title: The development of a thermostable CiP (Coprinus cinereus peroxidase) through in silico design.
abstract: Protein thermostability is a crucial issue in the practical application of enzymes, such as inorganic synthesis and enzymatic polymerization of phenol derivatives. Much attention has been focused on the enhancement and numerous successes have been achieved through protein engineering methods. Despite fruitful results based on random mutagenesis, it was still necessary to develop a novel strategy that can reduce the time and effort involved in this process. In this study, a rapid and effective strategy is described for increasing the thermal stability of a protein. Instead of random mutagenesis, a rational strategy was adopted to theoretically stabilize the thermo labile residues of a protein using computational methods. Protein residues with high flexibility can be thermo labile due to their large range of movement. Here, residue B factor values were used to identify putatively thermo labile residues and the RosettaDesign program was applied to search for stable sequences. Coprinus cinereus (CiP) heme peroxidase was selected as a model protein for its importance in commercial applications, such as the polymerization of phenolic compounds. Eleven CiP residues with the highest B factor values were chosen as target mutation sites for thermostabilization, and then redesigned using RosettaDesign to identify sequences. Eight mutants based on the redesigns, were produced as functional enzymes and two of these (S323Y and E328D) showed increased thermal stability over the wild-type in addition to conserved catalytic activity. Thus, this strategy can be used as a rapid and effective in silico design tool for obtaining thermostable proteins.
authors: Chemical Biotechnology Research Center, Korea Research Institute of Chemical Technology, Yuseong-gu, Daejeon, Korea.
authors: Su Jin
authors: SJ
authors: Kim
authors: Jeong Ah
authors: JA
authors: Lee
authors: Jeong Chan
authors: JC
authors: Joo
authors: Young Je
authors: YJ
authors: Yoo
authors: Yong Hwan
authors: YH
authors: Kim
authors: Bong Keun
authors: BK
authors: Song
mesh_terms: D003302:Coprinus / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D005470:Fluorometry; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009195:Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D009195:Peroxidase