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Hydrolysis of microcrystalline cellulose by cellobiohydrolase I and endoglucanase II from Trichoderma reesei: Adsorption, sugar production pattern, and synergism of the enzymes

Medve, József; Karlsson, Johan; Lee, Dora and Tjerneld, Folke LU (1998) In Biotechnology and Bioengineering 59(5). p.621-634
Abstract
Microcrystalline cellulose (10 g/L Avicel) was hydrolysed by two major cellulases, cellobiohydrolase I (CBH I) and endoglucanase II (EG II), of Trichoderma reesei. Two types of experiments were performed, and in both cases the enzymes were added alone and together, in equimolar mixtures. In time course studies the reaction time was varied between 3 min and 48 h at constant temperature (40degreesC) and enzyme loading (0.16 µmol/g Avicel). In isotherm studies the enzyme loading was varied in the range of 0.08-2.56 µmol/g at 4degreesC and 90 min. Adsorption of the enzymes and production of soluble sugars were followed by FPLC and HPLC, respectively. Adsorption started quickly (50% of maximum achieved after 3 min) but was not completed before... (More)
Microcrystalline cellulose (10 g/L Avicel) was hydrolysed by two major cellulases, cellobiohydrolase I (CBH I) and endoglucanase II (EG II), of Trichoderma reesei. Two types of experiments were performed, and in both cases the enzymes were added alone and together, in equimolar mixtures. In time course studies the reaction time was varied between 3 min and 48 h at constant temperature (40degreesC) and enzyme loading (0.16 µmol/g Avicel). In isotherm studies the enzyme loading was varied in the range of 0.08-2.56 µmol/g at 4degreesC and 90 min. Adsorption of the enzymes and production of soluble sugars were followed by FPLC and HPLC, respectively. Adsorption started quickly (50% of maximum achieved after 3 min) but was not completed before 60-90 min. For CBH I a linear relationship was observed between the production of soluble sugars and adsorption, showing that the average activity of the bound CBH I molecules does not change with increasing saturation. For EG II the corresponding curve levelled off which is explained by initial hydrolysis of loose ends on Avicel. The enzymes competed for binding sites, binding of EG II was considerably affected by CBH I, especially at high concentration. CBH I produced more soluble sugars than EG II, except at conversions below 1%. At 40degreesC when the enzymes were added together they produced 27-45% more soluble sugars than the sum of what they produced alone, i.e. synergistic action was observed (the final conversion after 48 h of hydrolysis was 3, 6, and 13% for EG II, CBH I, and their mixture, respectively). At 4degreesC, on the other hand, when the conversion was below 2.5%, almost no synergism could be observed. Molar proportions of the produced sugars were rather stable for CBH I (11-15%, 82-89%, and <6% for glucose, cellobiose, and cellotriose, respectively), while it varied considerably with both time and enzyme concentration for EG II. The observed stable but high glucose to cellobiose ratio for CBH I indicates that the processivity for this enzyme is not perfect. EG II produced significant amounts of glucose, cellobiose, and cellotriose, which are not the expected products of a typical endoglucanase activity on a solid substrate. We explain this by hypothesizing that EG II may show processivity due to its extended substrate binding site and the presence of its cellulose binding domain. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:621-634, 1998. (Less)
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Contribution to journal
publication status
published
subject
keywords
cellulase, cellobiohydrolase, endoglucanase, microcrystalline cellulose, cellulose hydrolysis, adsorption isotherm, synergism
in
Biotechnology and Bioengineering
volume
59
issue
5
pages
621 - 634
publisher
John Wiley & Sons
external identifiers
  • scopus:0032487130
ISSN
1097-0290
DOI
10.1002/(SICI)1097-0290(19980905)59:5<621::AID-BIT13>3.0.CO;2-C
language
English
LU publication?
yes
id
9b7f3517-832c-4a30-9af9-3fee8eeb68c4 (old id 125572)
date added to LUP
2007-07-09 08:14:43
date last changed
2017-09-10 03:30:36
@article{9b7f3517-832c-4a30-9af9-3fee8eeb68c4,
  abstract     = {Microcrystalline cellulose (10 g/L Avicel) was hydrolysed by two major cellulases, cellobiohydrolase I (CBH I) and endoglucanase II (EG II), of Trichoderma reesei. Two types of experiments were performed, and in both cases the enzymes were added alone and together, in equimolar mixtures. In time course studies the reaction time was varied between 3 min and 48 h at constant temperature (40degreesC) and enzyme loading (0.16 µmol/g Avicel). In isotherm studies the enzyme loading was varied in the range of 0.08-2.56 µmol/g at 4degreesC and 90 min. Adsorption of the enzymes and production of soluble sugars were followed by FPLC and HPLC, respectively. Adsorption started quickly (50% of maximum achieved after 3 min) but was not completed before 60-90 min. For CBH I a linear relationship was observed between the production of soluble sugars and adsorption, showing that the average activity of the bound CBH I molecules does not change with increasing saturation. For EG II the corresponding curve levelled off which is explained by initial hydrolysis of loose ends on Avicel. The enzymes competed for binding sites, binding of EG II was considerably affected by CBH I, especially at high concentration. CBH I produced more soluble sugars than EG II, except at conversions below 1%. At 40degreesC when the enzymes were added together they produced 27-45% more soluble sugars than the sum of what they produced alone, i.e. synergistic action was observed (the final conversion after 48 h of hydrolysis was 3, 6, and 13% for EG II, CBH I, and their mixture, respectively). At 4degreesC, on the other hand, when the conversion was below 2.5%, almost no synergism could be observed. Molar proportions of the produced sugars were rather stable for CBH I (11-15%, 82-89%, and &lt;6% for glucose, cellobiose, and cellotriose, respectively), while it varied considerably with both time and enzyme concentration for EG II. The observed stable but high glucose to cellobiose ratio for CBH I indicates that the processivity for this enzyme is not perfect. EG II produced significant amounts of glucose, cellobiose, and cellotriose, which are not the expected products of a typical endoglucanase activity on a solid substrate. We explain this by hypothesizing that EG II may show processivity due to its extended substrate binding site and the presence of its cellulose binding domain. © 1998 John Wiley &amp; Sons, Inc. Biotechnol Bioeng 59:621-634, 1998.},
  author       = {Medve, József and Karlsson, Johan and Lee, Dora and Tjerneld, Folke},
  issn         = {1097-0290},
  keyword      = {cellulase,cellobiohydrolase,endoglucanase,microcrystalline cellulose,cellulose hydrolysis,adsorption isotherm,synergism},
  language     = {eng},
  number       = {5},
  pages        = {621--634},
  publisher    = {John Wiley & Sons},
  series       = {Biotechnology and Bioengineering},
  title        = {Hydrolysis of microcrystalline cellulose by cellobiohydrolase I and endoglucanase II from Trichoderma reesei: Adsorption, sugar production pattern, and synergism of the enzymes},
  url          = {http://dx.doi.org/10.1002/(SICI)1097-0290(19980905)59:5<621::AID-BIT13>3.0.CO;2-C},
  volume       = {59},
  year         = {1998},
}