SDS-PAGE analysis of the purification of WcE392-rDSR.

<p>Gel A shows the purification protocol of the enzyme from <i>L. lactis</i> culture supernatant (1). A 2-fold concentrate obtained by ultrafiltration and the ultrafiltration permeate are shown on lanes 2 and 3, respectively. Lane 4 shows the sample eluted from Ni-NTA column. Gel B shows the approximately 60-fold concentrated enzyme sample obtained by ultrafiltration and dilution.</p

Enzymatic production of dextran into wheat bran.

<p>Composition of runs of the experimental design and the final dextran contents. Sucrose, fructose and dextran contents are given as a percentage of dry weight (dw-%). A hyphen (-) denotes that the values were below the detection limit.</p><p>Enzymatic production of dextran into wheat bran.</p

Predicted protein structure of WcE392-rDSR.

<p>Structural domains A, B, C, VI and V, as presented by Leemhuis et al.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116418#pone.0116418.ref001" target="_blank">1</a>], are shown separated by dotted lines.</p

Half-life (<i>t</i>_1/2) of WcE392-rDSR in the presence and absence of glycerol (0.5%, v/v) at various temperatures in 20 mM Na-acetate buffer with 2 mM CaCl₂, pH 5.4.

<p>Half-life (<i>t</i>_1/2) of WcE392-rDSR in the presence and absence of glycerol (0.5%, v/v) at various temperatures in 20 mM Na-acetate buffer with 2 mM CaCl₂, pH 5.4.</p

Influence of the sucrose and water contents in the initial reaction mixture and the incubation time on final dextran content in the experimental series shown as contour plots.

<p>Dextran production is enhanced by higher sugar content. In the 2 h plot, higher water content clearly increases dextran production but in the 4 h plot the effect can only be seen with higher sucrose content and in the 6 h plot the effect is completely muted. The quadratic effect of incubation time shows as highest dextran contents in the 4 h plot. Dextran amount is given as a percentage of dry weight.</p

Thermal stability of WcE392-rDSR.

<p>The stability was measured in the presence and absence of 0.5% (v/v) glycerol, after incubation at 25°C, 35°C and 40°C for 1, 2, 5 and 24 h.</p

Compositions of the doughs used for studying the effects of bran with dextran on bread properties.

<p>Compositions of the doughs used for studying the effects of bran with dextran on bread properties.</p

Effects of pH and temperature on WcE392-rDSR activity.

<p>Effect of pH was assayed in 20 mM Na-acetate (pH 4 to 6) and in 20 mM Tris-HCl (pH 6.5 to 7.0), and the effect of temperature in 20 mM Na-acetate, pH 5.4. Reactions were incubated for 15 min. The reaction mixtures were supplemented with 2 mM CaCl₂ and the Nelson-Somogyi method was used for assaying the activities. Error bars represent the standard errors of the mean (n = 3).</p

Specific activities and kinetic values of WcE392-rDSR.

<p>Specific activities and kinetic values of WcE392-rDSR.</p

Structure modeling and functional analysis of recombinant dextransucrase from <i>Weissella confusa</i> Cab3 expressed in <i>Lactococcus lactis</i>

<p>The dextransucrase gene from <i>Weissella confusa</i> Cab3, having an open reading frame of 4.2 kb coding for 1,402 amino acids, was amplified, cloned, and expressed in <i>Lactococcus lactis</i>. The recombinant dextransucrase, <i>Wc</i>Cab3-rDSR was expressed as extracellular enzyme in M17 medium with a specific activity of 1.5 U/mg which after purification by PEG-400 fractionation gave 6.1 U/mg resulting in 4-fold purification. <i>Wc</i>Cab3-rDSR was expressed as soluble and homogeneous protein of molecular mass, approximately, 180 kDa as analyzed by SDS-PAGE. It displayed maximum enzyme activity at 35°C at pH 5.0 in 50 mM sodium acetate buffer. <i>Wc</i>Cab3-rDSR gave <i>K</i>_m of 6.2 mM and <i>V</i>_m of 6.3 µmol/min/mg. The characterization of dextran synthesized by <i>Wc</i>Cab3-rDSR by Fourier transform infrared and nuclear magnetic resonance spectroscopic analyses revealed the structural similarities with the dextran produced by the native dextransucrase. The modeled structure of <i>Wc</i>Cab3-rDSR using the crystal structures of dextransucrase from <i>Lactobacillus reuteri</i> (protein data bank, PDB id: 3HZ3) and <i>Streptococcus mutans</i> (PDB id: 3AIB) as templates depicted the presence of different domains such as A, B, C, IV, and V. The domains A and B are circularly permuted in nature having (β/α)₈ triose phosphate isomerase-barrel fold making the catalytic core of <i>Wc</i>Cab3-rDSR. The structure superposition and multiple sequence alignment analyses of <i>Wc</i>Cab3-rDSR with available structures of enzymes from family 70 GH suggested that the amino acid residue Asp510 acts as a nucleophile, Glu548 acts as a catalytic acid/base, whereas Asp621 acts as a transition-state stabilizer and these residues are found to be conserved within the family.</p