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.

Explorations and Applications of Enzyme-linked Bioremediation of Synthetic Dyes

“…Protein thermostability is a crucial issue in the practical application of enzymes in dye bioremediation applications. Several studies have reported the application of protein engineering techniques to improve thermal stabilities of peroxidases [68,145,146], azoreductase [147], and laccases [148,149]. In silico design and site-directed mutagenesis of thermo-labile residues of Coprinus cinereus peroxidase (CiP) resulted in two variants (S323Y and E328D) with increased thermostability over the wild-type enzyme in addition to conserved catalytic activity [145].…”

“…Several studies have reported the application of protein engineering techniques to improve thermal stabilities of peroxidases [68,145,146], azoreductase [147], and laccases [148,149]. In silico design and site-directed mutagenesis of thermo-labile residues of Coprinus cinereus peroxidase (CiP) resulted in two variants (S323Y and E328D) with increased thermostability over the wild-type enzyme in addition to conserved catalytic activity [145]. Similarly, five rounds of mutagenesis/recombination followed by high-throughput screening yielded a variant 1B6, showing 300-fold higher half-life at 50°C than that exhibited by the homodimeric wild-type PpAzoR azoreductase from Pseudomonas putida [147].…”

Optimization of an Activity Assay of Coprinus Cinereus Peroxidase

“…This peroxidase (CIP) has high activity and broad substrate specificity similar to that of horse radish peroxidase (HRP), although there is less than 10%-16% sequence similarity between CIP and HRP, and thus has attained considerable attention since it was isolated 7,8 . CIP has higher thermostability than HRP 9,10 . It has been used successfully to remove phenolic compounds from wastewater [11][12][13][14][15][16] , to degrade benzene homologs and derivates 17,18 , to decolourate dyes [19][20][21] , and to produce functional polyaromatics [22][23][24][25][26] .…”

Big data, diminished design?

“…32–36 This is because the biological half-life of a protein is determined by many factors, in addition to the thermostability. For a protein to have a desirably long biological half-life, the protein must be thermostable enough at the body temperature (37°C) to have an in vitro half-life at 37°C which is at least not shorter than the desirable biological half-life.…”