D-Tagatose 3-Epimerase enzyme production and producing allulose(psicose) from fructose

In this study, D-Tagatose 3-Epimerase enzyme was produced by recombinant DNA technology and fructose was converted to a rare sugar, allulose (psicose), by using this enzyme. D-Tagatose 3-Epimerase enzyme gene was isolated from Escherichia coli JM109 and amplified by PCR. Escherichia coli JM109 was also used as host cell and enzyme gene was transferred to this microorganism with the help of a plasmid. The host cell was allowed to grow in the shaking incubator for 24 hours at 37 °C and after 24 hours the optical density of the cells was measured in a spectrophotometer at 600 nm wavelength. After the OD600 value reached 0.61, the protein expression was induced by the addition of 0.1 mM IPTG and the host cell culture was incubated at 18 °C for overnight. At the end of the incubation, the cells were collected by centrifugation and the enzyme production steps were performed respectively and the enzyme was produced. The produced enzyme was purified by means of His GraviTrap purification column and the pure enzyme was obtained. Then, the purified D-TE enzyme solution was added to 1%, 5% and 50% fructose solution at a rate of 5% and incubated at 20 °C, 40 °C, 60 °C, 70 °C and 80 °C for 24 hours. At this stage, during the first 8 hours, the samples were analyzed by HPLC every two hours and at the end of 24 hours, the amount of allulose converted from fructose was determined by D-TE activity. The most suitable temperature for D-TE enzyme activity was determined to be 60 °C and HPLC analysis of the samples of 50% fructose solutions kept for 2, 4, 6, 8 and 24 hours at this temperatures showed that the rate of conversion of fructose to allulose was 10.32%, 12.66%, 15.32%, 15.78% and 20.76% respectively.