D-Allulose 3-Epimerase of Bacillus Origin Displays Rich Thermostability and Remarkable Catalytic Efficiency

A Biocatalyst That Keeps Working for Weeks

For enzyme buyers and formulators, the bottleneck is rarely the reaction itself. It is enzyme lifetime. A biocatalyst that decays inside a reactor forces you into shorter production runs, higher enzyme loading, and more downstream cleanup. That is why a new D-allulose 3-epimerase from Bacillus sp. KCTC 13219 matters. This enzyme stays active at 50°C for 25 days. You read that correctly. Half-life measured in weeks, not hours.

Background: The Allulose Enzyme Problem

D-allulose is a rare sugar with real functional appeal. It provides bulk and sweetness without the glycemic load. Production depends on epimerases that convert fructose into allulose. Most known D-allulose 3-epimerases have moderate thermostability and mediocre catalytic speed. That makes industrial scale-up expensive. You either keep feeding fresh enzyme into the system or you accept lower conversion rates.

The research team looked for a better starting point. They turned to a plant probiotic strain, Bacillus sp. KCTC 13219, and cloned a novel D-allulose 3-epimerase called DaeB. Then they expressed it in Bacillus subtilis cells, a workhorse production host with a long safety record in food enzyme manufacturing.

What the Study Did

The study is straightforward in design. Identify the enzyme. Clone the gene. Express it in a food-grade host. Then measure the properties that matter for industrial users: thermal stability, catalytic rate, and conversion yield under high substrate concentration.

The results push DaeB into unusual territory. At 50°C, the enzyme’s half-life is 25 days. For context, many mesophilic epimerases lose activity within hours at that temperature. DaeB also reaches a kcat of 367 s⁻¹. That is a fast enzyme by any standard, especially for a rare sugar epimerase.

The researchers then tested it under realistic industrial conditions. Using 700 g/L fructose, DaeB produced about 200 g/L allulose. That is about 28% conversion in a very concentrated syrup. You do not need to dilute your feedstock to get useful yields.

Key Findings

Parameter	Result	Why It Matters
Enzyme name	DaeB	New biocatalyst candidate for allulose production
Source strain	Bacillus sp. KCTC 13219	Plant probiotic origin, not a random environmental isolate
Expression host	Bacillus subtilis	Established, food-compatible production system
Half-life at 50°C	25 days	Long operational stability in continuous or repeated-batch processes
Catalytic rate (kcat)	367 s⁻¹	Fast turnover, meaning less enzyme mass per kilogram of product
Fructose loading	700 g/L	Works in concentrated feed streams
Allulose yield	≈200 g/L	Practical product concentration for downstream recovery

What It Means for Manufacturers

The 25-day half-life is the headline metric. It changes how you design a production line. With this stability, you can run longer cycles without enzyme recharge. You can also use higher temperatures, which reduce viscosity and improve substrate solubility. That directly impacts reactor productivity, energy use, and enzyme cost per kilogram.

The kcat of 367 s⁻¹ tells you that DaeB does not waste time. High catalytic speed means you need less enzyme to hit your target conversion. For buyers comparing enzymes, that is a direct input-cost lever.

The 700 g/L fructose result matters just as much. Real-world feedstock is not dilute sugar water. You want to run as concentrated as possible to cut evaporation costs. DaeB does the job under those conditions. 200 g/L allulose from a single pass gives you a realistic starting point for chromatographic separation and crystallization.

There is also a practical expression advantage. Bacillus subtilis is not a novel, hard-to-scale system. It is already common in enzyme production. That should shorten the path from study to commercial supply.

Frequently Asked Questions

How long does DaeB actually stay active in production?

At 50°C, the half-life is 25 days. That means after 25 days, activity still sits at half the initial level. In a batch process, you could run for days or weeks without adding fresh enzyme.

What yield can I expect in a concentrated fructose feed?

At 700 g/L fructose, the enzyme generated roughly 200 g/L allulose. That is a meaningful conversion level for an industrial stream.

Was this enzyme produced in a food-safe host?

Yes. The researchers expressed DaeB in Bacillus subtilis, a widely used microbial production host in the food enzyme industry.

Closing

DaeB is not just another epimerase. It combines long thermal stability, fast turnover, and solid yield in a concentrated system. Those are exactly the traits formulators and ingredient buyers should look for when evaluating allulose production enzymes. The data is clear. The enzyme holds up. The rest is scale-up.