title: One-step combined focused epPCR and saturation mutagenesis for thermostability evolution of a new cold-active xylanase.
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.
authors.affiliation: Facultad de Medicina y Facultad de Ingeniería y Ciencias Aplicadas, Universidad de los Andes, San Carlos de Apoquindo, 2200 Santiago, Chile.
authors.forename: Juan Pablo
authors.initials: JP
authors.lastname: Acevedo
authors.affiliation: Max-Planck-Institut für Kohlenforschung, 45070 Mülheim, Germany; Chemistry Department, Philipps-University, 35032 Marburg, Germany.
authors.forename: Manfred T
authors.initials: MT
authors.lastname: Reetz
authors.affiliation: Centre for Biotechnology and Bioengineering, CeBiB, Department of Chemical Engineering and Biotechnology, University of Chile, Beauchef, 851 Santiago, Chile.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Asenjo
authors.affiliation: Institute for Biological and Medical Engineering, Schools of Engineering, Medicine and Biological Sciences, Pontificia Universidad Católica de Chile, Avenida Vicuña Mackenna, 4860 Santiago, Chile; Department of Chemical and Bioprocesses Engineering, School of Engineering, Pontificia Universidad Católica de Chile, Avenida Vicuña Mackenna, 4860 Santiago, Chile. Electronic address: lparrat@uc.cl.
authors.forename: Loreto P
authors.initials: LP
authors.lastname: Parra
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D003001:Cloning, Molecular; D003080:Cold Temperature; D019020:Directed Molecular Evolution / Q000379:methods*; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability / Q000235:genetics; D005798:Genes, Bacterial; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016296:Mutagenesis; D016133:Polymerase Chain Reaction / Q000379:methods*; D015202:Protein Engineering / Q000379:methods; D044096:Psychrobacter / Q000201:enzymology / Q000235:genetics; D040681:Structural Homology, Protein
chemical_list: D001426:Bacterial Proteins; D043364:Endo-1,4-beta Xylanases
keywords: Cold-active enzymes; Directed evolution; Thermostability; Xylanase; epPCR