One-Pot Production Of D-Allulose From Inulin By A Novel Identified Thermostable Exoinulinase From Aspergillus Piperis And Dorea Sp. D-Allulose 3-Epimerase

Inulin has been widely used as a cheap bioresource for producing many valuable products by enzymatic hydrolysis or microbial fermentation, such as high-fructose syrup and fructooligosaccharides. In this work, a onepot two-enzyme reaction system was developed to produce D-allulose from inulin using A. piperis exoinulinase and Dorea sp. D-Allulose 3-epimerase. The exoinulinase that was identified from Aspergillus piperis CBS 112811 was cloned and intracellularly expressed in Escherichia coli. The enzyme displayed the maximal activity as 3750 U mg(-1) at pH 6.0 and 55 degrees C. For the effects of different cations, Mn2+ simulated the enzyme activity by 41 %. When 10 g L-1 inulin was hydrolyzed by A. piperis exoinulinase, the conversion rate reached 98 % within 6 h. Furthermore, the optimum pH, temperature and the ratio of the two enzymes loaded for one-pot reaction were measured to be pH 6.0, 60 degrees C and 15/15 U mL(-1), respectively. The conversion rate of inulin to D-allulose reached 23.3 % after reaction for 4 h with 10 g L-1 inulin. When adding 100 g L-1 as a substrate, 21.4 g L-1 D-allulose was produced using the two-enzyme system.