Cloning and Characterization of the Lipase Genes from Candida rugosa (II)

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Project title

Code/計畫編號

NSC85-2611-B019-003

Translated Name/計畫中文名

Candida rugosa 脂肪脢的基因選殖與特性研究(II)

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=219380

Year

1996

Start date/計畫起

01-08-1995

Expected Completion/計畫迄

01-07-1996

Bugetid/研究經費

662千元

ResearchField/研究領域

生物技術(農)
食品科技(農)

Candida rugosa能生產菌體外脂肪酶、其脂肪酶基因已有五種被發表,其重要特性為CTG密碼產生Ser,而非一般之Leu。CTG密碼在五個脂肪酶中有3%的使用率,其中包括活性中心殘基209。首先,以PCR方法對LIP4殘基209進行定位基因突變,將CTG(Leu)的Codon改為TCG (Ser)送入E. coli進行表現,結果產生不可溶而不具活性之蛋白質。送入酵母菌---Saccharomyces cerevisiae進行誘導,以RT-PCR及Western blotting 的結果顯示LIP4在Saccharomyces cerevisiae中有RNA表現而無蛋白質表現。 Candida rugosa produces extracellular lipase (LIP) and five coding sequences of lipases have been identified. By comparing nucleotide and amino acid sequence, it has been demonstrated that the codon CTG codes for Ser instead of Leu. CTG is used with a high frequency (3% of the codons) in LIP, including that corresponding to the catalytic Ser. We have mutated the CTG codon of LIP4 into TCG at residue 209 by polymerase chain reaction (PCR) and expressed its gene product in E. coli BL21 (DE3). The protein expressing in E. coli was inactive and insoluble. We are transformed the gene into yeast Saccharomyces cerevisiae. After induction, RT-PCR and Western blotting were performed to identify its RNA expression and protein expression. There are RNA expression but no protein expression in this yeast expression system.

Keyword(s)

脂肪酶
基因選殖
啤酒酵母菌
Lipase
Gene cloning
Saccharomyces cerevisiae
Candida rugosa

DSpace CRIS

Cloning and Characterization of the Lipase Genes from Candida rugosa (II)

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