Production, Purification, Characterization, and Safety Evaluation of Recombinant D-Psicose

The enzyme bottleneck

If you buy or formulate with allulose, the question is always the same: can the enzyme step deliver enough yield at a cost you can defend? This paper, published in Microbial Cell Factories (DOI 10.1186/s12934-024-02487-x), answers with data.

Background

Allulose, also called D-psicose, is a rare sugar. For food formulators, it is a target ingredient. The production problem is the enzyme. DPEase, or D-psicose 3-epimerase, converts fructose into allulose, but the enzyme is often inefficient and hard to produce at scale. This study tackled both issues head-on.

The team built a recombinant DPEase, expressed it in E. coli, purified it, and then tested the resulting syrup for acute safety. It is a complete process story, not just an enzyme characterization.

What the study did

The researchers constructed a recombinant DPEase and expressed it in E. coli. They then optimized the purification process. The result was a 21.03-fold increase in specific activity. That is not a marginal tweak. It changes what the enzyme costs per unit of activity.

Next, they used the purified enzyme to convert fructose to allulose. The substrate was 25% fructose. The reaction ran at pH 7.5 and 55°C, with 10 mM Mn²⁺ as a cofactor. Under those conditions, the allulose concentration reached 5.60%, and the conversion rate hit 22.42%.

Finally, they ran an acute toxicity study on a D-fructose-D-allulose mixed syrup. In rats, it showed no toxicity. That is a useful safety signal for food-ingredient development.

Key findings

The table below captures the core numbers.

Parameter	Condition / Result
Enzyme expression host	E. coli
Purification optimization	21.03-fold higher specific activity
Substrate	25% fructose
Reaction pH	7.5
Reaction temperature	55°C
Mn²⁺ concentration	10 mM
Allulose concentration after reaction	5.60%
Fructose-to-allulose conversion rate	22.42%
Acute toxicity in rats	No toxicity observed

That 22.42% conversion rate is a benchmark, not a ceiling. With 25% starting fructose, you get 5.60 grams of allulose per 100 mL of reaction mix. That still means downstream separation. But a better enzyme shifts the balance. Less unreacted fructose to remove. Less water to evaporate. Lower operating cost.

What it means for manufacturers

For ingredient buyers, the immediate signal is process stability. A 21-fold improvement in specific activity means you need less enzyme to make the same amount of allulose. That directly attacks the cost per kilo. Enzyme cost is often the reason allulose sits at a premium. This work points to a way down.

The reaction conditions are practical, too. pH 7.5 and 55°C are not exotic. Many standard food-grade enzyme tanks already run in that range. Mn²⁺ at 10 mM is a simple supplement. You do not need special equipment or unusual buffers. That lowers the barrier for existing fructose processors.

The safety result also matters. A mixed fructose-allulose syrup with no acute toxicity in rats is a real data point. It supports the idea that a less-purified, more cost-effective syrup could be food-grade. That said, do not confuse acute toxicity with a full regulatory review. You still need your own compliance work for your target market.

FAQ

Is the syrup directly usable as a food ingredient? Not automatically. The study shows acute safety in rats, but commercial use depends on regulatory approval in your region, plus specifications for color, taste, and purity.

Why does a 22.42% conversion rate matter? It tells you how much allulose you get from a given fructose input. At 25% fructose, you get 5.60% allulose in the reaction mix. Higher conversion means less fructose to remove and better yield per batch.

What is DPEase? DPEase, or D-psicose 3-epimerase, is the enzyme that converts fructose into D-psicose, which is allulose. This study used a recombinant version expressed in E. coli, then purified it with a large gain in specific activity.

For now, the numbers in this paper give formulators a concrete reference point. If you are evaluating enzyme suppliers, ask for specific activity data and conversion curves under your own substrate conditions. This study shows what a well-optimized recombinant DPEase can do. The next step is scaling it into a process that works in your plant.