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

A new enzyme changes the allulose cost equation

Allulose formulators often hit the same wall. Enzyme stability at the temperatures and sugar loads that make production economical is hard to find. A new paper offers a candidate that stands up. Researchers cloned a novel D-allulose 3-epimerase, DaeB, from the plant probiotic strain Bacillus sp. KCTC 13219. They expressed it in Bacillus subtilis cells. The result is an enzyme with a 25-day half-life at 50°C and a kcat of 367 s⁻¹. Those numbers deserve attention.

The enzyme problem in allulose production

Allulose is a rare sugar. You do not extract it from fruit. You make it from fructose using an epimerase. The enzyme rearranges the hydroxyl group at C-3, converting fructose to allulose. The reaction is reversible, so yields depend on equilibrium. High fructose concentration pushes the equilibrium toward product, but it also stresses the enzyme. Heat helps solubility and reaction rate, but most biocatalysts lose activity quickly. Process engineers therefore spend more time managing enzyme stability than optimizing chemistry.

DaeB attacks that bottleneck directly. It comes from a Bacillus species tied to a plant probiotic environment, which often selects for tough enzymes. The researchers expressed it in B. subtilis, a production host enzyme manufacturers already handle at scale. That matters for commercialization. A novel enzyme is useful only if you can make it cheaply and consistently.

The work appeared in Microbial Cell Factories with DOI 10.1186/s12934-021-01550-1.

What the study did

The team cloned the gene encoding DaeB and put it into B. subtilis. They then characterized the purified enzyme under conditions relevant to industrial sugar processing. Instead of testing only dilute buffers, they pushed sugar concentrations to 700 g/L fructose. That is a realistic industrial feed, not a laboratory convenience.

They measured thermostability, catalytic rate, and conversion yield. The headline numbers:

• Half-life at 50°C: 25 days
• Turnover number (kcat): 367 s⁻¹
• Product yield from 700 g/L fructose: about 200 g/L allulose

These three data points tell one coherent story. A long half-life means less enzyme replacement. A high kcat means less enzyme protein for the same throughput. A high yield at high substrate load means the process can run concentrated, reducing downstream water removal costs.

Key findings

Property	DaeB value	Why it matters for manufacturing
Thermostability at 50°C	Half-life 25 days	Long operation cycles, lower enzyme cost
Catalytic turnover	kcat 367 s⁻¹	Fast conversion, smaller reactor volume
Fructose load	700 g/L	Concentrated feed, less water to evaporate
Allulose yield	~200 g/L from 700 g/L fructose	Realistic product concentration for downstream purification
Expression host	Bacillus subtilis	Existing fermentation infrastructure fits

The 25-day half-life stands out. Many epimerases lose half their activity within hours at 50°C. An enzyme that remains active for weeks lets batch processes run longer and continuous processes hold steady. The high kcat supports shorter residence times. Together, these properties lower both capital and operating costs.

What it means for manufacturers

For ingredient buyers, the practical message is not "buy this enzyme today." It is that enzyme suppliers now have a stronger option to develop. The data suggest DaeB can operate at the fructose concentrations used in commercial allulose plants. It can handle heat. It converts sugar fast. And it is expressed in B. subtilis, though the enzyme itself still requires separate regulatory review depending on your market.

Formulators should ask suppliers two questions. First, does their allulose enzyme show a half-life measured in days or hours? Second, do they have data at 700 g/L fructose? If not, the product may work only in dilute, low-yield conditions. DaeB sets a benchmark. It also shifts expectations about what an epimerase can tolerate.

The economics are straightforward. A 25-day half-life cuts enzyme consumption. A kcat of 367 s⁻¹ reduces reactor volume. A 200 g/L product concentration reduces evaporation energy. Each line item improves the cost per kilo of allulose. In a commodity sweetener market, those improvements decide who can sell at scale.

FAQ

Does DaeB work in continuous production? The paper does not test a continuous column. But a 25-day half-life at 50°C is exactly the stability profile that makes continuous operation feasible. You would still need immobilization and process validation.

Why is 700 g/L fructose important? Most lab studies use 10–100 g/L sugar. Industrial allulose production needs concentrated feed to avoid expensive water removal. DaeB produced about 200 g/L allulose at 700 g/L fructose, which is close to a commercial working concentration.

Is DaeB approved for food use? The study covers enzyme discovery and expression. Regulatory approval is a separate step. The expression host, Bacillus subtilis, is well known in food enzyme production, but that does not automatically approve the final enzyme.

The next step is scaling. DaeB gives process engineers a real reason to re-run their cost models. For buyers, the message is simple. Allulose enzymes are no longer the weakest link.