A Bacillus-Derived D-Allulose 3-Epimerase with Outstanding Thermostability and Catalytic Efficiency

Formulators hunting for a workhorse allulose-producing enzyme just got a new lead. A team cloned a novel D-allulose 3-epimerase (DaeB) from the plant probiotic strain Bacillus sp. KCTC 13219 and expressed it in Bacillus subtilis. The results are striking. At 50°C, the enzyme’s half-life stretches to 25 days. Its turnover number hits 367 s⁻¹. And when fed 700 g/L fructose, it cranks out roughly 200 g/L allulose.

That is not just another lab curiosity. This enzyme appears ready for industrial-scale sugar conversion.

Why Allulose Enzymes Matter

Allulose is a rare sugar with about 70% of sucrose’s sweetness but almost no calories. It does not spike blood glucose. It browns and melts like sugar. The catch? It is scarce in nature. Commercial production relies on enzymatic epimerization of fructose. The enzyme, D-allulose 3-epimerase (DAEase), rearranges a hydroxyl group at the C3 position. That simple shuffle turns fructose into allulose. But enzymes are picky. Many are thermolabile, slow, or both. A catalyst that keeps working for weeks at elevated temperatures changes the economics.

What the Study Did

The research team went hunting in bacterial genomes. They found a candidate in Bacillus sp. KCTC 13219, a strain originally isolated from plant environments. They cloned the gene for DaeB and expressed it in Bacillus subtilis, a workhorse industrial host. Then they characterized the purified enzyme.

They measured thermostability by incubating the enzyme at 50°C and sampling activity over time. The half-life was 25 days. That means after more than three weeks, the enzyme retained half its original activity. Most DAEases measured in days or hours degrade far faster. They also measured catalytic rate, or kcat, at 367 s⁻¹. That is a fast enzyme. Under realistic conditions with high fructose concentration, the conversion yielded about 200 g/L allulose from 700 g/L fructose. That is roughly a 28-29% conversion rate, typical for this reaction at equilibrium, but achieved with a catalyst that refuses to quit.

Key Findings at a Glance

Here is the evidence that matters for process design.

Parameter	DaeB Value	Why It Matters
Source organism	Bacillus sp. KCTC 13219	Plant-associated probiotic, safe lineage
Expression host	Bacillus subtilis	Established industrial fermentation
Half-life at 50°C	25 days	Enables long-batch or continuous operation
Turnover number (kcat)	367 s⁻¹	Fast conversion, lower enzyme loading
Substrate load	700 g/L fructose	High solids, realistic for commercial syrup
Product yield	~200 g/L allulose	Meaningful output per batch

That table tells a simple story. You can run this enzyme hot, keep it running for weeks, and push high substrate loads. That is exactly what a production facility wants.

What It Means for Manufacturers

First, enzyme costs drop. A 25-day half-life means you do not need to immobilize or continually replenish the catalyst. One batch of enzyme can power many reaction cycles. Even if immobilized preparations lose some activity during immobilization, the intrinsic stability offers a huge buffer.

Second, process temperature matters. At 50°C, fructose stays dissolved at high concentrations. The risk of microbial contamination falls. The reaction runs faster than at lower temperatures. DaeB was cloned and expressed in a GRAS-status Bacillus host, so regulatory approval pathways may be smoother than enzymes from less familiar organisms. No novel source worries. No exotic expression system.

Third, yields around 200 g/L from 700 g/L fructose are commercially plausible. While higher conversions are possible with borate complexation or continuous removal, even a simple batch process at this concentration gives a syrup that can be refined and purified. For ingredient buyers, this means a more stable supply chain and potentially lower allulose prices.

Frequently Asked Questions

How does DaeB compare to other DAEases?
Most reported DAEases have half-lives of hours to a few days at their optimal temperatures. DaeB’s 25-day half-life at 50°C is exceptional. Its kcat is also on the high end, meaning it converts substrate quickly. Both traits together are rare.

Can this enzyme be used in a continuous process?
That is the logical next step. With thermostability this high, packed-bed reactors with immobilized DaeB become practical. The enzyme would stay active for weeks under continuous flow. No published data yet on immobilization, but the intrinsic stability is a green light for trials.

What about regulatory approval?
The source strain is Bacillus sp., and the expression host is Bacillus subtilis. Both are common in food enzyme production. That does not guarantee approval, but it lowers the risk of novel toxicity or allergenicity concerns. Expect a smoother GRAS or food enzyme dossier than with a non-conventional source.

A New Benchmark

This paper puts a clear mark on the wall. A DAEase with a 25-day half-life and a 367 s⁻¹ kcat is not an incremental step. It is a practical tool. The 200 g/L yield at 700 g/L fructose confirms the enzyme works under real-world substrate loads. For formulators, that means a reliable path to allulose supply. For buyers, it means fewer process headaches and better cost projections. The next move belongs to the engineers who scale it up. But the biology is already on your side.