| Summary: | 碩士 === 國立臺灣海洋大學 === 食品科學系 === 102 === D-Psicose, the C3 epimer of D-fructose, is a rare monosaccharide in the nature. This rare sugar has special physiological functions and can be used as a food additive or sweetener. 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 dpe gene was previously cloned from the genomic DNA of Agrobacterium sp. ATCC 31750 and expressed in Escherichia coli. The poor expression of mutant Q53A Agrobacterium sp. ATCC 31750 DPE (AsDPE) caused the decrease of the total activity recovery. To increase the expression of AsDPE, the original alanine codon of GCG was replaced by the synonymous codon, GCA, GCC and GCT, respectively. The total activities for per gram of wet cells of Q53A-G, Q53A-A, Q53A-C and Q53A-T were 949 U/g, 836 U/g, 751 U/g and 822 U/g, respectively. The total activities for per liter of culture of Q53A-G, Q53A-A, Q53A-C and Q53A-T were 18,800 U/L、19,500 U/L、16,800 U/L 及 14,200 U/L, respectively, while the specific activities were not affected. Hence, the synonymous codons caused the changes on their total activities and the following total activity recoveries. In addition, AsDPE has been previously fused with the ATS peptide at the C-terminus (C’ATS AsDPE) and its precipitation during purification has been reduced. Besides, the mutant I33L/S213C A. tumefaciens DPE (AtDPE) showed significant increases in thermal stability, compared with wild-type. Therefore, this study has investigated the thermal stabilities and characteristics of C’ATS and I33L/S213C AsDPEs. In the presence of 0.1 mM Co2+, C’ATS and I33L/S213C AsDPEs can retain their stability at 50-55°C, and the thermal stability of C’ATS and I33L/S213C AsDPEs were better than that of wild-type AsDPE. The optimum temperature and pH for C’ATS and I33L/S213C AsDPEs were 60°C and pH 7.5-8.5, respectively, while those for wild-type AsDPE were 55°C and pH 7.5-8.5, respectively. LCATS AsDPE was constructed by carrying out I33L/S213C mutation on C’ATS AsDPE. The optimum temperature and pH for LCATS AsDPE were 65°C and pH 7.5-9.0. The half-life of LCATS AsDPE (73.7 min) at 65°C was 19.2-fold higher than that of wild-type. These results suggest that LCATS AsDPE has the potential to be used industrially for D-psicose production.
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