New insights in bacillus subtillis levansucrase mechanism and applications

B. subtilis levansucrase (SacB) is a widely studied glycoside hydrolase from Family 68 family. Although reports on SacB properties date back to the 70’s (Chambert & Gonzy-Tréboul, 1976), questions regarding levan synthesis mechanism are still open. These questions refer to the factors influencing reaction specificity, including the effect of sucrose and levan hydrolysis, product structure and levan molecular weight. In this conference we review recent findings regarding the modulating effect of SacB concentration on levan molecular weight distribution (Porras-Domínguez et al., 2015; Raga-Carbajal et al., 2016). In effect, we demonstrated that high enzyme concentrations (\u3e1.0 µM), direct levan synthesis exclusively to low molecular weight products (av 7.6 KDa), while low enzyme concentrations (\u3c 0.1µM) favor the synthesis of a high molecular weight levan fraction (\u3e2000 kDa). From a detailed HPAEC-PAD analysis of product evolution, a shift from a clear non-processive elongation mechanism at high protein concentrations to a -most likely- processive mechanism when low protein concentrations are used in the reaction. Trough calorimetric experiments we demonstrate that these changes in enzyme performance do not involve protein-protein interactions (Raga-Carbajal et al., 2016). We demonstrated, through an extensive characterization of the levan hydrolysis reaction by SacB, that the wide diversity of products derives also from fructosyl transfer to free sugars available from sucrose and levan hydrolysis. Actually, levan is an efficient fructosyl donor for fructosylation reactions, in which FOS such as levanbiose, inulobiose, blastose, …, are formed (Méndez-Lorenzo et al., 2015). The efficiency of SacB fructosylation with levan as donor was applied for the synthesis of blastose, a sucrose analogue with potential prebiotic properties. For this reaction, fructose was transferred to trehalose to produce a (2-6) fructosylated trehalose, which was later hydrolysed by trehalase to yield blastose (Miranda-Molina et al, 2017). Up to now there is not an efficient enzyme for the synthesis of levan-type FOS, in spite of intensive efforts to modify SacB or other levansucrases specificity by site directed mutagenesis. For this purpose, after a complete characterization of a combined bi-enzymatic reaction between SacB and an endolevanase produced by B.licheniformis. (LevB1) (Porras-Domínguez et al., 2014) we designed a fusion enzyme containing both activities. This fusion enzyme is able to produce levan-type FOS from sucrose, with molecular weights in the range of DP2 to DP10 including mainly 1-kestose, 6-kestose, neokestose, levanbiose and blastose, with 40% w/w yields. Chambert, R., & Gonzy-Tréboul, G. (1976). European Journal of Biochemistry / FEBS, 62(1), 55–64. Méndez-Lorenzo, L., Porras-Domínguez, J. R., Raga-Carbajal, E., Olvera, C., Rodríguez-Alegría, M. E., Carrillo-Nava, E.. López Munguía, A. (2015). PLoS ONE, 10(11), 1–15. Miranda-Molina, A., Castillo, E., & Lopez Munguia, A. (2017). Food Chemistry, 227, 202–210. Porras-Domínguez, J. R., Ávila-Fernández, Á., Miranda-Molina, A., Rodríguez-Alegría, M. E., & Munguía, A. L. (2015). Carbohydrate Polymers, 132(October), 338–344. Porras-Domínguez, J. R., Ávila-Fernández, Á., Rodríguez-Alegría, M. E., Miranda-Molina, A., Escalante, A., González-Cervantes, R., López Munguía, A. (2014). Process Biochemistry, 49(5), 783–790. Raga-Carbajal, E., Carrillo-Nava, E., Costas, M., Porras-Dominguez, J., López-Munguía, A., & Olvera, C. (2016). Glycobiology, 26(4), 377–385

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

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 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

<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

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

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

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