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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/17550

Title: Candida rugosa LIP之重組新式脂肪酶的表現及特性分析
Overexpression and characterization of novel chimeric lipase from Candida rugosa LIP
Authors: Chin-Yen Huang
黃晉彥
Contributors: NTOU:Institute of Bioscience and Biotechnology
國立臺灣海洋大學:生物科技研究所
Keywords: 脂肪酶;直接演化法;基質選擇性
lipase;candida rugosa;substrate specificity
Date: 2004
Issue Date: 2011-07-04
Abstract: 假絲酵母菌Candida rugosa又名Candida cylindracea,它所產生的五種菌體外脂肪酶(LIP1 ~ LIP5),皆含有534個胺基酸,且在序列上具有高度的相似性,但卻有不同的基質特異性,故Candida rugosa脂肪酶的序列與結構及基質特異性的關係是相當嚴謹的。因此可以用基因工程的方式改變CRL的序列,以研究序列與結構活性間的關係。 本論文以實驗室先前利用LIP4及LIP-F構築出13組新式脂肪酶中的NL-J,利用定點突變的方式將胺基酸序列由314F突變成314N,使其增加一處N-醣基化位置,而產生的蛋白質命名為NL-J-F314N,是具有活性的脂肪酶。經分析發現其分子量從原60-kDa變成62.5-kDa,且熱安定性相較於原NL-J上升5~10°C。 NL-L比起定點突變修飾後之NL-J-F314N高出約1倍的脂解酶(esterase)活性,而NL-L之脂解酶活性為野生型脂肪酶之10倍,且對於基質的選擇性與NL-J-F314N非常相似,皆較喜好中長鏈的受質。但在脂肪酶(lipase)活性上NL-J-F314N則有NL-L之1.5倍的活性,但二者都比野生型脂肪酶的活性低得多。 新式脂肪酶NL-J-F314N因為增加一醣基化位置而改變了酵素的整體結構,造成其熱穩性及基質選擇性的改變。
The yeast Candida rugosa produces five extracellular lipases (LIP1 ~ LIP5), all containing 534 amino acid. These lipases display high sequence homology but partially differ in substrate specificity, demonstrating a strong relationship between substrate specificity and protein sequences. Changing LIP sequence by site-directed protein engineering was undertaken in this study to examine the association between CRL sequences and enzyme activities. Thirteen chimeric lipases NL-A ~ NL-M derived from LIP4 and LIP-F was previously produced by homologous recombination. To add a glycosylation site, amino acid 314F in the NL-J was replaced into Asn by site-directed mutagenesis. The new lipase, NL-J-F314N exhibited a molecular weight about 62.5-kDa and was shown to be more thermostabe than NL-J. The esterase activity of NL-J-F314N was only half of NL-L, yet it was 5 fold stronger than commercial lipase. NL-L prefer C8 to C18 esters substrate and NL-J-F314N show the same activity as NL-L. The lipase activities of NL-J-F314N was higher 1.5 fold than NL-L, but they was lower than commercial lipase. The chimeric lipase NL-J-F314N showed higher thermostability and lipase activity than NL-J, indicating that an additional glycosylation site may change the overall enzyme structure and activity.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M92360018
http://ntour.ntou.edu.tw/ir/handle/987654321/17550
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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