Secreted d-allulose 3-epimerase for rare d-allulose biosynthesis and yeast-assisted isomer removal from d-allulose

Abstract

d-Allulose is a low-calorie rare sugar with multiple physiological functions, primarily produced through enzymatic isomerization with the key enzyme d-allulose 3-epimerase (DAEase). DAEase from a Clostridia bacterium (CDAE) was successfully expressed in food-grade Bacillus subtilis and substantially secreted into the extracellular medium (155.50 U/mL), achieving the highest reported extracellular activity among DAEases to date. The unique secretion behavior of CDAE without signal peptide was ascribed to cell membrane permeabilization and cell lysis. Furthermore, the secreted CDAE was employed for the synthesis of d-allulose from fructose or glucose via reversible isomerization. Yeast fermentation was implemented to selectively consume coexisting isomers in the product mixture. This process achieved a removal rate exceeding 99% for glucose and fructose while preserving d-allulose, resulting in a high-content d-allulose syrup (96.8% on a dry weight basis). Collectively, the efficient secretion of CDAE and cost-effective removal of isomers contribute to the economic production and separation of d-allulose.