Production, Purification, Characterization, and Safety Evaluation of Recombinant D-Psicose 3-Epimerase Constructed in E. coli

If you buy or formulate allulose, enzyme performance is the first thing to check

Allulose is a rare sugar. You don’t extract it from plants in commercial quantities. You produce it from fructose. The enzyme that does that conversion is D-psicose 3-epimerase, also called DPEase. Its activity decides your yield, your purification load, and your cost per kilo.

This paper, published with DOI 10.1186/s12934-024-02487-x, walks through the construction, expression, purification, characterization, and safety testing of a recombinant DPEase. It gives you concrete numbers to use when evaluating enzyme suppliers.

Why DPEase matters for allulose production

DPEase converts fructose into D-psicose, which is allulose, by epimerization at the C-3 position. Without a stable and active enzyme, you get low conversion and high downstream separation costs. With a good enzyme, you can run at practical substrate loads and still get enough allulose to justify chromatography or other separation steps.

Most commercial allulose processes rely on microbial enzymes. Expression in E. coli is common because the system grows fast, scales well, and produces high enzyme titers. But expression alone is not enough. Purification must remove host proteins, nucleic acids, and endotoxins before the enzyme can be considered for food use.

What the study did

The researchers constructed a recombinant DPEase in E. coli, expressed it, then optimized purification. They also characterized the purified enzyme’s reaction behavior and ran an acute toxicity study in rats using a D-fructose-D-allulose mixed syrup.

The work covers the pathway from lab enzyme to food-relevant safety data. That is a useful combination for anyone asking whether a new enzyme source is ready for scale-up.

Key findings

Purification optimization delivered a 21.03-fold increase in specific activity. That is the kind of jump that changes process economics. More activity per milligram of enzyme means less catalyst, smaller reactors, and fewer contaminants to remove.

Using 25% fructose as substrate, the enzyme produced 5.60% allulose at a conversion rate of 22.42%. The reaction ran at pH 7.5 and 55°C with 10 mM Mn²⁺. Manganese clearly played a supporting role, as it does for many epimerases.

The safety data matter too. Acute toxicity testing showed no toxicity from the mixed sugar syrup in rats. That supports further development for food use. It is not a full regulatory approval, but it is a necessary first step.

Parameter	Finding
Expression host	Recombinant E. coli
Specific activity gain after purification optimization	21.03-fold
Substrate	25% fructose
Reaction conditions	pH 7.5, 55°C, 10 mM Mn²⁺
Allulose concentration	5.60%
Fructose-to-allulose conversion	22.42%
Acute oral toxicity in rats	No toxicity observed

What it means for manufacturers

Start with the conversion number. 22.42% from a 25% fructose feed means roughly 5.6 grams of allulose per 100 mL of reaction mix. That is a realistic starting point for a batch. It is not the final yield after purification, but it lets you model separation loads and enzyme dosing.

The reaction conditions are factory-friendly. 55°C is warm enough to keep fructose soluble and reduce bioburden, yet cool enough for standard stainless steel equipment. pH 7.5 avoids extreme acidic or alkaline conditions, which simplifies buffering and downstream neutralization.

For ingredient buyers, the 21.03-fold activity improvement is a procurement signal. If one enzyme supplier uses a crude preparation and another uses an optimized recombinant enzyme like this one, the dosing rates will not be the same. Ask for specific activity data, not just enzyme weight.

The rat safety result also matters from a sourcing perspective. Suppliers that can document acute toxicity data for their enzyme and product stream have moved beyond the “just an enzyme in a test tube” stage.

FAQ

Does this paper prove the enzyme is safe for food?

No. It proves the mixed fructose-allulose syrup showed no acute toxicity in rats. Full food safety evaluation requires more than acute toxicity data. You also need chronic studies, genetic toxicity data, process contamination controls, and regulatory review. But this study checks one important box.

What should I ask an enzyme vendor after reading this?

Ask for specific activity expressed as units per milligram of protein. Then ask for conversion data at your planned substrate concentration. Use 25% fructose as a reference point. If a vendor cannot give you conversion and allulose concentration together, you are not comparing like with like.

Why does the Mn²⁺ in the reaction matter to me?

The enzyme needs 10 mM Mn²⁺ in this study. In a production process, you will need to account for manganese in the waste stream and in the final syrup. That affects your purification and food-additive compliance work. It is not a blocker, but it is a process cost.

Allulose is becoming a volume ingredient. The suppliers who can make DPEase cheaper, cleaner, and safer will have an edge. This paper provides a clear benchmark: 21-fold better enzyme activity after purification, 22.42% conversion on 25% fructose at 55°C and pH 7.5, and no acute toxicity signal in rats. Use those numbers as your starting point when you evaluate enzyme technologies or negotiate supply contracts.