Bacillus D-Allulose 3-Epimerase of Probiotic Origin Displays High Thermostability and Strong Activity

Opening

If you are evaluating enzymes for allulose production, you usually start with thermostability and activity. This new candidate from a probiotic Bacillus strain delivers on both. The paper, published in Microbial Cell Factories (DOI: 10.1186/s12934-021-01550-1), introduces DaeB, a D-allulose 3-epimerase cloned from Bacillus sp. KCTC 13219 and expressed in Bacillus subtilis cells.

Here is why this one stands out.

Background

Allulose is a rare sugar with a clean, sweet taste and almost no calories. Manufacturers make it by converting fructose using enzymes called D-allulose 3-epimerases. These enzymes do the chemical work, but they often struggle at the temperatures and sugar concentrations found in commercial production.

Heat is the enemy. If an enzyme loses activity quickly, operators need more catalyst, shorter runs, or costly cooling steps. That raises the cost of every kilogram of allulose produced.

DaeB appears to sidestep those problems. The research team behind this paper cloned the gene from a plant probiotic strain and expressed it in B. subtilis, a well-established production host for food enzymes.

What the study did

The researchers pulled the gene for DaeB from Bacillus sp. KCTC 13219. They inserted it into B. subtilis cells and then tested the enzyme under conditions that matter for industrial use.

They measured thermal stability. They measured catalytic speed. And they ran a high-load conversion test with concentrated fructose.

The results point to an enzyme built for scale.

Key findings

The biggest number is the 25-day half-life at 50°C. DaeB keeps half of its activity after 25 days at that temperature. That is a long operating window for an epimerase. Most process enzymes are lucky to hold up for hours or days under similar stress.

Activity also looks strong. The enzyme has a kcat of 367 s⁻¹. That means each active site turns over substrate quickly. Fast turnover reduces the amount of enzyme needed to hit production targets.

Then there is the substrate test. With 700 g/L fructose, DaeB produced about 200 g/L allulose. The abstract calls the biocatalyst’s potential for large-scale production “significant,” and these numbers support that.

Parameter	Reported value	Production implication
Thermal stability	25-day half-life at 50°C	Long catalyst life in batch or continuous processes
Catalytic activity	kcat = 367 s⁻¹	High turnover per enzyme unit
Substrate concentration	700 g/L fructose	Works with concentrated sugar feeds
Allulose output	~200 g/L from 700 g/L fructose	Roughly 28.6% conversion in the tested setup

What it means for manufacturers

For formulators, this enzyme could lower the cost of allulose production. A longer half-life means less enzyme replacement. A higher kcat means smaller reactor volumes or lower enzyme loadings. The ability to handle 700 g/L fructose fits with existing syrup infrastructure.

That combination matters for buyers. Ingredient purchasing decisions are not just about enzyme price per gram. They are about total process cost. DaeB’s stability can cut downtime. Its speed can cut capital costs. Its tolerance for concentrated substrate can reduce water removal later in the process.

This is not a laboratory curiosity. The data come from a published, peer-reviewed study using a realistic, high-substrate scenario.

FAQ

Why does a 25-day half-life matter in a production plant? At 50°C, the enzyme retains half its original activity after 25 days. That gives process designers options. They can run longer batches, reuse the enzyme, or extend continuous operation without reloading catalyst.

What does 200 g/L allulose from 700 g/L fructose mean in practice? It means the enzyme handles a concentrated feed and still gives a commercially relevant yield. The output stream is not pure allulose, so downstream separation remains part of the process. But the upstream conversion is strong enough to justify those separation costs.

Is this enzyme suitable for food production? The source strain is a plant probiotic, and the enzyme was expressed in B. subtilis. Both are positive signals for food applications. Commercial suitability still depends on enzyme purification, food-grade production methods, and regulatory approvals in your target market.

Closing

Allulose production depends on enzymes that can survive real plant conditions. DaeB brings a long half-life, fast turnover, and the ability to work in concentrated fructose. Those are the exact characteristics formulators and ingredient buyers should track when they compare biocatalyst options.