title: Highly thermostable fungal cellobiohydrolase I (Cel7A) engineered using predictive methods.
abstract: Building on our previous efforts to generate thermostable chimeric fungal cellobiohydrolase I (CBH I, also known as Cel7A) cellulases by structure-guided recombination, we used FoldX and a 'consensus' sequence approach to identify individual mutations present in the five homologous parent CBH I enzymes which further stabilize the chimeras. Using the FoldX force field, we calculated the effect on ΔG(Folding) of each candidate mutation in a number of CBH I structures and chose those predicted to be stabilizing in multiple structures. With an alignment of 41 CBH I sequences, we also used amino acid frequencies at each candidate position to calculate predicted effects on ΔG(Folding). A combination of mutations chosen using these methods increased the T(50) of the most thermostable chimera by an additional 4.7°C, to yield a CBH I with T(50) of 72.1°C, which is 9.2°C higher than that of the most stable native CBH I, from Talaromyces emersonii. This increased stability resulted in a 10°C increase in the optimal temperature for activity, to 65°C, and a 50% increase in total sugar production from crystalline cellulose at the optimal temperature, compared with native T.emersonii CBH I.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Russell S
authors.initials: RS
authors.lastname: Komor
authors.forename: Philip A
authors.initials: PA
authors.lastname: Romero
authors.forename: Catherine B
authors.initials: CB
authors.lastname: Xie
authors.forename: Frances H
authors.initials: FH
authors.lastname: Arnold
mesh_terms: D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D015202:Protein Engineering; D017510:Protein Folding; D055550:Protein Stability; D032901:Talaromyces; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D043366:Cellulose 1,4-beta-Cellobiosidase