Purpose: To synthesize allitol from D-psicose by a combination of novel ribitol dehydrogenase (RDH)
and formate dehydrogenase (FDH) under optimised production conditions.
Methods: RDH and FDH genes were cloned and introduced into pET-22b(+) vectors for expression in
Escherichia coli
to produce the corresponding enzymes. The effects of temperature, pH, shaking
velocity (75, 100, 125, and 150 rpm), and shaking type (horizontal and vortex) were optimised to
maximise the production yield of allitol. The final product was purified and subjected to nuclear magnetic
resonance (NMR) spectroscopy, infrared (IR) spectrometry, and liquid chromatography-mass
spectrometry (LC-MS) to confirm its structure.
Results: The optimal pH and temperature for the reaction were 7.5 and 40 °C, respectively. The results
revealed that allitol yield significantly increased with increase in reaction shaking velocity and reached a
maximum yield of 95.60 ± 0.54 % at 150 rpm shaking velocity after 6 h of reaction. When the reaction
was run under horizontal shaking, allitol yield increased from 100.00 ± 6.05 (without shaking) to 124.20
± 9.70 %. Twenty milligrams of D-psicose were successfully reduced to allitol under optimum conditions
with a high production yield of 16.7 ± 0.62 mg after 6 h. No by-products were formed during or after the
reaction. The produced allitol had a purity of 95 %, and its structure was confirmed by HPLC, IR, LC-MS,
and NMR spectral analyses.
Conclusion: Using D-psicose as a substrate, allitol with 95 % purity was successfully produced by the
combination of novel RDH and FDH.
