Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases.
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 degrees C) and more active (two- to fourfold) in hydrolysis of microcrystalline cellulose (Avicel) at 45 degrees 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 degrees 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.
Voutilainen
Sanni P
SP
VTT Technical Research Centre of Finland, P.O. Box 1000, FI-02044 VTT, Finland.
Puranen
Terhi
T
Siika-Aho
Matti
M
Lappalainen
Arja
A
Alapuranen
Marika
M
Kallio
Jarno
J
Hooman
Satu
S
Viikari
Liisa
L
Vehmaanperä
Jari
J
Koivula
Anu
A
D000164:Acremonium / Q000201:enzymology*; D001665:Binding Sites; D002475:Cellobiose / Q000494:pharmacology; D002482:Cellulose / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002597:Chaetomium / Q000201:enzymology*; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D032641:Eurotiales / Q000201:enzymology*; D006358:Hot Temperature; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014242:Trichoderma / Q000201:enzymology
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D002475:Cellobiose; D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase