Molecular Biology of Cellulolytic Fungi

Nevalainen, Helena; Penttilä, Merja

doi:10.1007/978-3-662-10364-7_18

Cited by 28 publications

(8 citation statements)

References 101 publications

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“…The only simplification in the above equations is that k,, has been assumed identical for all substrates. Eqns(1a-m) can now be combined to calculate the changes in the concentrations for all species that occurs during an infinitesimal time interval dt: G, = G, + dC [1] (2 a) G, = G, + dC [2] The simulation procedure is then as follows: step 1, an equilibration period using Eqn(2) to obtain a steady state, using a short time step (dt = IO-'s); step 2, at the steady state a much larger time step (0.1 -1 s) is used to simulate Eqn(3); step 3, equilibration with 10 short time steps using Eqn(2); steps 2 and 3 are repeated for the desired time period.…”

Section: Appendixmentioning

confidence: 99%

Cello‐Oligosaccharide Hydrolysis by Cellobiohydrolase II from Trichoderma Reesei

Harjunpää

Teleman

Koivula

et al. 1996

European Journal of Biochemistry

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The hydrolysis of soluble cello-oligosaccharides, with a degree of polymerisation of 4-6, catalysed by cellobiohydrolase I1 from Trichoderma reesei was studied using 'H-NMR spectroscopy and HPLC. The experimental progress curves were analysed by fitting numerically integrated kinetic equations, which provided cleavage patterns and kinetic constants for each oligosaccharide. This analysis procedure accounts for product inhibition and avoids the initial slope approximation. No glucose was detected at the beginning of the reaction indicating that only the internal glycosidic linkages are attacked. For cellotetraose only the second glycosidic linkage was cleaved. For cellopentaose and cellohexaose the second and the third glycosidic linkage from the non-reducing end were cleaved with approximately equal probability. The degradation rates of these cello-oligosaccharides, 1 -12 s-' at 27"C, are about 10-100 times faster than for the 4-methylumbelliferyl substituted analogs or for cellotriose. No intermediate products larger than cellotriose were released. The degradation rate for cellotetraose were higher than its off-rate, which accounts for the processive degradation of cellohexaose. A high cellohexaose/enzyme ratio caused slow reversible inactivation of the enzyme. sites of cleavage, hydrolysis stereochemistry, kinetic and binding constants as well as in in vitro mutagenesis studies [6-81. These studies have been performed mostly using 4-methylumbelliferyl p-D-glycosides derived from cellotriose, cellotetraose and cellopentaose. The binding data have been obtained by fluorescence titration techniques, which have provided association constants also for cello-oligosaccharides by means of competition experiments [9, 101. NMR spectroscopy is well suited to follow the stereochemistry of saccharides 11 I] and we have recently used it together with progress curve analysis to study the hydrolysis of cellotriose by 7: reesei CBHII [12]. Replacing the initial-rate approximation by the complete progress curve analysis was highly advantageous, since it allows the elucidation of more complex mechanisms and reduces the amount of enzyme needed .In the present work the hydrolysis of cellotetraose, cellopentame and cellohexaose by CBHII has been studied by means of proton NMR spectroscopy and HPLC while using progress curves to analyse the kinetic data.Correspondence to V. Harjunpaa, VTT Chemical Technology, P. 0.

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Section: Appendixmentioning

confidence: 99%

Cello‐Oligosaccharide Hydrolysis by Cellobiohydrolase II from Trichoderma Reesei

Harjunpää

Teleman

Koivula

et al. 1996

European Journal of Biochemistry

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“…Various stress responses are triggered in cells to alleviate the harmful effects of the limitation in the capacity of the secretory pathway. Mechanisms that sense the status of protein folding and transport in the endoplasmic reticulum (ER) 1 have been characterized in detail in the yeast Saccharomyces cerevisiae and in mammalian cells (reviewed in Refs. 6 -9).…”

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The Effects of Drugs Inhibiting Protein Secretion in the Filamentous Fungus Trichoderma reesei

Pakula

Laxell

Huuskonen

et al. 2003

Journal of Biological Chemistry

136

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To study the mechanisms of protein secretion as well as the cellular responses to impaired protein folding and transport in filamentous fungi, we have analyzed Trichoderma reesei cultures treated with chemical agents that interfere with these processes, dithiothreitol, brefeldin A, and the Ca 2؉ -ionophore A23187. The effects of the drugs on the kinetics of protein synthesis and transport were characterized using metabolic labeling of synthesized proteins. Cellobiohydrolase I (CBHI, Cel7A), the major secreted cellulase, was analyzed as a model protein. Northern analysis showed that under conditions where protein transport was inhibited (treatments with dithiothreitol or brefeldin A) the unfolded protein response pathway was activated. The active form of the hac1 mRNA that mediates unfolded protein response signaling was induced, followed by induction of the foldase and chaperone genes pdi1 and bip1. Concomitant with the activation of the unfolded protein response pathway, the transcript levels of genes encoding secreted proteins, like cellulases and xylanases, were drastically decreased, suggesting a novel type of feedback mechanism activated in response to impairment in protein folding or transport (repression under secretion stress (RESS)). By studying expression of the reporter gene lacZ under cbh1 promoters of different length, it was shown that the feedback response was mediated through the cellulase promoter.

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“…These are cellobiohydrolase (CBH), endoglucanase (EG), and p-glucosidase. Members of each of these enzyme groups have been characterised from Trichoderina reesei both at the enzyme and gene levels (reviewed by Nevalainen and Penttila, 1995).…”

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cDNA Cloning of a Trichoderma reesei Cellulase and Demonstration of Endoglucanase Activity by Expression in Yeast

Saloheimo

Nakari-Setälä

Tenkanen

et al. 1997

European Journal of Biochemistry

170

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A Trichoderma reesei cDNA encoding a previously unknown protein with a C-terminal cellulosebinding domain was obtained by complementation screening of a 7: reesei cDNA library in a secl yeast mutant impaired in protein secretion. The 7: reesei protein shows amino acid similarity over its entire length to the Agaricus hisporus cellulose-induced protein CELl whose function is not known. These two proteins form a new glycosyl hydrolase family, number 61. Expression of the 7: reesei cDNA in yeast showed that it encoded a protein with endoglucanase activity and thus the protein was named EGIV and the corresponding gene eg14. Polyclonal antibodies were prepared against EGIV produced in Escherichia c-oli and detected a 56-kDa protein in the 7: reesei culture supernatant. Northern hybridisation revealed that 7: reesei eg14 is regulated in the same manner as other cellulase genes of this fungus.

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Molecular Biology of Cellulolytic Fungi

Cited by 28 publications

References 101 publications

Cello‐Oligosaccharide Hydrolysis by Cellobiohydrolase II from Trichoderma Reesei

Cello‐Oligosaccharide Hydrolysis by Cellobiohydrolase II from Trichoderma Reesei

The Effects of Drugs Inhibiting Protein Secretion in the Filamentous Fungus Trichoderma reesei

cDNA Cloning of a Trichoderma reesei Cellulase and Demonstration of Endoglucanase Activity by Expression in Yeast

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