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

作者：Koyama Yoshiyuki; Hidaka Masafumi; Nishimoto Mamoru; Kitaoka Motomitsu^*

来源：Protein Engineering Design and Selection, 2013, 26(11): 755-761.

DOI：10.1093/protein/gzt049

• directed evolution
• galacto-N-biose
• lacto-N-biose I phosphorylase
• random mutation
• thermostability
• 96-well microplate-based screening

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.

• 出版日期2013-11