Studies of Mutation and Immobilization of Maltooligosyl Trehalose Synthase from Sulfolobus acidocaldarius

• Main Authors: Huang, Yu-Ting, 黃鈺婷
• Other Authors: Fang, Tsuei-Yun
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/70375507192919471295

碩士 === 國立臺灣海洋大學 === 食品科學系 === 104 === Maltooligosyl trehalose synthase (MTSase) catalyzes an intramolecular transglycosyl reaction to form maltooligosyltrehalose by converting the α-1.4-glucosidic linkage at the reducing end of maltooligosaccharide to an α-1.1-glucosidic linkage. MTSase acts with maltooligosyl trehalose trehalohydrolase (MTHase) which hydrolyzes the linkage between maltooligosaccharide group and trehalose group to produce trehalose. MTSase also hydrolyzes maltooligosaccharides and maltooligosyltrehaloses to liberate maltooligosaccharides and glucose. The hydrolysis reaction is unfavorable to trehalose production. In order to keep the transglycosyl activity and decrease hydrolytic activity of MTSase from Sulfolobus acidocaldarius, the mutant MTSase was constructed by site-directed mutagenesis. The wild-type and mutant MTSases were purified and studied to obtain their kinetic parameters. The kcat/kM value of mutant on transglycosyl reaction is similar to that of wild-type. The kcat/kM value of mutant on hydrolysis reaction reduces from 0.083 s-1∙mM-1 to 0.038 s-1∙mM-1. On the other hand, enzyme immobilization make enzyme become reusable. Therefore, the wild-type MTSase was immobilized and studied for the optimum immobilization conditions. The alginate and NHS-activated Sepharose 4 Fast Flow were used to immobilize MTSase. The optimum temperature for alginate -immobilized MTSase was 65oC. The optimum pH for alginate -immobilized MTSase was 5. However, the immobilizaion efficiency of alginate -immobilized MTSase was only 14%. The low immobilization efficiency may due to the leakage of MTSase from alginate beads. The immobilizaion efficiency of NHS-activated Sepharose 4 Fast Flow was 95%, the optimum temperature was 65oC, and the optimum pH was 5. Nevertheless, NHS-activated Sepharose 4 Fast Flow was expensive, and then immobilize cells containing MTSase by alginate entraption was also studied. The immobilizaion efficiency of immobilized cells containing MTSase was 37%, the optimum temperature was 70oC, and the optimum pH was 5. Whether immobilized MTSase or MTSase-containing immobilized cells, their thermostabilities are decreased. Although the immobilizaion efficiency using NHS-activated Sepharose 4 Fast Flow is higher than the others, the low cost and medium immobilizaion efficiency of MTSase-containing immobilized cells may be more suitable for trehalose production.