Journal
JOURNAL OF BIOTECHNOLOGY
Volume 154, Issue 1, Pages 46-53Publisher
ELSEVIER
DOI: 10.1016/j.jbiotec.2011.03.025
Keywords
Thermoanaerobacter tengcongensis; Directed evolution; Error-prone PCR; Endoglucanase; Molecular Dynamics
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Funding
- National Natural Science Foundation of China (NSFC) [30621005]
- Ministry of Science and Technology of China (MOST) [2007CB707801, 2009CB724705, 2006AA020201]
- Deutscher Akademischer Austausch Dienst (DAAD)
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Cel5A is a highly active endoglucanase from Thermoanaerobacter tengcongensis MB4, displaying an optimal temperature range between 75 and 80 degrees 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 degrees C and about 45 degrees C respectively, while 65 degrees 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. (C) 2011 Elsevier B. V. All rights reserved.
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