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Recombinant D-tagatose 3-epimerase production and converting fructose into allulose
(Wiley, 2022) Parildi, Erva; Kola, Osman; Ozcan, Bahri Devrim; Akkaya, Murat Reis; Dikkaya, Elif
In this study, D-tagatose 3-epimerase gene was amplified from Escherichia coli JM109 by PCR and re-cloned into E. coli JM109 using HincII digested pUC18 cloning vector. Digestion of recombinant plasmid by HincII and PCR amplification of the gene from recombinant plasmid produced 789 bp gene band on agarose gel which indicated the gene integration. The host cell was grown in a shaking incubator at 37 degrees C for 24 hr and then the optical density (OD) of the cells was measured in a spectrophotometer at 600 nm wavelengths. When the OD600 value reached 0.61, the protein expression was induced by the addition of 0.1 mM IPTG into the growth medium. Through His-select gel column purification, highly purified and stable DTE protein was produced. The conversion rate of D-fructose into D-allulose was determined by HPLC. The native and recombinant enzymes could effectively convert D-fructose into D-allulose with a turnover ratio of 20.76%. Novelty impact statement A recombinant enzyme, D-tagatose 3-epimerase, was produced and D-fructose was converted into D-allulose using this recombinant enzyme.