Immobilization of Recombinant D-Psicose 3-Epimerase from Agrobacterium sp. ATCC 31750 and Altering Its Thermostability and Catalytic Efficiency by Protein Engineering

• Main Authors: Lee, Tzu-Yi, 李姿儀
• Other Authors: Fang, Tsuei-Yun
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/cg4667

碩士 === 國立臺灣海洋大學 === 食品科學系 === 106 === D-Psicose (D-ribo-2-hexulose, or D-allulose) is a C3-epimer of D-fructose, and is a rare sugar found in nature in small quantities. It has high sweetness but low calorie content, improved food gelation, maintenance of blood glucose level and body fat reduction. Therefore, it has potential for development in food and medicine. At present, D-psicose is mostly produced by the enzyme conversion method using D-psicose 3-epimerase (DPE). However, DPE is quite sensitive to temperature and is not conducive to industrial mass production. In this study, protein engineering was used to improve the thermostability and characteristics of D-psicose 3-epimerase (AsDPE) from Agrobacterium sp. ATCC 31750. The mutation sites were chosen from previous studies, mutations F155Y/C'ATS (F155Y-ATS) and I33L/F155Y/S213C/C'ATS (F155Y-LCATS) were studied. The optimum temperature and pH of both mutants are 65°C and pH8.0, respectively. The thermostability of F155Y-LCATS is much higher than that of wild-type (WT). The residual activity of F155Y-LCATS was 73.0% and 42.9% at 60°C and 65°C , respectively , for 30 minutes, and the half-lives were 182 and 15.0 minutes, respectively. In terms of enzyme kinetics, the catalytic efficiencies of F155Y-ATS and F155Y-LCATS are better than WT, which are 2.4 and 1.8 times higher than WT, respectively. Co-expression of different molecular chaperones with C’ATS showed that chaperone expressed from pGro7 increased the amount of soluble protein from 50% to 60%, and the expression of C’ATS was also increased. AsDPE-containing cells were immobilized by calcium alginate by emulsification method. The optimal concentration of sodium alginate was 1%, the optimal cell mass was 80 g/L, and the immobilization efficiency was 70%. The mutant enzyme F155Y-LCATS was immobilized on a nickel-ion affinity column and D-psicose was produced by cyclic loading of D-fructose. The reaction reached equilibrium in 10 hours, the conversion rate was 23%. After five times reaction, the conversion rate is reduced to 8% due to partial enzymatic inactivation.