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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/17512

Title: Candida rugosa LIP-4之重組新式脂肪酶的表現及特性分析暨使用Error-prone PCR進行Candida rugosa脂肪酶蛋白質熱安定性改善工程
Characterization of novel chimeric lipase from Candida rugosa LIP-4 and improvement of thermostability of Candida rugosa lipase using Error-prone PCR
Authors: Hung-Wei Chen
陳宏偉
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;熱安定性
Candida rugosa lipase;thermostability;in vitro evolution
Date: 2003
Issue Date: 2011-07-04
Abstract: 摘要 Candida rugosa所產生五種相近的菌体外脂肪酶(LIP-1~LIP-5),在序列上具有高度的相似性,但卻有不同的基質特異性,由此可知Candida rugosa lipase的序列與結構及基質特異性的相關性可能是非常嚴謹的。因此本研究利用蛋白質工程,將LIP-4的序列改變,研究序列與蛋白質結構及基質特異性、熱穩定性之間的關係。 以本實驗室先前構建出13組新式脂肪酶的NL-J lipase,利用Error-prone PCR方式所形成的mutant NL-J library中,發現NJ2-3、NJ3-5的熱安定性較原始NL-J上升1.3倍之多,但彼此間的胺基酸差異僅有6個,於是推測這6個位置可能隱含熱安定性與序列結構間的關係。另外並以傳統cloning方式得到一NL-BJ ( 採用NL-B的前半段及NL-J的後半段接合而成 ),與NL-L purify protein一起測試esterase substrate specificity。從實驗結果中可發縣NL-L的基質特異性與LIP-4相近,NL-BJ與NL-J類似,比較4者間差異的胺基酸位置,歸類出可能造成的活性差異點,並綜合熱安定性差異的6個胺基酸,探討lipase序列與結構的關係。其中424突變位置正好位於構成lipase α3-7,8 helix上,可能因突變為帶電性胺基酸,使整體結構變動而使熱安定性上升。
Abstrate The yeast Candida rugosa produces five closely extracellular lipases , showing high homology in sequence but partial difference in the substrate specificity . The observation demonstrate that substrate specificity of CRL may be relative to the various CRL sequences . We can use protein engineering to change sequence of LIP-4 for studying the relationship of enzyme activity with CRL protein sequence . Thirteen chimeric new lipases NL-A~NL-M was created by homologous recombination in our labratory .Using Error-prone PCR produces mutant NL-J library from new chimeric NL-J DNA .Two mutant colonies , NJ2-3 and NJ3-5 , showing higher thermostability than original NL-J colony .The thermostability of two mutant colonies showing increase about 1.3 fold but the difference of NL-J with two colonies has six amino acid . A tradional cloning strategy was used to produce a new NL-BJ from N terminal sequence of NL-B and C terminal sequence of NL-J .After analysed new NL-BJ and chimeric NL-L protein , NL-L show similar esterase substrate specificity to LIP-4 toward C16>C12>C8>C4 and NL-BJ is the samw with NL-J towardC12>C8>C4>C6 . By examination of all six mutant amino acid position , the 424 mutant position may be the key role to improve lipase thermostability .
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M91360018
http://ntour.ntou.edu.tw/ir/handle/987654321/17512
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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