Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases

Sanni Voutilainen, Terhi Puranen, Matti Siika-aho, Arja Lappalainen, Marika Alapuranen, Jarno Kallio, Satu Hooman, Liisa Viikari, Jari Vehmaanperä, Anu Koivula (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

As part of the effort to find better cellulases for bioethanol production processes, we were looking for novel GH‐7 family cellobiohydrolases, which would be particularly active on insoluble polymeric substrates and participate in the rate‐limiting step in the hydrolysis of cellulose. The enzymatic properties were studied and are reported here for family 7 cellobiohydrolases from the thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum. The Trichoderma reesei Cel7A enzyme was used as a reference in the experiments. As the native T. aurantiacus Cel7A has no carbohydrate‐binding module (CBM), recombinant proteins having the CBM from either the C. thermophilum Cel7A or the T. reesei Cel7A were also constructed. All these novel acidic cellobiohydrolases were more thermostable (by 4–10°C) and more active (two‐ to fourfold) in hydrolysis of microcrystalline cellulose (Avicel) at 45°C than T. reesei Cel7A. The C. thermophilum Cel7A showed the highest specific activity and temperature optimum when measured on soluble substrates. The most effective enzyme for Avicel hydrolysis at 70°C, however, was the 2‐module version of the T. aurantiacus Cel7A, which was also relatively weakly inhibited by cellobiose. These results are discussed from the structural point of view based on the three‐dimensional homology models of these enzymes.
Original languageEnglish
Pages (from-to)515-528
Number of pages13
JournalBiotechnology and Bioengineering
Volume101
Issue number3
DOIs
Publication statusPublished - 2008
MoE publication typeA1 Journal article-refereed

Fingerprint

Thermoascus
Cellulose 1,4-beta-Cellobiosidase
Cloning
Cellulose
Organism Cloning
Hydrolysis
Enzymes
Chaetomium
Acremonium
Cellobiose
Cellulases
Recombinant proteins
Bioethanol
Trichoderma
Substrates
Fungi
Recombinant Proteins
Temperature
Experiments

Keywords

  • cellulose
  • cellobiohydrolase
  • Trichoderma reesei
  • Chaetomium thermophilum
  • Acremonium thermophilum
  • Thermoascus aurantiacus

Cite this

Voutilainen, Sanni ; Puranen, Terhi ; Siika-aho, Matti ; Lappalainen, Arja ; Alapuranen, Marika ; Kallio, Jarno ; Hooman, Satu ; Viikari, Liisa ; Vehmaanperä, Jari ; Koivula, Anu. / Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases. In: Biotechnology and Bioengineering. 2008 ; Vol. 101, No. 3. pp. 515-528.
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abstract = "As part of the effort to find better cellulases for bioethanol production processes, we were looking for novel GH‐7 family cellobiohydrolases, which would be particularly active on insoluble polymeric substrates and participate in the rate‐limiting step in the hydrolysis of cellulose. The enzymatic properties were studied and are reported here for family 7 cellobiohydrolases from the thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum. The Trichoderma reesei Cel7A enzyme was used as a reference in the experiments. As the native T. aurantiacus Cel7A has no carbohydrate‐binding module (CBM), recombinant proteins having the CBM from either the C. thermophilum Cel7A or the T. reesei Cel7A were also constructed. All these novel acidic cellobiohydrolases were more thermostable (by 4–10°C) and more active (two‐ to fourfold) in hydrolysis of microcrystalline cellulose (Avicel) at 45°C than T. reesei Cel7A. The C. thermophilum Cel7A showed the highest specific activity and temperature optimum when measured on soluble substrates. The most effective enzyme for Avicel hydrolysis at 70°C, however, was the 2‐module version of the T. aurantiacus Cel7A, which was also relatively weakly inhibited by cellobiose. These results are discussed from the structural point of view based on the three‐dimensional homology models of these enzymes.",
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Voutilainen, S, Puranen, T, Siika-aho, M, Lappalainen, A, Alapuranen, M, Kallio, J, Hooman, S, Viikari, L, Vehmaanperä, J & Koivula, A 2008, 'Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases', Biotechnology and Bioengineering, vol. 101, no. 3, pp. 515-528. https://doi.org/10.1002/bit.21940

Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases. / Voutilainen, Sanni; Puranen, Terhi; Siika-aho, Matti; Lappalainen, Arja; Alapuranen, Marika; Kallio, Jarno; Hooman, Satu; Viikari, Liisa; Vehmaanperä, Jari; Koivula, Anu (Corresponding Author).

In: Biotechnology and Bioengineering, Vol. 101, No. 3, 2008, p. 515-528.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases

AU - Voutilainen, Sanni

AU - Puranen, Terhi

AU - Siika-aho, Matti

AU - Lappalainen, Arja

AU - Alapuranen, Marika

AU - Kallio, Jarno

AU - Hooman, Satu

AU - Viikari, Liisa

AU - Vehmaanperä, Jari

AU - Koivula, Anu

PY - 2008

Y1 - 2008

N2 - As part of the effort to find better cellulases for bioethanol production processes, we were looking for novel GH‐7 family cellobiohydrolases, which would be particularly active on insoluble polymeric substrates and participate in the rate‐limiting step in the hydrolysis of cellulose. The enzymatic properties were studied and are reported here for family 7 cellobiohydrolases from the thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum. The Trichoderma reesei Cel7A enzyme was used as a reference in the experiments. As the native T. aurantiacus Cel7A has no carbohydrate‐binding module (CBM), recombinant proteins having the CBM from either the C. thermophilum Cel7A or the T. reesei Cel7A were also constructed. All these novel acidic cellobiohydrolases were more thermostable (by 4–10°C) and more active (two‐ to fourfold) in hydrolysis of microcrystalline cellulose (Avicel) at 45°C than T. reesei Cel7A. The C. thermophilum Cel7A showed the highest specific activity and temperature optimum when measured on soluble substrates. The most effective enzyme for Avicel hydrolysis at 70°C, however, was the 2‐module version of the T. aurantiacus Cel7A, which was also relatively weakly inhibited by cellobiose. These results are discussed from the structural point of view based on the three‐dimensional homology models of these enzymes.

AB - As part of the effort to find better cellulases for bioethanol production processes, we were looking for novel GH‐7 family cellobiohydrolases, which would be particularly active on insoluble polymeric substrates and participate in the rate‐limiting step in the hydrolysis of cellulose. The enzymatic properties were studied and are reported here for family 7 cellobiohydrolases from the thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum. The Trichoderma reesei Cel7A enzyme was used as a reference in the experiments. As the native T. aurantiacus Cel7A has no carbohydrate‐binding module (CBM), recombinant proteins having the CBM from either the C. thermophilum Cel7A or the T. reesei Cel7A were also constructed. All these novel acidic cellobiohydrolases were more thermostable (by 4–10°C) and more active (two‐ to fourfold) in hydrolysis of microcrystalline cellulose (Avicel) at 45°C than T. reesei Cel7A. The C. thermophilum Cel7A showed the highest specific activity and temperature optimum when measured on soluble substrates. The most effective enzyme for Avicel hydrolysis at 70°C, however, was the 2‐module version of the T. aurantiacus Cel7A, which was also relatively weakly inhibited by cellobiose. These results are discussed from the structural point of view based on the three‐dimensional homology models of these enzymes.

KW - cellulose

KW - cellobiohydrolase

KW - Trichoderma reesei

KW - Chaetomium thermophilum

KW - Acremonium thermophilum

KW - Thermoascus aurantiacus

U2 - 10.1002/bit.21940

DO - 10.1002/bit.21940

M3 - Article

VL - 101

SP - 515

EP - 528

JO - Biotechnology and Bioengineering

JF - Biotechnology and Bioengineering

SN - 0006-3592

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