Improving the thermostability and activity of Melanocarpus albomyces cellobiohydrolase Cel7B.
Two different types of approach were taken to improve the hydrolytic activity towards crystalline cellulose at elevated temperatures of Melanocarpus albomyces Cel7B (Ma Cel7B), a single-module GH-7 family cellobiohydrolase. Structure-guided protein engineering was used to introduce an additional tenth disulphide bridge to the Ma Cel7B catalytic module. In addition, a fusion protein was constructed by linking a cellulose-binding module (CBM) and a linker from the Trichoderma reesei Cel7A to the C terminus of Ma Cel7B. Both approaches proved successful. The disulphide bridge mutation G4C/M70C located near the N terminus, close to the entrance of the active site tunnel of Ma Cel7B, led to improved thermostability (DeltaT (m) = 2.5 degrees C). By adding the earlier found thermostability-increasing mutation S290T (DeltaT (m) = 1.5 degrees C) together with the disulphide bridge mutation, the unfolding temperature was increased by 4 degrees C (mutant G4C/M70C/S290T) compared to that of the wild-type enzyme, thus showing an additive effect on thermostability. Both disulphide mutants had increased activity towards microcrystalline cellulose (Avicel) at 75 degrees C, apparently solely because of their improved thermostability. The addition of a CBM also improved the thermostability (DeltaT (m) = 2.5 degrees C) and caused a clear (sevenfold) increase in the hydrolysis activity of Ma Cel7B towards Avicel at 70 degrees C.
Voutilainen
Sanni P
SP
VTT Technical Research Centre of Finland, P.O. Box 1000, Espoo, 02044 VTT, Finland.
Boer
Harry
H
Alapuranen
Marika
M
Jänis
Janne
J
Vehmaanperä
Jari
J
Koivula
Anu
A
D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D006868:Hydrolysis; D007700:Kinetics; D015202:Protein Engineering; D020037:Sordariales / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D005656:Fungal Proteins; D043366:Cellulose 1,4-beta-Cellobiosidase