title: A single mutation within a Ca(2+) binding loop increases proteolytic activity, thermal stability, and surfactant stability.
abstract: We improved the enzymatic properties of the oxidatively stable alkaline serine protease KP-43 through protein engineering to make it more suitable for use in laundry detergents. To enhance proteolytic activity, the gene encoding KP-43 was mutagenized by error-prone PCR. Screening identified a Tyr195Cys mutant enzyme that exhibited increased specific activity toward casein between pH 7 and 11. At pH 10, the mutant displayed 1.3-fold higher specific activity for casein compared to the wild-type enzyme, but the activity of the mutant was essentially unchanged toward several synthetic peptides. Furthermore, the Tyr195Cys mutation significantly increased thermal stability and surfactant stability of the enzyme under oxidizing conditions. Examination of the crystal structure of KP-43 revealed that Tyr195 is a solvent exposed residue that forms part of a flexible loop that binds a Ca(2+) ion. This residue lies 15-20Å away from the residues comprising the catalytic triad of the enzyme. These results suggest that the substitution at position 195 does not alter the structure of the active center, but instead may affect a substrate-enzyme interaction. We propose that the Tyr195Cys mutation enhances the interaction with Ca(2+) and affects the packing of the Ca(2+) binding loop, consequently increasing protein stability. The simultaneously increased proteolytic activity, thermal stability, and surfactant stability of the Tyr195Cys mutant enzyme make the protein an ideal candidate for laundry detergent application.
authors.affiliation: Biological Science Research, Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi 321-3497, Japan. okuda.mitsuyoshi@kao.co.jp
authors.forename: Mitsuyoshi
authors.initials: M
authors.lastname: Okuda
authors.forename: Tadahiro
authors.initials: T
authors.lastname: Ozawa
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Tohata
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Sato
authors.forename: Katsuhisa
authors.initials: K
authors.lastname: Saeki
authors.forename: Katsuya
authors.initials: K
authors.lastname: Ozaki
mesh_terms: D000468:Alkalies / Q000737:chemistry; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013501:Surface-Active Agents / Q000737:chemistry; D013696:Temperature; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
chemical_list: D000468:Alkalies; D001426:Bacterial Proteins; D013501:Surface-Active Agents; D014443:Tyrosine; D012697:Serine Endopeptidases; D003545:Cysteine; D002118:Calcium