Production of allulose from starch and starch derivatives using enzymes produced by recombinant dna technology

In this study, the production of D-allulose, a high-value rare sugar, from abundant and inexpensive starch using enzymes produced via recombinant DNA technology. In the first stage of the study, lyophilized Clostridium thermocellum and Thermoanaerobacterium thermosaccharolyticum strains were activated and their genomic DNA isolated. Specific primers were designed to amplify the genes encoding enzymes from the isolated DNA. The genes were cloned into pET series plasmid vectors and expressed in E. coli rosetta strain. The produced enzymes -phosphoglucose isomerase (PGI), allulose 6-phosphate 3-epimerase (A6PE), and allulose 6-phosphate phosphatase (A6PP)- were used in the starch-to-allulose conversion process. Initially, starch was hydrolyzed to glucose using commercial enzymes. Subsequently, D-allulose production was achieved by enzymatic conversion using the recombinant enzymes. Within the scope of this study, recombinant allulose 6-phosphate 3-epimerase and allulose 6-phosphate phosphatase were successfully produced and characterized for the first time in Türkiye. Each intermediate product formed during the starch-to-allulose conversion was analyzed by HPLC, and their 48-hour transformation profiles were graphically represented. The findings demonstrate that D-allulose can be efficiently and economically produced from starch, providing a promising alternative for industrial applications in the field of sugar substitutes. Keywords: Allulose, Recombinant DNA Technology, Enzymatic Conversion, Starch, Rare Sugar