Candida rugosa Lipases Protein Eengineering Using Directed Evolution (III)

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基本資料

Project title

Code/計畫編號

NSC94-2313-B019-004

Translated Name/計畫中文名

Candida rugosa脂肪酵素使用直接演化法執行蛋白質工程(III)

Project Coordinator/計畫主持人

Shye-Jye Tang

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Bioscience and Biotechnology

Website

https://www.grb.gov.tw/search/planDetail?id=1097152

Year

2005

Start date/計畫起

01-08-2005

Expected Completion/計畫迄

01-07-2006

Bugetid/研究經費

1040千元

ResearchField/研究領域

農業化學

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 NL-BL，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 NL-BL 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，本論文進一步將硫氧化蛋白 (thioredoxin) 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 LIP-F 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. NL-BL, 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 NL-BL 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 thermoal stability of lipase by introducing glycosylation, NLJF314N generated from alternating Phe314 into Asn, showed higher thermostability than NL-J. For further improvement of thermoal stability, NLJF314N fused with thioredoxin in the N terminal to generate Trx-NLJ-F3144N. The lipase exhibit more thermoal stability than NLJF314N and T1/2 of TrxJF314N was increased 1.6 folds.

DSpace CRIS

Candida rugosa Lipases Protein Eengineering Using Directed Evolution (III)

基本資料

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