Production, Purification, Characterization, and Safety Evaluation of a Constructed Recombinant D-Psicose 3-Epimerase

If you buy allulose or develop the enzymes behind it, this paper gives you a concrete benchmark. Published in Microbial Cell Factories under DOI 10.1186/s12934-024-02487-x, it covers the full route: enzyme construction, expression, purification, bioconversion, and a rat safety study. The results are practical enough to use in supplier conversations.

Background

Allulose, also called D-psicose, is a rare sugar that formulators want for its sugar-like functionality. The production bottleneck is usually the enzyme. You need something that converts fructose into allulose at scale, with enough activity to keep enzyme costs down. This study targets exactly that enzyme: D-psicose 3-epimerase, or DPEase.

The researchers built a recombinant DPEase and expressed it in E. coli. Then they optimized the purification step. They also tested the safety of the resulting D-fructose-D-allulose mixed syrup. That last part matters. Most commercial processes do not produce pure allulose in one step. Buyers often deal with a syrup stream that still contains fructose.

What the study did

The team produced recombinant DPEase in E. coli and worked through the purification process. After optimization, the purified enzyme showed a 21.03-fold increase in specific activity. That is a major improvement. More active enzyme per gram of protein means you can potentially use less enzyme per batch.

The bioconversion step used 25% fructose as the substrate. The reaction ran at pH 7.5, 55°C, and 10 mM Mn²⁺. Under those conditions, the allulose concentration reached 5.60% and the conversion rate hit 22.42%. Those numbers give you a realistic starting point for process design.

The safety evaluation is just as relevant. In an acute toxicity study, the D-fructose-D-allulose mixed syrup showed no toxicity in rats. That supports further development of the syrup stream, not just refined allulose powder.

Key findings

Process step	Finding	What it means
Enzyme source	Recombinant DPEase expressed in E. coli	A common, scalable expression host
Purification optimization	Specific activity increased 21.03-fold	Less enzyme protein needed per unit of activity
Substrate	25% fructose	A practical feed concentration for testing
Reaction conditions	pH 7.5, 55°C, 10 mM Mn²⁺	A clear baseline for process work
Allulose output	5.60%	Product concentration in the reaction broth
Conversion	22.42%	Residual fructose remains, so separation is required
Acute toxicity	No toxicity in rats	A useful safety signal for the mixed syrup

The 22.42% conversion figure is the one to remember. It tells you what a simple batch reaction can deliver under these conditions. It also tells you not to expect a single-pass, high-yield process. You still need downstream separation or recycling.

What it means for manufacturers

Use this paper as a reference when you evaluate enzyme suppliers. Ask how their enzyme compares to a 21.03-fold purification improvement. Ask what conversion they guarantee at 25% fructose, pH 7.5, and 55°C. If a supplier will not share those numbers, treat that as a red flag.

The manganese requirement deserves attention. The reaction uses 10 mM Mn²⁺. That is not a negligible amount. You will need to verify whether residual manganese can be removed from the final syrup or whether the supplier offers a metal-free enzyme variant.

The rat safety data supports this specific mixed syrup. It does not automatically cover every syrup made with a different enzyme or a different purification train. Your own qualification program should include your actual product stream.

FAQ

Is D-psicose the same as allulose? Yes. D-psicose is the older chemical name. The ingredient name used by most buyers today is allulose.

Can I use this enzyme in production right now? Probably not. This is a research study, not a commercial enzyme product. Use it to benchmark enzyme performance and safety expectations when you talk with suppliers.

What should a buyer do with this paper? Check your current supplier's enzyme activity and conversion data against these numbers. You now have a clear reference point.

That baseline is real. Compare your next enzyme datasheet against it.