National Food Research Institute, National Agriculture and Food Research Organization, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
Protein Eng Des Sel. 2013 Nov;26(11):755-61. doi: 10.1093/protein/gzt049. Epub 2013 Sep 24.
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.
半乳糖-1-磷酸-N-乙酰氨基葡萄糖磷酸化酶（GLNBP）是酶法合成乳-N-双糖 I 的关键酶。为了工业应用，我们通过易错聚合酶链反应构建的 GLNBP 随机突变文库，通过进化工程提高了 GLNBP 的热稳定性。其中，C236Y 和 D576V 突变体显示出相当大的热稳定性改善。C236Y 的结构分析表明 Tyr236 的羟基与 E319 的羧基形成氢键。C236Y 和 D576V 突变共同有助于热稳定性。C236Y/D576V 突变体的热稳定性比野生型高 20°C。
