Enhancement of the hydrolysis activity of β-galactosidase from Geobacillus stearothermophilus by saturation mutagenesis

Abstract

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.

A differentially conserved residue (Ile42) of GH42 β-galactosidase from Geobacillus stearothermophilus BgaB is involved in both catalysis and thermostability

“…Cell debris was removed by centrifugation at 12,000 × g for 10 min at 4°C. Recombinant His-tagged enzymes were purified using a Ni-chelating Sepharose Fast Flow column (Qiagen, Shanghai, China) based on a previous report (Dong et al, 2011). Homogeneous fractions were pooled and imidazole was removed using an Amicon Ultra Centrifugal Filter Device (EMD Millipore, Billerica, MA) with a 30-kDa cut-off membrane.…”

“…Substrate-free enzymes were used for measuring specific activities at the optimal temperatures and pH as previously described (Dong et al, 2011). Protein concentrations were determined by the method of Bradford (1976) with BSA as standard.…”

“…For the pH stability assay, samples were incubated at pH 6.5 and 55°C and then transferred to ice at different time points. Residual enzyme activity was assayed with ortho-nitrophenyl-β-galactoside (oNPG) as the substrate, as previously described (Dong et al, 2011).…”

“…A 1-min assay was used to measure the K m and maximum rate (V max ) values of BgaB at several different concentrations of oNPG. The inhibition constant (K i ) values were determined by fitting to Cornish-Bowden plots using various concentrations of the inhibitor galactose (from 0 to 20 mM) with oNPG as the substrate (Dong, et al, 2011).…”

“…An examination of the crystal structure of A4-β-Gal, which is the first known structure of a GH42 enzyme, revealed that this enzyme possesses a common (β/α) 8 -barrel (TIM barrel) structure and exhibits very strict recognition of the galactosyl moiety. Although the structure of BgaB has not been elucidated, we previously constructed a 3D structure of BgaB based on homology modeling using A4-β-Gal as the template (Dong et al, 2011). We predicted the substrate-binding mode of BgaB by comparative analysis with A4-β-Gal (Figure 1), and found that the nucleophilic Glu312 of A4-β-Gal does not work independently but requires assistance from an interaction network involving 2 other residues (Arg32 and Glu35) via H 2 O to achieve efficient catalysis.…”