# Thermostable D-Allulose 3-Epimerase for Long-Term Food-Compatible Continuous Production Systems
## 1 Introduction  
In recent decades, the global prevalence of metabolic disorders such as obesity, diabetes, and cardiovascular diseases has surged, primarily due to high-fat and high-sugar dietary patterns [1, 2]. In response, consumer preferences have shifted toward healthier sweetening alternatives, including erythroid, acesulfame and stevia, which offer low- or zero-calorie profiles [3, 4]. Among them, D-allulose (also known as D-psicose), a rare monosaccharide and the C-3 primer of D-fructose, has garnered increasing attention owing to its favorable nutritional and physiological properties. D-allulose exhibits approximately 70% of the sweetness and only 10% of the caloric value of sucrose (0.4 kcal/g vs. 4 kcal/g). In addition to its low caloric contribution, D-allulose has been shown to offer a range of health benefits, such as anti-obesity effects [5], modulation of cholesterol metabolism [6] and support for gut microbiota homeostasis [7]. These attributes position D-allulose as a highly promising functional sweetener for food and nutraceutical applications [8]. Despite its advantages, D-allulose is scarce in nature and its chemical synthesis typically requires harsh conditions and generates undesirable byproducts, limiting scalability [9; 10]. As a result, biocatalytic methods offer a cleaner, more sustainable, and cost-effective route [11]. Among these, enzymatic conversion using ketose 3-epimerases, particularly D-tagatose 3-epimerase (DTEase, EC 5.1.3.31) and D-allulose 3-epimerase (DAEase, EC 5.1.3.30), has become the most viable industrial-scale production method for D-allulose [12]. To date, more than 20 microbial DAEases have been identified from various genera such as _Arthobacter globiformis_ M30 [13], _Clostridium bolteae_[14], _Ruminococcus_ sp. [15] and _Bacillus subtilis_[16]. However, most of these enzymes display limited thermostability, with optimal activity typically below 50 \({}^{\circ}\)C. This low-temperature requirement constrains their application in high-temperature processes, which are desirable in the industry for improving substrate solubility, reducing viscosity and suppressing microbial contamination [17]. Furthermore, many reported DAEases require divalent metal cofactors such as Co\({}^{2+}\) or Mn\({}^{2+}\) for activation. These ions, despite their catalytic roles, are either toxic or disallowed in food processing, thereby limiting the practical use of such enzymes in food-related manufacturing processes [18]. Consequently, there remains a pressing need for the discovery and engineering of novel DAEases that combine high thermostability with activation by food-grade metal ions such as Mg\({}^{2+}\) or Ca\({}^{2+}\). In recent years, various immobilization strategies have been explored to improve the performance of DAEases [19], yet challenges remain. [PERSON] et al. [20] developed a DNA-origami-based co-immobilization platform integrating DAEase and glucose isomerase, enabling one-pot cascade conversion of glucose to D-allulose with minimal byproduct formation (reduced by approximately 80%). [PERSON] et al. [21] reported a ZnCo-MOF-based immobilized DAEase with enhanced thermostability (80% activity retention at 70 \({}^{\circ}\)C) and reusability (over 95% after eight cycles), attributed to Zn\({}^{2+}\)/Co\({}^{2+}\) coordination-induced structural stabilization. However, the high cost and complexity of such nanomaterials limit their industrial applicability. Alternatively, several studies have explored traditional immobilization platforms, such as chitosan beads and ion-exchange resins [13; 22], enabling medium-term continuous operation for 60-120 days. Nevertheless, few systems have demonstrated stable catalytic performance beyond 100 days under food-compatible conditions, underscoring the need for robust and durable immobilized biocatalysts. In this study, we characterized and immobilized a novel thermostable D-allulose 3-epimerase (TcDAEase) from _Thermogemmatispora carboxiditorans_.