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Optimizing lactose hydrolysis by computer-guided modification of the catalytic site of a wild-type enzyme

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Abstract

Lactose intolerance is a serious global health problem. A lactose hydrolysis enzyme, thermostable \(\upbeta \)-galactosidase, BgaB (from Geobacillus stearothermophilus) has attracted the attention of industrial biologists because of its potential application in processing lactose-containing products. However, this enzyme experiences galactose product inhibition. Through homology modeling and molecular dynamics (MD) simulation, we have identified the galactose binding sites in the thermostable \(\upbeta \)-galactosidase BgaB (BgaB). The binding sites are formed from Glu303, Asn310, Trp311, His354, Arg109, Phe341, Try272, Asn147, Glu148, and H354; these residues are all important for enzyme catalysis. A ligand–receptor binding model has been proposed to guide site-directed BgaB mutagenesis experiments. Based upon the model and the MD simulations, we recommend mutating Arg109, Phe341, Trp311, Asn147, Asn310, Try272, and His354 to reduce galactose product inhibition. In vitro site-directed mutagenesis experiments confirmed our predictions. The success rate for mutagenesis was 66.7 %. The best BgaB mutant, F341T, can hydrolyze lactose completely, and is the most promising enzyme for use by the dairy industry. Thus, our study is a successful example of optimizing enzyme catalytic chemical reaction by computer-guided modifying the catalytic site of a wild-type enzyme.

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Acknowledgments

This study was supported in part by the Major Scientific and Technological Special Project of the Ministry of Science and Technology of China (2010ZX09102-305), the National Natural Science Foundation of China (31125021, 31171636), the National High Technology Research and Development Program of China (2011AA100905), and the Anhui provincial university natural science research Project (KJ2013B187)

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Authors and Affiliations

  1. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, People’s Republic of China

    Yi-Ning Dong, Haiqin Chen, Xiaoming Liu, Yuanda Song, Wei Chen & Hao Zhang

  2. School of Pharmaceutical Sciences & Institute of Human Virology, Sun Yat-sen University, Guangzhou, 510006, People’s Republic of China

    Ling Wang, Qiong Gu & Jun Xu

  3. Department of Chemistry, University of Massachusetts, 701 Lederle Graduate Research Tower, 710 North Pleasant Street, Amherst, MA, 01003-9336, USA

    Arnold T. Hagler

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  1. Yi-Ning Dong
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Correspondence to Haiqin Chen or Jun Xu.

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Ling Wang and Yi-Ning Dong contributed equally to this study.

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Dong, YN., Wang, L., Gu, Q. et al. Optimizing lactose hydrolysis by computer-guided modification of the catalytic site of a wild-type enzyme. Mol Divers 17, 371–382 (2013). https://doi.org/10.1007/s11030-013-9437-y

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