Computer-aided search for a cold-active cellobiose 2-epimerase

Abstract

Cellobiose 2-epimerase (CE) is a promising industrial enzyme that can catalyze bioconversion of lactose to its high-value derivatives, namely epilactose and lactulose. A need exists in the dairy industry to catalyze lactose bioconversions at low temperatures to avoid microbial growth. We focused on the discovery of cold-active CE in this study. A genome mining method based on computational prediction was used to screen the potential genes encoding cold-active enzymes. The CE-encoding gene from Roseburia intestinalis, with a predicted high structural flexibility, was expressed heterologously in Escherichia coli. The catalytic property of the recombinant enzyme was extensively studied. The optimum temperature and pH of the enzyme were 45°C and 7.0, respectively. The specific activity of this enzyme to catalyze conversion of lactose to epilactose was measured to be 77.3 ± 1.6 U/mg. The kinetic parameters, including turnover number (k cat ), Michaelis constant (K m ), and catalytic efficiency (k cat /K m ) using lactose as a substrate were 117.0 ± 7.7 s −1 , 429.9 ± 57.3 mM, and 0.27 mM −1 s −1 , respectively. In situ production of epilactose was carried out at 8°C: 20.9% of 68.4 g/L lactose was converted into epilactose in 4 h using 0.02 mg/mL (1.5 U/mL, measured at 45°C) of recombinant enzyme. The enzyme discovered by this in silico method is suitable for low-temperature applications.

Molecular Characterization of a Mesophilic Cellobiose 2-Epimerase That Maintains a High Catalytic Efficiency at Low Temperatures

“…The specific epimerization rate catalyzed by Roin-CE at 8 °C is 177.0 g of epilactose/(L•h•g of purified enzyme). 21 The final yield of epilactose catalyzed by Trbr-CE is ∼24% with 200 mM lactose as the substrate, which is lower than in the cases of Fljo-CE-and Pehe-CE-catalyzed biotransformation at 8 °C (33.6 and 30.5%, respectively). 32 The activity of Trbr-CE was influenced by the initial concentration of substrates and the other components in whey.…”

“…The specific initial epimerization rates of bioconversions using 200 mM (68.4 g/L) and 400 mM (136.8 g/L) lactose as substrates are 190.8 and 284.5 g of epilactose/(L·h·g of purified enzyme), respectively. The specific epimerization rate catalyzed by Roin-CE at 8 °C is 177.0 g of epilactose/(L·h·g of purified enzyme) . The final yield of epilactose catalyzed by Trbr-CE is ∼24% with 200 mM lactose as the substrate, which is lower than in the cases of Fljo-CE- and Pehe-CE-catalyzed biotransformation at 8 °C (33.6 and 30.5%, respectively) .…”

“…In general, all of the CEs showed the strongest binding affinities toward epilactose compared with those toward lactose and lactulose (Figure 5), which agree with the experimental result that epilactose possesses the smallest share in the reaction mixture when the CE-catalyzed reaction of lactose reaches equilibrium. 14,20,21,33 Other than the differences between inherent energies of ligands, the binding preference of enzymes toward epilactose might also contribute to the thermodynamic balance of reactions catalyzed by CE.…”

“…The cultivation of the recombinant cells containing Trbr-CE and the recombinant enzyme purification were carried out as previously described for other enzymes. 21 Sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE) (12% wt/vol acrylamide) was carried out to measure the subunit molecular mass of purified Trbr-CE. The gel was dyed with 0.1% Coomassie Brilliant Blue staining and was destained with a mixture of ethanol, acetic acid, and water (5:10:85, vol/vol/vol).…”

“…coli BL21 (DE3) for expression. The cultivation of the recombinant cells containing Trbr-CE and the recombinant enzyme purification were carried out as previously described for other enzymes …”