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  • 1
    ISSN: 1432-0983
    Keywords: Trichoderma ; Transformation ; Hygromycin B resistance ; Benomyl resistance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have developed a transformation system for Trichoderma hamatum and Trichoderma harzianum Rifai, using dominant markers for selection based on the Escherichia coli hygromycin B phosphotransferase gene (hph) and the β-tubulin gene (bml) from Neurospora crassa, respectively. Transformation frequencies and protoplast regeneration were low in both species. All the T. hamatum hygromycin-resistant transformants analysed were mitotically stable, in contrast to those of T. harzianum derived by benomyl resistance, in which only 50% of the transformants analysed were stable. Molecular analysis of transformants showed the integration of the transforming DNA into the genome and indicated that the number and sites of integration varied among the transformants.
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  • 2
    ISSN: 1432-0991
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Chitobiase (EC 3.2.1.29), from the culture filtrate ofTrichoderma harzianum, was purified in sequential steps by ammonium sulfate precipitation, ion exchange chromatography, and gel filtration. The physical and biochemical properties of the enzyme have been determined. The native enzyme has a molecular weight of 118 kDa when determined by gel filtration, and 64 kDa by SDS-PAGE. The enzyme catalyzed the hydrolysis of N,N-diacetylchitobiose andp-nitrophenyl-β-N-acetyl glucosamine with apparent Km of 575 µM and 235 µM, respectively. The pH optimum for the enzyme was pH 5.5, and maximum activity was obtained at 50°C. Glucosamine and N-acetylglyucosamine strongly inhibited the enzyme.
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  • 3
    ISSN: 1432-0991
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Aspergillus fumigatus produces substantial extracellular cellulases on several cellulosic substrates including simple sugars. Low glucose potentiates enzyme production, but most cellulose-induced cellulases are repressed by high glucose. As production of cellulase in a wide substrate range is unusual, the cellulolytic complex of this thermophilic fungus was investigated. A β-glucosidase was separated by gel filtration and ion-exchange chromatography. It migrated in native polyacrylamide gel as a single protein (130 kDa), which split under denaturing conditions into two smaller proteins having molecular masses of 90 kDa and 45 kDa. However, only the 90-kDa protein was active. Conventional chromatographic procedures were unsuccessful for the separation of these two proteins. Therefore, the 130-kDa protein was studied for its kinetic properties. It hydrolyzed p-nitrophenyl-β-D-glucopyranoside (p-NPG) and cellobiose, but not β-glucans, laminarin, and p-nitrophenyl-β-D-xilopyranoside. The optimal pH and temperature of p-NPG and cellobiose hydrolysis were 5.0 and 4.0, and 65°C and 60°C, respectively. The K m values, determined for cellobiose and p-NPG of hydrolysis, were 0.075 mM and 1.36 mM, respectively. Glucose competitively inhibited the hydrolysis of p-NPG. The Ki was 3.5 mM.
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  • 4
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The effect of carbon sources on the level of β-1,3-glucanases in the culture filtrates of Trichoderma harzianum (Tc) was investigated. Enzyme activity was detected in all carbon sources, but highest levels were found when laminarin and purified cell walls were used. Three isoforms of β-1,3-glucanase were produced during growth of the fungus on purified cell walls. Two isoforms were produced on chitin, chitosan, N-acetylglucosamine and laminarin, while only one was detected when the fungus was grown on cellulose and glucose. A 36-kDa β-1,3-glucanase (GLU36) was secreted from T. harzianum (Tc) grown on all carbon sources tested as demonstrated by Western blot analysis. We found that a significant increase in the level of GLU36 in the culture filtrate follows glucose exhaustion, suggesting that this enzyme is controlled by carbon catabolite repression.
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  • 5
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: During our screening of amylolytic microorganisms from Brazilian fruits, we isolated a yeast strain classified as Cryptococcus flavus. When grown on starch-containing medium this strain exhibited the highest amylase production after 24 h of cultivation. The extracellular amylase from C. flavus was purified from the culture broth by a single step using chromatography on a Sephacryl S-100 column. The enzyme was purified 16.14-fold with a yield of 50.21% of the total activity. The purified enzyme was a glycoprotein with an apparent molecular mass of 75 and 84.5 kDa as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and gel filtration, respectively. The enzyme lost approximately 50% of the molecular mass after treatment with glycosidases. The major end products of starch, amylose, amylopectin, pullulan and glycogen were maltose and maltotriose. The Km value for the pure enzyme was 0.056 mg ml−1 with soluble starch as the substrate. Enzyme activity was optimal at pH 5.5 and 50°C. The enzyme retained 90% of the activity after incubation at 50°C for 60 min and was inhibited by Cu2+, Fe2+ and Hg2+.
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  • 6
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Trichoderma asperellum produces at least two extracellular β-1,3-glucanases upon induction with cell walls from Rhizoctonia solani. A β-1,3-glucanase was purified by gel filtration and ion exchange chromatography. A typical procedure provided 35.7-fold purification with 9.5% yield. The molecular mass of the purified exo-β-1,3-glucanases was 83.1 kDa as estimated using a 12% (w/v) SDS–electrophoresis slab gel. The enzyme was only active toward glucans containing β-1,3-linkages and hydrolyzed laminarin in an exo-like fashion to form glucose. The Km and Vmax values for exo-β-1,3-glucanase, using laminarin as substrate, were 0.087 mg ml−1 and 0.246 U min−1, respectively. The pH optimum for the enzyme was pH 5.1 and maximum activity was obtained at 55°C. Hg2+ strongly inhibited the purified enzyme.
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  • 7
    ISSN: 1573-0972
    Keywords: α-Amylase ; fusion protein ; glucoamylase ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A fusion gene containing the Bacillus subtilis α-amylase gene and Aspergillus awamori glucoamylase cDNA was expressed in Saccharomyces cerevisiae. The resulting bifunctional fusion protein having both α-amylase and glucoamylase activities secreted into the culture medium was purified to apparent homogeneity by affinity chromatography and gel filtration on Sephadex G-100. The enzyme had an apparent molecular mass of 150 kDa and showed an optimum pH and temperature of 6.0 and 60 °C, respectively. The main hydrolysis products from soluble starch were glucose and maltose.
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