Protein engineering and immobilization of recombinant D-psicose 3-epimerase from Agrobacterium sp. ATCC 31750 to increase its thermostability

• Main Authors: Fang, Hong-Yi, 方弘懿
• Other Authors: Fang, Tsuei-Yun
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/70237426069183697756

碩士 === 國立臺灣海洋大學 === 食品科學系 === 104 === D-Psicose, the C3-epimer of fructose, is one of the rare sugar, since it exists in trace amount in nature. It has several physiological functions can apply to food or pharmaceutical industry. D-Psicose can be produced by DTEase family enzymes (D-tagatose 3-epimerase family enzymes) which can convert D-fructose into D-psicose. Among the DTEase family enzymes, DPE (D-psicose 3-epimerase) which has better affinity to D-fructose, is more suitable for producing D-psicose. However, currently found DPEs have poor thermostability that discourages the process of D-psicose production. Therefore, it is necessary to improve thermostability of DPE for increasing its applicability to industry. Gycine and proline have higher possibility to increase thermostability based on the studies on the weak interaction in proteins and comparing the amino acid compositions between mesophilic and thermophilic proteins. Furthermore, computational method also used to screen the thermostable mutants by predicting thermodynamic stability via amino acid sequence and protein structure. In this study, Agrobacterium sp. ATCC 31750 DPE (AsDPE) structure was modeled by SWISS-MODEL, and then online softwares ENCoM and PoPMuSiC were used to evaluate the possible mutations in order to increase its thermostability. Besides, I66L mutation which has improved the catalytic efficiency of A. tumefaciens ATCC 33970 DPE (AtDPE) was also combined to I33L/S213C and I33L/S213C/C’ATS AsDPEs. The results show the half life of I33L/I66L/S213C is about twenty times longer than that of wild-type at 65°C. Chemical chaperones, the compounds keep proteins from aggregation are also used to study the solubility of AsDPE. The bioavailable chemical chaperones like arginine, sorbitol, and trehalose were added respectively during induction, but neither of them can enhance AsDPE solubility. I33L/S213C/C’ATS AsDPE (LCATS) was then expressed in the non-endotoxin-producing strain ClearColi BL21 (DE3). These cells treated with hexadecyltrimethyl ammonium bromide were immobilized by calcium alginate in order to raise industrial applicability such as thermostability or reusability. As a result, the LCATS-containing immobilized cells can reach reaction equilibrium in 1.5 h at about 30% convertion rate. Moreover, the LCATS-containing immobilized cells can be used ten cycles in D-psicose production without losing enzyme activity at 60°C.