Fig 1.
Degree of polymerization (DP) of levans designated as ‘n’ can be very high–over 10 000 [31]. For simplicity, levans are shown unbranched and fructooligosaccharides (FOS) derived from endo-levanase reaction are shown to contain only fructose.
Fig 2.
Clustal Omega alignment of the Bacteroides thetaiotaomicron endo-levanase BT1760 with putative levanase (D4IW69) of Butyrivibrio fibrisolvens 16/4 (BF 16/4).
Identical residues between the sequences are shown on black background and marked with an asterisk. Similar residues are designated below the alignment with dots. Predicted N-terminal signal peptides are shown on grey background. Predicted nucleophiles, proton donors and the ‘RDP’ motifs are shown on red background. Cc–Clustal consensus.
Fig 3.
Comparison of fructan PUL of Bacteroides thetaiotaomicron (Bt) VPI-5482 [7] with genomic locus of Bacteroides xylanisolvens (Bx) CL03T12C04 harboring a close homologue of the B. thetaiotaomicron endo-levanase BT1760.
Colour code is used to designate homologous proteins of the two loci. ORFs of levan-hydrolyzing proteins of B. thetaiotaomicron and their homologues in B. xylanisolvens are in blue. Intervening unrelated genes [7] are in white.
Table 1.
Size-distribution of levans used in the study.
Fig 4.
Levan degradation by Escherichia coli expressing endo-levanase BT1760 of Bacteroides thetaiotaomicron from the pURI3-BT1760Cter plasmid.
(A) 5 μL of the liquid culture of recombinant E. coli was spotted onto a LB plate containing Amp, IPTG and 5 g/L of Lsc3-produced HMW levan and grown overnight at room temperature. Agar discs were cut off from regions marked with black rings, melted by heating and 0.5 μL of the sample was applied to a thin layer chromatography (TLC) plate. (B) TLC analysis of levan degradation from regions 3–6 of the agar plate in panel A and on lanes 7 and 8 products from 5 g/L levan by 1 h and 4 days of reaction with the lysate from recombinant E. coli. The chromatogram was developed in chloroform: methanol: water (90:65:15, v/v/v). Reference sugars on the TLC plate: fructose (F) and 1-kestose (K) on lane 1; sucrose (S) and nystose (N) on lane 2. F1-F5; levan degradation products with respective degree of polymerization.
Fig 5.
The effect of temperature on catalytic activity of the endo-levanase BT1760.
(A) Temperature optimum of BT1760. Release of reducing sugars from levan by BT1760 was recorded at varied temperatures and relative activities were calculated. 100% of activity corresponds to 123.6 ± 6.4 U/mg, measured at 37°C. (B) Thermostability of BT1760. The endo-levanase BT1760 was incubated at indicated temperatures for 30 min, and residual endo-levanase activity was then measured at 37°C by recording reducing sugar release from levan. 100% of activity corresponds to 124.0 ± 1.8 U/mg. The average and standard deviation values are calculated form at least two independent measurements with two parallel samples analysed. Detailed description of the methods is presented in Materials and Methods.
Fig 6.
Initial velocities of reducing sugar release by the endo-levanase BT1760 from various levans added at 5 g/L.
* Synthesized by Pseudomonas syringae pv. tomato levansucrase Lsc3 or its mutant Asp300Asn (D300N) from sucrose or raffinose (Raf). Mean values and standard deviation were calculated from at least three independent experiments. For additional information on levans, see Table 1.
Table 2.
Kinetic constants of endo-levanase BT1760 at degradation of various levans calculated from initial velocities of reducing sugar release.
Fig 7.
Time course of degradation of six different levans by BT1760 (A-F). Levans synthesized by Lsc3 or its mutant Asp300Asn (D300N) are designated by an asterisk. Degradation of the substrate (levan) is illustrated by the start region of developed thin layer chromatography plates inserted below each graph. Release of fructose presented as a line diagramme shows the percentage of fructose from total sugars detected in the sample during 72 h (4320 min) of the reaction. Total amount of FOS (g/L; DP from 2 to 13) produced by each time point is presented as grey bars. Up to four parallel samples were analysed to calculate the average values and standard deviations. Details of the reaction conditions and used methods are given in Materials and Methods section.
Fig 8.
Time course of degradation of six different levans by the BT1760 into products of varied DP (A-F). Levans synthesized by Lsc3 or its mutant Asp300Asn (D300N) are designated by an asterisk. Reaction products were analysed using HPLC. Up to four parallel samples were analysed to calculate the average values and standard deviations. Details of the reaction conditions and used methods are presented in Materials and Methods section.
Table 3.
Parameters of FOS production by endo-levanase BT1760 from six different levans at optimal reaction duration.
Fig 9.
Acid-resistance of levans and dahlia inulin.
Fructans (10 g/L) were incubated in 0.01 M hydrochloric acid (pH 2.0) at 37°C for 24 h and sampled over this period for reducing sugar and thin layer chromatography (TLC) analysis. The bars indicate the extent of hydrolysis of the fructan calculated as percentage of released reducing sugars from the total amount of reducing sugars in completely hydrolysed sample. Standard deviation values shown on bars are calculated from at least two independent experiments with at least two parallel samples. TLC shows fructan hydrolysis products at 7 and 24 h of incubation. Samples spotted onto a TLC plate are numbered from 1 to 7 according to respective numbers on the bar diagramme. The chromatogram was developed in chloroform: acetic acid: water (60:70:10; v/v/v). Sugar markers (M) on TLC: 7 g/L levan (L), 8 mM nystose (N), 8 mM 1-kestose (K), 30 mM sucrose (S) and 30 mM fructose (F). Details of the methods are presented in Materials and Methods section.