Abstract
| Original language | English |
|---|---|
| Pages (from-to) | 261-272 |
| Number of pages | 12 |
| Journal | Applied Microbiology and Biotechnology |
| Volume | 83 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - 2009 |
| MoE publication type | A1 Journal article-refereed |
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Keywords
- Cellulase
- Cellulose
- Protein engineering
- Saccharomyces cerevisiae expression
- Site-directed mutagenesis
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Improving the thermostability and activity of Melanocarpus albomyces cellobiohydrolase Cel7B. / Voutilainen, Sanni; Boer, Harry; Alapuranen, M.; Jänis, J.; Vehmaanperä, Jari; Koivula, Anu (Corresponding Author).
In: Applied Microbiology and Biotechnology, Vol. 83, No. 2, 2009, p. 261-272.Research output: Contribution to journal › Article › Scientific › peer-review
TY - JOUR
T1 - Improving the thermostability and activity of Melanocarpus albomyces cellobiohydrolase Cel7B
AU - Voutilainen, Sanni
AU - Boer, Harry
AU - Alapuranen, M.
AU - Jänis, J.
AU - Vehmaanperä, Jari
AU - Koivula, Anu
PY - 2009
Y1 - 2009
N2 - Two different types of approach were taken to improve the hydrolytic activity towards crystalline cellulose at elevated temperatures of Melanocarpus albomyces Cel7B (Ma Cel7B), a single-module GH-7 family cellobiohydrolase. Structure-guided protein engineering was used to introduce an additional tenth disulphide bridge to the Ma Cel7B catalytic module. In addition, a fusion protein was constructed by linking a cellulose-binding module (CBM) and a linker from the Trichoderma reesei Cel7A to the C terminus of Ma Cel7B. Both approaches proved successful. The disulphide bridge mutation G4C/M70C located near the N terminus, close to the entrance of the active site tunnel of Ma Cel7B, led to improved thermostability (ΔT m = 2.5°C). By adding the earlier found thermostability-increasing mutation S290T (ΔT m = 1.5°C) together with the disulphide bridge mutation, the unfolding temperature was increased by 4°C (mutant G4C/M70C/S290T) compared to that of the wild-type enzyme, thus showing an additive effect on thermostability. Both disulphide mutants had increased activity towards microcrystalline cellulose (Avicel) at 75°C, apparently solely because of their improved thermostability. The addition of a CBM also improved the thermostability (ΔT m = 2.5°C) and caused a clear (sevenfold) increase in the hydrolysis activity of Ma Cel7B towards Avicel at 70°C.
AB - Two different types of approach were taken to improve the hydrolytic activity towards crystalline cellulose at elevated temperatures of Melanocarpus albomyces Cel7B (Ma Cel7B), a single-module GH-7 family cellobiohydrolase. Structure-guided protein engineering was used to introduce an additional tenth disulphide bridge to the Ma Cel7B catalytic module. In addition, a fusion protein was constructed by linking a cellulose-binding module (CBM) and a linker from the Trichoderma reesei Cel7A to the C terminus of Ma Cel7B. Both approaches proved successful. The disulphide bridge mutation G4C/M70C located near the N terminus, close to the entrance of the active site tunnel of Ma Cel7B, led to improved thermostability (ΔT m = 2.5°C). By adding the earlier found thermostability-increasing mutation S290T (ΔT m = 1.5°C) together with the disulphide bridge mutation, the unfolding temperature was increased by 4°C (mutant G4C/M70C/S290T) compared to that of the wild-type enzyme, thus showing an additive effect on thermostability. Both disulphide mutants had increased activity towards microcrystalline cellulose (Avicel) at 75°C, apparently solely because of their improved thermostability. The addition of a CBM also improved the thermostability (ΔT m = 2.5°C) and caused a clear (sevenfold) increase in the hydrolysis activity of Ma Cel7B towards Avicel at 70°C.
KW - Cellulase
KW - Cellulose
KW - Protein engineering
KW - Saccharomyces cerevisiae expression
KW - Site-directed mutagenesis
U2 - 10.1007/s00253-008-1848-9
DO - 10.1007/s00253-008-1848-9
M3 - Article
VL - 83
SP - 261
EP - 272
JO - Applied Microbiology and Biotechnology
JF - Applied Microbiology and Biotechnology
SN - 0175-7598
IS - 2
ER -