Directed evolution to enhance thermostability of galacto-N-biose/lacto-N-biose I phosphorylase.
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 20°C higher thermostability than the wild type.
National Food Research Institute, National Agriculture and Food Research Organization, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
Yoshiyuki
Y
Koyama
Masafumi
M
Hidaka
Mamoru
M
Nishimoto
Motomitsu
M
Kitaoka
D000595:Amino Acid Sequence; D019020:Directed Molecular Evolution / Q000379:methods*; D004795:Enzyme Stability; D006358:Hot Temperature; D008969:Molecular Sequence Data; D006005:Phosphorylases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment
D006005:Phosphorylases; C540259:galacto-N-biose-lacto-N-biose I phosphorylase, Bifidobacterium longum
96-well microplate-based screening; directed evolution; galacto-N-biose/lacto-N-biose I phosphorylase; random mutation; thermostability