A D-Allulose 3-Epimerase of Bacillus Origin Exhibits High Thermostability and Noteworthy Catalytic Efficiency

Allulose production lives or dies by enzyme performance. The enzyme converts fructose to allulose. If it cannot survive heat and syrup load, process economics fall apart. A newly characterized D-allulose 3-epimerase, DaeB, from Bacillus sp. KCTC 13219, makes the case that stability and speed can come in one package.

Why thermostability drives allulose economics

Rare sugar enzymes work under punishing conditions. Reactors run hot to keep sugar solutions fluid and drive the reaction. Concentrated fructose syrup is thick and sticky. If an epimerase loses activity quickly, you face frequent enzyme additions, batch interruptions, and higher production costs.

Thermostability is not a luxury. It is a cost parameter.

What the study did

The researchers cloned the DaeB gene from Bacillus sp. KCTC 13219, a plant probiotic strain, and expressed it in Bacillus subtilis cells. Using a known production strain shortens the path from lab to commercial fermentation. Then they measured thermostability, catalytic activity, and conversion at high fructose concentration.

Key findings from the study

The headline number is the half-life. DaeB held up for 25 days at 50°C. At a fructose load of 700 g/L, it generated about 200 g/L allulose. Its turnover number, kcat, came in at 367 s⁻¹. Put those together and you have an enzyme that runs long, works fast, and handles heavy syrup.

Metric	DaeB value	Why it matters
Half-life at 50°C	25 days	Long operating window, less enzyme replacement
Turnover number (kcat)	367 s⁻¹	Fast catalysis per unit of enzyme
Fructose load tested	700 g/L	Handles concentrated commercial feed streams
Allulose produced	~200 g/L	Direct reference point for process design

What this means for formulators and buyers

Look at operating cost first. A longer-lasting enzyme means fewer refills and less downtime. A faster enzyme means more throughput per reactor volume. Both reduce the cost per kilo of allulose.

The high substrate load is the other practical signal. At 700 g/L, the syrup is difficult to mix and pump. DaeB still produced around 200 g/L allulose. Process engineers can start mass balance calculations from that number, then adjust for their own reactor type and downstream setup.

The study authors say the biocatalyst has great potential for large-scale production. The data support that claim.

FAQ

Q: What makes DaeB different from other epimerases?

A: The combination of stability and speed. A 25-day half-life at 50°C is a long operating window. A kcat of 367 s⁻¹ means the enzyme does not waste time. You get both in this one.

Q: What fructose load was tested?

A: 700 g/L. That produced about 200 g/L allulose. It is a realistic concentrated feed stream for commercial consideration.

Q: Which host expressed the enzyme?

A: The team expressed DaeB in Bacillus subtilis cells. The gene came from Bacillus sp. KCTC 13219.

Every allulose project has a cost model. Enzyme life sits at the center of it. DaeB extends that life, keeps a high reaction rate, and tolerates the concentrated conditions you would actually run. That makes it a serious candidate for the next round of allulose process development. Run your own pilot test. Let the numbers decide.