucosidase that attacks ribosomal RNA at a highly conserved adenine residue. Our recent crystallographic studies show that not only adenine and formycin, but also pterin-based rings can bind in the active site of ricin. For a better understanding of the means by which ricin recognizes adenine rings, the geometries and interaction energies were calculated for a number of complexes between ricin and tautomeric modifications of formycin, adenine, pterin, and guanine. These were studied by molecular mechanics, semi-empirical quantum mechanics, and ab initio quantum mechanical methods. The calculations indicate that the formycin ring binds better than adenine and pterin better than formycin, a result that is consistent with the crystallographic data. A tautomer of pterin that is not in the low energy form in either the gas phase or in aqueous solution has the best interaction with the enzyme. The net interaction energy, defined as the interaction energy calculated in vacuo between the receptor and an inhibitor minus the solvation energy of the inhibitor, provides a good prediction of the ability of the inhibitor to bind to the receptor. The results from experimental and molecular modeling work suggest that the ricin binding site is not flexible and may only recognize a limited range of adenine-like rings. Proteins 31:33-41, 1998. Published 1998 Wiley-Liss, Inc.This article is a US Government work and, as such, is in the public domain in the United States of America.
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  • 16
    ISSN: 0887-3585
    Keywords: mutagenesis ; protein stability ; salt bridge ; protein folding ; malic enzyme ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A double mutant (R9E/M17K) of pigeon liver malic enzyme with glutamate and lysine replaced for arginine and methionine at positions 9 and 17, respectively, was found to be much more stable in urea and thermal denaturation, but was enzymatically less active than the wild-type enzyme (WT). Unfolding of the enzyme by urea produced a large red shifting of the protein fluorescence maximum from 320 to 360 nm, which was completely reversible upon dilution. Analysis of the denaturation curves monitored by enzyme activity lost suggested that a putative intermediate was involved in the denaturation process. The half unfolding urea concentration, measured by fluorescence spectral changes, increased from 2.24 M for WT to 3.13 M for R9E/M17K. The melting temperature increased by approximately 10°C for R9E/M17K compared with that for WT. Kinetic analysis of the thermal inactivation at 58°C also conformed to a three-state model with the rate constant for the intermediate state of R9E/M17K (k2 = 0.03 min-1) being much smaller than the WT value (k2= 2.39 min-1). Results obtained from single mutants indicated that the decreasing enzyme activity of R9E/M17K was exclusively due to R9 mutation, which increased the KmMn and KmMal by at least one order of magnitude compared with WT. Consequently, a decrease occurred in the specificity constant [kcat/(KmMnKmNADPKmMal)] for the R9 mutants at least four orders of magnitude smaller than the WT. M17K has similar properties to the WT, while R9E is more labile than the WT enzyme. The above results indicate that the extra stability gained by the double mutant possibly occurs through the introduction of an extra ion-pair between E9 and K17, which freezes the double mutant in the putative intermediate state. Examination of the N-terminal amino acid sequence of pigeon liver malic enzyme reveals that position 15 is also a lysine residue. Since the R9E mutant, which has an extra Glu9-Lys15 ion-pair, is less stable than the WT, we conclude that the contribution to malic enzyme stability is specific for the Glu9-Lys17 ion-pair. Proteins 31:61-73, 1998. © 1998 Wiley-Liss, Inc.
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  • 17
    ISSN: 0887-3585
    Keywords: α domains ; β domains ; α/β domains ; α+β domains ; resubstitution ; jackknife ; SCOP database ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Can the coupling effect among different amino acid components be used to improve the prediction of protein structural classes? The answer is yes according to the study by Chou and Zhang (Crit. Rev. Biochem. Mol. Biol. 30:275-349, 1995), but a completely opposite conclusion was drawn by Eisenhaber et al. when using a different dataset constructed by themselves (Proteins 25:169-179, 1996). To resolve such a perplexing problem, predictions were performed by various approaches for the datasets from an objective database, the SCOP database (Murzin, Brenner, Hubbard, and Chothia. J. Mol. Biol. 247:536-540, 1995). According to SCOP, the classification of structural classes for protein domains is based on the evolutionary relationship and on the principles that govern the 3D structure of proteins, and hence is more natural and reliable. The results from both resubstitution tests and jackknife tests indicate that the overall rates of correct prediction by the algorithm incorporated with the coupling effect among different amino acid components are significantly higher than those by the algorithms without using such an effect. It is elucidated through an analysis that the main reasons for Eisenhaber et al. to have reached an opposite conclusion are the result of (1) misusing the component-coupled algorithm, and (2) using a conceptually incorrect rule to classify protein structural classes. The formulation and analysis presented in this article are conducive to clarify these problems, helping correctly to apply the prediction algorithm and interpret the results. Proteins 31:97-103, 1998. © 1998 Wiley-Liss, Inc.
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  • 18
    ISSN: 0887-3585
    Keywords: molecular dynamics ; X-ray crystallography ; essential dynamics ; lysozyme ; hinge bending ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A comparison of a series of extended molecular dynamics (MD) simulations of bacteriophage T4 lysozyme in solvent with X-ray data is presented. Essential dynamics analyses were used to derive collective fluctuations from both the simulated trajectories and a distribution of crystallographic conformations. In both cases the main collective fluctuations describe domain motions. The protein consists of an N- and C-terminal domain connected by a long helix. The analysis of the distribution of crystallographic conformations reveals that the N-terminal helix rotates together with either of these two domains. The main domain fluctuation describes a closure mode of the two domains in which the N-terminal helix rotates concertedly with the C-terminal domain, while the domain fluctuation with second largest amplitude corresponds to a twisting mode of the two domains, with the N-terminal helix rotating concertedly with the N-terminal domain. For the closure mode, the difference in hinge-bending angle between the most open and most closed X-ray structure along this mode is 49 degrees. In the MD simulation that shows the largest fluctuation along this mode, a rotation of 45 degrees was observed. Although the twisting mode has much less freedom than the closure mode in the distribution of crystallographic conformations, experimental results suggest that it might be functionally important. Interestingly, the twisting mode is sampled more extensively in all MD simulations than it is in the distribution of X-ray conformations. Proteins 31:116-127, 1998. © 1998 Wiley-Liss, Inc.
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  • 19
    ISSN: 0887-3585
    Keywords: acid denatured state ; ANS fluorescence ; Arrhenius plot ; kinetics ; molten globule intermediate ; TFE denatured state ; protein folding ; human stefin B ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: It has been shown that human stefin B exhibits molten globule intermediates when denatured by acid or GuHCl. In the presence of TFE, it transforms into a highly helical state. In our first study on its folding mechanism (Žerovnik et al., Proteins 32:296-303), the kinetics measured by circular dichroism (CD) and fluorescence were correlated. In the present work the kinetics of folding were monitored by tyrosine fluorescence, ANS fluorescence, and, for certain reactions, far ultraviolet (UV) CD. The folding was started from the unfolded state in 3.45 M GuHCl, the acid denatured state at pH 1.8 ± 0.2, an acid molten globule intermediate I1 (pH 3.3 ± 0.1, low salt), a more structured acid molten globule intermediate I2 (pH 3.3 ± 0.1, 0.42 M NaCl), and the TFE state (pH 3.3 ± 0.1, 42% TFE). It has been found that all denatured states, including GuHCl, TFE, acid denatured and acid molten globule intermediate I1, fold with the same kinetics, provided that the final conditions are identical. This does not apply to the second acid molten globule intermediate I2, which demonstrates a higher rate of folding by a factor of 270. Different energy of activation and pH dependence were found for folding from states I1 or I2. Proteins 32:304-313, 1998. © 1998 Wiley-Liss, Inc.
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  • 20
    ISSN: 0887-3585
    Keywords: molecular evolution ; protein evolution ; mutation matrices ; Metropolis kinetics ; Boltzmann statistics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: New computational models of natural site mutations are developed that account for the different selective pressures acting on different locations in the protein. The number of adjustable parameters is greatly reduced by basing the models on the underlying physical-chemical properties of the amino acids. This allows us to use our method on small data sets built of specific protein types. We demonstrate that with this approach we can represent the evolutionary patterns in HIV envelope proteins far better than with more traditional methods. Proteins 32:289-295, 1998. © 1998 Wiley-Liss, Inc.
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  • 21
    ISSN: 0887-3585
    Keywords: antivirals ; Zovirax ; drug target ; drug binding ; enzyme structure ; intermolecular interactions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Antiherpes therapies are principally targeted at viral thymidine kinases and utilize nucleoside analogs, the triphosphates of which are inhibitors of viral DNA polymerase or result in toxic effects when incorporated into DNA. The most frequently used drug, aciclovir (Zovirax), is a relatively poor substrate for thymidine kinase and high-resolution structural information on drugs and other molecules binding to the target is therefore important for the design of novel and more potent chemotherapy, both in antiherpes treatment and in gene therapy systems where thymidine kinase is expressed. Here, we report for the first time the binary complexes of HSV-1 thymidine kinase (TK) with the drug molecules aciclovir and penciclovir, determined by X-ray crystallography at 2.37 Å resolution. Moreover, from new data at 2.14 Å resolution, the refined structure of the complex of TK with its substrate deoxythymidine (R = 0.209 for 96% of all data) now reveals much detail concerning substrate and solvent interactions with the enzyme. Structures of the complexes of TK with four halogen-containing substrate analogs have also been solved, to resolutions better than 2.4 Å. The various TK inhibitors broadly fall into three groups which together probe the space of the enzyme active site in a manner that no one molecule does alone, so giving a composite picture of active site interactions that can be exploited in the design of novel compounds. Proteins 32:350-361, 1998. © 1998 Wiley-Liss, Inc.
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  • 22
    ISSN: 0887-3585
    Keywords: molecular dynamics simulations ; mutagenesis ; aminoacyl-tRNA synthetase ; ATP ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Histidyl-tRNA synthetase (HisRS) differs from other class II aminoacyl-tRNA synthetases (aaRS) in that it harbors an arginine at a position where the others bind a catalytic Mg2+ ion. In computer experiments, four mutants of HisRS from Escherichia coli were engineered by removing the arginine and introducing a Mg2+ ion and residues from seryl-tRNA synthetase (SerRS) that are involved in Mg2+ binding. The mutants recreate an active site carboxylate pair conserved in other class II aaRSs, in two possible orders: Glu-Asp or Asp-Glu, replacing Glu-Thr in native HisRS. The mutants were simulated by molecular dynamics in complex with histidyl-adenylate. As controls, the native HisRS was simulated in complexes with histidine, histidyl-adenylate, and histidinol. The native structures sampled were in good agreement with experimental structures and biochemical data. The two mutants with the Glu-Asp sequence showed significant differences in active site structure and Mg2+ coordination from SerRS. The others were more similar to SerRS, and one of them was analyzed further through simulations in complex with histidine, and His+ATP. The latter complex sampled two Mg2+ positions, depending on the conformation of a loop anchoring the second carboxylate. The lowest energy conformation led to an active site geometry very similar to SerRS, with the principal Mg2+ bridging the α- and β-phosphates, the first carboxylate (Asp) coordinating the ion through a water molecule, and the second (Glu) coordinating it directly. This mutant is expected to be catalytically active and suggests a basis for the previously unexplained conservation of the active site Asp-Glu pair in class II aaRSs other than HisRS. Proteins 32:362-380, 1998. © 1998 Wiley-Liss, Inc.
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  • 23
    ISSN: 0887-3585
    Keywords: P1 nuclease ; X-ray crystallography ; substrate recognition ; catalytic mechanism ; thiophosphorylated oligonucleotides ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The reaction mechanism of nuclease P1 from Penicillium citrinum has been investigated using single-stranded dithiophosphorylated di-, tetra-, and hexanucleotides as substrate analogs. The complexes crystallize in tetragonal and orthorhombic space groups and have been solved by molecular replacement. The high resolution structures give a clear picture of base recognition by P1 nuclease at its two nucleotide-binding sites, especially the 1.8 Å structure of a P1-tetranucleotide complex which can be considered a P1-product complex. The observed binding modes are in agreement with a catalytic mechanism where the two closely spaced zinc ions activate the attacking water while the third, more exposed zinc ion stabilizes the leaving 03' oxyanion. Stacking as well as hydrogen bonding interactions with the base 5' to the cleaved phosphodiester bond are important elements of substrate binding and recognition. Modelling of a productive P1-substrate complex based on the solved structures suggests steric hindrance as the likely reason for the resistance of Rp-phosphorothioates and phosphorodithioates. Differences with the highly homologous nuclease S1 from Aspargillus oryzae are discussed. Proteins 32:414-424, 1998. © 1998 Wiley-Liss, Inc.
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  • 24
    ISSN: 0887-3585
    Keywords: theory of protein folding ; folding funnel ; folding thermodynamics ; folding kinetics ; conformation space ; sequence/structure compatibility ; thermal denaturation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: It is hard to construct theories for the folding of globular proteins because they are large and complicated molecules having enormous numbers of nonnative conformations and having native states that are complicated to describe. Statistical mechanical theories of protein folding are constructed around major simplifying assumptions about the energy as a function of conformation and/or simplifications of the representation of the polypeptide chain, such as one point per residue on a cubic lattice. It is not clear how the results of these theories are affected by their various simplifications. Here we take a very different simplification approach where the chain is accurately represented and the energy of each conformation is calculated by a not unreasonable empirical function. However, the set of amino acid sequences and allowed conformations is so restricted that it becomes computationally feasible to examine them all. Hence we are able to calculate melting curves for thermal denaturation as well as the detailed kinetic pathway of refolding. Such calculations are based on a novel representation of the conformations as points in an abstract 12-dimensional Euclidean conformation space. Fast folding sequences have relatively high melting temperatures, native structures with relatively low energies, small kinetic barriers between local minima, and relatively many conformations in the global energy minimum's watershed. In contrast to other folding theories, these models show no necessary relationship between fast folding and an overall funnel shape to the energy surface, or a large energy gap between the native and the lowest nonnative structure, or the depth of the native energy minimum compared to the roughness of the energy landscape. Proteins 32:425-437, 1998. © 1998 Wiley-Liss, Inc.
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  • 25
    ISSN: 0887-3585
    Keywords: protein inhibitors ; serine proteinases ; protein loop ; canonical conformation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Canonical loops of protein inhibitors of serine proteinases occur in proteins having completely different folds. In this article, conformations of the loops have been analyzed for inhibitors belonging to 10 structurally different families. Using deviation in Cα-Cα distances as a criterion for loop similarity, we found that the P3-P3′ segment defines most properly the length of the loop. When conformational differences among loops of individual inhibitors were compared using root mean square deviation (rmsd) in atomic coordinates for all main chain atoms (Δr method) and rmsd operating in main chain torsion angles (Δt method), differences of up to 2.1 Å and 72.3°, respectively, were observed. Such large values indicate significant conformational differences among individual loops. Nevertheless, the overall geometry of the inhibitor-proteinase interaction is very well preserved, as judged from the similarity of Cα-Cα distances between Cα of catalytic Ser and Cα of P3-P3′ residues in various enzyme-inhibitor complexes. The mode of interaction is very well preserved both in the chymotrypsin and subtilisin families, as distances calculated for subtilisin-inhibitor complexes are almost always within the range of those for chymotrypsin-inhibitor complexes. Complex formation leads to conformational changes of up to 160° for χ1 angle. Side chains of residue P1 and P2′ adopt in different complexes a similar orientation (χ1 angle = -60° and -180°, respectively). To check whether the canonical conformation can be found among non-proteinase-inhibitor Brookhaven Protein Data Bank structures, two selection criteria - the allowed main chain dihedral angles and Cα-Cα distances for the P3-P3′ segment - were applied to all these structures. This procedure detected 132 unique hexapeptide segments in 121 structurally and functionally unrelated proteins. Partial preferences for certain amino acids occurring at particular positions in these hexapeptides could be noted. Further restriction of this set to hexapeptides with a highly exposed P1 residue side chain resulted in 40 segments. The possibility of complexes formation between these segments and serine proteinases was ruled out in molecular modeling due to steric clashes. Several structural features that determine the canonical conformation of the loop both in inhibitors and in other proteins can be distinguished. They include main chain hydrogen bonds both within the P3-P3′ segment and with the scaffold region, P3-P4 and P3′-P4′ hydrophobic interactions, and finally either hydrophobic or polar interactions involving the P1′ residue. Proteins 32:459-474, 1998. © 1998 Wiley-Liss, Inc.
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  • 26
    ISSN: 0887-3585
    Keywords: megakaryocyte growth and development factor ; thrombopoietin ; cytokine ; equilibrium denaturation ; conformation ; protein folding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The effect of pH and urea on the conformation of recombinant human megakaryocyte growth and development factor (rHuMGDF) was determined by circular dichroism, intrinsic fluorescence spectroscopy, and equilibrium ultracentrifugation. The conformation of rHuMGDF was dependent on pH and urea concentration. Multiple folding forms were evidenced by multiple pH-induced transitions and urea-induced equilibrium transitions that deviated from a simple two-state process. In neutral to alkaline pH, rHuMGDF exists as a monomer, but an acid-induced conformational state self-associates to form a soluble aggregate. A folding intermediate(s) was observed with a more stable secondary structure than tertiary structure and was dependent on the pH of the urea-induced denaturation. The differences in the stabilities of the folding states were most distinct in the pH range of 4.5 to 6.5. The presence of intermediates in the folding pathway of rHuMGDF are similar to findings of previous studies of related growth factors that share a common three-dimensional structure. Proteins 32:495-503, 1998. © 1998 Wiley-Liss, Inc.
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  • 27
    ISSN: 0887-3585
    Keywords: sugar ; acetamido group ; mimicry ; inhibition ; lysozyme ; CDR loop ; VHH ; heavy-chain immunoglobulin ; solvent accessible surface area ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Whereas antibodies have demonstrated the ability to mimic various compounds, classic heavy/light-chain antibodies may be limited in their applications. First, they tend not to bind enzyme active site clefts. Second, their size and complexity present problems in identifying key elements for binding and in using these elements to produce clinically valuable compounds. We have previously shown how cAb-Lys3, a single variable domain fragment derived from a lysozyme-specific camel antibody naturally lacking light chains, overcomes the first limitation to become the first antibody structure observed penetrating an enzyme active site. We now demonstrate how cAb-Lys3 mimics the oligosaccharide substrate functionally (inhibition constant for lysozyme, 50 nM) and structurally (lysozyme buried surface areas, hydrogen bond partners, and hydrophobic contacts are similar to those seen in sugar-complexed structures). Most striking is the mimicry by the antibody complementary determining region 3 (CDR3) loop, especially Ala104, which mimics the subsite C sugar 2-acetamido group; this group has previously been identified as a key feature in binding lysozyme. Comparative simplicity, high affinity and specificity, potential to reach and interact with active sites, and ability to mimic substrate suggest that camel heavy-chain antibodies present advantages over classic antibodies in the design, production, and application of clinically valuable compounds. Proteins 32:515-522, 1998. © 1998 Wiley-Liss, Inc.
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  • 28
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Recently, James Bowie addressed the question of how to normalize correctly the distribution of observed helix-helix packing angles in proteins (Bowie, Nature Struct. Biol. 4:915-917, 1997). A hitherto unrealized yet significant bias toward crossed packing angles was revealed. However, the derived random reference distribution of packing angles requires that helices have to be assumed as infinite in length. Here, we complement Bowie's analysis by consideration of the more realistic case where helices are of finite length. As a result, the statistical bias toward near perpendicular packings appears to be even stronger. Proteins 33:457-459, 1998. © 1998 Wiley-Liss, Inc.
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  • 29
    ISSN: 0887-3585
    Keywords: MS/MS electrospray mass spectrometry ; CD ; human immunodeficiency virus (HIV) ; glycoprotein 41,000 (gp41) ; N-terminal domain ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The N-terminal domain of human immunodeficiency virus (HIV)-1 glycoprotein 41,000 (FP; residues 1-23; NH2-AVGIGALFLGFLGAAGSTMGARS-CONH2) is involved in the fusion and cytolytic processes underlying viral-cell infection. Here, we use circular dichroism (CD) spectroscopy, along with electrospray ionization (ESI) mass spectrometry and tandem (MS/MS) mass spectrometry during the course of hydrogen/deuterium exchange, to probe the local conformations of this synthetic peptide in two membrane mimics. Since amino acids that participate in defined secondary structure (i.e., α-helix or β-sheet) exchange amido hydrogens more slowly than residues in random structures, deuterium exchange was combined with CD spectroscopy to map conformations to specific residues. For FP suspended in the highly structure-promoting solvent hexafluoroisopropanol (HFIP), CD spectra indicated high α-helix and disordered structures, whereas ESI and MS/MS mass spectrometry indicated that residues 5-15 were α-helical and 16-23 were disordered. For FP suspended in the less structure-promoting solvent trifluoroethanol (TFE), CD spectra showed lower α-helix, with ESI and MS/MS mass spectrometry indicating that only residues 9-15 participated in the α-helix. These results compare favorably with previous two-dimensional nuclear magnetic resonance studies on the same peptide. Proteins Suppl. 2:38-49, 1998. © 1998 Wiley-Liss, Inc.
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  • 30
    ISSN: 0887-3585
    Keywords: MALDI mass spectrometric peptide mapping ; membrane proteins ; in situ gel digestion ; porin ; permeability transition ; noncovalent complexes ; protein interactions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Mass spectrometric peptide mapping, particularly by matrix-assisted laser desorption-ionization (MALDI-MS), has recently been shown to be an efficient tool for the primary structure characterization of proteins. In combination with in situ proteolytic digestion of proteins separated by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), mass spectrometric peptide mapping permits identification of proteins from complex mixtures such as cell lysates. In this study we have investigated several ion channel membrane proteins (porins) and their supramolecular assembly in mitochondrial membranes by peptide mapping in solution and upon digestion in the gel matrix. Porins are integral membrane proteins serving as nonspecific diffusion pores or as specific systems for the transport of substrates through bacterial and mitochondrial membranes. The well-characterized porin from Rhodobacter capsulatus (R.c.-porin) has been found to be a native trimeric complex by the crystal structure and was used as a model system in this study. R.c.-porin was characterized by MALDI-MS peptide mapping in solution, and by direct in situ-gel digestion of the trimer. Furthermore, in this study we demonstrate the direct identification of the noncovalent complex between a mitochondrial porin and the adenine nucleotide translocator from rat liver, by MALDI-MS determination of the specific peptides due to both protein sequences in the SDS-PAGE gel band. The combination of native gel electrophoresis and mass spectrometric peptide mapping of the specific gel bands should be developed as a powerful tool for the molecular identification of protein interactions. Proteins Suppl. 2:63-73, 1998. © 1998 Wiley-Liss, Inc.
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  • 31
    ISSN: 0006-3592
    Keywords: phosphoglucomutase ; site-directed mutagenesis ; kinetic constants ; Pm promoter ; metabolic engineering ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Mutants of Escherichia coli deficient in phosphoglucomutase accumulate amylose when the cells are grown on maltose or galactose as carbon source. In the presence of physiological levels of phosphoglucomutase, most of the sugar is catabolized, leading to strongly reduced levels of amylose accumulation. By varying the expression level of heterologous phosphoglucomutase, we show that the minimum level needed to block amylose accumulation corresponds to a phosphoglucomutase activity of 150-600 nmole substrate transformed per min per mg of total soluble protein. Mutant phosphoglucomutases with strongly reduced Vmax values and increased Km values for the substrate glucose-1-phosphate or the co-substrate glucose-1,6-diphosphate, could also reduce amylose accumulation, but much higher enzyme expression levels were required. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:299-302, 1998.
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  • 32
    ISSN: 0006-3592
    Keywords: glycerol ; Enterobacter agglomerans ; 3-hydroxypropionaldehyde ; catabolic limitation ; inhibition ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Batch fermentation of glycerol to 1,3-propanediol (1,3PPD) by Enterobacter agglomerans CNCM 1210 showed the lethal accumulation of 3-hydroxypropionaldehyde (3-HPA) when performed under initial substrate content higher than 40 g/L. Assigned to the inhibition by the NAD/NADH ratio of the 3-HPA converting enzyme: 1,3PPD dehydrogenase, intracellular assays were conducted in an attempt to identify the metabolic mechanisms involved in the increase of that ratio. An overflow metabolism through the 1,3PPD formation pathway was established, while a catabolic limitation in the oxidative branch at the level of glyceraldehyde-3-phosphate dehydrogenase occurred. Uncoupled activities of synthesis and consumption of reducing equivalents are thus suspected to provoke the increase of the NAD/NADH ratio and the subsequent accumulation of 3-HPA. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:303-305, 1998.
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  • 33
    ISSN: 0006-3592
    Keywords: lycopene ; Candida utilis ; carotenoids ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The Erwinia uredovora crtE, crtB, and crtI genes, which are responsible for the synthesis of carotenoid lycopene from farnesyl pyrophosphate, were expressed in Candida utilis under the control of the promoters and terminators derived from the C. utilis GAP, PGK, and PMA genes, respectively. The yeast transformant carrying the carotenoid biosynthesis genes produced 758 μg/g dry weight of lycopene along with 407 μg/g dry weight of phytoene in the stationary phase. It was observed in the C. utilis transformant that ergosterol content was decreased to 65% of that in the parent strain that accumulated 6.04 mg/g dry weight of ergosterol. It is therefore possible that the carbon flux for the ergosterol biosynthesis has been branched at farnesyl pyrophosphate to generate a new pathway for the lycopene production in this yeast transformant. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:306-308, 1998.
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  • 34
    ISSN: 0006-3592
    Keywords: l-ascorbic acid ; vitamin C ; 2-keto-l-gulonic acid ; l-sorbose dehydrogenase ; l-sorbosone dehydrogenase ; Gluconobacter ; chemical mutation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We isolated Gluconobacter oxydans T-100 that had an activity to produce 2-KLGA from d-sorbitol; however, the yield of 2-KLGA was quite insufficient. Therefore, enzymes involved in the biosynthesis of l-sorbosone and 2-KLGA, l-sorbose dehydrogenase (SDH) and l-sorbosone dehydrogenase (SNDH), respectively, were purified from G. oxydans T-100. A genomic library of G. oxydans T-100 was screened to clone both genes for SDH and SNDH based on their amino acid sequences. SNDH and SDH were encoded in sequential open reading frames with 1497 and 1596 nucleotides, respectively, which were verified by the expression in Escherichia coli. The amino acid sequence of SDH and SNDH showed close similarity with E. coli choline dehydrogenase (CDH) and betaine-aldehyde dehydrogenase (BADH), respectively, which cooperatively play a key role for conferring osmotic tolerance. Because the yield of 2-KLGA by G. oxydans introduced with the genes for SDH and SNDH were insufficient, replacement of the promoter with that of Escherichia coli tufB1 in combination with chemical mutagenesis by N-methyl-N′-nitro-N-nitrosoguanidine resulted in improvement of the production level. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:309-315, 1998.
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  • 35
    ISSN: 0006-3592
    Keywords: ATP allocation ; celluloytic microorganisms ; consolidated bioprocessing ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Under anaerobic, carbon limited conditions, celluloytic fermentative microorganisms face a metabolic choice with respect to the allocation of relatively scarce ATP: to invest it in cells or in hydrolytic enzymes. A model is proposed that defines an allocation parameter reflecting the fractional expenditure of ATP on cell synthesis relative to the total ATP available (gross ATP synthesized less maintenance). This parameter is then incorporated into an ATP-centered model of anaerobic cellulose fermentation based on the ethanol fermentation of yeast and the cellulase system of Trichoderma reesei. Results indicate that high processing rates are possible via a consolidated bioprocessing strategy, especially at high cellulase specific activities, and that cell/cellulase allocation represents an interesting system in which to study, and perhaps exploit, microbial evolution and metabolic control. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:316-320, 1998.
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  • 36
    ISSN: 0006-3592
    Keywords: yeast cell wall porosity and permeability ; β-1,3-glucanase ; selective protein release ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In this article, we consider the impact on downstream process design resulting from the use of metabolically engineered yeast strains. We address the issue of how manipulation of cell wall permeability can improve the release and subsequent recovery of heterologous products produced in yeast. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:321-324, 1998.
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  • 37
    ISSN: 0006-3592
    Keywords: denitritification ; denitratification ; anoxic filter ; kinetic model ; distributed fraction of reductase ; parametric sensitivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Denitritification and denitratification in anoxic filters were performed to generate experimental data. Also, a kinetic model of denitratification that accounts for intrinsic biokinetics and hydrodynamic behavior of the biofilter is proposed. In denitritification, the simulated results are in good agreement with the experimental data; and a higher nitrite influent concentration gives a higher nitrite reduction efficiency if the denitrifying loading is kept the same. In denitratification, the intermediate nitrite tends to accumulate, and a higher denitrifying loading results in a higher nitrite effluent concentration. By inserting biological and physical parameter values into the kinetic model, the variations in distributed fractions of nitrate-reductase (f) and nitrite-reductase (1-f) with different denitrifying loadings can be estimated by fitting in experimental data. The estimated f increased with an increase in denitrifying loading, implying that a higher denitrifying loading results in a higher nitrite effluent concentration. From parametric sensitivity analyses, the parameter f is more sensitive than other biological and physical parameters. Accordingly, the proposed kinetic model of denitratification can be used to predict the treatment performance of anoxic filters appropriately. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:52-61, 1998.
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  • 38
    ISSN: 0006-3592
    Keywords: enzymatic ; solid-to-solid conversion ; peptide synthesis ; proteases ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have studied a thermolysin-catalyzed solid-to-solid dipeptide synthesis using equimolar amounts of Z-Gln-OH and H-Leu-NH2 as model substrates. The high substrate concentrations make this an effective alternative to enzymatic peptide synthesis in organic solvents. Water content was varied in the range of 0 to 600 mL water per mol substrate and enzyme concentration in the range of 0.5 to 10 g/mol of substrates. High yields around 80% conversion and initial rates from 5 to 20 mmol s-1 kg-1 were achieved. The initial rate increases 10-fold on reducing the water content, to reach a pronounced optimum at 40 mL water per mol substrate. Below this, the rate falls to much lower values in a system with no added water, and to zero in a rigorously dried system. This behavior is discussed in terms of two factors: At higher water contents the system is mass transfer limited (as shown by varying enzyme content), and the diffusion distances required vary. At low water levels, effects reflect the stimulation of the enzymatic activity by water. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:68-72, 1998.
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  • 39
    ISSN: 0006-3592
    Keywords: apoptosis ; necrosis ; bcl-2 ; amino acids ; cell culture ; cell death ; hybridoma ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The transfection of murine hybridomas with the apoptosis suppressor gene bcl-2 has been reported to result in the extension of batch culture duration, leading to significant improvements in culture productivity. In the present study, the effect of deprivation, individually, of each amino acid found in culture medium was examined to characterize the chemical environment of the culture in terms of its propensity to induce apoptosis. When cells were deprived of each amino acid, individually for 48 h, the majority of cell deaths in each case occurred by apoptosis, with essential amino acids being clearly most effective. For nearly all the amino acids, the viability of the bcl-2 cell line cultures was greater than 70% after 48 h, representing a substantial improvement in viability over control cell line cultures. Time course studies revealed that the induction of death could be divided into two phases. Initially, following the deprivation of a single essential amino acid, there was a period of time during which all the control cell line cultures retained high viability. The duration of this phase varied from 15 h in the case of lysine deprivation, through to 40 h in the case methionine deprivation. In the second phase of deprivation, the cultures exhibited an abrupt and rapid collapse in viability. The time taken for the viability to fall to 50% was similar for each amino acid. In every case, the duration of both phases of the bcl-2 cultures was considerably extended. Specific utilization rates were increased during the control cultures relative to the bcl-2 cultures for both the growth phase (ranging between 2% and 57% higher than the bcl-2 cultures) and the death phase (ranging between 172% to 1900% higher than the bcl-2 culture). © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:90-98, 1998.
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  • 40
    ISSN: 0006-3592
    Keywords: Monod kinetics ; mixed substrate growth ; continuous culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In natural environments, heterotrophic microorganisms encounter complex mixtures of carbon sources, each of which is present only at very low concentrations. Under such conditions no significant growth could be expected if cells utilized only one of the available carbon compounds as suggested by the principle of diauxic growth. Indeed, there is much evidence that microbial cells utilize many carbon sources simultaneously. In order to predict bacterial growth under such conditions we developed a model describing the specific growth rate as a function of the individual concentrations of several simultaneously utilized carbon substrates. Together with multisubstrate models previously published, this model was evaluated for its ability to describe growth of Escherichia coli during the simultaneous utilization of mixtures of sugars in carbon-limited continuous culture. Using the μmax and Ks constants determined for single substrate growth with six different sugars, the model was able for most experiments to adequately describe the specific growth rate of the culture, i.e., the experimentally set dilution rate, from the measured concentrations of the individual sugars. The model provides an explanation why bacteria can still grow relatively fast under environmental conditions where the concentrations of carbon substrates are usually extremely low. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:99-107, 1998.
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  • 41
    ISSN: 0006-3592
    Keywords: thermophilic β-glycosidase ; catalytic membranes ; nonisothermal bioreactors ; thermodialysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Catalytic membranes, obtained by immobilizing thermophilic β-glycosidase onto nylon supports, were used in a nonisothermal bioreactor to study the effect of temperature gradients on the rate of enzyme reaction. Two experimental approaches were carried out to explain the molecular mechanisms by which the temperature gradients affect enzyme activity. The results showed that the thermophilic enzyme behaved as the mesophilic β-galactosidase, exhibiting an activity increase which was linearly proportional to the transmembrane temperature difference. The efficiency of the system proposed was determined by calculating two constants, α and β, which represent respectively the percentage increase of enzyme activity when a temperature difference of 1°C or a temperature gradient of 1°C cm-1 were applied across the catalytic membrane. The increase of enzyme activity in nonisothermal bioreactors entailed a proportional reduction of production times. The advantages in using thermophilic enzymes immobilized in nonisothermal bioreactors are also discussed. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:108-115, 1998.
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  • 42
    ISSN: 0006-3592
    Keywords: mixed-substrate growth ; mathematical model ; competing species ; dynamical analogy ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: There is a similarity between the metabolic dynamics of a microbial species growing on a mixture of two substrates and the dynamics of growth of two competing populations. Specifically, the enzymes catalyzing the uptake and catabolism of substrates exhibit phenomena analogous to extinction and coexistence.“Extinction” of the enzymes associated with one of the substrates results in sequential utilization of the substrates (diauxie) (Monod, 1942). “Coexistence” of the enzymes associated with the substrates results in simultaneous utilization of the substrates (Egli, 1995). Here, we formulate a simple model that shows the basis for this dynamical similarity: The equations describing the evolution of the enzyme levels are dynamical analogs of the Lotka-Volterra model for two competing species. The analogy suggests ways of capturing the experimentally observed preculture-dependent growth patterns, i.e., growth patterns that vary depending on the physiological state of the preculture. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:116-121, 1998.
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  • 43
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: No abstract.
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  • 44
    ISSN: 0006-3592
    Keywords: lipase ; organic solvent ; flavour esters ; interfacial activation ; enzyme conformers ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In order to improve the lipase-catalyzed synthesis of flavour esters, we have used the reported strategy of interfacial activation-based molecular (bio)imprinting [Mingarro et al. 1995. Proc. Natl. Acad. Sci. U.S.A. 92: 3308], later called trapping in the presence of amphiphile interfaces (TPI) [Mingarro et al. 1996. Biochemistry 35: 9935]. Five lipases of fungal and mammalian origin typically used for esterification process have been explored to improve production by TPI treatment. A marked enhancement of enzymatic activity has been observed in all TPI-treated lipases assayed and the activation factor obtained was up to 90-fold. The dependence on chain length of acyl donors in the esterification of geranyl alcohol has been investigated, showing clear differences between activated and nonactivated lipase. The results indicate that this rational approach leads to conversion yields that are remarkably higher, not only than its counterpart pH-optimized control lipase, but also the “protected” lipase by conventional methods (lyoprotectans or salts). We propose this strategy as a promising tool to be used in more industrial biotransformations. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:122-127, 1998.
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  • 45
    ISSN: 0006-3592
    Keywords: smooth muscle ; polyglycolic acid ; biodegradable ; tissue engineering ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The engineering of functional smooth muscle (SM) tissue is critical if one hopes to successfully replace the large number of tissues containing an SM component with engineered equivalents. This study reports on the effects of SM cell (SMC) seeding and culture conditions on the cellularity and composition of SM tissues engineered using biodegradable matrices (5 × 5 mm, 2-mm thick) of polyglycolic acid (PGA) fibers. Cells were seeded by injecting a cell suspension into polymer matrices in tissue culture dishes (static seeding), by stirring polymer matrices and a cell suspension in spinner flasks (stirred seeding), or by agitating polymer matrices and a cell suspension in tubes with an orbital shaker (agitated seeding). The density of SMCs adherent to these matrices was a function of cell concentration in the seeding solution, but under all conditions a larger number (approximately 1 order of magnitude) and more uniform distribution of SMCs adherent to the matrices were obtained with dynamic versus static seeding methods. The dynamic seeding methods, as compared to the static method, also ultimately resulted in new tissues that had a higher cellularity, more uniform cell distribution, and greater elastin deposition. The effects of culture conditions were next studied by culturing cell-polymer constructs in a stirred bioreactor versus static culture conditions. The stirred culture of SMC-seeded polymer matrices resulted in tissues with a cell density of 6.4 ± 0.8 × 108 cells/cm3 after 5 weeks, compared to 2.0 ± 1.1 × 108 cells/cm3 with static culture. The elastin and collagen synthesis rates and deposition within the engineered tissues were also increased by culture in the bioreactors. The elastin content after 5-week culture in the stirred bioreactor was 24 ± 3%, and both the elastin content and the cellularity of these tissues are comparable to those of native SM tissue. New tissues were also created in vivo when dynamically seeded polymer matrices were implanted in rats for various times. In summary, the system defined by these studies shows promise for engineering a tissue comparable in many respects to native SM. This engineered tissue may find clinical applications and provide a tool to study molecular mechanisms in vascular development. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 46-54, 1998.
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  • 46
    ISSN: 0006-3592
    Keywords: Escherichia coli ; protein production ; secretion ; plasmid stability ; fed-batch ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Periplasmic secretion of overexpressed Bacillus stearothermophilus α-amylase was analyzed in batch and fed-batch cultivations of Escherichia coli MG1655:pCSS4-p and the mutant strain CWML2:pCSS4-p. Under all conditions investigated, growth and product formation of MG1655:pCSS4-p were severely impaired by heterologous protein expression and/or processing, while E. coli CWML2:pCSS4-p was found to be more robust and to accumulate 2- to 3-fold higher maximum α-amylase levels. While this strain is itself potentially interesting for applications, its properties also illustrate the potential of the selection procedure that was employed to obtain it from its progenitor MG1655 (Weikert, C., Sauer, U., Bailey, J. E., 1997. Microbiol. 143: 1567-1574. Application of this procedure to existing industrial strains may lead to significantly improved process organisms. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:386-391, 1998.
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  • 47
    ISSN: 0006-3592
    Keywords: denitrification ; biodegradation ; kinetics ; 1,1,1-trichloroethane ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A denitrifying consortium capable of degrading carbon tetrachloride (CT) was shown to also degrade 1,1,1-trichloroethane (TCA). Fed-batch experiments demonstrated that the specific rate of TCA degradation by the consortium was comparable to the specific rate of CT degradation (approximately 0.01 L/gmol/min) and was independent of the limiting nutrient. Although previous work demonstrated that 4-50% of CT transformed by the consortium was converted to chloroform (CF), no reductive dechlorination products were detected during TCA degradation, regardless of the limiting nutrient. The lack of chlorinated TCA degradation products implies that the denitrifying consortium possesses an alternate pathway for the degradation of chlorinated solvents which does not involve reductive dechlorination. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:393-399, 1998.
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  • 48
    ISSN: 0006-3592
    Keywords: nar promoter ; oxygen-dependent inducible promoter ; anaerobic/microaerobic conditions ; recombinant E. coli ; fed-batch cultivation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The nar promoter of Escherichia coli is maximally induced under anaerobic or microaerobic conditions in the presence of nitrate. We previously demonstrated in batch experiments that the intact nar promoter of E. coli cloned into a pBR322-based plasmid serves as a high-level expression system in a nar mutant of E. coli (Lee et al., 1996b). In this study, we extend characterization of the nar promoter expression system to the fed-batch culture mode, which is widely used in industrial-scale fermentation. From these experiments, it was found that the specific β-galactosidase activity expressed from the lacZ gene fused to the nar promoter was maximal when host cells were grown under aerobic conditions [dissolved oxygen, (DO) = 80%] to absorbance at 600 nm (OD600) = 35 before induction of the nar promoter by lowering DO to 1-2% with alternating microaerobic and aerobic conditions. Approximately 15 h after induction, the OD600 of the culture reached 135 and the specific β-galactosidase activity increased to 40,000 Miller units, equivalent to approximately 35% of the total cellular proteins. The specific β-galactosidase activity before induction was approximately 1,000 Miller units, giving an induction ratio of approximately 40. Based on these results, we conclude that the nar promoter provides a convenient and effective high level expression system under conditions of fed-batch culture. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:400-406, 1998.
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  • 49
    ISSN: 0006-3592
    Keywords: Alcaligenes eutrophus ; hydrogenase ; NADH regeneration ; HLADH ; organic solvent ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A soluble NAD-dependent hydrogenase contained in Alcaligenes eutrophus was evaluated as a coenzyme regenerating catalyst in an organic-aqueous two-phase (predominantly organic) system. The horse-liver alcohol-dehydrogenase (HLADH) catalyzed reduction of cyclohexanone to cyclohexanol was used as a model reaction. The impact of different solvents (selected to span a large variety of principal properties) on the stability and activity of the HLADH, using substrate-driven regeneration, was studied. Solvents suitable for the HLADH were then selected for an evaluation of the hydrogenase-driven coenzyme regeneration. Hydrophobic solvents such as heptane, toluene, and 1,1,1-trichloroethane were found to be suitable for the coupled reactions catalyzed by HLADH and hydrogenase. Nonimmobilized cells, permeabilized with cetyl-trimethyl-ammonium bromide, were the most efficient preparation for the regeneration of NADH. The use of this preparation in heptane (10% water) was optimized with respect to the yield obtained in the HLADH-catalyzed reduction of cyclohexanone. Using the optimized conditions, yields of 99% cyclohexanol were obtained. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 79-86, 1988.
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  • 50
    ISSN: 0006-3592
    Keywords: CHO cells ; glycosylation engineering ; antisense ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Novel glycoproteins, inaccessible by other techniques, can be obtained by metabolic engineering of the oligosaccharide biosynthesis pathway. Furthermore, alteration of cell-surface oligosaccharides can change the properties of receptors involved in cell-cell adhesion. Sialyl Lewis X (sLex) is a cell-surface oligosaccharide determinant which is specifically expressed on granulocytes and monocytes and which interacts with selectins to influence leukocyte trafficking, thrombosis, inflammation, and cancer. Antisense technology targeting fucosyltransferase VI (Fuc-TVI), an enzyme necessary for the synthesis of the sLex in engineered Chinese hamster ovary (CHO) cells, has reduced Fuc-TVI activity, sLex synthesis, and adhesion to endothelial cells. Antisense methodology to reduce targeted activity in oligosaccharide biosynthesis or other pathways is an important addition to CHO cell metabolic engineering capabilities. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:445-450, 1998.
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  • 51
    ISSN: 0006-3592
    Keywords: aqueous two-phase separation ; protein partitioning ; T4 lysozyme ; electrochemical partitioning ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Protein partitioning in aqueous two-phase systems based on phase-forming polymers is strongly affected by the net charge of the protein, but a thermodynamic description of the charge effects has been hindered by conflicting results. Many of the difficulties could be because of problems in isolating electrochemical effects from other interactions of phase components.We explored charge effects on protein partitioning in poly(ethylene glycol)-dextran two-phase systems by using two series of genetically engineered charge modifications of bacteriophage T4 lysozyme produced in Escherichia coli. The two series, one in the form of charged-fusion tails and the other in the form of charge-change point mutations, provided matching net charges but very different polarity. Partition coefficients of both series were obtained and interfacial potential differences of the phase systems were measured. Multi-angle laser light scattering measurements were also performed to determine second virial coefficients. A semi-empirical model accounting for the roles of both charge and non-charge effects on protein partitioning behavior is proposed, and the results predicted from the model are compared to the results from the experiments. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:461-470, 1998.
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  • 52
    ISSN: 0006-3592
    Keywords: polycyclic aromatic hydrocarbon ; biodegradation ; surfactants ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The objectives of this study were to isolate and evaluate microorganisms with the ability to degrade high molecular weight polycyclic aromatic hydrocarbons (PAHs) in the presence of synthetic surfactants. Stenotrophomonas maltophilia VUN 10,010, isolated from PAH-contaminated soil, utilized pyrene as a sole carbon and energy source and also degraded other high molecular weight PAHs containing up to seven benzene rings. Various synthetic surfactants were tested for their ability to improve the PAH degradation rate of strain VUN 10,010. Anionic and cationic surfactants were highly toxic to this strain, and the Tween series was used as a growth substrate. Five nonionic surfactants (Brij 35, Igepal CA-630, Triton X-100, Tergitol NP-10, and Tyloxapol) were not utilized by, and were less toxic to, strain VUN 10,010. MSR and log Km values were determined for fluoranthene, pyrene, and benzo[a]pyrene in the presence of these nonionic surfactants and their apparent solubility was increased by a minimum of 250-fold in the presence of 10 g L-1 of all surfactants. The rate of pyrene degradation by strain VUN 10,010 was enhanced by the addition of four of the nonionic surfactants (5-10 g L-1); however, 5 g L-1 Igepal CA-630 inhibited pyrene degradation and microbial growth. The specific growth rate of VUN 10,010 on pyrene was increased by 67% in the presence of 10 g L-1 Brij 35 or Tergitol NP-10. The addition of Brij 35 and Tergitol NP-10 to media containing a single high molecular weight PAH (four and five benzene rings) as the sole carbon source increased the maximum specific PAH degradation rate and decreased the lag period normally seen for PAH degradation. The addition of Tergitol NP-10 to VUN 10,010 cultures which contained a PAH mixture (three to seven benzene rings) substantially improved the overall degradation rate of each PAH and increased the specific growth rate of VUN 10,010 by 30%. Evaluation of the use of VUN 10,010 for degrading high molecular weight PAHs in leachates from surfactant-flushed, weathered, PAH-contaminated sites is warranted. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:482-494, 1998.
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  • 53
    ISSN: 0006-3592
    Keywords: biocompatibility ; microfabrication ; biohybrid organs ; immunoisolation ; Islets of Langerhans ; silicon ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A microfabricated silicon-based biocapsule for the immunoisolation of cell transplants is presented. The biocapsule-forming process employs bulk micromachining to define cell-containing chambers within single crystalline silicon wafers. These chambers interface with the surrounding biological environment through polycrystalline silicon filter membranes. The membranes are surface micromachined to present a high density of uniform pores, thus affording sufficient permeability to oxygen, glucose, and insulin. The pore dimensions, as small as 20 nm, are designed to impede the passage of immune molecules and graft-borne viruses. The underlying filter-membrane nanotechnology has been successfully applied in controlled cell culture systems (Ferrari et al., 1995), and is under study for viral elimination in plasma fractionation protocols. Here we report the encouraging results of in vitro experiments investigating the biocompatibility of the microfabricated biocapsule, and demonstrate that encapsulated rat neonatal pancreatic islets significantly outlive and outperform controls in terms of insulin-secretion capability over periods of several weeks. These results appear to warrant further investigations on the potential of cell xenografts encapsulated within microfabricated, immunoisolating environments for the treatment of insulin-dependent diabetes. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 118-120, 1998.
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  • 54
    ISSN: 0006-3592
    Keywords: sucrose monoester synthesis ; lipase-catalyzed acylation ; water activity (a w) ; regioselectivity ; salt hydrate pair ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Sucrose monoesters of a fatty acid were synthesized by using lipase in a solvent-free system. When lipase from Mucor miehei was used as a catalyst with capric acid as the donor and sugar as the acceptor, sucrose 6-monocaprate was predominantly produced in a yield of 25.3%. The yield of product was significantly increased by the direct addition of a suitable pair of solid salt hydrates to the reaction mixture to control the water activity (aw). Among the salt hydrate pairs investigated, the barium hydroxide, 8/1H2O pair resulted in the highest yield of the product. This salt addition method was also successfully employed for acylation of primary hydroxyl groups in various unprotected mono- and disaccharides such as glucose, galactose, fructose, trehalose, mannose, maltose, and lactose. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 121-125, 1998.
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  • 55
    ISSN: 0006-3592
    Keywords: membrane mass spectrometer ; kinetic measurements ; anaerobic biofilm ; acetate ; inhibition ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A small, stirred, 14.4-mL tank reactor was designed to serve as a measurement cell for short-term investigation of microbial kinetics. A mass spectrometer membrane probe allowed the measurement of the dissolved gases of hydrogen, methane, oxygen, and carbon dioxide. pH was measured by an electrode and controlled by addition of acid or alkali. The highly sensitive measurement of gases with low solubility allowed rapid measurements at very low conversion. In kinetic experiments, a stepwise increase of substrate concentration (method A) and continuous feed of substrate (method B) were used, allowing quick estimation of substrate kinetics. Acetate conversion in mixed culture biofilms from a fluidized bed reactor was investigated. Substrate inhibition was found to be negligible in the concentration range studied. Experiments at various pH values showed that the undissociated acid form was the kinetic determinant. Kinetic parameters for Haldane kinetics of protons were KSH = 1.3 × 10-5 mol m-3 and KIH = 8.1 × 10-3 mol m-3. With free acid (HAc) as the rate determining species, the kinetic parameters for method A were KSHAc = 0.005 mol m-3 and KIHAc = 100 mol m-3 and for method B were KSHAc = 0.2 mol m-3 and KIHAc = 50 mol m-3. The maximum biomass activity occurred at around pH 6.5. Acetate was exclusively converted to methane and CO2 at pH > 6. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 127-135, 1998.
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  • 56
    ISSN: 0006-3592
    Keywords: down-flow fluidization ; bed expansion ; biofilm ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This article describes the bed expansion characteristics of a down-flow anaerobic fluidized bed reactor treating a synthetic wastewater. Experiments were carried out in a 0.08 m diameter and 1 m length PVC column. The carrier used was ground perlite (an expanded volcanic rock). Particles characteristics were 0.968 mm in diameter, specific density of 213 kg · m-3 and Umf (minimal fluidization velocity): 2.3 m · h-1. Experimental data of terminal velocities and bed expansion parameters at several biofilm thicknesses were compared to different models predicting the bed expansion of up-flow and down-flow fluidized beds.Measured bed porosities at different liquid superficial velocities for the different biofilm thicknesses were in agreement with the Richardson-Zaki model, when Ut (particle terminal velocity) and n (expansion coefficient) were calculated by linear regression of the experimental data. Terminal velocities of particles at different biofilm thicknesses calculated from experimental bed expansion data, were found to be much smaller than those obtained when Cd (drag coefficient) is determined from the standard drag curve (Lapple and Sheperd, 1940) or with others' correlations (Karamanev and Nikolov, 1992a,b). This difference could be explained by the fact that free-rising particles do not obey Newton's law for free-settling, as proposed by Karamanev and Nikolov (1992a,b) and Karamanev et al. (1996). In the present study, the same free-rising behavior was observed for all particles (densities between 213 and 490 kg · m-3). © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 136-144, 1998.
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  • 57
    ISSN: 0006-3592
    Keywords: bioavailability ; PAH ; biodegradation ; dissolution ; hydrodynamic ; mixing ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The influence of hydrodynamic conditions on the dissolution rate of crystalline naphthalene as a model polycyclic aromatic hydrocarbon (PAH) was studied in stirred batch reactors with varying impeller speeds. Mass transfer from naphthalene melts of different surface areas to the aqueous phase was measured and results were modeled according to the film theory. Results were generalized using dimensionless numbers (Reynolds, Schmidt, and Sherwood). In combined mass transfer and biodegradation experiments, the effect of hydrodynamic conditions on the degradation rate of naphthalene by Pseudomonas 8909N was studied. Experimental results were mathematically described using mass-transfer and microbiological models. The experiments allowed determination of mass-transfer and microbiological parameters separately in a single run. The biomass formation rate under mass transfer limited conditions, which is related to the naphthalene biodegradation rate, was correlated to the dimensionless Reynolds number, indicating increased bioavailability at increased mixing in the reactor liquid. The methodology presented in which mass transfer processes are quantified under sterile conditions followed by a biodegradation experiment can also be adapted to more complex and realistic systems, such as particulate, suspended PAH solids or soils with intrapartically sorbed contaminants when the appropriate mass-transfer equations are incorporated. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 145-154, 1998.
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  • 58
    ISSN: 0006-3592