A Novel D-Allulose 3-Epimerase from Bacillus sp. KCTC 13219 Displays High Thermostability and Notable Catalytic Efficiency

The half-life that changes allulose economics

Allulose production lives and dies by enzyme stability. If your epimerase loses activity after a shift, you spend more money, time, and buffer on replacement. A recent paper in Microbial Cell Factories (DOI: 10.1186/s12934-021-01550-1) reports an enzyme that behaves differently. DaeB, a D-allulose 3-epimerase from Bacillus sp. KCTC 13219, kept working at 50°C with a half-life of 25 days. It also turned over substrate at a kcat of 367 s⁻¹. Those numbers matter for anyone scaling up allulose.

Why epimerase thermostability matters

D-allulose is a rare sugar. Epimerases convert fructose into allulose by flipping the configuration at carbon 3. The reaction works, but the enzymes often fail in hot, concentrated sugar streams. Industrial fructose syrups are dense. They need high temperatures to move and react. An enzyme that folds quickly loses value. Heat-tolerant biocatalysts let manufacturers push substrate loads and extend batch cycles. The new enzyme comes from a plant probiotic strain of Bacillus. That source appears to carry an unusually sturdy enzyme.

What the study did

The researchers cloned DaeB from Bacillus sp. KCTC 13219. They expressed it in Bacillus subtilis cells. Then they tested how long the enzyme held up and how fast it worked. They didn’t just run a dilute lab reaction. They used 700 g/L fructose. That is close to industrial conditions. DaeB produced about 200 g/L allulose from that feed. The combination of speed and heat tolerance stands out.

What the data show

Finding	Reported value	Why it matters
Source organism	Bacillus sp. KCTC 13219	The strain is a plant probiotic isolate.
Expression host	Bacillus subtilis cells	A practical microbial system for enzyme production.
Thermostability	25-day half-life at 50°C	Reactors stay productive for weeks.
Catalytic speed	kcat = 367 s⁻¹	Fast conversion per enzyme molecule.
High-load conversion	~200 g/L allulose from 700 g/L fructose	A realistic benchmark for process design.

Read the table as a package. A long half-life alone can hide a sluggish enzyme. A fast kcat alone can hide poor thermostability. DaeB pairs both. The high loading test shows the enzyme can tolerate a real sugar stream. That is exactly what a process chemist wants to see before piloting a new biotransformation.

What this means for your production line

For enzyme buyers, the practical point is simple. A 25-day half-life means fewer enzyme reloads during a production campaign. It means less downtime. It also means you can consider longer fed-batch cycles without watching activity crash. The kcat of 367 s⁻¹ suggests that productivity will not sit behind a slow catalyst. And the 200 g/L yield at 700 g/L fructose gives a concrete benchmark for your mass balance calculations.

The expression host matters too. Using Bacillus subtilis as the production host lowers the barrier to scale-up. The authors state that this biocatalyst has strong potential for large-scale D-allulose production. The reported data support that claim.

Frequently asked questions

What is DaeB?

DaeB is a D-allulose 3-epimerase cloned from Bacillus sp. KCTC 13219. It catalyzes the conversion of fructose to D-allulose. The gene was expressed in Bacillus subtilis cells for this study.

How stable is DaeB at production temperatures?

DaeB has a half-life of 25 days at 50°C. Half of the starting activity remains after 25 days at that temperature. That is an unusually long window for a sugar epimerase.

Does DaeB work at industrial sugar concentrations?

Yes. In the study, a 700 g/L fructose solution yielded roughly 200 g/L allulose. That demonstrates tolerance to high substrate concentration, not just activity in dilute buffer.

The allulose market rewards enzymes that hold up under pressure. DaeB appears to be one of those enzymes. Its long half-life, fast turnover, and high-load performance give formulators and ingredient buyers a concrete reason to ask suppliers for data on this D-allulose 3-epimerase family. The next question is how well it behaves in your reactor. The laboratory numbers say it deserves that question.