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Enhancement of the thermostability and activity of mesophilic Clostridium cellulovorans EngD by in vitro DNA recombination with Clostridium thermocellum CelE

Paper ID Volume ID Publish Year Pages File Format Full-Text
21349 43218 2010 6 PDF Available
Title
Enhancement of the thermostability and activity of mesophilic Clostridium cellulovorans EngD by in vitro DNA recombination with Clostridium thermocellum CelE
Abstract

The thermal stability and catalytic activity of endoglucanase (EngD) from mesophilic Clostridium cellulovorans were improved by evolutionary molecular engineering. Thermostable mutants were isolated after staggered extension process (StEP) with celE from thermophilic Clostridium thermocellum performed to conduct family shuffling and overlay screening of the resultant mutant library. The relative activity of the best-evolved clone has been improved of about 2 times higher at 50 °C and showed a higher kcat/Km value than its engD parental clone. We determined that these variants had two amino acid substitutions (L157N, Q158E) and confirmed their effects by substituting these amino acids in the parental gene by site-directed mutagenesis. These substitutions resulted in an increase in hydrophilic or charged residues. Our results demonstrate that in vitro recombination is an effective approach to improve the thermostability and enzymatic activity of a mesophilic enzyme.

Keywords
Clostridium cellulovorans; Clostridium thermocellum; Cellulase; Thermostability; in vitro DNA recombination
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Enhancement of the thermostability and activity of mesophilic Clostridium cellulovorans EngD by in vitro DNA recombination with Clostridium thermocellum CelE
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 109, Issue 4, April 2010, Pages 331–336
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