Thermomonospora

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David Wilson - One of the best experts on this subject based on the ideXlab platform.

  • Fed-batch production of Thermomonospora fusca endoglucanase by recombinant streptomyces lividans
    Biotechnology and bioengineering, 1998
    Co-Authors: Eun-ki Kim, Diana C. Irwin, Dong-hoon Shin, David Wilson
    Abstract:

    The factors affecting the production of a Thermomonospora fusca endoglucanase by a recombinant Streptomyces lividans strain were studied in a fermentor with glucose addition controlled by a pH-stat. The recombinant plasmid was stable for 35 generations with constant endoglucanase productivity. Glucose and peptone were used as the carbon and nitrogen sources. Addition of Tween-80 increased endoglucanase production twofold. A significant decrease in endoglucanase production was observed at low aeration. During fed-batch cultivation, pulse feeding (6 g/L) of a glucose-ammonium sulfate solution was optimal for endoglucanase production. With higher concentrations of glucose (15 g/L), a significant amount of organic acid, including acetic acid, was produced, which inhibited cell growth and endoglucanase production. Under optimum conditions, 1.7 U/mL of endoglucanase were produced. Copyright 1998 John Wiley & Sons, Inc.

  • Regulation of Biosynthesis of Individual Cellulases in Thermomonospora fusca
    Journal of bacteriology, 1998
    Co-Authors: Nikolay A. Spiridonov, David Wilson
    Abstract:

    Regulation of the biosynthesis of the six cellulases comprising the cellulolytic system of the thermophilic soil bacterium Thermomonospora fusca ER1 was studied. The levels of the individual enzymes produced on different noninducing and inducing carbon sources were determined. The lowest level of cellulase synthesis (3 nM) was observed with xylose as a carbon source, and the highest level (247 to 1,670 nM for different enzymes) was found in cultures grown on microcrystalline cellulose. Endocellulases and exocellulases showed distinctly different regulation patterns. Differences in the regulation of individual enzymes appear to be determined by the specific structural organization of the upstream regulatory sequences of their genes.

  • structure and mechanism of endo exocellulase e4 from Thermomonospora fusca
    Nature Structural & Molecular Biology, 1997
    Co-Authors: Joshua Sakon, David Wilson, Diana C. Irwin, P A Karplus
    Abstract:

    Cellulase E4 from Thermomonospora fusca is unusual in that it has characteristics of both exo- and endo-cellulases. Here we report the crystal structure of a 68K M(r) fragment of E4 (E4-68) at 1.9 A resolution. E4-68 contains both a family 9 catalytic domain, exhibiting an (alpha/alpha)6 barrel fold, and a family III cellulose binding domain, having an antiparallel beta-sandwich fold. While neither of these folds is novel, E4-68 provides the first cellulase structure having interacting catalytic and cellulose binding domains. The complexes of E4-68 with cellopentaose, cellotriose and cellobiose reveal conformational changes associated with ligand binding and allow us to propose a catalytic mechanism for family 9 enzymes. We also provide evidence that E4 has two novel characteristics: first it combines exo- and endo-activities and second, when it functions as an exo-cellulase, it cleaves off cellotetraose units.

  • binding capacities for Thermomonospora fusca e3 e4 and e5 the e3 binding domain and trichoderma reesei cbhi on avicel and bacterial microcrystalline cellulose
    Bioresource Technology, 1997
    Co-Authors: M K Bothwell, S D Daughhetee, G Y Chaua, David Wilson, Larry P. Walker
    Abstract:

    Abstract Equilibrium binding of Thermomonospora fusca E 3 , E 4 and E 5 , the E 3 binding domain (CBDE 3 ), and Trichoderma reesei CBHI on Avicel PH102 and bacterial microcrystalline cellulose (BMCC) was studied. The maximum adsorption levels, E b,m , for all four cellulases and the binding domain were 9–30 times higher on BMCC than on Avicel. The association constants for the individual cellulases were dependent upon the substrate; however, no obvious patterns were noted. A comparison of the T. fusca E bm s showed a decreasing power function relationship between molecular weight and maximum adsorption levels. This was particularly true for the cellulases binding on Avicel. The T. fusca binding results strongly suggest that binding capacity is a function of the cellulase size and the pore structure of the cellulose.

  • Synergism between pure Thermomonospora fusca and Trichoderma reesei cellulases
    Biomass and Bioenergy, 1993
    Co-Authors: M K Bothwell, David Wilson, L.p. Walke, D.c. Irwin, M. Price
    Abstract:

    Mixtures of Trichoderma reesei cellobiohydrolase I (CBHI) or crude enzyme extract and Thermomonospora fusca endoglucanases E2 or E3 were investigated to determine which gave maximum rates of hydrolysis, maximum reducing sugar concentration (RSC) at 4 h, and maximum degree of synergistic effect (DSE). Mixtures of T. fusca E3 and T. reesei crude enzyme extract (mass ratios of 0.1 to 1) produced the highest RSC (0.020 mg ml−1) and fastest initial reaction rate (0.009 mg ml−1 h−1) of the mixtures investigated. With exception of the DSEs observed for mixtures of T. fusca E2 and T. reesei CBHI, mixtures analyzed showed maximum RSC, rate of hydrolysis, and DSE over a range of enzyme mass ratios. These properties were sensitive to changes in the ratio outside of the optimal ratio range but were independent of the ratio within the optimal range. The DSE results of mixtures of T. fusca E2 and T. reesei CBHI were not conclusive. The ratio ranges which produced the maximum RSC, initial reaction velocity, and DSE did not completely overlap for all mixtures investigated.

Miroslav Petricek - One of the best experts on this subject based on the ideXlab platform.

Klaus Piontek - One of the best experts on this subject based on the ideXlab platform.

  • Crystallization and preliminary crystallographic analysis of two beta-mannanase isoforms from Thermomonospora fusca KW3.
    Acta Crystallographica Section D Biological Crystallography, 1996
    Co-Authors: M. Hilge, Sergio M. Gloor, Kaspar H. Winterhalter, W. Zimmermann, Klaus Piontek
    Abstract:

    Three β-mannanase isoforms were isolated from the supernatant of a thermophilic actinomycete culture from Thermomonospora fusca KW3. Two of the isoforms (Q1, Q 1.1) were crystallized by the hanging-drop method at room temperature using ammonium sulfate as a precipitant. The isoforms form rod-shaped colorless crystals. Both belong to the orthorhombic space group P212121. The cell dimensions are a = 46.7, b = 61.1, and c = 128.2 A for isoform Q1, and a = 43.8, b = 46.2, and c = 132.8 A for isoform Q1.1. The asymmetric unit of either isoform contains one mannanase molecule. Native data have been collected to 2.2 A resolution for Q1 and to 1.65 A resolution for Q1.1 using synchrotron radiation.

  • Crystallization and preliminary crystallographic analysis of two beta-mannanase isoforms from Thermomonospora fusca KW3.
    Acta crystallographica. Section D Biological crystallography, 1996
    Co-Authors: M. Hilge, W. Zimmermann, S Gloor, K Winterhalter, Klaus Piontek
    Abstract:

    Three beta-mannanase isoforms were isolated from the supernatant of a thermophilic actinomycete culture from Thermomonospora fusca KW3. Two of the isoforms (Q1, Q 1.1) were crystallized by the hanging-drop method at room temperature using ammonium sulfate as a precipitant. The isoforms form rod-shaped colorless crystals. Both belong to the orthorhombic space group P2(1)2(1)2(1). The cell dimensions are a = 46.7, b = 61.1, and c = 128.2 A for isoform Q1, and a = 43.8, b = 46.2, and c = 132.8 A for isoform Q1.1. The asymmetric unit of either isoform contains one mannanase molecule. Native data have been collected to 2.2 A resolution for Q1 and to 1.65 A resolution for Q1.1 using synchrotron radiation.

Chris Kroupis - One of the best experts on this subject based on the ideXlab platform.

  • Dimerization of Thermomonospora fusca beta-1,4-endoglucanase E2.
    Biochemistry, 1993
    Co-Authors: Kathleen Mcginnis, Chris Kroupis
    Abstract:

    Unboiled Thermomonospora fusca endoglucanase E2 electrophoresed on SDS-polyacrylamide gels migrated in the range of 80-90 kDa, but when boiled it migrated in the 40-42-kDa range. Sedimentation equilibrium centrifugation as well as chemical cross-linking experiments confirmed that E2 is a dimer. The dimer was reversibly dissociated at low pH. The E2 dimer was stable up to 70 degrees C, but began to dissociate at this temperature after a 30-60-min incubation. A nondimerizing mutant was obtained using region-specific chemical mutagenesis. DNA sequencing of this mutant revealed a single base change that substituted Gly for Glu-263. Chemical modification of carboxylic acid residues in E2 disrupted the dimer interaction.

L Janda - One of the best experts on this subject based on the ideXlab platform.

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