Directed evolution to enhance thermostability of galacto-N-biose/lacto-N- biose I phosphorylase

Yoshiyuki Koyama, Masafumi Hidaka, Mamoru Nishimoto, Motomitsu Kitaoka

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16 Citations (Scopus)


Galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) is the key enzyme in the enzymatic production of lacto-Nbiose I. For the purpose of industrial use, we improved the thermostability of GLNBP by evolutionary engineering in which five substitutions in the amino acid sequence were selected from a random mutagenesis GLNBP library constructed using error-prone polymerase chain reaction. Among them, C236Y and D576V mutants showed considerably improved thermostability. Structural analysis of C236Y revealed that the hydroxyl group of Tyr236 forms a hydrogen bond with the carboxyl group of E319. The C236Y and D576V mutations together contributed to the thermostability. The C236Y/D576V mutant exhibited 208C higher thermostability than the wild type.

Original languageEnglish
Pages (from-to)755-761
Number of pages7
JournalProtein Engineering, Design and Selection
Issue number11
Publication statusPublished - 2013 Nov


  • 96-well microplate-based screening
  • Directed evolution
  • Galacto-N-biose
  • Lacto-N-biose I phosphorylase
  • Random mutation
  • Thermostability


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