10-04: A Family of Thermostable Fungal Cellulases Created by Structure-Guided Recombination

SCHEMA structure-guided recombination of fungal cellulases has yielded a collection of novel, highly thermostable CBH2 chimeras.  An appreciable fraction of a sample set of cellulase chimeras were secreted by a heterologous host in catalytically active form.  Many of these chimeras have half-lives of thermal inactivation that are greater than the most stable parent cellulase.  We predict that the collection of cellulase chimeras contains hundreds of highly stable cellulases.  All of the active sequences chosen from the chimeras predicted to be thermostable based on the sample set sequence-stability data retained more activity than the most stable parent upon incubation at elevated temperature.  These validated thermostable cellulases have high sequence diversity, differing from their closest natural homologs at up to 63 amino acid positions.  Selected thermostable chimeras hydrolyzed phosphoric acid swollen cellulose at temperatures between 7 and 15°C higher than the parent enzymes.  These chimeras also hydrolyzed as much or more cellulose than the parent cellulases in long-time cellulose hydrolysis assays and had pH/activity profiles as broad, or broader than, the parent enzymes.  Generating this group of diverse, thermostable fungal cellulases is the first step in building an inventory of thermostable cellulases from which optimized enzyme mixtures for biomass conversion can be formulated.