Fig 1.
Initial levanase reaction rate of B. subtilis levansucrase as a function of low molecular weight levan concentration.
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.
Fig 2.
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.
The concentrations were: 6 to 296 mMF for HMw levan and 5 to 51 mMF for LMw levan.
Fig 3.
Calorimetric trace of LMw levan hydrolysis by B. subtilis levansucrase (SacB) in a VP-ITC.
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)).
Fig 4.
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.
These are the same concentrations employed in the ITC experiment depicted in Fig 3.
Fig 5.
Evolution of low molecular weight levan during hydrolysis by SacB (500 nM) as determined by GPC.
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 3 and 4.
Fig 6.
Evolution of low molecular weight levan distribution during hydrolysis by SacB (500 nM) as determined by HPAEC-PAD.
Samples correspond to reactions also described by Figs 3, 4 and 5. 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).
Fig 7.
B.subtilis 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.
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.