Production, purification, characterization, and safety evaluation of a recombinant D-psicose 3-epimerase

Allulose buyers and enzyme suppliers should watch this paper. It shows a working route for making D-psicose 3-epimerase in E. coli, and it backs the finished syrup with an animal safety study. The headline number is 21.03. That is the fold improvement in specific activity for the purified enzyme after process optimization. This is not a lab curiosity. It directly affects yield per gram of enzyme and downstream cost.

Why DPEase is the critical enzyme

Allulose, also called D-psicose, is a low-calorie rare sugar. It gives food formulators the bulk and sweetness of sugar without the same glycemic response. Industrial production depends on enzymatic isomerization of fructose. The enzyme D-psicose 3-epimerase, or DPEase, performs that conversion at the C-3 position. But DPEase can be expensive to produce and tricky to purify. Engineered expression and better recovery steps are what make the economics work at scale.

What the study did

The team constructed a recombinant DPEase and expressed it in Escherichia coli. They then optimized both the expression and purification workflow. After optimization, the purified enzyme’s specific activity rose 21.03-fold. That is a meaningful jump for anyone pricing enzyme doses.

The researchers also tested the enzyme under fixed reaction conditions. They used 25% fructose as the substrate. The reaction ran at pH 7.5 and 55°C, with 10 mM Mn²⁺ present. Under those conditions, the allulose concentration reached 5.60%. The conversion rate hit 22.42%. That means roughly 22% of the available fructose became allulose in that reaction mixture. The rest remains fructose, so downstream separation is still required.

Safety data came from an acute toxicity study in rats. The test material was a mixed syrup of D-fructose and D-allulose. The result: no toxicity. That supports the safety profile of the syrup, at least under acute exposure.

Key findings

Here are the numbers that matter most for process review.

Parameter	Finding
Enzyme expression host	Escherichia coli
Substrate	25% fructose
Reaction pH	7.5
Reaction temperature	55°C
Cofactor	10 mM Mn²⁺
Allulose concentration in reaction	5.60%
Fructose-to-allulose conversion rate	22.42%
Purified enzyme specific activity	21.03-fold improvement after optimization
Acute toxicity study	No toxicity in rats for D-fructose/D-allulose mixed syrup

What this means for manufacturers

The 22.42% conversion rate is not a theoretical maximum. It is a measured result from a defined reactor condition. You can use that number as a baseline for mass balance work. If your incoming fructose stream is 25% solids, you know what a single-pass enzyme reaction might give you before concentration and chromatography.

The expression host matters for supply security. E. coli fermentation is familiar to enzyme producers. It scales in standard stainless steel tanks. The 21.03-fold specific activity improvement makes the enzyme more cost-competitive because you need less protein to achieve the same conversion. For ingredient buyers, that can translate into a lower cost per kilogram of allulose.

The reaction conditions are practical. pH 7.5 is close to neutral. 55°C is hot enough to discourage microbial growth, but not so hot that sugar degradation becomes a serious issue. Mn²⁺ at 10 mM is the cofactor. Be careful with that. Manganese can carry into the final product stream. You will need to test for residual metal and decide whether removal or sequestration is necessary for your application.

The toxicity result matters for procurement, too. An acute rat study does not replace a full regulatory package. But it gives your quality group a document to review. When a supplier claims their allulose syrup is safe, you can ask for this kind of evidence. The D-fructose/D-allulose mixed syrup showed no toxicity in the study. That is a useful data point for initial risk assessment.

Frequently asked questions

Is 5.60% allulose concentration high enough for commercial production?

It is not a finished product concentration. It is a reaction titer. Commercial allulose processes run moderate conversions and then use chromatographic separation to isolate allulose. The 5.60% number matters because it shows the enzyme’s real output under defined conditions. It gives you a starting point for yield calculations.

Why should I care about a 21.03-fold specific activity improvement?

Specific activity is activity per unit of protein. A 21.03-fold improvement means you need far less purified protein to hit the same fructose conversion. That lowers enzyme production costs and reduces the load on downstream purification steps.

Does the rat safety study prove allulose is safe to sell?

No single study proves market safety. This is one piece of toxicological evidence. Regulatory approval depends on the target market and the full dossier. But the acute toxicity result supports the safety profile of the mixed fructose-allulose syrup and gives buyers something concrete to evaluate.

The full paper, DOI 10.1186/s12934-024-02487-x, contains the methods behind these numbers. Use the 21.03-fold improvement as a benchmark when comparing enzyme suppliers. Use the 22.42% conversion rate as a planning figure for process design. The data is not a magic bullet, but it is a solid starting point for procurement conversations.