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

• Published in: Biotechnology and bioengineering 2008-10, Vol.101 (3), p.515-528
• Main Authors: Voutilainen, Sanni P, Puranen, Terhi, Siika-aho, Matti, Lappalainen, Arja, Alapuranen, Marika, Kallio, Jarno, Hooman, Satu, Viikari, Liisa, Vehmaanperä, Jari, Koivula, Anu
• Format: Article
• Language: English
• Subjects: Acremonium; Acremonium - enzymology; Acremonium thermophilum; Binding Sites; Biological and medical sciences; Biotechnology; Carbohydrates; cellobiohydrolase; Cellobiose - pharmacology; Cellulose; Cellulose - metabolism; Cellulose 1,4-beta-Cellobiosidase - chemistry; Cellulose 1,4-beta-Cellobiosidase - genetics; Cellulose 1,4-beta-Cellobiosidase - metabolism; Chaetomium; Chaetomium - enzymology; Chaetomium thermophilum; Cloning; Cloning, Molecular; Enzyme Inhibitors - pharmacology; Enzyme Stability; Enzymes; Eurotiales - enzymology; Fundamental and applied biological sciences. Psychology; Fungi; Hot Temperature; Hypocrea jecorina; Models, Molecular; Polymer chemistry; Polymers; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Thermoascus aurantiacus; Trichoderma - enzymology; Trichoderma reesei
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.21940

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. Biotechnol. Bioeng. 2008;101: 515-528.