Bacillus-Derived D-Allulose 3-Epimerase Exhibits Rich Thermostability and Remarkable Catalytic Efficiency

If you produce allulose, you know the enzyme often gives out before the sugar does. This paper describes DaeB, a d-allulose 3-epimerase cloned from the plant probiotic strain Bacillus sp. KCTC 13219 and expressed in Bacillus subtilis. The results are hard to ignore. At 50°C, the enzyme holds a half-life of 25 days. It turns over substrate at 367 s⁻¹. And from 700 g/L fructose, it makes about 200 g/L allulose. That combination could change how you plan bioconversion runs.

Why enzyme stability matters for allulose

Allulose production relies on epimerases that convert fructose into allulose. Most epimerases degrade quickly under industrial conditions. Heat, high sugar loads, and long reaction times all shorten enzyme life. When the enzyme dies early, you need more of it, you stop the reactor to reload, and your cost per kilo climbs. A stable enzyme lets you run longer and use less biocatalyst. That is the practical promise of DaeB.

What the study did

Researchers isolated a novel d-allulose 3-epimerase gene from Bacillus sp. KCTC 13219, a strain originally associated with plant probiotic effects. They named the enzyme DaeB. Instead of expressing it in E. coli, they used Bacillus subtilis cells. That choice matters. B. subtilis has a good safety record and secretes proteins well. For food-ingredient makers, an enzyme produced in a generally regarded as safe host simplifies the path to regulatory acceptance and downstream purification.

The team then characterized the purified enzyme. They measured its half-life at 50°C, its catalytic turnover rate, and its ability to convert high concentrations of fructose. The study appeared in Microbial Cell Factories under DOI 10.1186/s12934-021-01550-1.

Key findings

DaeB outperforms many reported epimerases in simple, head-to-head laboratory terms. The table below summarizes what the paper reports.

Parameter	DaeB result
Source organism	Bacillus sp. KCTC 13219
Expression host	Bacillus subtilis cells
Half-life at 50°C	25 days
Turnover number (kcat)	367 s⁻¹
Fructose substrate load	700 g/L
Allulose produced	~200 g/L

The 25-day half-life is the headline number. Most epimerases lose activity within hours or days at elevated temperatures. DaeB stays active for weeks. The kcat value of 367 s⁻¹ means the enzyme acts quickly, not just for a long time. Speed plus stability gives you productivity. The conversion test also used a realistic industrial substrate load. Few papers test at 700 g/L fructose. This one did, and the enzyme still generated roughly 200 g/L allulose.

What this means for manufacturers

The numbers point to lower enzyme usage. If DaeB remains active for weeks, you can reduce the amount of enzyme charged to each reactor. You can also run at higher temperatures without fearing rapid inactivation. Higher temperatures lower viscosity, improve mixing, and reduce the risk of microbial contamination. All of those factors matter when you scale from pilot tanks to production volume.

The expression system also deserves attention. Bacillus subtilis is not a lab-only oddity. It grows on inexpensive media and does not produce endotoxins. For food formulators, that simplifies downstream processing. You get a cleaner enzyme prep with fewer host-cell contaminants to remove. Ingredient buyers should watch for DaeB-based products because the economics of allulose production could shift toward continuous or semi-continuous processing.

One caveat: the paper reports laboratory performance, not a full industrial demonstration. You still need to test DaeB in your own broth, your own equipment, and your own time scale. But the starting point is unusually strong.

FAQ

Where does DaeB come from?
It comes from Bacillus sp. KCTC 13219, a plant-associated probiotic strain. The gene was cloned and expressed in Bacillus subtilis cells.

How much allulose can DaeB make?
In the study, DaeB converted 700 g/L fructose into about 200 g/L allulose. That is a realistic high-load condition, not a dilute lab setup.

Why is a 25-day half-life so important?
Long half-life means the enzyme stays active for weeks at 50°C. You can extend reaction times, use less enzyme, and avoid frequent reactor stops. That directly cuts processing cost.

The allulose market runs on enzyme performance. DaeB brings a rare combination of heat tolerance, catalytic speed, and high-substrate capacity. If these results hold up in production conditions, formulators should take notice. The next step is clear: run your own trial.