Evolution of fructose liberation from LMw levan hydrolysis by SacB (500 nM) at 25°C, with LMw levan (0.85 mM) in a total reaction volume of 1.4 mL in the working buffer.

<p>These are the same concentrations employed in the ITC experiment depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143394#pone.0143394.g003" target="_blank">Fig 3</a>.</p

Evolution of low molecular weight levan during hydrolysis by SacB (500 nM) as determined by GPC.

<p>The reaction took place at 25°C with LMw levan (0.85 mM) in a total reaction volume of 1.4 mL in the working buffer. These are the same concentrations employed in the ITC experiment depicted in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143394#pone.0143394.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143394#pone.0143394.g004" target="_blank">4</a>.</p

Evolution of low molecular weight levan distribution during hydrolysis by SacB (500 nM) as determined by HPAEC-PAD.

<p>Samples correspond to reactions also described by Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143394#pone.0143394.g003" target="_blank">3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143394#pone.0143394.g004" target="_blank">4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143394#pone.0143394.g005" target="_blank">5</a>. The chromatograms are shown according to levans molecular weights:A) Mono-, di- and fructo-oligosaccharides with elution times of 1.6–20 min, B) intermediate size levans with elution times of 20–50 min, C) Chromatogram showing all reaction products obtained from levan. Fructose (F), blastose (B), 1-kestose (1k), 6-kestose (6k), levanobiose (Lb), inulobiose (Ib) and neokestose (nk).</p

Initial levanase reaction rate of <i>B</i>. <i>subtilis</i> levansucrase as a function of low molecular weight levan concentration.

<p>The dash line represents the non-linear regression of the data using the Michaelis-Menten model. All reactions were carried out with 2.4 μM of SacB, at 37°C in the working buffer. Levan concentration is reported according to its total fructose concentration considering and average molecular weight of 8.3 kDa.</p

Kinetic behavior of SacB levanase activity towards high (HMw) and low (LMw) molecular weight levans, measured as initial rates at 37°C with 2.4 μM of enzyme in the working buffer.

<p>The concentrations were: 6 to 296 mM_F for HMw levan and 5 to 51 mM_F for LMw levan.</p

<i>B</i>.<i>subtilis</i> Levansucrase reaction products profile as observed by HPAEC-PAD: A) Products obtained from sucrose B) Products obtained from levan as donor and glucose as acceptor, C) Products obtained from levan as donor and fructose as acceptor, D) Levan hydrolysis products.

<p>Fructose (F), blastose (B), 1-kestose (1k), 6-kestose (6k), levanobiose (Lb), inulobiose (Ib) and neokestose (nk) were identified from standards. The DPn region is shown according to inulo-oligosaccharide standards.</p

Calorimetric trace of LMw levan hydrolysis by <i>B</i>. <i>subtilis</i> levansucrase (SacB) in a VP-ITC.

<p>The enzymatic reaction was carried out at 25°C. The reaction took place in the 1.4 mL calorimetric cell, containing 500 nM SacB in the working buffer. The reaction started by the addition of 40 μL of a 30.16 mM LMw levan solution placed in the calorimeter syringe. After this titration the LMw levan concentration in the cell was 0.85 mM. (*) indicates the onset of the “second reaction” For comparison, the inset show a calorimetric trace for a single reaction to completion (for the hydrolysis of L-arginine ethyl ester catalyzed by tripsin from Martínez (2015)).</p