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

Abstract

The synthesis of complex oligosaccharides is desired for their potential as prebiotics, and their role in the pharmaceutical and food industry. Levansucrase (LS, EC 2.4.1.10), a fructosyl-transferase, can catalyze the synthesis of these compounds. LS acquires a fructosyl residue from a donor molecule and performs a non-Lenoir transfer to an acceptor molecule, via β-(2→6)-glycosidic linkages. Genome mining was used to uncover new LS enzymes with increased transfructosylating activity and wider acceptor promiscuity, with an initial screening revealing five LS enzymes. The product profiles and activities of these enzymes were examined after their incubation with sucrose. Alternate acceptor molecules were also incubated with the enzymes to study their consumption. LSs from Gluconobacter oxydans and Novosphingobium aromaticivorans synthesized fructooligosaccharides (FOSs) with up to 13 units in length. Alignment of their amino acid sequences and substrate docking with homology models identified structural elements causing differences in their product spectra. Raffinose, over sucrose, was the preferred donor molecule for the LS from Vibrio natriegens, N. aromaticivorans, and Paraburkolderia graminis. The LSs examined were found to have wide acceptor promiscuity, utilizing monosaccharides, disaccharides, and two alcohols to a high degree.

Investigating the Potential of Phenolic Compounds and Carbohydrates as Acceptor Substrates for Levansucrase‐Catalyzed Transfructosylation Reaction**

“…LS4’s active site was deeper and had more positive electrostatic potential. Comparing the least binding energy conformation of sucrose with the LSs, sucrose was rotated 45° downwards with LS1 but was orientated in the same way as the B. subtilis LS with LS2 and LS4 [1b] . In addition, while previous studies have reported that the fructose residue of the donor sucrose molecule binds the −1 subsite with high affinity, it was suggested that the docking of acceptor molecules could be variable because of the +1 subsite's relaxed binding nature.…”

“…The differences in the reaction selectivity of LSs are attributed to their amino acid sequences, which dictate their active subsite structure [1b,3] . The cavities of LS from G. oxydans (LS1), V. natriegens (LS2) and P. graminis (LS4) were compared to that of the deep negatively charged pocket of Bacillus subtilis LS.…”

Discovery and Enzymatic Screening of Genome-Mined Microbial Levanases to Produce Second-Generation β-(2,6)-Fructooligosaccharides: Catalytic Properties

“…6 These causes justify the quest for new sources and approaches for the synthesis of tailor-made levan-type FOSs that can effect broader physiological changes. 3,7,8 Levan, a β- (2,6) polymer of fructose, naturally occurs in various plant sources (e.g., ryegrass) and can also be obtained from a wide selection of bacteria during their assimilation of sucrose by the action of levansucrase (EC 2.4.1.10). 9−11 This polysaccharide has been identified as a promising source for the production of levan-type FOSs 10,12,13 by the action of endo-levanases (EC 3.2.1.65), glycosyl hydrolases belonging to the GH32 family.…”

“…The intake of prebiotics has been credited with a wide range of physiological benefits, the most widely recognized being enhanced gastrointestinal health. − Levan-type fructooligosaccharides (β-(2,6)-FOSs and neo-FOSs) are receiving increased interest due to higher selectivity and prebiotic potential as compared to their β-(2,1)-counterparts , as well as anti-adhesion activity against pathogens . These causes justify the quest for new sources and approaches for the synthesis of tailor-made levan-type FOSs that can effect broader physiological changes. ,, …”

Production of Sucrolytic Enzyme by Bacillus licheniformis by the Bioconversion of Pomelo Albedo as a Carbon Source

“…Sucrose hydrolysis is catalyzed by sucrolytic enzymes to produce a mixture of glucose and fructose (also known as invert sugar). Sucrolytic enzymes are of various types such as invertases (sucrases) [2], levansucrases [3], dextransucrases [4], glucansucrases [5], and inulosucrases [6]. They use sucrose as the donor of fructosyl moiety to be transferred to various kinds of acceptors, such as water (sucrose hydrolysis), sucrose, and fructan [7].…”