title: Efficient screening of fungal cellobiohydrolase class I enzymes for thermostabilizing sequence blocks by SCHEMA structure-guided recombination.
abstract: We describe an efficient SCHEMA recombination-based approach for screening homologous enzymes to identify stabilizing amino acid sequence blocks. This approach has been used to generate active, thermostable cellobiohydrolase class I (CBH I) enzymes from the 390 625 possible chimeras that can be made by swapping eight blocks from five fungal homologs. Constructing and characterizing the parent enzymes and just 32 'monomeras' containing a single block from a homologous enzyme allowed stability contributions to be assigned to 36 of the 40 blocks from which the CBH I chimeras can be assembled. Sixteen of 16 predicted thermostable chimeras, with an average of 37 mutations relative to the closest parent, are more thermostable than the most stable parent CBH I, from the thermophilic fungus Talaromyces emersonii. Whereas none of the parent CBH Is were active >65°C, stable CBH I chimeras hydrolyzed solid cellulose at 70°C. In addition to providing a collection of diverse, thermostable CBH Is that can complement previously described stable CBH II chimeras (Heinzelman et al., Proc. Natl Acad. Sci. USA 2009;106:5610-5615) in formulating application-specific cellulase mixtures, the results show the utility of SCHEMA recombination for screening large swaths of natural enzyme sequence space for desirable amino acid blocks.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Pete
authors.initials: P
authors.lastname: Heinzelman
authors.forename: Russell
authors.initials: R
authors.lastname: Komor
authors.forename: Arvind
authors.initials: A
authors.lastname: Kanaan
authors.forename: Philip
authors.initials: P
authors.lastname: Romero
authors.forename: Xinlin
authors.initials: X
authors.lastname: Yu
authors.forename: Shannon
authors.initials: S
authors.lastname: Mohler
authors.forename: Christopher
authors.initials: C
authors.lastname: Snow
authors.forename: Frances
authors.initials: F
authors.lastname: Arnold
mesh_terms: D000595:Amino Acid Sequence; D002482:Cellulose / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D005658:Fungi / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase