Heinzelman, Pete and Komor, Russell and Kanaan, Arvind and Romero, Philip and Yu, Xinlin and Mohler, Shannon and Snow, Christopher and Arnold, Frances (2010) Efficient screening of fungal cellobiohydrolase class I enzymes for thermostabilizing sequence blocks by SCHEMA structure-guided recombination. Protein Engineering, Design and Selection, 23 (11). pp. 871-880. ISSN 1741-0126 http://resolver.caltech.edu/CaltechAUTHORS:20101108-110520683
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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.
|Additional Information:||© The Author 2010. Published by Oxford University Press. Received August 18, 2010; revised August 18, 2010; accepted August 24, 2010. Published online September 16, 2010. Edited by Stephen Withers. Funding: This work was supported by grants from the Army-Industry Institute for Collaborative Biotechnologies and the Caltech Innovation Institute.|
|Subject Keywords:||biofuel; cellulase; directed evolution; protein recombination; protein thermostability; CBH I; Cel7A|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Tony Diaz|
|Deposited On:||19 Nov 2010 22:59|
|Last Modified:||26 Dec 2012 12:36|
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