王惠鈞臺灣大學：生化科學研究所Wen, YiYiWen文彝2007-11-262018-07-062007-11-262018-07-062004http://ntur.lib.ntu.edu.tw//handle/246246/52743在蛋白質科學的研究中，蛋白質的耐熱性是個非常有趣的研究議題。耐熱的酵素特別在工業界有廣泛的應用。過去曾有無數研究嘗試去增加蛋白質的耐熱性。在本篇論文中，提出了一種嶄新的策略去設計製造具有耐熱性的酵素。 Sso7d是一種可能具有chaperone功能的耐熱性蛋白。 將Sso7d與一種在動物食品工業中具有重要價值的酵素植酸脢(phytase)接在一起，期望能製造出具有耐熱性之植酸脢。經過一連串的嘗試希望能得到可溶的Sso7d-phytase，終於純化出四種具有不同連接長度的的Sso7d-phytase融合蛋白。最後利用活性測試及圓二色光譜去研究Sso7d-phytase的耐熱性。 活性測試的結果顯示與Sso7d融合並不會影響其最佳催化溫度，但會些微增進植酸脢對熱處理的耐受度。另一方面，圓二色光譜的結果則顯示了與Sso7d融合會幫助植酸脢在熱變性後的回復摺疊。回復的程度會受到融合蛋白間連接長度縮短與ATP/Mg2+的存在而增加。 本研究暗示了Sso7d可能具有chaperone的功能，並且利用Sso7d融合蛋白去設計製造具耐熱性的蛋白質是種可行的方法。The thermostability of protein is an interesting topic of the research in protein science. Thermostable enzyme especially has broad applications in industry. Numerous attempts have been done to improve protein thermostability. In this thesis, a novel strategy to engineer thermostable enzyme was proposed. Sso7d, a thermostable protein with potential chaperone function was linked to the N-terminus of phytase, a specific phosphotase of great importance in animal food industry. Series of trials has been done to get soluble Sso7d-phytase fusion protein. At last four kinds of Sso7d-phytase with different linker-length were obtained. Activity assay and CD spectroscopy were employed to analyze the thermostablity of Sso7d-phytase. The results of activity assay showed that fusion with Sso7d does not affect the optimal catalytic temperature of phytase, but slightly enhances the heat tolerance. On the other hand, the CD data demonstrated that Sso7d-fusion helps the refolding of phytase after thermal denaturation. The degree of renaturation elevates with the shortening of flexible linkage and the presence of ATP/Mg2+. The study suggests that Sso7d has the chaperone ability and fusion with Sso7d is a practical way to engineer thermostable protein.ACKNOLEDGEMENTS 1 中文摘要 2 ABSTRACT 3 Table of contents 4 List of Tables 7 List of Figures 8 CHAPTER 1 INTRODUCTION 10 1.1 Thermostability of Enzymes 10 1.2 Sso7d 12 1.3 Phytic acid and phytase 13 1.4 The Aim and Strategy of this Study 15 CHAPTER 2 MATERIALS AND METHODS 21 2.1 Overall Strategy 21 2.2 Materials 21 2.2.1 Instruments 21 2.2.2 Materials 22 2.2.3 Reagents 23 2.3 General Molecular Biology Methods 25 2.3.1 Preparation of LB broth, agarose gels and LB-agar plates 25 2.3.2 Agarose gel electrophoresis 26 2.3.3 Preparation of competent E. coli cells 26 2.3.4 Minipreparation for purification of plasmid plasmid DNA 27 2.3.5 Polymerase chain reaction (PCR) 27 2.3.6 Restriction enzyme digestion of DNA 28 2.3.7 Ligation of insert DNA fragment and vector plasmid 28 2.3.8 Ligation transformation of competent E. coli DH52189191 bytesapplication/pdfen-US耐熱熱穩定性植酸植酸脢Sso7dthermostabilityphytasephytic acid, thermostableotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/52743/1/ntu-93-R91242006-1.pdf