Insight into the potential factors influencing the catalytic direction in cellobiose 2-epimerase by crystallization and mutagenesis.

Cellobiose 2-epimerase (CE) is commonly recognized as an epimerase as most CEs mainly exhibit an epimerization activity towards disaccharides. In recent years, several CEs have been found to possess bifunctional epimerization and isomerization activities. They can convert lactose into lactulose, a high-value disaccharide that is widely used in the food and pharmaceutical industries. However, the factors that determine the catalytic direction in CEs are still not clear. In this study, the crystal structures of three newly discovered CEs, CsCE (a bifunctional CE from Caldicellulosiruptor saccharolyticus), StCE (a bifunctional CE from Spirochaeta thermophila DSM 6578) and BtCE (a monofunctional CE from Bacillus thermoamylovorans B4166), were determined at 1.54, 2.05 and 1.80 Šresolution, respectively, in order to search for structural clues to their monofunctional/bifunctional properties. A comparative analysis of the hydrogen-bond networks in the active pockets of diverse CEs, YihS and mannose isomerase suggested that the histidine corresponding to His188 in CsCE is uniquely required to catalyse isomerization. By alignment of the apo and ligand-bound structures of diverse CEs, it was found that bifunctional CEs tend to have more flexible loops and a larger entrance around the active site, and that the flexible loop 148-181 in CsCE displays obvious conformational changes during ligand binding. It was speculated that the reconstructed molecular interactions of the flexible loop during ligand binding helped to motivate the ligands to stretch in a manner beneficial for isomerization. Further site-directed mutagenesis analysis of the flexible loop in CsCE indicated that the residue composition of the flexible loop did not greatly impact epimerization but affects isomerization. In particular, V177D and I178D mutants showed a 50% and 80% increase in isomerization activity over the wild type. This study provides new information about the structural characteristics involved in the catalytic properties of CEs, which can be used to guide future molecular modifications.

Enhancement of isomerization activity and lactulose production of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus.

Industrial application of Caldicellulosiruptor saccharolyticus cellobiose 2-epimerase (CsCE) for lactulose synthesis is limited by low enzyme activity and formation of epilactose as by-product. After four sequential rounds of random mutagenesis and screening, an optimal mutant G4-C5 was obtained. Compared with wild type (WT) enzyme, mutant G4-C5 demonstrated 2.8- and 3.0-fold increases in specific activity and kcat/Km for lactulose production, respectively, without compromising thermostability. DNA sequencing of mutant G4-C5 revealed five amino acid substitutions, namely, R5M, I52V, A12S, K328I and F231L, which were located on the protein surface, except for the mutation I52V. The yield of lactulose catalyzed by mutant G4-C5 increased to approximately 76% with no obvious epilactose detected, indicating that mutant G4-C5 was more suitable for lactulose production than the WT enzyme.

Cloning, expression and structural stability of a cold-adapted ß-galactosidase from Rahnella sp. R3.

A novel gene was isolated for the first time from a psychrophilic gram-negative bacterium Rahnella sp. R3. The gene encoded a cold-adapted ß-galactosidase (R-ß-Gal). Recombinant R-ß-Gal was expressed in Escherichia coli BL21 (DE3), purified and characterized. R-ß-gal belongs to the glycosyl hydrolase family 42. Circular dichroism spectrometry of the structural stability of R-ß-Gal with respect to temperature indicated that the secondary structures of the enzyme were stable to 45°C. In solution, the enzyme was a homo-trimer and was active at temperatures as low as 4°C. The enzyme did not require the presence of metal ions to be active, but Mg(2+), Mn(2+), and Ca(2+) enhanced its activity slightly, whereas Fe(3+), Zn(2+) and Al(3+) appeared to inactive it. The purified enzyme displayed K(m) values of 6.5 mM for ONPG and 2.2mM for lactose at 4°C. These values were lower than the corresponding K(m)s reported for other cold-adapted ß-Gals.

Production of 1-lactulose and lactulose using commercial ß-galactosidase from Kluyveromyces lactis in the presence of fructose.

Production of 1-lactulose and lactulose using commercial ß-galactosidase DSM Maxilact® 5000 in the presence of fructose was investigated. Experiments were performed at 40 °C and pH 7.5. Lactose starting concentration was constantly 250 g/l. A novel transgalactosylation product 1-lactulose was detected besides lactulose. Effects of fructose concentration, reaction time and enzyme concentration on transgalactosylation reactions were discussed. In all reactions, the yield ratio 1-lactulose:lactulose was close to 3:1 due to the regioselectivity of ß-galactosidase. The maximum production of 1-lactulose and lactulose was approximately 22 and 8 g/l, respectively, when fructose concentration was increased to 100 g/l. Lactose hydrolysis was significantly retarded since fructose strongly attracted water molecules. Higher enzyme concentration can accelerate transgalactosylation reactions without affecting the maximum production of transgalactosylation products. Fructose was a more preferred galactosyl acceptor than lactose, since the synthesis of galactooligosacchairdes was found to be absolutely inhibited in the presence of fructose.

Enzymatic synthesis and identification of oligosaccharides obtained by transgalactosylation of lactose in the presence of fructose using ß-galactosidase from Kluyveromyces lactis.

The enzymatic transgalactosylation of lactose in the presence of fructose using ß-galactosidase from Kluyveromyces lactis (KlßGal) leading to the formation of oligosaccharides was investigated in detail. The reaction mixture was analyzed by high performance liquid chromatography with differential refraction detector (HPLC-RI) and two main transgalactosylation products were discovered. To elucidate their overall structures, the products were isolated and purified using preparative liquid chromatography and analyzed by LC/MS, one-dimensional (1D) and two-dimensional (2D) NMR studies. Allo-lactulose(ß-d-galactopyranosyl-(1→1)-d-fructose) with two main isomers in D(2)O was identified to be the major transgalactosylation product while lactulose(ß-d-galactopyranosyl-(1→4)-d-fructose) turned out to be the minor one, indicating that KlßGal was regioselective with respect to the primary C-1 hydroxyl group of fructose. The maximum yields of allo-lactulose and lactulose were 47.5 and 15.4g/l, respectively, at 66.5% lactose conversion (200g/l initial lactose concentration).