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Efficient screening of fungal cellobiohydrolase class I enzymes for thermostabilizing sequence blocks by SCHEMA structure-guided recombination

Efficient screening of fungal cellobiohydrolase class I enzymes for thermostabilizing sequence... 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 >65C, stable CBH I chimeras hydrolyzed solid cellulose at 70C. 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;106:56105615) 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Protein Engineering, Design and Selection Oxford University Press

Efficient screening of fungal cellobiohydrolase class I enzymes for thermostabilizing sequence blocks by SCHEMA structure-guided recombination

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References (23)

Publisher
Oxford University Press
Copyright
The Author 2010. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissionsoxfordjournals.org
Subject
Original articles
ISSN
1741-0126
eISSN
1741-0134
DOI
10.1093/protein/gzq063
pmid
20847102
Publisher site
See Article on Publisher Site

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 >65C, stable CBH I chimeras hydrolyzed solid cellulose at 70C. 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;106:56105615) 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.

Journal

Protein Engineering, Design and Selection – Oxford University Press

Published: Nov 16, 2010

Keywords: biofuel cellulase directed evolution protein recombination protein thermostability CBH I Cel7A

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