title: Enhancement of thermostability of fungal deglycating enzymes by directed evolution.
abstract: Fructosyl peptide oxidases are valuable for the determination of glycoproteins such as hemoglobin A1c. For practical use in clinical diagnosis, we applied directed evolution to improve the thermostability of these enzymes. After two rounds of random mutagenesis and high-throughput screening, six thermostabilizing amino acid substitutions were identified. Therefore, site-directed and cassette mutageneses were applied to combine these six stabilizing mutations. The simultaneous mutants showed that the stabilizing effect of the amino acid replacement was cumulative. The sextuple mutant enzyme, R94K/G184D/F265L/N272D/H302R/H388Y, had a half-life of thermal inactivation at 50 degrees C that was 79.8-fold longer than that of the parental fructosyl peptide oxidase. The thermostable variants also showed increased tolerance to digestion by a protease. The sextuple mutant enzyme did not lose its activity on incubation with neutral protease, while the wild-type enzyme almost completely lost its activity. Furthermore, three amino acid substitutions were introduced into another fructosyl peptide oxidase with a different substrate specificity. The half-life of inactivation at 50 degrees C was 3.61-fold longer than that of the parent enzyme. These engineered fructosyl peptide oxidases will be useful for industrial application to clinical diagnosis.
authors.affiliation: Research and Development Division, Kikkoman, 399 Noda, Noda City, Chiba Prefecture 278-0037, Japan. khirokawa@mail.kikkoman.co.jp
authors.forename: Kozo
authors.initials: K
authors.lastname: Hirokawa
authors.forename: Atsushi
authors.initials: A
authors.lastname: Ichiyanagi
authors.forename: Naoki
authors.initials: N
authors.lastname: Kajiyama
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001203:Ascomycota / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D016296:Mutagenesis; D015202:Protein Engineering; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D000594:Amino Acid Oxidoreductases; C520096:fructosyl-peptide oxidase