In Vitro Recombination of a Family of Genes of Candida rugosa Lipases

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Project title

Code/計畫編號

NSC89-2313-B019-051

Translated Name/計畫中文名

使用in Vitro Recombination 產生基因重組Candida rugosa脂肪脢

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=551872

Year

2000

Start date/計畫起

01-08-2000

Expected Completion/計畫迄

01-07-2001

Bugetid/研究經費

800千元

ResearchField/研究領域

食品科技(農)
生物技術(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 orcreation of hybrid enzymes)方式獲得LIP1-LIP4hybrid lipases ，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 LIP1 and LIP4 by progressive trunction of their coding sequencing. The chimeric lipase library are identified and characterized their enzyme activities.

Keyword(s)

念珠菌
脂肪脢
基因重組
生體外
酵素活性
Candida rugosa
Lipase
Gene recombination
In vitro
Enzyme activity

DSpace CRIS

In Vitro Recombination of a Family of Genes of Candida rugosa Lipases

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