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  One-Step Combined Focused epPCR and Saturation Mutagenesis for Thermostability Evolution of a New Cold-Active Xylanase

Acevedo, J. P., Reetz, M. T., Asenjo, J. A., & Parra, L. P. (2017). One-Step Combined Focused epPCR and Saturation Mutagenesis for Thermostability Evolution of a New Cold-Active Xylanase. Enzyme and Microbial Technology, 100(5), 60-70. doi:10.1016/j.enzmictec.2017.02.005.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-002D-6F2E-B Version Permalink: http://hdl.handle.net/21.11116/0000-0004-599F-6
Genre: Journal Article


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Acevedo, Juan Pablo1, Author              
Reetz, Manfred T.2, 3, Author              
Asenjo, Juan A. 4, Author
Parra, Loreto P.5, 6, Author              
1Facultad de Medicina y Facultad de Ingenieria y Ciencias Aplicadas, Universidad de los Andes, San Carlos de Apoquindo, 2200 Santiago, Chile, ou_persistent22              
2Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society, ou_1445588              
3Chemistry Department, Philipps-University, 35032 Marburg, Germany, ou_persistent22              
4Centre for Biotechnology and bioengineering; CeBIB, Department of chemical Engineering and biotechnology, University of Chile, Beauchef, 851 Santiago, Chile, ou_persistent22              
5Institute for Biological and Medical Engineering, Schools of Engineering, Medicine and Medicine and Biological Sciences, Pontificia Unsiversidad Católica de Chile, Avenida Vicuna Mackenna, 4860 Santiago, chile, ou_persistent22              
6Department of Chemical and Bioprocesses Engineering, School of Engineering, Pontificia Universidad Católica de Chile, Avenida Vicũna Mackenna, 4860 Santiago, Chile, ou_persistent22              


Free keywords: Xylanase, Cold-active enzymes, Thermostability, Directed evolution, epPCR
 Abstract: Enzymes active at low temperature are of great interest for industrial bioprocesses due to their high efficiency at a low energy cost. One of the particularities of naturally evolved cold-active enzymes is their increased enzymatic activity at low temperature, however the low thermostability presented in this type of enzymes is still a major drawback for their application in biocatalysis. Directed evolution of cold-adapted enzymes to a more thermostable version, appears as an attractive strategy to fulfill the stability and activity requirements for the industry. This paper describes the recombinant expression and characterization of a new and highly active cold-adapted xylanase from the GH-family 10 (Xyl-L), and the use of a novel one step combined directed evolution technique that comprises saturation mutagenesis and focused epPCR as a feasible semi-rational strategy to improve the thermostability. The Xyl-L enzyme was cloned from a marine-Antarctic bacterium, Psychrobacter sp. strain 2–17, recombinantly expressed in E. coli strain BL21(DE3) and characterized enzymatically. Molecular dynamic simulations using a homology model of the catalytic domain of Xyl-L were performed to detect flexible regions and residues, which are considered to be the possible structural elements that define the thermolability of this enzyme. Mutagenic libraries were designed in order to stabilize the protein introducing mutations in some of the flexible regions and residues identified. Twelve positive mutant clones were found to improve the T5015value of the enzyme, in some cases without affecting the activity at 25◦C. The best mutant showed a 4.3◦C increase in its T5015. The efficiency of the directed evolution approach can also be expected to work in the protein engineering of stereoselectivity.


Language(s): eng - English
 Dates: 2016-08-262017-02-092017-02-132017-05-01
 Publication Status: Published in print
 Pages: 11
 Publishing info: -
 Table of Contents: -
 Rev. Method: Peer
 Identifiers: DOI: 10.1016/j.enzmictec.2017.02.005
 Degree: -



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Source 1

Title: Enzyme and Microbial Technology
  Other : Enzyme Microb. Technol.
Source Genre: Journal
Publ. Info: Amsterdam : Elsevier
Pages: - Volume / Issue: 100 (5) Sequence Number: - Start / End Page: 60 - 70 Identifier: ISSN: 0141-0229
CoNE: https://pure.mpg.de/cone/journals/resource/954925471335