Structure, function and engineering of thermostable GH-7 family cellobiohydrolases

Anu Koivula, Sanni Voutilainen, Harry Boer, T. Puranen, Matti Siika-aho, M. Alapuranen, Liisa Viikari, Jari Vehmaanperä

Research output: Contribution to conferenceConference articleScientific

Abstract

Cellulases are important industrial enzymes, which can be used e.g. in the pulp and paper, textile, and detergent industry. They are currently under intensive study particularly due to their applicability in total hydrolysis of cellulosic biomass for production of bioethanol and other chemicals. Thermostable enzymes are desirable for many cellulase applications, as they are generally robust, tolerating various harsh process conditions. Thermophilic organisms are a potential source for thermostable enzymes, or as an alternative the thermostability of an enzyme can be improved by structure-based rational mutagenesis or directed evolution methods. Glycosyl hydrolase family 7 (GH-7; see http://afmb.cnrs-mrs.fr/CAZY/citing.html) cellobiohydrolases seem to be especially important in the hydrolysis of highly crystalline cellulose, and are found only in the fungal kingdom. Here, we present the characterisation of the enzymatic properties of three novel thermostable cellobiohydrolases originating from the thermophililic fungi Acremonium thermophilum, Chaetomium thermophilum and Thermoascus aurantiacus. These GH-7 family enzymes were expressed in the industrially relevant production host Trichoderma reesei, and the kinetics on small soluble substrates, cellobiose inhibition, crystalline cellulose hydrolysis and thermostability of the purified enzymes were determined. Enzymatic properties of the cellobiohydrolases were compared to those of T.reesei cellobiohydrolase Cel7A, one of the most thoroughly studied fungal cellobiohydrolases. In addition, both random and site-directed mutagenesis approaches have been used to improve the performance of the Melanocarpus albomyces Cel7B at elevated temperatures. The biochemical results are also discussed from the structural point of view based on the three-dimensional structures and homology models of these enzymes. Acknowledgements: This work has been supported by grants from the EU ("Technological improvement for ethanol production from lignocellulose" project; coordinator L. Viikari, VTT), the Academy of Finland (SV) and the Finnish Glycoscience Graduate School (SV). References : [1] Voutilainen, S., Boer, H., Linder, M., Puranen, T., Rouvinen, J., Vehmaanperä, J. and Koivula, A. (2007) Heterologous expression and random mutagenesis to improve the thermostability of Melanocarpus albomyces Cel7B cellobiohydrolase. Enz. Microb. Technol. 41, 234-243. [2] Voutilainen, S., Puranen, T., Siika-aho, M., Lappalainen, A., Alapuranen, M., Kallio, J., Hooman, S., Viikari, L., Vehmaanperä, J. and Koivula, A. (2008) Cloning, expression and characterization of novel thermostable family 7 cellobiohydrolases. Biotechn. Bioeng.101, 515-528. [3] Parkkinen, T., Koivula, A., Vehmaanperä, J. and Rouvinen J. (2008) Crystal structure of Melanocarpus albomyces cellobiohydrolase Cel7B in complex with cello-oligomers show high flexibility in the substrate binding. Protein Sci. 17, 1383-1394. [4] Voutilainen, S., Boer, H., Alapuranen, M., Jänis, J., Vehmaanperä, J. and Koivula, A. (2008) Improving the thermostability and activity of Melanocarpus albomyces cellobiohydrolase Cel7B. - Submitted for publication.
Original languageEnglish
Publication statusPublished - 2009
MoE publication typeNot Eligible
EventProStab2009 Meeting - Graz, Austria
Duration: 14 Apr 200917 Apr 2009

Seminar

SeminarProStab2009 Meeting
CountryAustria
CityGraz
Period14/04/0917/04/09

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cellulose 1,4-beta-cellobiosidase
engineering
thermal stability
enzymes
hydrolysis
mutagenesis
cellulose
thermophilic microorganisms
Chaetomium
Acremonium
Trichoderma reesei
bioethanol
lignocellulose
cellobiose
cellulases
glycosidases
site-directed mutagenesis
ethanol production
crystal structure
endo-1,4-beta-glucanase

Cite this

Koivula, A., Voutilainen, S., Boer, H., Puranen, T., Siika-aho, M., Alapuranen, M., ... Vehmaanperä, J. (2009). Structure, function and engineering of thermostable GH-7 family cellobiohydrolases. Paper presented at ProStab2009 Meeting, Graz, Austria.
Koivula, Anu ; Voutilainen, Sanni ; Boer, Harry ; Puranen, T. ; Siika-aho, Matti ; Alapuranen, M. ; Viikari, Liisa ; Vehmaanperä, Jari. / Structure, function and engineering of thermostable GH-7 family cellobiohydrolases. Paper presented at ProStab2009 Meeting, Graz, Austria.
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title = "Structure, function and engineering of thermostable GH-7 family cellobiohydrolases",
abstract = "Cellulases are important industrial enzymes, which can be used e.g. in the pulp and paper, textile, and detergent industry. They are currently under intensive study particularly due to their applicability in total hydrolysis of cellulosic biomass for production of bioethanol and other chemicals. Thermostable enzymes are desirable for many cellulase applications, as they are generally robust, tolerating various harsh process conditions. Thermophilic organisms are a potential source for thermostable enzymes, or as an alternative the thermostability of an enzyme can be improved by structure-based rational mutagenesis or directed evolution methods. Glycosyl hydrolase family 7 (GH-7; see http://afmb.cnrs-mrs.fr/CAZY/citing.html) cellobiohydrolases seem to be especially important in the hydrolysis of highly crystalline cellulose, and are found only in the fungal kingdom. Here, we present the characterisation of the enzymatic properties of three novel thermostable cellobiohydrolases originating from the thermophililic fungi Acremonium thermophilum, Chaetomium thermophilum and Thermoascus aurantiacus. These GH-7 family enzymes were expressed in the industrially relevant production host Trichoderma reesei, and the kinetics on small soluble substrates, cellobiose inhibition, crystalline cellulose hydrolysis and thermostability of the purified enzymes were determined. Enzymatic properties of the cellobiohydrolases were compared to those of T.reesei cellobiohydrolase Cel7A, one of the most thoroughly studied fungal cellobiohydrolases. In addition, both random and site-directed mutagenesis approaches have been used to improve the performance of the Melanocarpus albomyces Cel7B at elevated temperatures. The biochemical results are also discussed from the structural point of view based on the three-dimensional structures and homology models of these enzymes. Acknowledgements: This work has been supported by grants from the EU ({"}Technological improvement for ethanol production from lignocellulose{"} project; coordinator L. Viikari, VTT), the Academy of Finland (SV) and the Finnish Glycoscience Graduate School (SV). References : [1] Voutilainen, S., Boer, H., Linder, M., Puranen, T., Rouvinen, J., Vehmaanper{\"a}, J. and Koivula, A. (2007) Heterologous expression and random mutagenesis to improve the thermostability of Melanocarpus albomyces Cel7B cellobiohydrolase. Enz. Microb. Technol. 41, 234-243. [2] Voutilainen, S., Puranen, T., Siika-aho, M., Lappalainen, A., Alapuranen, M., Kallio, J., Hooman, S., Viikari, L., Vehmaanper{\"a}, J. and Koivula, A. (2008) Cloning, expression and characterization of novel thermostable family 7 cellobiohydrolases. Biotechn. Bioeng.101, 515-528. [3] Parkkinen, T., Koivula, A., Vehmaanper{\"a}, J. and Rouvinen J. (2008) Crystal structure of Melanocarpus albomyces cellobiohydrolase Cel7B in complex with cello-oligomers show high flexibility in the substrate binding. Protein Sci. 17, 1383-1394. [4] Voutilainen, S., Boer, H., Alapuranen, M., J{\"a}nis, J., Vehmaanper{\"a}, J. and Koivula, A. (2008) Improving the thermostability and activity of Melanocarpus albomyces cellobiohydrolase Cel7B. - Submitted for publication.",
author = "Anu Koivula and Sanni Voutilainen and Harry Boer and T. Puranen and Matti Siika-aho and M. Alapuranen and Liisa Viikari and Jari Vehmaanper{\"a}",
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Koivula, A, Voutilainen, S, Boer, H, Puranen, T, Siika-aho, M, Alapuranen, M, Viikari, L & Vehmaanperä, J 2009, 'Structure, function and engineering of thermostable GH-7 family cellobiohydrolases' Paper presented at ProStab2009 Meeting, Graz, Austria, 14/04/09 - 17/04/09, .

Structure, function and engineering of thermostable GH-7 family cellobiohydrolases. / Koivula, Anu; Voutilainen, Sanni; Boer, Harry; Puranen, T.; Siika-aho, Matti; Alapuranen, M.; Viikari, Liisa; Vehmaanperä, Jari.

2009. Paper presented at ProStab2009 Meeting, Graz, Austria.

Research output: Contribution to conferenceConference articleScientific

TY - CONF

T1 - Structure, function and engineering of thermostable GH-7 family cellobiohydrolases

AU - Koivula, Anu

AU - Voutilainen, Sanni

AU - Boer, Harry

AU - Puranen, T.

AU - Siika-aho, Matti

AU - Alapuranen, M.

AU - Viikari, Liisa

AU - Vehmaanperä, Jari

PY - 2009

Y1 - 2009

N2 - Cellulases are important industrial enzymes, which can be used e.g. in the pulp and paper, textile, and detergent industry. They are currently under intensive study particularly due to their applicability in total hydrolysis of cellulosic biomass for production of bioethanol and other chemicals. Thermostable enzymes are desirable for many cellulase applications, as they are generally robust, tolerating various harsh process conditions. Thermophilic organisms are a potential source for thermostable enzymes, or as an alternative the thermostability of an enzyme can be improved by structure-based rational mutagenesis or directed evolution methods. Glycosyl hydrolase family 7 (GH-7; see http://afmb.cnrs-mrs.fr/CAZY/citing.html) cellobiohydrolases seem to be especially important in the hydrolysis of highly crystalline cellulose, and are found only in the fungal kingdom. Here, we present the characterisation of the enzymatic properties of three novel thermostable cellobiohydrolases originating from the thermophililic fungi Acremonium thermophilum, Chaetomium thermophilum and Thermoascus aurantiacus. These GH-7 family enzymes were expressed in the industrially relevant production host Trichoderma reesei, and the kinetics on small soluble substrates, cellobiose inhibition, crystalline cellulose hydrolysis and thermostability of the purified enzymes were determined. Enzymatic properties of the cellobiohydrolases were compared to those of T.reesei cellobiohydrolase Cel7A, one of the most thoroughly studied fungal cellobiohydrolases. In addition, both random and site-directed mutagenesis approaches have been used to improve the performance of the Melanocarpus albomyces Cel7B at elevated temperatures. The biochemical results are also discussed from the structural point of view based on the three-dimensional structures and homology models of these enzymes. Acknowledgements: This work has been supported by grants from the EU ("Technological improvement for ethanol production from lignocellulose" project; coordinator L. Viikari, VTT), the Academy of Finland (SV) and the Finnish Glycoscience Graduate School (SV). References : [1] Voutilainen, S., Boer, H., Linder, M., Puranen, T., Rouvinen, J., Vehmaanperä, J. and Koivula, A. (2007) Heterologous expression and random mutagenesis to improve the thermostability of Melanocarpus albomyces Cel7B cellobiohydrolase. Enz. Microb. Technol. 41, 234-243. [2] Voutilainen, S., Puranen, T., Siika-aho, M., Lappalainen, A., Alapuranen, M., Kallio, J., Hooman, S., Viikari, L., Vehmaanperä, J. and Koivula, A. (2008) Cloning, expression and characterization of novel thermostable family 7 cellobiohydrolases. Biotechn. Bioeng.101, 515-528. [3] Parkkinen, T., Koivula, A., Vehmaanperä, J. and Rouvinen J. (2008) Crystal structure of Melanocarpus albomyces cellobiohydrolase Cel7B in complex with cello-oligomers show high flexibility in the substrate binding. Protein Sci. 17, 1383-1394. [4] Voutilainen, S., Boer, H., Alapuranen, M., Jänis, J., Vehmaanperä, J. and Koivula, A. (2008) Improving the thermostability and activity of Melanocarpus albomyces cellobiohydrolase Cel7B. - Submitted for publication.

AB - Cellulases are important industrial enzymes, which can be used e.g. in the pulp and paper, textile, and detergent industry. They are currently under intensive study particularly due to their applicability in total hydrolysis of cellulosic biomass for production of bioethanol and other chemicals. Thermostable enzymes are desirable for many cellulase applications, as they are generally robust, tolerating various harsh process conditions. Thermophilic organisms are a potential source for thermostable enzymes, or as an alternative the thermostability of an enzyme can be improved by structure-based rational mutagenesis or directed evolution methods. Glycosyl hydrolase family 7 (GH-7; see http://afmb.cnrs-mrs.fr/CAZY/citing.html) cellobiohydrolases seem to be especially important in the hydrolysis of highly crystalline cellulose, and are found only in the fungal kingdom. Here, we present the characterisation of the enzymatic properties of three novel thermostable cellobiohydrolases originating from the thermophililic fungi Acremonium thermophilum, Chaetomium thermophilum and Thermoascus aurantiacus. These GH-7 family enzymes were expressed in the industrially relevant production host Trichoderma reesei, and the kinetics on small soluble substrates, cellobiose inhibition, crystalline cellulose hydrolysis and thermostability of the purified enzymes were determined. Enzymatic properties of the cellobiohydrolases were compared to those of T.reesei cellobiohydrolase Cel7A, one of the most thoroughly studied fungal cellobiohydrolases. In addition, both random and site-directed mutagenesis approaches have been used to improve the performance of the Melanocarpus albomyces Cel7B at elevated temperatures. The biochemical results are also discussed from the structural point of view based on the three-dimensional structures and homology models of these enzymes. Acknowledgements: This work has been supported by grants from the EU ("Technological improvement for ethanol production from lignocellulose" project; coordinator L. Viikari, VTT), the Academy of Finland (SV) and the Finnish Glycoscience Graduate School (SV). References : [1] Voutilainen, S., Boer, H., Linder, M., Puranen, T., Rouvinen, J., Vehmaanperä, J. and Koivula, A. (2007) Heterologous expression and random mutagenesis to improve the thermostability of Melanocarpus albomyces Cel7B cellobiohydrolase. Enz. Microb. Technol. 41, 234-243. [2] Voutilainen, S., Puranen, T., Siika-aho, M., Lappalainen, A., Alapuranen, M., Kallio, J., Hooman, S., Viikari, L., Vehmaanperä, J. and Koivula, A. (2008) Cloning, expression and characterization of novel thermostable family 7 cellobiohydrolases. Biotechn. Bioeng.101, 515-528. [3] Parkkinen, T., Koivula, A., Vehmaanperä, J. and Rouvinen J. (2008) Crystal structure of Melanocarpus albomyces cellobiohydrolase Cel7B in complex with cello-oligomers show high flexibility in the substrate binding. Protein Sci. 17, 1383-1394. [4] Voutilainen, S., Boer, H., Alapuranen, M., Jänis, J., Vehmaanperä, J. and Koivula, A. (2008) Improving the thermostability and activity of Melanocarpus albomyces cellobiohydrolase Cel7B. - Submitted for publication.

M3 - Conference article

ER -

Koivula A, Voutilainen S, Boer H, Puranen T, Siika-aho M, Alapuranen M et al. Structure, function and engineering of thermostable GH-7 family cellobiohydrolases. 2009. Paper presented at ProStab2009 Meeting, Graz, Austria.