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  • 1
    Electronic Resource
    Electronic Resource
    Crystallization and preliminary X-ray crystallographic analysis of RNase HIII from Bacillus subtilis (2001)
    Copenhagen : International Union of Crystallography (IUCr)
    Acta crystallographica 57 (2001), S. 438-440 
    Details
    ISSN: 1399-0047
    Source: Crystallography Journals Online : IUCR Backfile Archive 1948-2001
    Topics: Chemistry and Pharmacology , Geosciences , Physics
    Notes: The genome of Bacillus subtilis contains three different genes encoding RNase H homologs: RNases HI, HII and HIII. RNase HIII from B. subtilis degrades RNA in RNA–DNA hybrids in an Mg2+-dependent manner like Escherichia coli RNase HI. However, they belong to different classes; the former belongs to the `class II' or `large' RNase H family, while the latter belongs to the `class I' or `small' RNase H family. RNase HIII of B. subtilis has been overexpressed in E. coli and crystallized at 296 K using sodium formate as a precipitant. The native X-ray diffraction data have been collected to 2.8 Å resolution using synchrotron radiation. The crystals are hexagonal, belonging to the space group P61, with unit-cell parameters a = b = 86.89, c = 214.49 Å, α = β = 90.0, γ = 120.0°. A self-rotation function calculation indicated the presence of two monomers of the recombinant RNase HIII in the crystallographic asymmetric unit, giving a VM of 3.43 Å3 Da−1 and a solvent content of 64.2%.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1107/S0907444901000440
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  • 2
    Electronic Resource
    Electronic Resource
    Isolation and characterization of the tobramycin biosynthetic gene cluster from Streptomyces tenebrarius (2004)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 230 (2004), S. 0 
    Details
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The biosynthetic gene cluster for tobramycin, a 2-deoxystreptamine-containing aminoglycoside antibiotic, was isolated from Streptomyces tenebrarius ATCC 17920. A genomic library of S. tenebrarius was constructed, and a cosmid, pST51, was isolated by the probes based on the core regions of 2-deoxy-scyllo-inosose (DOI) synthase, and l-glutamine:DOI aminotransferase and l-glutamine:scyllo-inosose aminotransferase. Sequencing of 33.9 kb revealed 24 open reading frames (ORFs) including putative tobramycin biosynthetic genes. We demonstrated that one of these ORFs, tbmA, encodes DOI synthase by in vitro enzyme assay of the purified protein. The catalytic residues of TbmA and dehydroquinate synthase were studied by homology modeling. The gene cluster found is likely to be involved in the biosynthesis of tobramycin.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1016/S0378-1097(03)00881-4
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  • 3
    Electronic Resource
    Electronic Resource
    Optical Resolution of Racemic 1-Phenylethylamine Catalyzed by Aminotransferase and Dehydrogenase (1996)
    Oxford, UK : Blackwell Publishing Ltd
    Annals of the New York Academy of Sciences 799 (1996), S. 0 
    Details
    ISSN: 1749-6632
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Natural Sciences in General
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1749-6632.1996.tb33280.x
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  • 4
    Electronic Resource
    Electronic Resource
    Kinetic resolution of α-methylbenzylamine with ο-transaminase screened from soil microorganisms: Application of a biphasic system to overcome product inhibition (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 348-358 
    Details
    ISSN: 0006-3592
    Keywords: enzymatic resolution ; ο-transaminase ; biphasic system ; product inhibition ; α-methylbenzylamine ; biocompatibility ; extracting capacity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Two microorganisms showing high ο-transaminase activity (Klebsiella pneumoniae JS2F and Bacillus thuringiensis JS64) were screened by the enrichment method using (S)-α-methylbenzylamine (α-MBA) as a sole nitrogen source. Optimal carbon and nitrogen sources for enzyme induction and the properties of ο-transaminases were investigated. ο-Transaminase from B. thuringiensis JS64 was highly enantioselective (E = 75.3) for (S)-enantiomer of α-MBA and showed remarkable stability. However, ο-transaminase showed severe product inhibition by acetophenone. An aqueous/organic two-phase system was introduced to overcome this problem. Through solvent screening, cyclohexanone and ethyl acetate were selected as the best organic phases. The acetophenone-extracting capacity of the solvent and the biocompatibility of the solvent to the cell were important determinants in the reaction rate at high concentrations of α-MBA. The reaction rate of ο-transamination was strongly influenced by the volume ratio of organic phase to aqueous phase as well as agitation speed in the biphasic mixture. Using the optimal volume ratio (Vorg:Vaq = 1:4) in the biphasic system with cyclohexanone, the reaction rate of ο-transaminase under vigorous mixing conditions increased ninefold compared with that in the monophasic aqueous system. At the same optimal conditions, using whole cells, 500 mM α-MBA could be resolved successfully to above 95% enantiomeric excess of (R)-α-MBA with ca. 51% conversion. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 348-358, 1997.
    Additional Material: 10 Ill.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Effect of the hydration state of supports before lyophilization on subtilisin-A activity in organic media (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 687-692 
    Details
    ISSN: 0006-3592
    Keywords: hydration state of support ; colyophilized enzyme ; lyophilization ; subtilisin-A ; enzymatic optical resolution ; organic solvent ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Subtilisin-A was colyophilized with various types of support materials, such as Amberlite IRC-50, Celite545, chitosan, DEAE-cellulose, DOWEX-1, zeolite, glass bead, and polystyrene. The colyophilized enzyme was used for the optical resolution of racemic 1-phenylethylamine with 2,2,2-trifluoroethylbutyrate in 3-methyl-3-pentanol. The enzyme activity in organic media changed dramatically according to the hydration state of the support materials before lyophilization. This effect was especially marked with supports of high water capacity (aquaphilicity), such as chitosan and DEAE-cellulose. By hydrating these supports of high aquaphilicity prior to lyophilization, subtilisin-A activity in organic media increased ca. 4-8 times, depending upon the supports used. This result suggests that the hydration state of aquaphilic support materials for colyophilization is critical to determining enzyme activity in organic solvents. © 1996 John Wiley & Sons, Inc.
    Additional Material: 3 Ill.
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  • 6
    Electronic Resource
    Electronic Resource
    Kinetic modeling of ω-transamination for enzymatic kinetic resolution of α-methylbenzylamine (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 60 (1998), S. 534-540 
    Details
    ISSN: 0006-3592
    Keywords: ω-transaminase ; kinetic modeling ; kinetic resolution ; product inhibition ; α-methylbenzylamine ; sensitivity analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A kinetic model for ω-transaminase from Bacillus thuringiensis JS64 was developed by using the King-Altman method to simulate the kinetic resolution of α-methylbenzylamine (α-MBA). Starting from a ping-pong bi-bi mechanism, a complete kinetic model including substrate inhibition only in the reverse reaction (i.e., transamination between acetophenone and L-alanine) was developed. The asymmetric synthesis of (S)-α-MBA proved to be difficult due to a much lower maximum reverse reaction rate than the maximum forward reaction rate, thermodynamically exergonic forward reaction (i.e., transamination between (S)-α-MBA and pyruvate), and the severe product and substrate inhibition of the reverse reaction. Experimental values for kinetic parameters show that the product inhibition constant of (S)-α-MBA is the most important parameter on determining the resolution reaction rate, suggesting that the resolution reaction rate will be very low unless (S)-α-MBA strongly inhibits the reverse reaction. Using the kinetic model, the kinetic resolution of α-MBA in aqueous buffer was simulated, and the simulation results showed a high degree of consistency with experimental data over a range of reaction conditions. Various simulation results suggest that the crucial bottleneck in the kinetic resolution of α-MBA lies mainly in the accumulation of acetophenone in reaction media as the reaction proceeds, whereas L-alanine exerts a little inhibitory effect on the reaction. The model predicts that removing acetophenone produced during the reaction can enhance the reaction rate dramatically. Indeed, the biphasic reaction system is capable of extracting acetophenone from the aqueous phase, showing a much higher reaction rate compared to a monophasic reaction system. The kinetic model was also useful in predicting the properties of other, better enzymes as well as the optimal concentrations of amino acceptor and enzyme in the resolution reaction. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 534-540, 1998.
    Additional Material: 5 Ill.
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  • 7
    Electronic Resource
    Electronic Resource
    Kinetic analysis of the effects of plasmid multimerization on segregational instability of CoIE1 type plasmids in Escherichia coli B/r (1991)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 37 (1991), S. 1076-1086 
    Details
    ISSN: 0006-3592
    Keywords: plasmids-mathematical models ; multimerization ; segregational instability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of plasmid multimerization on segregational instability was investigated using a structured, segregated model of genetically modified Escherichia coli cells. By including the multimerization of plasmids, the model can predict the proportion of each multimer in the total plasmid population. Simulation results suggest that the plasmid copy number is controlled by the total plasmid content (i.e., total number of plasmid origins) in the host cell and that multimerization reduces the total number of independent, monomeric segregation units. However, multimerization is found to have a minor effect on decreasing plasmid segregational stability for multicopy plasmids with average copy number per cell greater than about 25. Also model predictions were used to test whether or not a nonrandom plasmid distribution at cell fission could cause segregational instability. Even in the case of severely biased partitioning, plasmids whose copy number is above 45 per cell do not show significant segregational instability. The results suggest that when the ColE1-type plasmid does not encode and express any large or disruptive foreign proteins, the copy number of 45 per cell may be the threshold at which only growth rate-dependent instability is responsible for overall plasmid instability.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260371113
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  • 8
    Electronic Resource
    Electronic Resource
    Justification of continuous packed-bed reactor for retroviral vector production from amphotropic ΨCRIP murine producer cell (2000)
    Springer
    Cytotechnology 34 (2000), S. 151-158 
    Details
    ISSN: 1573-0778
    Keywords: anchorage-dependent cell ; cell culture ; packed-bedreactor ; retroviral vector ; viral production
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract To indentify a plausible large-scale production system forretroviral vector, three culture systems, i.e., batch culturewith medium exchange, microcarrier culture, and packed-bedreactor culture were compared. In batch cultures with mediumexchange, high cell concentrations were maintained for about amonth, and the harvested retroviral titer remained constant. Inmicrocarrier cultures, although cell growth was rapid, theretroviral titer was unexpectedly low, suggesting that the lowtiter was due either to serious damage to the retroviral vectoror to a reduction in the production rate of retroviral vector,caused by mechanical shear forces. Although the retroviral titer(maximum titer, 1.56 × 106) in the packed-bedreactor was a little bit lower than that obtained in the batchculture with medium exchange (maximum titer, 1.91 ×106), continuous production made it possible to increasethe cumulative titer up to 16-fold of that from the batchculture with medium exchange. Moreover, as the packed-bedreactor system requires less labor and shows excellentvolumetric productivity in comparison to batch cultures withmedium exchanges, it will be an appropriate production systemfor retroviral vector in large quantities.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008120313175
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  • 9
    Electronic Resource
    Electronic Resource
    Analysis of pBR322 replication kinetics and its dependency on growth rate (1990)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 36 (1990), S. 233-242 
    Details
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Accurate estimates of plasmid copy number in a cell are a prerequisite for predicting plasmid stability and protein production. A refined version of a structured model for the pBR322 plasmid replication mechanism is described. The model is capable of accurately predicting pBR322 plasmid copy number in Escherichia coli B/r for a wide range of growth rates. The refinements include better estimates of promoter strength, the degradation rate of RNA species, binding constant of RNAI-RNAII reaction, and dependency of promoter strength on growth rate. The predictions of the model are verified by recent experimental observations but differ from some previous reports. This model can also be used to predict the binding constant of the RNAI-RNAII reaction of ColE1 type plasmids. At 37°C, the binding constant is estimated to be 77 ± 11 × 10-13 mL/molecule-h for pBR322.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260360304
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  • 10
    Electronic Resource
    Electronic Resource
    A structured, segregated model for genetically modified Escherichia coli cells and its use for prediction of plasmid stability (1990)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 36 (1990), S. 581-592 
    Details
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A structured, segregated model is presented for an asynchronously growing population of genetically modified Escherichia coli cells. A finite representation method was modified so that 272 cells could be used to represent a microbial population. The concept of a “limbo” compartment was introduced to allow random plasmid distribution to daughter cells upon cell division while restricting the number of computer cells included in the calculation. This scheme enabled us to predict plasmid instability and distribution of plasmid-originated properties in a population without a priori determination of growth rates or probability of forming plasmid-free cells from plasmid-containing cells. Predictions of population behavior using a single-cell model requires no adjustable parameters. The results comparing different induction strategies suggest that in continuous culture, there exists an optimum efficiency of partial induction that maximizes the long-term productivity of the gene product due to plasmid stability. With the optimum efficiency of partial induction, constant induction appears to prove more stable than cycling induction.
    Additional Material: 13 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260360605
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