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

Journal: Food chemistry
Published Date: Apr 4, 2025

Abstract

Previously, a glycoside hydrolase (GH) family 2 β-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 galacto-oligosaccharides (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 β-galactosidases.

Authors

  • HaiYan Yu
    Department of Food Science and Technology, Shanghai Institute of Technology, Shanghai 201418, China.
  • Yulu Wang
    College of Public Health, Zhengzhou University, Zhengzhou, P. R. China.
  • Zhisen Yang
    Department of Neurology of The First Affiliated Hospital of USTC, MOE Key Laboratory for Membraneless Organelles and Cellular Dynamics, Hefei National Research Center for Physical Sciences at the Microscale, Biomedical Sciences and Health Laboratory of Anhui Province, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230027, China.
  • Jiabao Ying
    Laboratory of Biomanufacturing and Food Engineering, Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
  • Feifei Guan
    Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
  • Bolin Liu
    Laboratory of Biomanufacturing and Food Engineering, Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
  • Miao Miao
    College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
  • Abeer Mohamed
    Laboratory of Biomanufacturing and Food Engineering, Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
  • Xue Wei
    Laboratory of Biomanufacturing and Food Engineering, Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
  • Yuji Yang
    Laboratory of Biomanufacturing and Food Engineering, Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
  • Xin Liu
    Peking University Institute of Advanced Agricultural Sciences, Shandong Laboratory of Advanced Agricultural Sciences, Weifang, Shandong, China.
  • Linfeng Sun
    Centre for Quantum Physics, Key Laboratory of Advanced Optoelectronic Quantum Architecture and Measurement (MOE), School of Physics, Beijing Institute of Technology, Beijing 100081, China.
  • Zhengqiang Jiang
    Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China. Electronic address: zhqjiang@cau.edu.cn.
  • Shaoqing Yang
    College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China. Electronic address: ysq@cau.edu.cn.
  • Fengjiao Xin
    Laboratory of Biomanufacturing and Food Engineering, Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, China; Institute of Food Science Technology Nutrition and Health (Cangzhou), Chinese Academy of Agricultural Sciences, Cangzhou 061001, China. Electronic address: 2002hongzhi30@163.com.

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