One-step combined focused epPCR and saturation mutagenesis for thermostability evolution of a new cold-active xylanase
tEnzymes active at low temperature are of great interest for industrial bioprocesses due to their highefficiency at a low energy cost. One of the particularities of naturally evolved cold-active enzymes istheir increased enzymatic activity at low temperature, however the low thermostability presente...
| Published in: | Enzyme and Microbial Technology |
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| Main Authors: | , , , |
| Format: | Article in Journal/Newspaper |
| Language: | English |
| Published: |
Elsevier
2017
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| Subjects: | |
| Online Access: | https://doi.org/10.1016/j.enzmictec.2017.02.005 https://repositorio.uchile.cl/handle/2250/168933 |
| Summary: | tEnzymes active at low temperature are of great interest for industrial bioprocesses due to their highefficiency at a low energy cost. One of the particularities of naturally evolved cold-active enzymes istheir increased enzymatic activity at low temperature, however the low thermostability presented inthis type of enzymes is still a major drawback for their application in biocatalysis. Directed evolutionof cold-adapted enzymes to a more thermostable version, appears as an attractive strategy to fulfill thestability and activity requirements for the industry. This paper describes the recombinant expression andcharacterization of a new and highly active cold-adapted xylanase from the GH-family 10 (Xyl-L), andthe use of a novel one step combined directed evolution technique that comprises saturation mutage-nesis and focused epPCR as a feasible semi-rational strategy to improve the thermostability. The Xyl-Lenzyme was cloned from a marine-Antarctic bacterium, Psychrobacter sp. strain 2–17, recombinantlyexpressed in E. coli strain BL21(DE3) and characterized enzymatically. Molecular dynamic simulationsusing a homology model of the catalytic domain of Xyl-L were performed to detect flexible regions andresidues, which are considered to be the possible structural elements that define the thermolability ofthis enzyme. Mutagenic libraries were designed in order to stabilize the protein introducing mutationsin some of the flexible regions and residues identified. Twelve positive mutant clones were found toimprove the T5015value of the enzyme, in some cases without affecting the activity at 25◦C. The bestmutant showed a 4.3◦C increase in its T5015. The efficiency of the directed evolution approach can alsobe expected to work in the protein engineering of stereoselectivity. |
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