Effect of spacer arms on the immobilization of protein switch

• Main Authors: Kuen-You Hong, 洪堃祐
• Other Authors: Sung-Chyr Lin
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/19113311404744845136

碩士 === 國立中興大學 === 化學工程學系所 === 102 === Protein switch RG13 is an artificial fusion protein with specificity to maltose, and its capability of hydrolyzing β-lactam depends on the concentration of maltose. Previous research uses epoxy-modified regenerated cellulose membrane as a support for covalent immobilization of RG13 to test its potential as a biosensor. In this study, two diamines, ethylenediamine and hexamethylenediamine, are introduced as spacer-arms to test effects of different lengths of spacer-arms on covalent immobilization of RG13. First, immobilization under different enzyme concentrations gives the optimal enzyme concentration 0.1mg/mL, which yields the highest specific activities for immobilization on both spacer-arms. Next, substrates are added during enzyme immobilization to protect the active sites. With addition of 50mM maltose, highest increases in specific activities are found to be 3.56 and 3.17 folds for ethylenediamine and hexamethylenediamine individually. Then, addition of 75mM penicillin G gives the highest enzyme load and total activity performances for both spacer-arms. Finally, enzyme concentration 0.1mg/mL with addition of 50mM maltose and 75mM penicillin G are chosen as the optimal condition for RG13 immobilization on epoxy-modified RC membrane with different spacer-arms. For enzyme immobilized with spacer-arm ethylenediamine, estimated enzyme load is 0.047±0.006 mg/cm2, activity is 0.153±0.005 U and specific activity is 3.303±0.415 U/mg. For enzyme immobilized with spacer-arm hexamethylene- diamine, estimated enzyme load is 0.052±0.011 mg/cm2, activity is 0.142±0.002 U and specific activity is 2.079±0.610 U/mg. Both of the performances are better than that with addition of single substrate during immobilization. And, the immobilized RG13 still shows specificity to maltose. In the repeated batch operation, after 10 cycles of operations, 77.2% of activity is retained for the RG13 on ethylenediamine, which is slightly higher than the activity without maltose in the system. And, 82.1% for the RG13 on hexamethylenediamine, which equals to that without maltose. In conclusion, the performance of immobilized RG13 is slightly better on a shorter spacer-arm, and the possible reasons may come from the hydrophobicity, length and flexibility of spacer arms that lead to different behaviors when the enzyme is immobilized.