Abstract
Bacteroides thetaiotaomicron, an abundant commensal of the human gut, degrades numerous complex carbohydrates. Recently, it was reported to grow on a β-2,6-linked polyfructan levan produced by Zymomonas mobilis degrading the polymer into fructooligosaccharides (FOS) with a cell surface bound endo-levanase BT1760. The FOS are consumed by B. thetaiotaomicron, but also by other gut bacteria, including health-promoting bifidobacteria and lactobacilli. Here we characterize biochemical properties of BT1760, including the activity of BT1760 on six bacterial levans synthesized by the levansucrase Lsc3 of Pseudomonas syringae pv. tomato, its mutant Asp300Asn, levansucrases of Zymomonas mobilis, Erwinia herbicola, Halomonas smyrnensis as well as on levan isolated from timothy grass. For the first time a plant levan is shown as a perfect substrate for an endo-fructanase of a human gut bacterium. BT1760 degraded levans to FOS with degree of polymerization from 2 to 13. At optimal reaction conditions up to 1 g of FOS were produced per 1 mg of BT1760 protein. Low molecular weight (<60 kDa) levans, including timothy grass levan and levan synthesized from sucrose by the Lsc3Asp300Asn, were degraded most rapidly whilst levan produced by Lsc3 from raffinose least rapidly. BT1760 catalyzed finely at human body temperature (37°C) and in moderately acidic environment (pH 5–6) that is typical for the gut lumen. According to differential scanning fluorimetry, the Tm of the endo-levanase was 51.5°C. All tested levans were sufficiently stable in acidic conditions (pH 2.0) simulating the gastric environment. Therefore, levans of both bacterial and plant origin may serve as a prebiotic fiber for B. thetaiotaomicron and contribute to short-chain fatty acids synthesis by gut microbiota. In the genome of Bacteroides xylanisolvens of human origin a putative levan degradation locus was disclosed.
Highlights
Seven different levans were used in this study: 1) levan synthesized from 1.2 M (410.8 g/L) sucrose by levansucrase Lsc3 of Pseudomonas syringae pv. tomato; 2) levan synthesized from 0.3 M (151.3 g/L) raffinose by Lsc3 of P. syringae; 3) levan synthesized from 1.2 M (410.8 g/L) sucrose by the Lsc3Asp300Asn (D300N) mutant of P. syringae; 4) levan of Zymomonas mobilis 113S
Levans of Z. mobilis, E. herbicola and H. smyrnensis are all produced from sucrose. 1-kestose, nystose, stachyose and inulin from dahlia were from SigmaAldrich (Germany), FOS-preparations P95 and Synergy1 were from Beneo (Belgium), xylooligosaccharide mixture was from Sweet Town Biotech (Taiwan) and raffinose was from Naxo (Estonia)
We aligned the sequences of INU2 and endo-levanase BT1760 of Bacteroides thetaiotaomicron and disclosed only a low (20.7%) overall identity between these proteins
Summary
Billions of microorganisms live in and on our bodies. Among these microbes, the gut community comprises a major part and largely affects our well-being and health [1,2]. The gut bacteria feed mostly on complex polysaccharides (food fiber, microbiota-accessible carbohydrates) that are resistant to degradation by gastric acid and host digestive enzymes. For example short-chain fatty acids (SCFA) and some other metabolites of microbial origin, have numerous beneficial functions for the host [1,4]. Rapid accumulation of new data on gut microbiota and its multiple functions has fueled search of prebiotics for other potentially beneficial gut bacteria such as Faecalibacterium and Bacteroides which are abundant commensals in healthy adults [8,9,10,11]
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