Mol Divers (2013) 17:371–382 DOI 10.1007/s11030-013-9437-y FULL-LENGTH PAPER Optimizing lactose hydrolysis by computer-guided modification of the catalytic site of a wild-type enzyme Yi-Ning Dong · Ling Wang · Qiong Gu · Haiqin Chen · Xiaoming Liu · Yuanda Song · Wei Chen · Arnold T. Hagler · Hao Zhang · Jun Xu Received: 23 January 2013 / Accepted: 11 March 2013 / Published online: 13 April 2013 © Springer Science+Business Media Dordrecht 2013 Abstract Lactose intolerance is a serious global health problem. A lactose hydrolysis enzyme, thermostable β- 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 prod- uct inhibition. Through homology modeling and molecular dynamics (MD) simulation, we have identified the galac- tose binding sites in the thermostable β-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 pro- posed to guide site-directed BgaB mutagenesis experiments. Based upon the model and the MD simulations, we recom- mend mutating Arg109, Phe341, Trp311, Asn147, Asn310, Try272, and His354 to reduce galactose product inhibition. Electronic supplementary material The online version of this article (doi:10.1007/s11030-013-9437-y) contains supplementary material, which is available to authorized users. Ling Wang and Yi-Ning Dong contributed equally to this study. Y. -N. Dong · H. Chen (B )· X. Liu · Y. Song · W. Chen · H. Zhang State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, People’s Republic of China e-mail: haiqinchen@jiangnan.edu.cn L. Wang · Q. Gu · J. Xu (B ) School of Pharmaceutical Sciences & Institute of Human Virology, Sun Yat-sen University, Guangzhou 510006, People’s Republic of China e-mail: junxu@biochemomes.com A. T. Hagler Department of Chemistry, University of Massachusetts, 701 Lederle Graduate Research Tower, 710 North Pleasant Street, Amherst, MA 01003-9336, USA 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 com- pletely, and is the most promising enzyme for use by the dairy industry. Thus, our study is a successful example of optimiz- ing enzyme catalytic chemical reaction by computer-guided modifying the catalytic site of a wild-type enzyme. Keywords Protein design · Lactose hydrolysis · Homology modeling · Molecular dynamics simulations · Site-directed mutagenesis Introduction Lactose is milk’s main carbohydrate. β-Galactosidases con- vert the β-1,4-d-galactosidic linkage of lactose (galactosyl β (1 → 4) glucose) to glucose and galactose [1] and hydrolyze terminally non-reducing β-d-galactose residues in oligo- and polysaccharides [2]. Lactose-intolerant peo- ple cannot digest significant amounts of lactose because of the low level of β-d-galactosidase (lactase) in the jejunum. The resulting malabsorption of dietary lactose can pro- duce unpleasant gastrointestinal symptoms (e.g., fermenta- tive diarrhea, bloating, and excessive flatulence). Generally, lactose intolerance is a lifelong inherited condition; however, it can be the temporary result of a jejuna mucosa infection [3]. A significant fraction of the global population is lac- tose deficient: up to 15 % of people from northern European descent, up to 80 % of people of African and Latin descent, and up to 100 % of American Indians and Asians [4]. To avoid lactose intolerance problems, lactase in milk and related products is usually enzymatically hydrolyzed. Con- sequently, β-d-galactosidase (EC 3.2.1.23, β-d-galactoside galactohydrolase, lactase) is one of the most common industrial enzymes [5, 6]. However, because many 123