“Whoa! It's like Spotify but for academic articles.”

Instant Access to Thousands of Journals for just $40/month

Get 2 Weeks Free

Enhancement of thermostability of fungal deglycating enzymes by directed evolution



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°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°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.



Applied Microbiology and BiotechnologySpringer Journals

Published: Apr 1, 2008

DOI: 10.1007/s00253-008-1363-z

Free Preview of First Page

Loading next page...

You're reading a free preview. Subscribe to read the entire article.

And millions more from thousands of peer-reviewed journals, for just $40/month

Get 2 Weeks Free

To be the best researcher, you need access to the best research

  • With DeepDyve, you can stop worrying about how much articles cost, or if it's too much hassle to order — it's all at your fingertips. Your research is important and deserves the top content.
  • Read from thousands of the leading scholarly journals from Springer, Elsevier, Nature, IEEE, Wiley-Blackwell and more.
  • All the latest content is available, no embargo periods.