A D-Allulose 3-Epimerase Originating from Bacillus Shows High Thermostability and Notable Activity

A 25-day half-life changes the cost equation

D-allulose production is an enzyme game. The enzyme must convert fructose into D-allulose quickly, stay active under heat, and tolerate heavy sugar loads. Many enzymes fail on at least one of those. Not this one.

The study, published with DOI 10.1186/s12934-021-01550-1, describes a newly cloned D-allulose 3-epimerase called DaeB. It comes from the plant probiotic strain Bacillus sp. KCTC 13219. The researchers expressed it in Bacillus subtilis cells and put it through standard enzyme tests. The results are worth a hard look.

Why formulators care about DAEases

D-allulose is a rare sugar with real commercial demand. It delivers sweetness with far fewer calories than sucrose. That makes it attractive for beverages, bakery goods, and confectionery. But you do not get D-allulose from nature in bulk. You make it by isomerizing fructose.

The enzyme responsible is D-allulose 3-epimerase. Its economics depend on three things: heat stability, reaction speed, and substrate tolerance. Heat stability decides how long a batch of catalyst lasts. Reaction speed decides throughput. Substrate tolerance decides how much sugar you can load into a reactor without losing conversion. DaeB looks strong on all three.

What the study did

The team cloned the DaeB gene from Bacillus sp. KCTC 13219. Then they expressed it in Bacillus subtilis cells. That host choice matters because B. subtilis is already common in commercial enzyme production. Scale-up does not require an exotic system.

The researchers then measured the enzyme’s half-life at 50°C, its catalytic constant, and its conversion under high fructose loading. Those are the numbers that matter for industrial design.

Key findings

The data point that jumps out is stability. At 50°C, DaeB had a half-life of 25 days. That is not a typo. Twenty-five days.

It also works fast. The kcat is 367 s⁻¹. That is a serious number for a biocatalyst. And it handles heavy feed streams. At 700 g/L fructose, the enzyme produced about 200 g/L D-allulose. That level of substrate loading is close to what a plant would actually run.

Property	DaeB result	Why it matters
Thermostability	Half-life of 25 days at 50°C	Longer operating life, less enzyme replacement
Catalytic rate	kcat = 367 s⁻¹	Faster conversion per unit of enzyme
Substrate loading	700 g/L fructose → ~200 g/L allulose	Industrial sugar concentrations without losing output

The combination is what makes this enzyme interesting. Thermostability alone is good. Speed alone is good. Substrate tolerance alone is good. Together, they point to a process that can run longer, faster, and on more concentrated syrups.

What it means for manufacturers

If you currently buy DAEase enzymes, compare them to DaeB. The half-life alone could change your enzyme dosage. A catalyst that stays active for 25 days at 50°C cuts replacement costs and reduces downtime. The high kcat means you may need less enzyme per batch. The high fructose tolerance means you can run more concentrated feed streams. That shrinks evaporation costs.

There are caveats. This is a characterization study, not a pilot plant trial. Real-world conditions include impurities, pH shifts, and metal ions. Those can shorten enzyme life. But the baseline performance is strong enough to justify pilot testing. The expression host also removes one scale-up barrier.

FAQ

What exactly is DaeB? DaeB is a D-allulose 3-epimerase cloned from Bacillus sp. KCTC 13219. It converts fructose into D-allulose.

How stable is DaeB? At 50°C, it has a half-life of 25 days. That is exceptionally stable for this class of enzyme.

What yields can be expected? At a very high fructose concentration of 700 g/L, DaeB produced roughly 200 g/L D-allulose. That puts the reaction in a practical industrial range.

The numbers are hard to ignore

Read the paper. Then run your own side-by-side test against your current enzyme. The thermostability alone justifies a closer look. The speed and substrate tolerance make it more compelling. Large-scale D-allulose production needs a catalyst that can take industrial conditions. This one is a serious candidate.