A Bacillus-Derived D-Allulose 3-Epimerase Exhibits Rich Thermostability and Notable Catalytic Potency

Enzyme stability is the bottleneck in allulose production. Most epimerases fall apart too quickly at industrial temperatures. A new paper changes the picture.

What you need to know about DAEases

Allulose is a rare sugar with about 70% of sucrose's sweetness and nearly zero calories. Formulators use it in beverages, bakery, and confectionery. Manufacturers make it by enzymatic isomerization of fructose using D-allulose 3-epimerase (DAEase). The enzyme's thermal stability decides the economics. A short half-life means frequent reloads, more downtime, and higher enzyme cost. That's why thermostability draws so much attention from processors.

What the study did

The team cloned a novel DAEase called DaeB from Bacillus sp. KCTC 13219, a plant probiotic strain. Then they expressed it in Bacillus subtilis cells. Why does that matter? Because B. subtilis is a workhorse for food-grade enzyme production. You skip the fuss of adapting to a new expression host.

They then tested the enzyme under conditions that approximate industrial sugar conversion.

Key findings

Here's where the numbers get interesting. DaeB has a half-life of 25 days at 50°C. Most DAEases lose activity within hours to days. That's a stability leap. The catalytic rate is equally notable: kcat = 367 s⁻¹. At 700 g/L fructose, the enzyme produced about 200 g/L allulose. That's roughly 28% conversion in a single pass at high substrate load.

Parameter	Measured value	Why it matters
Half-life at 50°C	25 days	Longer reactor runs, less enzyme replacement
Catalytic rate (kcat)	367 s⁻¹	Fast turnover, shorter residence times
Substrate concentration	700 g/L fructose	Industrial-level feed, less water to remove downstream
Allulose output	~200 g/L	Viable product titer for commercial recovery

Each of these numbers reinforces the others. High kcat means you need less enzyme. Long half-life means that enzyme keeps working. High substrate load means smaller downstream processing loads.

What this means for manufacturers

The practical takeaway is simple. Run a bioconversion reactor longer without stopping to recharge enzyme. That alone cuts labor, cleaning, and validation costs. The high substrate tolerance is also a relief. Concentrated fructose feeds mean smaller vessels and less evaporation at the back end.

For ingredient buyers, the implication is future pricing. Enzymes are a major cost component in allulose production. A catalyst with this stability profile could lower the minimum selling price of bulk allulose over time. It also reduces supply risk. Fewer enzyme batches mean fewer quality-control headaches.

The expression host adds another layer of confidence. Bacillus subtilis has a long history in food enzyme manufacturing. Regulatory acceptance should be smoother than for a novel host.

Questions formulators ask

Is DaeB ready for commercial use?

The data show strong potential, but production scale-up remains unproven. The paper demonstrates the biocatalyst's performance in controlled trials.

How does DaeB compare to existing commercial epimerases?

The combination of a 25-day half-life and a 367 s⁻¹ kcat sits at the top of reported values for this enzyme class, according to the authors.

Does high substrate load hurt yield?

At 700 g/L fructose, the enzyme still converted roughly 28% of substrate to allulose. That suggests tolerance to osmotic stress and product inhibition. A good sign for industrial feeds.

The road ahead

This paper gives food formulators a concrete reason to watch enzyme supply developments. DaeB comes from a strain already present in plant probiotics, expressed in a food-safe host, and delivers stability numbers that change the cost model. The study is published under DOI 10.1186/s12934-021-01550-1. Scale-up is the next test. If it holds, allulose pricing could get friendlier.