| Summary: | 碩士 === 國立臺灣海洋大學 === 食品科學系 === 103 === D-Psicose is a rare monosaccharide in the nature which has special physiological functions being used as a food additive or sweetener but expensive. It has been announced as generally recognized as safe (GRAS) by Food and Drug Administration (FDA) in 2012. D-Psicose 3-epimerase (DPE) can catalyze the epimerization of D-fructose and D-psicose. The Agrobacterium sp. ATCC 31750 D-psicose 3-epimerase (AsDPE) dpe gene had been previously cloned and expressed in Escherichia coli BL21 CodonPlus (DE3)-RIL. According to the 98% homology between AsDPE and Agrobacterium tumefaciens (AtDPE), molecular modeling of the mutations of AtDPE was performed to choose the candidates for site-directed mutagenesis. The results have shown that mutant F185K and F185Y AsDPEs has specific activity of 68 U/mg and 23 U/mg, respectively.
AsDPE was then expressed in the nonendotoxin producing strain CleanColi BL21 (DE3) and the AsDPE containing immobilized cells were prepared for the reuse of the enzyme and improve enzyme’s thermostability. However, immobilized cells didn’t improve AsDPE’s thermostability as expected. Continuous production of D-psicose from D-fructose using AsDPE containing immobilized cells has been performed in packed-bed bioreactor. Substrate of 50% D-fructose in 50 mM Tris-HCl buffer (pH 8.0) plus 0.1 mM Co2+ was passed through the bioreactor at 55oC, and the convertion rate was about 30%. Under the reaction conditions, the epimerization reaction between D-fructose and D-psicose reached equilibrium after 2 h.
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