Proteinase　K　(PROK)　from　Parengyodontium　album　hydrolyzes　keratin,　a　major　protein　component　of　poultry　feathers,　which　are　an　inexpensive　and　renewable　protein　resource.　Based　on　structural　studies　for　analysis　of　amino　acid　flexibility　near　the　catalytic　center,　identification　of　highly　conserved　residues,　and　experimental　screening,　we　obtained　a　mutant　R218S　with　residual　activity　1.6-fold　higher　than　that　of　PROK　after　incubation　at　60　°C　for　1　h.　Molecular　dynamics　simulation　indicated　that　substitution　of　Arg218　with　Ser　leads　to　three　hydrogen　bonds　being　introduced　into　the　structure,　stabilizing　the　β-sheet　in　which　Ser218　is　located,　and　thus　improvement　of　thermostability.　Additionally,　the　mutant　R218S　had　a　15%　increase　in　specific　activity　compared　to　PROK　and　improvement　in　the　rate　and　thoroughness　of　feather　degradation　compared　with　PROK.　We　confirmed　the　positive　effects　of　enhancing　catalytic　center　rigidity　on　enzyme　thermostability,　a　finding　which　may　have　broad　applications.　©　2019　Elsevier　B.V.