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Improved activity and thermostability of Bacillus pumilus lipase by directed evolution

Paper ID Volume ID Publish Year Pages File Format Full-Text
23570 43452 2013 7 PDF Available
Title
Improved activity and thermostability of Bacillus pumilus lipase by directed evolution
Abstract

To improve enzymatic activity of Bacillus pumilus lipases, DNA shuffling was applied to two lipase genes from local B. pumilus isolates. Using a high-throughput activity assay, the mutant with highest activity was selected. This chimeric mutant (L3-3), carrying two crossover positions and three point mutations, has a specific activity 6.4 and 8.2 times higher than the two parent enzymes. The mutant also is more tolerant to various detergents and organic solvents, and has a 9 times longer half-life at 50 °C. Homology modeling of mutant L3-3, based on the highly homologous B. subtilis lipase A, shows that the increased thermostability is likely due to structural rigidification and reduced surface hydrophobicity. Increased specific activity may result from the location of mutations close to the active site. Together, our results show that it is possible to evolve, by DNA shuffling, B. pumilus lipase variants with improved applicability as biocatalysts, even if the two parent enzymes are highly similar.

► DNA shuffling with two lipase genes from local Bacillus pumilus isolates. ► High-throughput screening for activity; mutant with highest activity characterized. ► Specific activity increased up to 8-fold, half-life at 50 °C increased 9-fold. ► Homology modeling to discuss the role of sequence changes.

Keywords
Bacillus pumilus lipase; Biocatalyst; Directed evolution; DNA shuffling; Thermostability; 3D homology model
First Page Preview
Improved activity and thermostability of Bacillus pumilus lipase by directed evolution
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 164, Issue 1, 10 March 2013, Pages 123–129
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering