Abstract
Under specific reaction conditions, levansucrase from Bacillus subtilis (SacB) catalyzes the synthesis of a low molecular weight levan through the non-processive elongation of a great number of intermediates. To deepen understanding of the polymer elongation mechanism, we conducted a meticulous examination of the fructooligosaccharide profile evolution during the levan synthesis. As a result, the formation of primary and secondary intermediates series in different reaction stages was observed. The origin of the series was identified through comparison with product profiles obtained in acceptor reactions employing levanbiose, blastose, 1-kestose, 6-kestose, and neo-kestose, and supported with the isolation and NMR analyses of some relevant products, demonstrating that all of them are inherent products during levan formation from sucrose. These results allowed to establish the network of fructosyl transfer reactions involved in the non-processive levan synthesis. Overall, our results reveal how the relaxed acceptor specificity of SacB during the initial steps of the synthesis is responsible for the formation of several levan series, which constitute the final low molecular weight levan distribution.
Highlights
Fructans are fructosyl polymers produced by several plants and microorganisms whose biological function as energy reservoir and microbial defense agent has been demonstrated[1,2]
The observation of reaction intermediates using this type of analysis allowed us to elucidate the SacB levan elongation mechanisms; namely, a processive and a non-processive mechanism for high molecular weight (HMW) and low-molecular weight (LMW) levan synthesis respectively[19]
In order to gain a better understanding about the mode of action of SacB levansucrase in the non-processive elongation of levans, we meticulously examined the synthesis products following the formation and evolution of intermediates by HPAE-PAD in reactions with sucrose as a single substrate and in the presence of different inherent acceptors of the reaction
Summary
Distinct intermediate series emerge throughout the non-processive synthesis of levans. Bearing in mind that blastose and levanbiose are the major low-DP products in the final product profile of SacB-catalyzed reaction, both disaccharides were tested as acceptors to elucidate their role in the formation of intermediates observed during the LMW levan synthesis, either in the primary or secondary groups. Our evidence highlights the capability of SacB to synthetize a complete series of neo-FOS from the corresponding precursor, i.e. 6-neo-nystose; during LMW levan synthesis only the tri- and tetra-saccharides accumulate, to be later consumed This truncated polymerization may be related to the low acceptor specificity of neo-kestose, which explains its accumulation among the primary trisaccharides. In the late phase, when glucose and fructose reach an adequate concentration to become fructosyl acceptors, ercose, inulobiose, blastose, and levanbiose are produced, including the two latter as initiators of the blasto-FOS and oligolevan series respectively, both part of the secondary intermediate group of products. Further work to study the effect of oligosaccharide DPs in the reaction catalyzed by the B. subtilis levansucrase is in progress
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