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Directed evolution of a thermophilic endoglucanase (Cel5A) into highly active Cel5A variants with an expanded temperature profile

Paper ID Volume ID Publish Year Pages File Format Full-Text
23764 43471 2011 8 PDF Available
Title
Directed evolution of a thermophilic endoglucanase (Cel5A) into highly active Cel5A variants with an expanded temperature profile
Abstract

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.

Keywords
Thermoanaerobacter tengcongensis; Directed evolution; Error-prone PCR; Endoglucanase; Molecular Dynamics
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Directed evolution of a thermophilic endoglucanase (Cel5A) into highly active Cel5A variants with an expanded temperature profile
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 154, Issue 1, 10 June 2011, Pages 46–53
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us