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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/17479

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脂肪酶蛋白質工程
Overexpression and characterization of novel chimeric lipase from Candida rugosa LIP-4 and Protein engineering of Candida rugosa lipase using Error-prone PCR
Authors: Hsu-Feng Liu
劉旭峰
Contributors: NTOU:Institute of Bioscience and Biotechnology
國立台灣海洋大學:生物科技研究所
Keywords: 脂肪酶
LIP-4;lipase;Candida rugosa;Error-prone PCR
Date: 2002
Issue Date: 2011-07-04
Abstract: Candida rugosa所產生的五種菌体外脂肪酶(LIP-1~LIP-5),皆為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,由此可知CRL的序列與結構及基質特異性的相關性可能是非常嚴謹的。因此可利用蛋白質工程,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。 以基因重組的方式,産生13組的新式脂肪酶NL-A ~ NL-M,但在E. coli系統中,只有NL-L有活性反應,而NL-B及NL-J卻會在酵母菌系統中,因有醣基化(glycosylation)修飾,而回復其活性,其中NL-JF與NL-J只有三個胺基酸不同,卻因此造成沒有活性,推測可能是NL-JF蛋白質序列中的S436L有較大的可能性。 在熱穩定性(Thermostability)的分析上,NL-J雖然不是非常穩定,但因NL-L的熱穩定性還算不錯,且NL-J與NL-L僅只有8個胺基酸有所不同,固可對此8個胺基酸作蛋白質工程或將NL-J的314F突變成N使其有多一個醣基化(glycosylation)修飾,研究是否可改變成具有高度熱穩定性(Thermostability),便可再提升其應用性。 而NL-J雖與LIP-4的胺基酸只有28個不同,且有95%的相同度(identity),但NL-J的脂肪酶活性相較於LIP-4卻有非常明顯的增強,相對於不同的受質約有增強為1~4倍左右,對Tributyrin (C4:0)而言,NL-J是LIP-4的1.7倍左右,Tricapryin (C8:0)是2.2倍,Tristearin (C18:0)是2.1倍左右,Triolein (C18:1)是3.3倍左右,對Olive oil而言是4.2倍左右。而在Esterase Activity中雖然整体而言NL-J均較LIP-4低,但卻比Commercial lipase高,約有2 ~ 3倍的活性增強。而在NL-B與NL-L方面,雖然皆與LIP-4只有20個胺基酸不同,且皆有95%的相同度(identity),但在脂肪酶及酯解酶的相對活性卻也是迥然不同。 在結束以重組基因方式,所產生的各式脂肪酶的一連串測試後,可得知CRL的序列與結構及基質特異性的相關性是真的非常嚴謹,之後便以In vitro evolution中的錯誤傾向聚合酶鏈反應(Error-prone PCR)的方式來對LIP-4進行研究,以其能更進一步的了解CRL的序列與結構及基質特異性的關性,實驗後發現有88.75 %的LIP-4會經由Error-prone PCR的突變而導致失去活性,只剩11.25 %仍保有活性。
The yeast Candida rugosa produces five extracellular lipases (LIP-1 ~ LIP-5) which encode 534 amino acid, showing high homology in sequence but partial difference in the substrate specificity. These observation demonstrate that substrate specificity of CRL may be relative to the various CRL sequences. We can using protein engineering to change sequence of LIP-4. In order to study the relationship of enzyme activity with CRL protein sequence. Using homologous recombination produces thirteen chimeric new lipases NL-A ~ NL-M. Only NL-L show activity in E. coli system. In addition strategy of NL-B and NL-J show activity in Saccharomyces cerevisiae system, that modify these LIPs protein engineering and we to manipulate the LIP-4 gene with glycosylation. As comparison of NL-J, NL-JF does not show enzyme activity and its sequence only has difference of three amino acid. Thermostability of NL-J is showed the more stable than the other CRL. The difference of NL-J with LIP-4 has twenty-eight amino acid, and they has identity of 95%. The lipase activity of NL-J is more active than LIP-4, showing increase about 1 ~ 4 fold. The lipase activity of NL-J is increased in the substrates of tributyrin (C4:0), tricapryin (C8:0), tristearin (C18:0), triolein (C18:1) and olive oil about 1.7, 2.2, 2.1, 3.3 and 4.2 fold, respectively. The esterase activity of NL-J is less active than LIP-4 but NL-J increases 2 ~ 3 fold than commercial lipase in the esterase activity. Both NL-B and NL-L only have twenty amino acid differently, and they show ninety-five percent identity. However, they are different in the substrate specificity of lipase and esterase. Error-prone PCR is performed in the LIP-4. A LIP-4 random mutation library was generated by using Error-prone PCR, using tributyrin plate to screen, 11.25 % clones show enzyme activity. This result demonstrates that LIP-4 sequence is critical and mutation in the 88.75 % position of LIP-4 may inactivate the enzyme activity.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0000000176
http://ntour.ntou.edu.tw/ir/handle/987654321/17479
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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