臺灣博碩士論文加值系統

酵母菌Candida rugosa所產生的五種菌體外脂肪酶 (LIP1~ LIP5)，在序列上具有高度的相似性，但卻有不同的基質特異性，因此可以用蛋白質工程的方式改變CRL的序列，以研究序列與結構活性間的關係。實驗室先前以基因重組的方式，重組LIPF和LIP4産生13組的新式脂肪酶NL-A ~ NL-M。所產生的各式脂肪酶的經測試後，可顯示CRL的序列結構及基質特異性的具有相關性。
本論文進一步使用直接演化法 (direct evolution) 對重組新式脂肪酶基因進行改造，探討以了解CRL的序列與結構及基質特異性的相關性。將NL-B和NL-L重組形成新式脂肪酶，對於tributyrin (C4:0)具有高活行，但對olive oil則表現出低活性。結果顯示新式脂肪酶與NL-B和NL-L具有不同的基直特異性。另外，使用錯誤傾向聚合酶鏈反應 (error-prone PCR) 以NL-J為模版 (template) 進行研究，發現序列的突變，對tributyrin (C4:0) plate的酵素活性都有不同的改變。
實驗室先前利用定點突變的方式將新式脂肪酶中的NL-J突變成NLJF314N，其胺基酸序列由F314突變成N314，熱安定性相較於原NL-J 來的高。為提高熱安定性，本論文進一步將Heat-shock protein融合在NLJF314N的N端，發現其耐熱性較原NLJF314N效果提升了1.6倍。

The yeast Candida rugosa produces five extracellular lipases (LIP1 ~ LIP5) , showing high homology in sequence but partial difference in the substrate specificity. Changing LIP sequence by site-directed protein engineering was undertaken in this study to examine the relationship between CRL sequences and enzyme activities. Thirteen chimera lipases NL-A ~ NL-M derived from LIP4 and LIPF was previously produced by homologous recombination. Compared as these novel lipases enzyme activity, we demonstrate that lipases has a strong relationship between substrate specificity and protein sequences.
To understand future relationship between CRL activity and substrate specificity, the method of direct evolution to generate novel lipases gene was application in this study. NEW lipase, produced from the recombination of NL-B and NL-L, showed the different substrate specificity as compared with NL-B as well as NL-L. The lipase activity of NEW lipase was high in the substrate of tributyrin (C4:0) and low in olive oil. Random mutation lipase generated from NL-J by error-prone PCR were observed in the tributyrin (C4:0) plate and They showed different lipase activity.
To improve thermal stability of lipase by introducing glycosylation, NLJF314N generated from alternating Phe314 into Asn, showed higher thermostability than NL-J. For further improvement of thermal stability, NLJF314N fused with heat-shock protein in the N terminal to generate Hsp-NLJ-F3144N. The lipase exhibit more thermal stability than NLJF314N and T1/2 of HspJF314N was increased 1.6 folds.

目錄………………………………………………………………………1
中文摘要…………………………………………………………………2
英文摘要…………………………………………………………………3
序論………………………………………………………………………4
一、脂肪酶的用…………………………………………………………4
二、脂肪酶的生化特性…………………………………………………6
三、脂肪酶的結構特性…………………………………………………8
四、Candida rugosa lipase (CRL)…………………………………10
五、C. rugosa脂肪酶的結構特性……………………………………12
六、研究原起……………………………………………………………16
七、酵素直接演化法……………………………………………………19
八、實驗方向……………………………………………………………21

材料與方法………………………………………………………………23
一、材料…………………………………………………………………23
二、實驗方法……………………………………………………………28

實驗結果…………………………………………………………………39
論…………………………………………………………………………43
參考獻……………………………………………………………………49
圖…………………………………………………………………………56
附錄………………………………………………………………………65

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