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. 2016 Sep 16:6:33438.
doi: 10.1038/srep33438.

A highly efficient sorbitol dehydrogenase from Gluconobacter oxydans G624 and improvement of its stability through immobilization

Tae-Su Kim  1 , Sanjay K S Patel  1 , Chandrabose Selvaraj  1 , Woo-Suk Jung  2 , Cheol-Ho Pan  2 , Yun Chan Kang  3 , Jung-Kul Lee  1
Affiliations

A highly efficient sorbitol dehydrogenase from Gluconobacter oxydans G624 and improvement of its stability through immobilization

Tae-Su Kim et al. Sci Rep. .

Abstract

A sorbitol dehydrogenase (GoSLDH) from Gluconobacter oxydans G624 (G. oxydans G624) was expressed in Escherichia coli BL21(DE3)-CodonPlus RIL. The complete 1455-bp codon-optimized gene was amplified, expressed, and thoroughly characterized for the first time. GoSLDH exhibited Km and kcat values of 38.9 mM and 3820 s(-1) toward L-sorbitol, respectively. The enzyme exhibited high preference for NADP(+) (vs. only 2.5% relative activity with NAD(+)). GoSLDH sequencing, structure analyses, and biochemical studies, suggested that it belongs to the NADP(+)-dependent polyol-specific long-chain sorbitol dehydrogenase family. GoSLDH is the first fully characterized SLDH to date, and it is distinguished from other L-sorbose-producing enzymes by its high activity and substrate specificity. Isothermal titration calorimetry showed that the protein binds more strongly to D-sorbitol than other L-sorbose-producing enzymes, and substrate docking analysis confirmed a higher turnover rate. The high oxidation potential of GoSLDH for D-sorbitol was confirmed by cyclovoltametric analysis. Further, stability of GoSLDH significantly improved (up to 13.6-fold) after cross-linking of immobilized enzyme on silica nanoparticles and retained 62.8% residual activity after 10 cycles of reuse. Therefore, immobilized GoSLDH may be useful for L-sorbose production from D-sorbitol.

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Figures

Figure 1
Figure 1. Purification and characterization of GoSLDH.
(A) Determination of the molecular mass of Gluconobacter oxydans G624 GoSLDH by SDS-PAGE. SDS-PAGE analysis of GoSLDH expressed in E. coli BL21 (DE3)-CodonPlus RIL was carried out on a 12% gel. Lane M: molecular standard marker, insoluble protein (Lane 1), soluble protein (Lane 2) and purified protein (Lane 3) GoSLDH. (B) Effect of pH on the activity of purified GoSLDH. Filled circles in Tris-HCl (100 mM) buffer (pH 7.0–9.0) and filled diamonds in glycine-NaOH (100 mM) buffer (pH 9.0–10.5). (C) Effect of temperature on the activity of purified GoSLDH. (D) Effect of substrate concentration on the activity of purified GoSLDH.
Figure 2
Figure 2. Substrate docking of GoSLDH and PfMDH with NAD(P)+ and D-sorbitol.
(A) Distance between the Lys294 (N atom) and D-sorbitol (H atom) in GoSLDH. (B) Distance between the Lys295 (N atom) and D-sorbitol (H atom) in PfMDH. All other atoms are colored according to standard coloring. Amino acid residues are shown by the tube model, whereas bound D-sorbitol is represented by the ball-and-stick model. Active-site coordinating bond lengths are shown in Å. Images were generated using Maestro 10.2.
Figure 3
Figure 3. Electrochemical property of GoSLDH.
(A) Reaction mechanism of GoSLDH. (B) Electrocatalytic voltammograms for D-sorbitol oxidation by dehydrogenases. The oxidation of D-sorbitol by GoSLDH (blue line), PfMDH (red line), and blank (gray dot line) are shown.
Figure 4
Figure 4. Thermodynamic contributions to D-sorbitol binding by GoSLDH (Lys294Gln) and PfMDH (Lys195Gln) determined by isothermal titration calorimetry.
(A) Purified GoSLDH. (B) Purified PfMDH. The inset graph shows thermograms of GoSLDH (Lys294Gln) and PfMDH (Lys195Gln).
Figure 5
Figure 5
Stability of free (filled circle) and immobilized GoSLDH (open circle) at 25 °C (A) and reusability of immobilized GoSLDH on SiO2 particles (B).
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References

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