The glycoside hydrolase family 42 (GH42) of thermophilic microorganisms consists of thermostable β-galactosidases that display significant variations in their temperature optima and stabilities. In this study, we compared the substrate binding modes of 2 GH42 β-galactosidases, BgaB from Geobacillus stearothermophilus and A4-β-Gal from Thermus thermophilus A4. The A4-β-Gal has a catalytic triad (Glu312-Arg32-Glu35) with an extended hydrogen bond network that has not been observed in BgaB. In this study, we performed site-saturation mutagenesis of Ile42 in BgaB (equivalent to Glu312 in A4-β-Gal) to study the effects of different residues on thermostability, catalytic function, and the extended hydrogen bond network. Our experimental results suggest that substitution of Ile42 with polar AA enhanced the thermostability but decreased the catalytic efficiency of BgaB. Polar AA substitution for Ile42 simultaneously affected thermostability, catalytic efficiency, and the hydrogen bond network, suggesting that Ile42 is responsible for functional discrimination between members of the GH42 family. These observations could lead to a novel strategy for investigating the functional evolution of the GH42 β-galactosidases.

Thermostable β-galactosidase (BgaB) from Geobacillus stearothermophilus is characterized by its thermoactivity in the hydrolysis of lactose to produce lactose-free milk products. However, BgaB has limited activity toward lactose. We established a method for screening evolved mutants with high hydrolysis activity based on prediction of substrate binding sites. Seven amino acid residues were identified as candidates for substrate binding to galactose. To study the hydrolysis activity of these residues, we constructed mutants by site-saturation mutagenesis of these residue sites, and each variant was screened for its hydrolysis activity. The first round of mutagenesis showed that changes in amino acid residues of Arg109, Tyr272, and Glu351 resulted in altered hydrolysis activity, including greater activity toward ortho-nitrophenyl-β-d-galactopyranoside (oNPG). The mutants R109V and R109L displayed changes in the optimum pH from 7.0 to 6.5, and the mutant R109V/L displayed different substrate affinity and catalytic efficiency (k(cat)/K(m)). Mutant R109G showed complete loss of BgaB enzymatic activity, suggesting that Arg109 plays a significant role in maintaining hydrolysis activity. The optimum pH of mutant E351R increased from 7.0 to 7.5 and this mutant showed a prominent increase in catalytic efficiency with oNPG and lactose as substrates.

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 product inhibition. Through homology modeling and molecular dynamics (MD) simulation, we have identified the galactose 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 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.