Enhancing the synthesis efficiency of galacto-oligosaccharides of a β-galactosidase from Paenibacillus barengoltzii by engineering the active and distal sites

文献类型： 外文期刊

第一作者： Yu, Haiyan

作者： Yu, Haiyan;Wang, Yulu;Ying, Jiabao;Liu, Bolin;Mohamed, Abeer;Wei, Xue;Yang, Yuji;Xin, Fengjiao;Yang, Zhisen;Liu, Xin;Sun, Linfeng;Guan, Feifei;Miao, Miao;Jiang, Zhengqiang;Yang, Shaoqing;Wang, Yulu;Xin, Fengjiao

作者机构：

关键词： beta-Galactosidase; GOS synthesis; Cryo-EM structure; Machine learning; Catalytic mechanism

期刊名称：FOOD CHEMISTRY （ 影响因子：9.8； 五年影响因子：9.7 ）

ISSN： 0308-8146

年卷期： 2025 年 483 卷

页码：

收录情况： SCI

摘要： Previously, a glycoside hydrolase (GH) family 2 beta-galactosidase (PbBGal2A) from Paenibacillus barengoltzii is characterized for its high transglycosylation capability. Here, the cryo-electron microscopy (cryo-EM) structure of PbBGal2A was determined, revealing an enlarged acidic catalytic pocket that facilitate the binding of carbohydrate substrates. Three structure-based strategies as well as machine learning MECE platform (method for enhancing the catalytic efficiency) were employed to identify active and distal mutations with enhanced galactooligosaccharides (GOS) synthesis and their synergistic effects were evaluated. The best H331V mutation yielded a maximum GOS production of 76.57 % at 4 h when 35 % (w/v) of lactose was used as a substrate. Molecular dynamics (MD) simulation analysis further indicated that distal mutations increase the rigidity of the loops surrounding the catalytic pocket. This research sheds light on the structural and catalytic mechanisms of PbBGal2A, highlighting the importance of both active and distal mutations in the efficient design of customized beta-galactosidases.

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