Expression of Talaromyces emersonii cellobiohydrolase Cel7A in Saccharomyces cerevisiae and rational mutagenesis to improve its thermostability and activity.
We report here a successful expression of a single-module GH-7 family cellobiohydrolase Cel7A from a thermophilic fungus Talaromyces emersonii (Te Cel7A) in Saccharomyces cerevisiae. The heterologous expression system allowed structure-guided protein engineering to improve the thermostability and activity of Te Cel7A. Altogether six different mutants aimed at introducing additional disulphide bridges to the catalytic module of Te Cel7A were designed. These included addition of five individual S-S bridges in or between the loops extending from the beta-sandwich fold, and located either near the active site tunnel or forming the tunnel in Te Cel7A. A triple mutant containing the three best S-S mutations was also engineered. Three out of five single S-S mutants all had clearly improved thermostability which was also reflected as improved Avicel hydrolysis efficiency at 75 degrees C. The best mutant was the triple mutant whose unfolding temperature was improved by 9 degrees C leading to efficient microcrystalline cellulose hydrolysis at 80 degrees C. All the additional S-S bonds contributed mainly to the thermostability of the Te Cel7A, but one of the mutants (N54C/P191C) also showed, somewhat surprisingly, improved activity even at room temperature.
Voutilainen
Sanni P
SP
VTT Technical Research Centre of Finland, PO Box 1000, FI-02044 VTT, Finland.
Murray
Patrick G
PG
Tuohy
Maria G
MG
Koivula
Anu
A
D002482:Cellulose / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D055550:Protein Stability; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D032901:Talaromyces / Q000201:enzymology*; D013696:Temperature
D050505:Mutant Proteins; D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase