Improving the thermostability and activity of Melanocarpus albomyces cellobiohydrolase Cel7B

Sanni Voutilainen, Harry Boer, M. Alapuranen, J. Jänis, Jari Vehmaanperä, Anu Koivula (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

40 Citations (Scopus)

Abstract

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.
Original languageEnglish
Pages (from-to)261-272
Number of pages12
JournalApplied Microbiology and Biotechnology
Volume83
Issue number2
DOIs
Publication statusPublished - 2009
MoE publication typeA1 Journal article-refereed

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Cellulose 1,4-beta-Cellobiosidase
Cellulose
Disulfides
Mutation
Protein Engineering
Trichoderma
Temperature
Catalytic Domain
Hydrolysis
Enzymes
Proteins

Keywords

  • Cellulase
  • Cellulose
  • Protein engineering
  • Saccharomyces cerevisiae expression
  • Site-directed mutagenesis

Cite this

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title = "Improving the thermostability and activity of Melanocarpus albomyces cellobiohydrolase Cel7B",
abstract = "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.",
keywords = "Cellulase, Cellulose, Protein engineering, Saccharomyces cerevisiae expression, Site-directed mutagenesis",
author = "Sanni Voutilainen and Harry Boer and M. Alapuranen and J. J{\"a}nis and Jari Vehmaanper{\"a} and Anu Koivula",
year = "2009",
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language = "English",
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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 journalArticleScientificpeer-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 -