Journal
PROTEIN ENGINEERING DESIGN & SELECTION
Volume 26, Issue 11, Pages 755-761Publisher
OXFORD UNIV PRESS
DOI: 10.1093/protein/gzt049
Keywords
directed evolution; galacto-N-biose; lacto-N-biose I phosphorylase; random mutation; thermostability; 96-well microplate-based screening
Funding
- Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN)
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Galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) is the key enzyme in the enzymatic production of lacto-N-biose 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 20C higher thermostability than the wild type.
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