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

Andrea Hill,Tarun J Narwani,Salwa Karboune,Alexandre G De Brevern

doi:10.3390/ijms21155402

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.

Highlights

Biosynthetic routes for the synthesis of novel carbohydrates is an attractive course
The conversion rate of the sucrose decreased over the time course, which can be the consequence of a lower sucrose concentration and/or product inhibition
The rate of sucrose consumption by LS from G. oxydans, N. aromaticivorans, P. graminis, and B. indica subsp. indica was constant throughout the initial stage of the reaction, with final consumptions of 78%, 53%, 81%, and 72%, respectively, at 50 h

Summary

Introduction

Biosynthetic routes for the synthesis of novel carbohydrates is an attractive course. Enzymatic glycosylation reactions can proceed via a regio- and stereo-selective manner, unlike chemical synthesis, which requires the use of protecting groups [1]. Levansucrase (EC 2.4.1.10, LS) is a β-fructosyl transferase capable of catalyzing the non-Lelior-type transfructosylation reaction generating prebiotic fructooligosaccharides (FOSs) and the fructan polysaccharide levan. Levan is composed of β-(2-6)-linked fructosyl residues. It has its own functional properties for food and pharmaceutical industries and can be hydrolyzed to FOSs [2,3,4,5]. FOSs have interesting properties as prebiotics, having bifidogenic and anticarcinogenic effects, which make their synthesis desirable [6,7,8]

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Conclusion