D-allulose is a natural rare sugar with important physiological properties that is used in food, health care items, and even the pharmaceutical industry. In the current study, a novel D-allulose 3-epimerase gene (Bp-DAE) from the probiotic strainBlautia producawas discovered for the production and characterization of an enzyme known as Bp-DAE that can epimerize D-fructose into D-allulose. Bp-DAE was strictly dependent on metals (Mn2+and Co2+), and the addition of 1 mM of Mn2+could enhance the half-life of Bp-DAE at 55 °C from 60 to 180 min. It exhibited optimal activity in a pH of 8 and 55 °C, and theKmvalues of Bp-DAE for the different substrates D-fructose and D-allulose were 235.7 and 150.7 mM, respectively. Bp-DAE was used for the transformation from 500 g/L D-fructose to 150 g/L D-allulose and exhibited a 30% of conversion yield during biotransformation. Furthermore, it was possible to employ the food-grade microbial speciesBacillus subtilisfor the production of D-allulose using a technique of whole-cell catalysis to circumvent the laborious process of enzyme purification and to obtain a more stable biocatalyst. This method also yields a 30% conversion yield.Keywords:D-allulose 3-epimerase;D-allulose;Blautia produca;probiotics;biochemical characterization;biotransformation
D-Allose, a rare sugar, has gained significant attention not only as a low-calorie sweetener but also for its anticancer, antitumor, anti-inflammatory, antioxidant, and other pharmaceutical properties. Despite its potential, achieving high-level biosynthesis of D-allose remains challenging due to inefficient biocatalysts, low conversion rates, and the high cost of substrates. Here, we explored the food-grade coexpression of Blautia produca D-allulose 3-epimerase (Bp-DAE) and Bacillus subtilis L-rhamnose isomerase (BsL-RI) within a single cell using B. subtilis WB800N as the host. Using this system, D-allose was synthesized via a simple, cost-effective, one-pot enzymatic process, employing whole cells as catalysts and D-fructose as the substrate. The system exhibited optimal activity at 65 °C, pH 8.5, with 1 mM Mn2+ and 20 g/L of whole-cell dry weight. Initial production reached 12.5 g/L D-allose with a 12.5% yield from 100 g/L D-fructose. Optimization of dual promoter combinations enhanced production, achieving 15.0, 29.1, and 43.2 g/L D-allose from 100, 200, and 300 g/L D-fructose, with yields of 15.00, 14.55, and 14.40%, respectively. This D-allose production biocatalyst offers a scalable and economically viable platform for the industrial production of rare sugar.
危险品化学品经营许可证（带存储）