The gene sequence of RpDAE (NCBI ACCESSION: WP_020816056.1) was codon-optimized for E. coli expression and fused with a modified His-based tag (HE tag) containing eight repeat histidine-glutamate residues (HEHEHEHEHEHEHEHE) at C-terminus, which is capable of immobilized metal ion affinity chromatography purification. The sequence was synthesized (GenScript, Nanjing, China) and subcloned into the pET-21a (+) between NdeI and XhoI restriction sites. Recombinant plasmid pET-RpDAE was cloned and transformed into E. coli. BL21 (DE3) for protein expression.

Recombinant strains were inoculated into 10 ml of Luria-Bertani (LB) medium. When needed, ampicillin was added into LB medium at the concentration of 100 μg/ml. Then, strains were cultured at 37°C with shaking at 200 rpm overnight. The seed was transferred into 100 ml of LB medium and after cultivated at 37°C with shaking at 200 rpm. When cells grown to the proper optical density (OD₆₀₀ = 0.7), 0.5 mM isopropyl-β-d-1-thiogalactopyranoside (IPTG) was added, and induced recombinant cells were further cultured at 15°C with shaking at 180 rpm for 16 h.

Recombinant cells were harvested by centrifugation at 8,000g for 5 min at 4°C. Subsequently, cells were washed thrice in lysis buffer (50 mM Tris-HCl, 100 mM NaCl, pH 7.5). Then, cells were lysed by sonication at 30 amplitudes (pulse on for 3 s and pulse off for 3 s) for 30 min over the ice. The cell debris was removed by centrifugation at 10,000g for 5 min at 4°C, and the supernatant was obtained for further purification. Enzyme with HE tag was trapped on His Trap™ FF column (Cytiva, MA, USA) at a flow rate of 0.5 ml/min. Wash buffer (50 mM Tris-HCl, 10 mM imidazole, 0.5 M NaCl, pH 7.5) was used to elute unbound proteins, and the target enzyme was eluted by elution buffer (50 mM Tris-HCl, 300 mM imidazole, 0.5 M NaCl, pH 7.5). Eluant was further dialyzed to remove imidazole with 50 mM Tris-HCl (pH 7.5) and concentrated by Amicon^® Ultra filter (10 kDa) (Merck, USA). The protein concentration was measured by Bradford Assay (Thermo Fisher, MA, USA). The purified protein was loaded onto sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel for the determination of molecular mass and purity.

The enzyme activity was determined by quantitative determination of product converted from the substrate. Data for this study was collected by high-performance liquid chromatography (HPLC) system, linked to a 2424 evaporative light scattering detector and an xBridge BEH amide column (all from Waters, MA, USA). The temperatures of the detector and column were set at 65°C and 35°C, respectively. The mobile phase was acetonitrile and water mixture (80:20, v/v, with 0.1% v/v ammonia added) at a flow rate of 1 ml/min. The reaction system incorporated D-fructose (50 g/L), 1 mM Co²⁺, appropriate amount of RpDAE or RpDAE-ZIF67, and 50 mM KH₂PO₄/Na₂HPO₄ (pH 7.5). The reaction was performed at 60°C for 5 min and terminated by boiling for 5 min. One unit (U) of RpDAE activity was defined as the amount of enzyme required to catalyze 1 μmol of D-allulose within the unit time (min) under the reaction condition.

To determine the influence of metal ions on the RpDAE activity, the activity was examined under standard enzyme assay except for supplemented with different metal ions (Mg²⁺, Cu²⁺, Co²⁺, Ni²⁺, Mn²⁺, Ca²⁺, and Zn²⁺) at the final concentration of 1 mM. The activity measured without metal ions was defined as 100% relative activity. The substrate specificity of RpDAE was tested by adding different ketoses (D-allulose, D-fructose, D-sorbose, and D-tagatose) into the reaction system as substrates. The reaction conditions were standard.

To determine the bioconversion from D-fructose to D-allulose, 2 μM purified RpDAE was added with 1 mM Co²⁺ and D-fructose (500 g/L) in 50 mM KH₂PO₄/Na₂HPO₄ buffer (pH 7.5) at 50°C. Samples were taken at designated time intervals and diluted tenfold. The yields of the accumulative D-allulose were detected by HPLC.

To study the effect of temperature on RpDAE activity, RpDAE was added in KH₂PO₄/Na₂HPO₄ buffer (pH 7.5) at temperatures varying from 40°C to 80°C. To investigate the effect of the pH on the activity of RpDAE, the reaction was conducted at 60°C across a pH range of 6–10 in MES buffer (50mM, pH 6.0) or KH₂PO₄/Na₂HPO₄ buffer (50 mM, pH 7.0–10.0).

RpDAE-ZIF67 was synthesized by in situ approach in the aqueous solution. Experimentally, 2 ml of purified RpDAE (2 mg/ml) and 2 ml of cobalt nitrate hexahydrate (0.04 M) were mixed with 2-methylimidazole (1.2 M, 2 ml) in distilled water and stirred for 1 h at ambient temperature. The sample solution was aged for 7 h and collected by centrifugation at 6,000 rpm for 20 min. Subsequently, samples were washed three times with distilled water. It was freeze-dried for 12 h. PXRD data were collected by SmartLab 9 kW X-ray diffractometer (Rigaku, Tokyo, Japan) with Cu-Kα radiation at 2θ from 5° to 40°. FT-IR measured was performed by Nicolet iS20 spectroscopy (Thermo Scientific, MA, USA) in the range of 400–2,500 cm⁻¹.

The genome mining approach has turned out to be a promising way toward the detection of novel industrial enzymes, such as lipase (Vorapreeda et al., 2015), ß-glucosidase (Zou et al., 2012), and laccase (Fang et al., 2011). To explore novel DAE applicable to biosynthesis D-allulose, the amino acid sequence of Clostridium cellulolyticum DAE (GenBank: ACL75304.1) with significant thermostability was chosen as the template to BLAST in the NCBI database. The sequence that encoded a deduced DAE (NCBI: WP_020816056.1) from Ruminiclostridium papyrosolvens C7 with 59% amino acid identity with C. cellulolyticum DAE was selected. R. papyrosolvens strain was initially isolated from mud in Massachusetts, and its whole-genome shotgun sequence data was uploaded to NCBI database in 2013 (Zepeda et al., 2013), with the NCBI accession number PRJNA201398.

As presented in comparison of amino acid sequence (Figure 1A), deduced DAE from R. Papyrosolvens showed maximum homology identity with DAE from Dorea sp. (59.4%, GenBank: CDD07088.1), followed by DAE from Clostridium sp. (59.2%, GenBank: EDP19602.1). Except for DAEs from Pirellula sp., R. baltica, S. aureus, and S. fredii origin, there was more than 40% of homology identity between RpDAE and other characterized DAEs, while existed relatively low (less than 40%) homology identity with most D-tagatose-3-epimerases and l-ribulose-3-epimerases. Phylogenetic analysis with previously characterized KEases revealed its evolutionary relationship with DAE from genera of T. caenicola (Figure 1B).

So far, the crystal structures of A. tumefaciens ATCC33970 DAE (PDB: 2HK0) (Kim et al., 2006b) and C. cellulolyticum H10 DAE (PDB: 3VNI) (Chan et al., 2012) have been successfully resolved, and they are homotetramers with similar monomeric structures; each subunit possesses a distinct TIM barrel structure forming by eight β-sheets and α-helices, and the catalytic activity center is strictly conserved (Jia et al., 2021). Multiple amino acid sequences alignment was performed between characterized DAEs and RpDAE (Figure 2). Crucial residues of RpDAE for the catalytic activity, including metal coordination sites (Glu150, Asp183, His209, and Glu244) and residues binding the O-1, O-2, O-3 of the substrate (Glu156, His186, and Arg215), were conserved with other reported DAEs. In general, these analyses indicate that RpDAE belongs to the DAE family.

RpDAE fused with a modified histidine (HE) tag was recombinantly overexpressed in E. coli. BL21 (DE3) by IPTG induction. HE tag can function as promoters of both affinity (Hofström et al., 2011) and solubility (Han et al., 2018). After cell disruption, the supernatants were purified by immobilized metal affinity chromatography. SDS-PAGE visualization confirmed expression and purification of proteins of about 35 kDa (Figure 3), which was in accordance with the theoretical molecular weight of RpDAE (34.89 kDa, containing HE tag). Target proteins were mostly expressed in a soluble form under the conditions described above (Figure 3, Lane 2).

To capture the effect of metal ions on RpDAE activity, diverse divalent metal ions were added into the reaction system at the final concentration of 1 mM. As illustrated in Figure 4A, RpDAE displayed activity in the absence of metal ions and enhanced in the presence of Co²⁺ and Mn²⁺, by 1.78- and 1.56-fold, respectively. In contrast, the addition of Cu²⁺, Ca²⁺, Zn²⁺, and Ni²⁺ inhibited enzyme activity, and the inhibitory effect of Zn²⁺ was the most significant, over 80%. Moreover, Mg²⁺ had little effect on the relative activity of RpDAE. Monosaccharide epimerase employing deprotonation/reprotonation mechanism often required metal ions as co-factor to participate in the catalysis (Van Overtveldt et al., 2015), and the optimum co-factor for RpDAE was determined to be Co²⁺.

To explore the substrate specificity of RpDAE, four kinds of ketoses were used, containing D-allulose, D-fructose, D-tagatose, and D-sorbose. RpDAE displayed the highest activity in the presence of D-allulose, which was 40% higher in relation to D-fructose (Figure 4B). On the contrary, RpDAE displayed low epimerization activity toward D-tagatose and D-sorbose.

The large-scale bioconversion of D-allulose was performed with D-fructose (500 g/L), 1 mM Co²⁺, and 50 mM KH₂PO₄/Na₂HPO₄ (pH 7.5), along with 0.2 g/L RpDAE at 50°C. The reaction rate for D-allulose production was 107.5 g/h for the first hour. Finally, the equilibrium ratio of D-allulose and D-fructose was measured to be 29.9:70.1, and D-allulose (approximately 149.5 g/L) were obtained from D-fructose (500 g/L) without byproducts after 150 min (Figure 4C). At present, many multinational enterprises are researching and developing the biological production of D-allulose. In 2021, the European Food Safety Authority certified the safety of the two kinds of food enzyme DAEs, which were produced by genetically modified E. coli K-12 W3110 (pWKLP) strain (Matsutani Chemical Industry Co., Ltd.) (Lambré et al., 2021a) and Corynebacterium Glutamicum FIS002 strain (CJ-Tereos Sweeteners Europe SAS) (Lambré et al., 2021b), respectively. The efficiency of biocatalysts is crucial in industrial production. Generally, the conversion rates of different DAEs reported in the literature were between 22% and 32.9% (Jia et al., 2021), and the highest conversion yield was achieved by A. tumefaciens ATCC33970 DAE (Kim et al., 2006a). Compared to reported DAEs, the catalytic performance of RpDAE at the substrate scale of 500 g/L was at middle-upper levels, revealing as a potential candidate in the biological production of D-allulose.

The enzyme activity of RpDAE was dependent on temperature and pH conditions. The influence of the temperatures on RpDAE activity was depicted in Figure 5A, and the activity was assayed with a temperature range of 40°C–80°C at pH 7.5. RpDAE displayed more than 82.9% relative activity at the temperature range between 55°C and 70°C. The maximum enzymatic activity of RpDAE was recorded at 60°C, which was higher than optimum temperature of C. cellulolyticum DAE (Mu et al., 2011). To further determine the activation energy (E _a ) of epimerization reaction at pH 7.5, activities measured at 40°C–60°C were plotted as ln (relative activity) versus 1000/T, and Arrhenius equation [lnk = (−E _a /RT) + lnA] was used to calculate the E _a of 23.51 kJ/mol (illustration of Figure 5A).

The effect of pH on RpDAE was examined at 60°C over a pH range from 6.0 to 10.0, and the optimal pH occurred at a weak level of alkaline, which was pH 7.5 (Figure 5B). Most characterized DAEs showed optimum epimerization activity at pH 7.0 to 9.0, and the exception was Dorea sp. DAE with an optimal pH at 6.0 (Zhang et al., 2015). RpDAE showed more than 87% of relative activity at pH 7.0, demonstrating application potentials in D-allulose production, because neutral conditions help to reduce browning of monosaccharide, thereby reducing yield loss.

Immobilized enzymes are widely utilized in the food industry. On the one hand, immobilized enzymes are heterogeneous catalysts so that can be simply separated from the reaction medium and obtain a pure product without contamination. On the other hand, they can be applied multiple times to the production process, thus reducing costs (Yushkova et al., 2019). Benefiting from high biocompatibility and mild synthesis conditions, ZIFs are the most widely used in situ immobilization matrices (Lian et al., 2017). RpDAE-ZIF67 was synthesized through crystallization of ZIF67 around RpDAE in aqueous solution (Figure 6A), which is a straightforward, rapid, and cost-effective process (Patil and Yadav, 2018). To remove loosely attached RpDAE on the surface, the obtained RpDAE-ZIF67 particles were washed three times with deionized water. The synthesized ZIF67 and RpDAE-ZIF67 were characterized by PXRD. As shown in Figure 6B, the conspicuous reflections of synthesized ZIF67 at 2θ = 7.5°, 10.5°, 12.9°, 14.8°, 16.6°, 18.1°, 22.2°, 24.6°, 25.7°, 26.8°, 29.7°, 30.7°, 31.6°, and 32.5° were associated with (0 1 1), (0 0 2), (1 1 2), (0 2 2), (0 1 3), (2 2 2), (1 1 4), (2 3 3), (2 2 4), (1 3 4), (0 4 4), (3 3 4), (2 4 4), and (2 3 5), respectively, of the simulated ZIF67 single-crystal planes (Banerjee et al., 2008) (Guo et al., 2016). Similar diffraction patterns were also observed in RpDAE-ZIF67, which indicated that the crystal structure of ZIF67 remained unaffected after the enzyme being incorporated. Figure 6C showed the FT-IR spectra of RpDAE and RpDAE-ZIF67. The vibrational bands of bare ZIF67 in the range of 600–1,500 cm⁻¹ correspond to the characteristic stretch and bending modes of imidazole rings. Furthermore, the band at 1,574 cm⁻¹ can be attributed to the stretching mode of C=N in 2-methylimidazole. The above bands were all well represented in the spectrum of RpDAE-ZIF67. In addition, RpDAE-ZIF67 had a new absorption band at 1,658 cm⁻¹ compared to bare ZIF67, corresponding to the stretching vibration mode of C=O in the amide I bond, confirming the existence of DAE within ZIF67.

The relative activities of encapsulated DAE were evaluated, and Figure 5A demonstrated nearly identical activities of RpDAE and RpDAE-ZIF67 under 60°C. However, at higher temperatures (over 65°C), the encapsulated DAE displayed higher relative activity than free DAE. At 80°C, the free DAE showed only 55.3% of relative activity, whereas the encapsulated DAE retained 76.1% of relative activity. These results demonstrated that encapsulating enzymes in MOFs prevented conformational transitions at high temperatures and improved the thermostability. As shown in Figure 5B, encapsulated DAE reached maximum activity at pH 8.0 and showed the higher tolerance in alkaline compared with free DAE. Another purpose of immobilization is to make the biocatalyst easy to recover and reuse, which is a key factor for economic viability. Hence, the reusability of RpDAE was examined in consecutive epimerization reactions. Accordingly, the encapsulated DAE was separated from the reaction system by centrifugation. As shown in Figure 6D, the encapsulated DAE was successfully cycled for five times, and the residual activity was determined to be 56%. Wang et al. immobilized laccase within ZIF67 by the one-pot synthesis strategy, which maintained 59% residual enzyme activity after five reaction cycles (Wang et al., 2020). The gradual loss of activity during recycling may cause by mechanical damage to the enzyme. The reusability can be attributed to the small window size of the cages, physically preventing the leaching of the enzymes from ZIF67.