Document Detail

Directed evolution of a thermophilic endoglucanase (Cel5A) into highly active Cel5A variants with an expanded temperature profile.
MedLine Citation:
PMID:  21501637     Owner:  NLM     Status:  Publisher    
Cel5A is a highly active endoglucanase from Thermoanaerobacter tengcongensis MB4, displaying an optimal temperature range between 75 and 80°C. After three rounds of error-prone PCR and screening of 4700 mutants, five variants of Cel5A with improved activities were identified by Congo Red based screening method. Compared with the wild type, the best variants 3F6 and C3-13 display 135±6% and 193±8% of the wild type specific activity for the substrate carboxymethyl cellulose (CMC), besides improvements in the relative expression level in Escherichia coli system. Remarkable are especially the improvements in activities at reduced temperatures (50% of maximum activity at 50°C and about 45°C respectively, while 65°C for the wild type). Molecular Dynamics simulations performed on the 3F6 and C3-13 variants show a decreased number of intra-Cel5A hydrogen bonds compared to the wild type, implying a more flexible protein skeleton which correlates well to the higher catalytic activity at lower temperatures. To investigate functions of each individual amino acid position site-directed (saturation) mutagenesis were generated and screened. Amino acid positions Val249 and Ile321 were found to be crucial for improving activity and residue Ile13 (encoded by rare codon AUA) yields an improved expression level in E. coli.
Chaoning Liang; Marco Fioroni; Francisco Rodríguez-Ropero; Yanfen Xue; Ulrich Schwaneberg; Yanhe Ma
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2011-4-8
Journal Detail:
Title:  Journal of biotechnology     Volume:  -     ISSN:  1873-4863     ISO Abbreviation:  -     Publication Date:  2011 Apr 
Date Detail:
Created Date:  2011-4-19     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8411927     Medline TA:  J Biotechnol     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
Copyright © 2011. Published by Elsevier B.V.
State Key Laboratory of Microbial Resource, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing 100101, People's Republic of China; Graduate School, Chinese Academy of Sciences, Beijing 100049, People's Republic of China.
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