Identify Domains Involving Activities of Candida rugosa Lipases and Create Novel Lipases from Lipase Library Generated by Using in Vitro Evolution

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簡歷

基本資料

Project title

Code/計畫編號

NSC90-2313-B019-028

Translated Name/計畫中文名

鑑定Candida rugosa脂肪脢活性區域和使用in Vitro Evolution產生新酵素

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=670376

Year

2001

Start date/計畫起

01-08-2001

Expected Completion/計畫迄

01-07-2002

Bugetid/研究經費

900千元

ResearchField/研究領域

農業化學
食品科技(農)

Candida rugosa能產生菌體外脂肪酵素(CRL)，此類lipase廣用於食品、清潔劑、藥物、或精密化學品的合成上。然而Candida rugosa非使用universal codon，其CUG產生Ser非一般的Leu。因此，CRL無法在E. coli或yeast表現具活性的酵素。經定位突變的方式，克服codon usage的難題，獲得具活性的LIP4醇素。LIP4 對長鏈的基質有高esterase活性，但對tributyrin、triolein、and olive oil表現較低活性。研究顯示它與其它CRL的活性不相同。計畫中使用ITCHY(iterative trunction or creation of hybrid enzymes)方式獲得LIPF-LIP4 hybrid lipases，產生NL-A~M13種chimeric lipases。E. coli expression system獲得LIP4和NL-L具活性，但在yeast中則LIP4, NL-B, NL-G, NL-K, and NL-L 具有活性。現正鑑定轉殖株的活性中。 The yeast Candida rugosa produces extracellular lipase (CRL) extensively used for industrial purposes. C. rugosa utilizes a unusual Ser codon CUG, encoding Leu in universal codon, that hampers those genes heterologous expression in E. coli and yeast. Here we have reported that the recombinant LIP4 with replacements of 19 CUG codons into serine codons are functionally overexpressed in E. coli and Pichia pastoris. LIP showed high esterase activities toward long-chain ester and lower lipase activities toward tributyrin, triolein and olive oil. These observations demonstrated that LIP4 shows distinguished catalytic activities from the commercial preparations of CRLs. Using DNA muagenesis ia an essential approach to understand the CRL for the structure-function relationship, the enzyme reaction mechanism, and designation of novel lipase to its new application. We use ITCHY (iterative truncation or the creation of hybrid enzymes) to generate the N- and C- terminal fragment library of LIPF and LIP4 by progressive trunction of their coding sequencing to generate the chimeric lipases NL-A~M. The chimeric lipase NL-L expressing in E. coli and NL-B, NL-K, and NL-L expressing in yeast show enzyme activity .The chimeric lipases are identified and characterized their enzyme activities.

Keyword(s)

酵素活性
酵素製備
脂肪脢
假絲酵母
Enzyme activity
Enzyme preparation
Lipase
Candida sp.
Candida rugosa

DSpace CRIS

Identify Domains Involving Activities of Candida rugosa Lipases and Create Novel Lipases from Lipase Library Generated by Using in Vitro Evolution

基本資料

Description