A D-Allulose 3-Epimerase of Bacillus Origin Exhibits High Thermostability and Notable Activity

Why this enzyme matters for allulose production

If you formulate bulk sweeteners, enzyme stability is the metric that decides whether a process makes money. A one-week half-life forces you to reload biocatalyst constantly. That raises cost and downtime. A new enzyme from a plant probiotic Bacillus strain flips those assumptions. It keeps working for weeks. Put simply, this is the kind of catalyst that makes allulose production at high substrate load more practical.

Background: the bottleneck in allulose manufacturing

Allulose is a rare sugar. It has the same taste and texture as sucrose but fewer calories. The challenge is enzymatic conversion. Most D-allulose 3-epimerases are heat-sensitive and slow. Industrial runs need both high temperatures and high substrate concentrations to keep yields reasonable. A little stability goes a long way, but many enzymes fail there. This paper tackles exactly that problem.

What the study did

Researchers cloned a novel D-allulose 3-epimerase from Bacillus sp. KCTC 13219. They named it DaeB. Then they expressed it in Bacillus subtilis cells. That choice matters for food manufacturers because B. subtilis is already accepted in industrial enzyme production.

The team characterized the enzyme under conditions that imitate real processing. They measured thermostability, reaction rate, and conversion yield. They did not settle for small substrate loads. The experiments went up to 700 g/L fructose.

Key findings at a glance

Parameter	Reported result	Formulator takeaway
Thermostability	Half-life of 25 days at 50°C	Extended operation without frequent reloading
Catalytic rate	kcat of 367 s⁻¹	Fast enough for practical cycle times
Substrate load	700 g/L fructose	High-solids feedstocks are viable
Product yield	~200 g/L allulose	Product concentration is worth recovering

The half-life number is the headline. 25 days at 50°C does heavy lifting for process economics. The catalytic rate is also notable. A kcat of 367 s⁻¹ means each enzyme molecule converts hundreds of substrate molecules per second. That combination is rare. You get both speed and endurance.

What it means for manufacturers

Your current process might use a less stable enzyme, forcing lower temperatures. DaeB allows you to run at 50°C without worrying about rapid deactivation. Higher temperature also helps solubility and enzyme activity. That can shorten reaction times.

The high substrate load result points to a workable industrial route. With 700 g/L fructose, the reaction produced about 200 g/L allulose. That is in the range where downstream purification becomes economical. You are not trying to recover tiny concentrations. This reduces the cost per kilogram.

Expression in Bacillus subtilis is another plus. You do not need to validate a new expression host. The production organism is familiar to enzyme manufacturers. That can shorten scale-up development.

The enzyme’s origin as a plant probiotic strain adds a clean-label story. Consumers may not care about the source, but formulators who audit ingredient supply chains will.

Frequently asked questions

Is DaeB the same as existing D-allulose 3-epimerases?

No. The team cloned a new enzyme from Bacillus sp. KCTC 13219. They named it DaeB. It is a distinct gene sequence with the thermostability and activity profile above.

Can I use this enzyme directly in my existing allulose process?

The study is at characterization stage, not a commercial product. But the data suggest you can test it under your own conditions. The 50°C half-life and high substrate tolerance make it a strong candidate for pilot trials.

What about downstream processing?

The ~200 g/L allulose yield from 700 g/L fructose simplifies separation. The residual fructose and allulose can be separated with established chromatography or filtration methods. The yield itself is the key advantage.

The practical read

DaeB stands out for one simple reason: it does not die fast. A 25-day half-life at 50°C is rare. When you combine that with a high kcat and a workable product yield, the enzyme deserves serious attention. If you buy enzymes or design allulose production lines, ask your supplier about DaeB. The data in this paper gives you solid ground for a pilot evaluation. It might be the stability boost your process needs.