Protein Engineering of Recombinant D-Psicose 3-Epimerase from Agrobacterium sp. ATCC 31750 to Increase Its Thermostability and Production of Psicose from Fructose by Immobilized Strains

• Main Authors: Lee, Hsu-Chieh, 李旭傑
• Other Authors: Fang, Tsuei-Yun
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/f77d5j

碩士 === 國立臺灣海洋大學 === 食品科學系 === 105 === D-Psicose (D-ribo-2-hexulose, or D-allulose) is a hexulose and C3-epimer of fructose, which is one of the rare sugars. It has high sweetness but low calories, and also can inhibit the activities of α-glycosidase and fatty acid synthase; therefore, it can be applied to food and pharmaceutical industries. Currently, the enzymatic conversion is the major method to produce D-psicose. D-Psicose 3-epimerase (DPE) (EC 5.1.3.30) can convert D-fructose into D-psicose, however, most DPEs are sensitive to temperature and that discourages industrial production of D-psicose. In this study, online softwares ENCoM and PoPMuSiC were used to evaluate the possible mutations of Agrobacterium sp. ATCC 31750 DPE (AsDPE) in order to increase its thermostability, and mutants S15R, T103F, T103Y, and T249P were selected. In addition, using molecular modeling was applied to select mutants K17D, F155Y, and G19D/S54R, A164D/T200R. Co-expression of molecular chaperones was used to increase the amount of soluble protein of the expressed AsDPE in E.coli ClearColi BL21 (DE3). Finally, the AsDPE-containing cells were embedded by calcium alginate for immobilizatin. The results showed that the optimum temperature and pH of wild-type (WT) and the mutant AsDPEs were 60°C and pH 8, respectively. However, the activities of all mutant AsDPEs are reduced. As to thermostabliity, WT had only 17% activity after incubation of 6 minutes at 65°C, mutant F155Y incubated with the same time still had 82% activity, and the half-life of mutant F155Y AsDPE at 65°C is 32.8 min. After the co-expression of different molecular chaperones with AsDPE, the pGro7 chaperone can increase the expression of solube protein from 35.8% to 52.6%, and the activity and the amount of protein expressed were also increased. Mutant F155Y plasmid and pGro7 chaperone plasmid were co-transformed into E. coli ClearColi BL21 (DE3) and then the cells were immobilized. The optimum cell mass was 40 g wet cell/L and the concentration of calcium alginate was 6%. The packed bed reactor was used to produce D-psicose continuously, the reaction reached an equilibrium after 5.2 h with conversion yield of 25.4%.