Intrinsic Levanase Activity of Bacillus subtilis 168 Levansucrase (SacB)

Abstract

Levansucrase catalyzes the synthesis of fructose polymers through the transfer of fructosyl units from sucrose to a growing fructan chain. Levanase activity of Bacillus subtilis levansucrase has been described since the very first publications dealing with the mechanism of levan synthesis. However, there is a lack of qualitative and quantitative evidence regarding the importance of the intrinsic levan hydrolysis of B. subtilis levansucrase and its role in the levan synthesis process. Particularly, little attention has been paid to the long-term hydrolysis products, including its participation in the final levan molecules distribution. Here, we explored the hydrolytic and transferase activity of the B. subtilis levansucrase (SacB) when levans produced by the same enzyme are used as substrate. We found that levan is hydrolyzed through a first order exo-type mechanism, which is limited to a conversion extent of around 30% when all polymer molecules reach a structure no longer suitable to SacB hydrolysis. To characterize the reaction, Isothermal Titration Calorimetry (ITC) was employed and the evolution of the hydrolysis products profile followed by HPLC, GPC and HPAEC-PAD. The ITC measurements revealed a second step, taking place at the end of the reaction, most probably resulting from disproportionation of accumulated fructo-oligosaccharides. As levanase, levansucrase may use levan as substrate and, through a fructosyl-enzyme complex, behave as a hydrolytic enzyme or as a transferase, as demonstrated when glucose and fructose are added as acceptors. These reactions result in a wide variety of oligosaccharides that are also suitable acceptors for fructo-oligosaccharide synthesis. Moreover, we demonstrate that SacB in the presence of levan and glucose, through blastose and sucrose synthesis, results in the same fructooligosaccharides profile as that observed in sucrose reactions. We conclude that SacB has an intrinsic levanase activity that contributes to the final levan profile in reactions with sucrose as substrate.

Investigating the Product Profiles and Structural Relationships of New Levansucrases with Conventional and Non-Conventional Substrates

“…These LSs may have demonstrated exolevanase activity once the oligolevan/levan accumulation was high enough to be hydrolyzed in the presence of the preferred substrate sucrose. This exolevanase activity was previously demonstrated by BS SacB (LS from B. subtilis), where hydrolysis (without sucrose present) continued until β(2-1) was reached [24]. In the presence of sucrose, these hydrolysis products then have the potential to be used as acceptor molecules in transfructosylation reactions.…”

Size-Dependent Variability in Flow and Viscoelastic Behavior of Levan Produced by Gluconobacter albidus TMW 2.1191

“…However, it is yet unknown to what extent the size of levan influences its rheological properties. It was previously shown that levans produced by G. albidus TMW 2.1191 without external pH control are comparatively smaller in size at prolonged fermentation times, which might be due to (a combination of) continuous acidic hydrolysis, the intrinsic β-fructosidase activities of levansucrases or possibly additionally expressed β-fructosidases [10,20,35,36]. Accordingly, in the present study, the levan recovered from NaG medium after 48 h of incubation exhibited the lowest hydrodynamic volume/radius, at which an elevated nitrogen content was determined for this levan in comparison to the levans produced in Na-acetate buffers (pH 4.0 and 5.0).…”

Size-Dependent Rheological Variability of Levan Produced by Gluconobacter Albidus TMW 2.1191

“…However, it is yet unknown to which extent the size of levan in fact influences its rheological properties. It was previously shown that levans produced by G. albidus TMW 2.1191 without external pH control are comparatively smaller in size at prolonged fermentation times, which might be due to (a combination of) continuous acidic hydrolysis, the intrinsic β-fructosidase activities of levansucrases or possibly additionally expressed β-fructosidases [9,19,32,33]. Accordingly, the levan recovered from NaG medium after 48 h of incubation in the present study exhibited the lowest hydrodynamic volume/radius.…”