Hirokawa Kozo, Ichiyanagi Atsushi, Kajiyama Naoki
Research and Development Division, Kikkoman, 399 Noda, Noda City, Chiba Prefecture 278-0037, Japan.
Appl Microbiol Biotechnol. 2008 Apr;78(5):775-81. doi: 10.1007/s00253-008-1363-z. Epub 2008 Feb 1.
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.
果糖基肽氧化酶对于测定诸如糖化血红蛋白A1c等糖蛋白很有价值。为了在临床诊断中实际应用，我们应用定向进化来提高这些酶的热稳定性。经过两轮随机诱变和高通量筛选，确定了六个提高热稳定性的氨基酸替换。因此，应用定点诱变和盒式诱变来组合这六个稳定突变。同时突变体表明氨基酸替换的稳定作用是累积的。六重突变酶R94K/G184D/F265L/N272D/H302R/H388Y在50℃下的热失活半衰期比亲本果糖基肽氧化酶长79.8倍。热稳定变体对蛋白酶消化的耐受性也有所提高。六重突变酶与中性蛋白酶孵育时不会失去活性，而野生型酶几乎完全失去活性。此外，在另一种具有不同底物特异性的果糖基肽氧化酶中引入了三个氨基酸替换。在50℃下的失活半衰期比亲本酶长3.61倍。这些工程化的果糖基肽氧化酶将有助于在临床诊断的工业应用中发挥作用。
