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.
Highlights
28 This study for enzymes based on physical, covalent, cross-linking or affinity interactions
A catalytically efficient and stable sorbitol dehydrogenase (SLDH) is required to improve the bioconversion of D-sorbitol to L-sorbose[25,26,27,28,29,30]
A previous study reported the fermentative production of L-sorbose from D-sorbitol by G. oxydans G62431
Summary
28 This study for enzymes based on physical, covalent, cross-linking or affinity interactions. Among these methods, covalent immobilization resulted in higher stability through strong attachments[14,15,16,17,18,19,20,21,22,23,24]. Stability of the SLDH was improved through immobilization on silica (SiO2) nanoparticles, resulting in high reusability. There is a need to produce a catalytically efficient and stable SLDH to improve the production of L-sorbose from D-sorbitol because of its broad industrial applications. GoSLDH is a promising candidate for the efficient production of L-sorbose from D-sorbitol
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