Fungal thermostable cellobiohydrolases. Characterization and protein engineering studies

Dissertation

Research output: ThesisDissertationCollection of Articles

Abstract

Cellulases are important industrial enzymes, and they are already today utilized for example in the pulp and paper and textile industries. Due to their application potential in so-called second generation bioethanol production, starting from lignocellulosic raw materials, cellulases are currently in the centre of attention. Especially cellobiohydrolases are the key enzymes in total hydrolysis of biomass based processes. The currently known cellobiohydrolases from GH family 7 are, however, not highly active under industrial conditions. In this work two different approaches were taken to improve the hydrolysis of crystalline cellulose. Firstly, thermotolerance and activity of two known fungal cellobiohydrolases from GH7 family was improved by protein engineering and secondly novel cellobiohydrolases were studied. Engineering of GH7 cellobiohydrolases has been hindered because of difficulties in heterologous expression in a bacterial or yeast host. In this study a functional yeast expression system was developed for two single-module cellobiohydrolases. This heterologous expression system enabled protein engineering by random mutagenesis and site-directed mutagenesis. Random mutagenesis, carried out with error-prone PCR and followed by functional screening with an automated, thermostability screening method, was used for improving the thermostability of Melanocarpus albomyces Cel7B (Ma Cel7B). The stability and activity of Ma Cel7B was further improved through structure-guided protein engineering by introducing an additional disulphide bridge and a carbohydrate binding module. Rational mutagenesis was also used for engineering Talaromyces emersonii Cel7A (Te Cel7A). Altogether five individual S-S bridges were introduced to improve the stability of Te Cel7A. Three out of these five single S-S mutants had a clearly improved thermostability. These mutations were combined in a triple mutant, which had significantly improved unfolding temperature (by 9°C) and ability to hydrolyse microcrystalline cellulose at 80°C. In addition to the mutagenesis studies of known cellobiohydrolases, we found novel GH-7 family cellobiohydrolases, which have high activity on insoluble polymeric substrates. Three family 7 cellobiohydrolases originating from thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus and Chaetomium thermophilum were characterized and compared to the widely studied Trichoderma reesei Cel7A enzyme. The comparison revealed that all these novel cellobiohydrolases are promising for application purposes, because they were more thermostable than T. reesei Cel7A and more active in the hydrolysis of microcrystalline cellulose (Avicel) at 45°C.
Original languageEnglish
QualificationDoctor Degree
Awarding Institution
  • University of Jyväskylä
Supervisors/Advisors
  • Koivula, Anu, Supervisor
Award date4 Feb 2011
Place of PublicationEspoo
Publisher
Print ISBNs978-951-38-7425-4
Electronic ISBNs978-951-38-7426-1
Publication statusPublished - 2010
MoE publication typeG5 Doctoral dissertation (article)

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cellulose 1,4-beta-cellobiosidase
protein engineering
mutagenesis
thermal stability
Trichoderma reesei
cellulose
cellulases
hydrolysis
engineering
enzymes
thermophilic fungi
yeasts
Chaetomium
screening
Acremonium
carbohydrate binding
mutants
pulp and paper industry
textile industry
site-directed mutagenesis

Keywords

  • cellulase
  • protein engineering
  • thermostability
  • heterologous expression
  • Saccharomyces cerevisiae
  • high-throughput screening
  • random
  • mutagenesis
  • site-directed mutagenesis
  • disulphide bridge

Cite this

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title = "Fungal thermostable cellobiohydrolases. Characterization and protein engineering studies: Dissertation",
abstract = "Cellulases are important industrial enzymes, and they are already today utilized for example in the pulp and paper and textile industries. Due to their application potential in so-called second generation bioethanol production, starting from lignocellulosic raw materials, cellulases are currently in the centre of attention. Especially cellobiohydrolases are the key enzymes in total hydrolysis of biomass based processes. The currently known cellobiohydrolases from GH family 7 are, however, not highly active under industrial conditions. In this work two different approaches were taken to improve the hydrolysis of crystalline cellulose. Firstly, thermotolerance and activity of two known fungal cellobiohydrolases from GH7 family was improved by protein engineering and secondly novel cellobiohydrolases were studied. Engineering of GH7 cellobiohydrolases has been hindered because of difficulties in heterologous expression in a bacterial or yeast host. In this study a functional yeast expression system was developed for two single-module cellobiohydrolases. This heterologous expression system enabled protein engineering by random mutagenesis and site-directed mutagenesis. Random mutagenesis, carried out with error-prone PCR and followed by functional screening with an automated, thermostability screening method, was used for improving the thermostability of Melanocarpus albomyces Cel7B (Ma Cel7B). The stability and activity of Ma Cel7B was further improved through structure-guided protein engineering by introducing an additional disulphide bridge and a carbohydrate binding module. Rational mutagenesis was also used for engineering Talaromyces emersonii Cel7A (Te Cel7A). Altogether five individual S-S bridges were introduced to improve the stability of Te Cel7A. Three out of these five single S-S mutants had a clearly improved thermostability. These mutations were combined in a triple mutant, which had significantly improved unfolding temperature (by 9°C) and ability to hydrolyse microcrystalline cellulose at 80°C. In addition to the mutagenesis studies of known cellobiohydrolases, we found novel GH-7 family cellobiohydrolases, which have high activity on insoluble polymeric substrates. Three family 7 cellobiohydrolases originating from thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus and Chaetomium thermophilum were characterized and compared to the widely studied Trichoderma reesei Cel7A enzyme. The comparison revealed that all these novel cellobiohydrolases are promising for application purposes, because they were more thermostable than T. reesei Cel7A and more active in the hydrolysis of microcrystalline cellulose (Avicel) at 45°C.",
keywords = "cellulase, protein engineering, thermostability, heterologous expression, Saccharomyces cerevisiae, high-throughput screening, random, mutagenesis, site-directed mutagenesis, disulphide bridge",
author = "Sanni Voutilainen",
year = "2010",
language = "English",
isbn = "978-951-38-7425-4",
series = "VTT Publications",
publisher = "VTT Technical Research Centre of Finland",
number = "754",
address = "Finland",
school = "University of Jyv{\"a}skyl{\"a}",

}

Fungal thermostable cellobiohydrolases. Characterization and protein engineering studies : Dissertation. / Voutilainen, Sanni.

Espoo : VTT Technical Research Centre of Finland, 2010. 103 p.

Research output: ThesisDissertationCollection of Articles

TY - THES

T1 - Fungal thermostable cellobiohydrolases. Characterization and protein engineering studies

T2 - Dissertation

AU - Voutilainen, Sanni

PY - 2010

Y1 - 2010

N2 - Cellulases are important industrial enzymes, and they are already today utilized for example in the pulp and paper and textile industries. Due to their application potential in so-called second generation bioethanol production, starting from lignocellulosic raw materials, cellulases are currently in the centre of attention. Especially cellobiohydrolases are the key enzymes in total hydrolysis of biomass based processes. The currently known cellobiohydrolases from GH family 7 are, however, not highly active under industrial conditions. In this work two different approaches were taken to improve the hydrolysis of crystalline cellulose. Firstly, thermotolerance and activity of two known fungal cellobiohydrolases from GH7 family was improved by protein engineering and secondly novel cellobiohydrolases were studied. Engineering of GH7 cellobiohydrolases has been hindered because of difficulties in heterologous expression in a bacterial or yeast host. In this study a functional yeast expression system was developed for two single-module cellobiohydrolases. This heterologous expression system enabled protein engineering by random mutagenesis and site-directed mutagenesis. Random mutagenesis, carried out with error-prone PCR and followed by functional screening with an automated, thermostability screening method, was used for improving the thermostability of Melanocarpus albomyces Cel7B (Ma Cel7B). The stability and activity of Ma Cel7B was further improved through structure-guided protein engineering by introducing an additional disulphide bridge and a carbohydrate binding module. Rational mutagenesis was also used for engineering Talaromyces emersonii Cel7A (Te Cel7A). Altogether five individual S-S bridges were introduced to improve the stability of Te Cel7A. Three out of these five single S-S mutants had a clearly improved thermostability. These mutations were combined in a triple mutant, which had significantly improved unfolding temperature (by 9°C) and ability to hydrolyse microcrystalline cellulose at 80°C. In addition to the mutagenesis studies of known cellobiohydrolases, we found novel GH-7 family cellobiohydrolases, which have high activity on insoluble polymeric substrates. Three family 7 cellobiohydrolases originating from thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus and Chaetomium thermophilum were characterized and compared to the widely studied Trichoderma reesei Cel7A enzyme. The comparison revealed that all these novel cellobiohydrolases are promising for application purposes, because they were more thermostable than T. reesei Cel7A and more active in the hydrolysis of microcrystalline cellulose (Avicel) at 45°C.

AB - Cellulases are important industrial enzymes, and they are already today utilized for example in the pulp and paper and textile industries. Due to their application potential in so-called second generation bioethanol production, starting from lignocellulosic raw materials, cellulases are currently in the centre of attention. Especially cellobiohydrolases are the key enzymes in total hydrolysis of biomass based processes. The currently known cellobiohydrolases from GH family 7 are, however, not highly active under industrial conditions. In this work two different approaches were taken to improve the hydrolysis of crystalline cellulose. Firstly, thermotolerance and activity of two known fungal cellobiohydrolases from GH7 family was improved by protein engineering and secondly novel cellobiohydrolases were studied. Engineering of GH7 cellobiohydrolases has been hindered because of difficulties in heterologous expression in a bacterial or yeast host. In this study a functional yeast expression system was developed for two single-module cellobiohydrolases. This heterologous expression system enabled protein engineering by random mutagenesis and site-directed mutagenesis. Random mutagenesis, carried out with error-prone PCR and followed by functional screening with an automated, thermostability screening method, was used for improving the thermostability of Melanocarpus albomyces Cel7B (Ma Cel7B). The stability and activity of Ma Cel7B was further improved through structure-guided protein engineering by introducing an additional disulphide bridge and a carbohydrate binding module. Rational mutagenesis was also used for engineering Talaromyces emersonii Cel7A (Te Cel7A). Altogether five individual S-S bridges were introduced to improve the stability of Te Cel7A. Three out of these five single S-S mutants had a clearly improved thermostability. These mutations were combined in a triple mutant, which had significantly improved unfolding temperature (by 9°C) and ability to hydrolyse microcrystalline cellulose at 80°C. In addition to the mutagenesis studies of known cellobiohydrolases, we found novel GH-7 family cellobiohydrolases, which have high activity on insoluble polymeric substrates. Three family 7 cellobiohydrolases originating from thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus and Chaetomium thermophilum were characterized and compared to the widely studied Trichoderma reesei Cel7A enzyme. The comparison revealed that all these novel cellobiohydrolases are promising for application purposes, because they were more thermostable than T. reesei Cel7A and more active in the hydrolysis of microcrystalline cellulose (Avicel) at 45°C.

KW - cellulase

KW - protein engineering

KW - thermostability

KW - heterologous expression

KW - Saccharomyces cerevisiae

KW - high-throughput screening

KW - random

KW - mutagenesis

KW - site-directed mutagenesis

KW - disulphide bridge

M3 - Dissertation

SN - 978-951-38-7425-4

T3 - VTT Publications

PB - VTT Technical Research Centre of Finland

CY - Espoo

ER -