Efficient screening of fungal cellobiohydrolase class I enzymes for thermostabilizing sequence blocks by SCHEMA structure-guided recombination
العنوان: | Efficient screening of fungal cellobiohydrolase class I enzymes for thermostabilizing sequence blocks by SCHEMA structure-guided recombination |
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المؤلفون: | Russell S. Komor, Christopher D. Snow, Frances H. Arnold, Philip A. Romero, Shannon Mohler, Xinlin Yu, Arvind Kanaan, Pete Heinzelman |
المصدر: | Protein Engineering, Design and Selection. 23:871-880 |
بيانات النشر: | Oxford University Press (OUP), 2010. |
سنة النشر: | 2010 |
مصطلحات موضوعية: | Models, Molecular, Bioengineering, Cellulase, Biology, Protein Engineering, Biochemistry, law.invention, law, Enzyme Stability, Cellulose 1,4-beta-Cellobiosidase, Amino Acid Sequence, Cellulose, Molecular Biology, Peptide sequence, chemistry.chemical_classification, Thermophile, fungi, Fungi, Temperature, Protein engineering, Recombinant Proteins, Amino acid, Enzyme, chemistry, biology.protein, Recombinant DNA, Sequence space (evolution), Biotechnology |
الوصف: | 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. |
تدمد: | 1741-0134 1741-0126 |
URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::f04816be826c3166bbda1a7922c2c62a https://doi.org/10.1093/protein/gzq063 |
حقوق: | OPEN |
رقم الأكسشن: | edsair.doi.dedup.....f04816be826c3166bbda1a7922c2c62a |
قاعدة البيانات: | OpenAIRE |
تدمد: | 17410134 17410126 |
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