Candida rugosa Lipases Protein Engineering Using Directed Eevolution (II)

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基本資料

Project title

Code/計畫編號

NSC93-2313-B019-017

Translated Name/計畫中文名

Candida rugosa脂肪酵素使用直接演化法執行蛋白質工程(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=990788

Year

2004

Start date/計畫起

01-08-2004

Expected Completion/計畫迄

01-07-2005

Bugetid/研究經費

1024千元

ResearchField/研究領域

農業化學

Candida rugosa所產生的五種菌体外脂肪脢(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，產生13組的新式脂肪脢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。在熱穩定性(Thermostability)的分析上，NL-J不是非常穩定，但因NL-L的熱穩定性高。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的脂肪脢活性相較於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。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。因NL-J熱安定性差，使用定點突變將F314N，提高醣基化程度；使用error-prone PCR經55度處理篩選可提高熱安定性之酵素。得到兩突變基因，酵素活性正鑑定中。 The yeast Candida rugosa produces five extracellular lipases (LIP-1 ~ LIP-5) which encode 534 amino acid, showing high homology in sequence but diversity in the substrate specificity. These observations demonstrate that substrate specificity of CRL may be relative to the various CRL sequences. Using homologous recombination produces thirteen chimeric new lipases NL-A ~ NL-M. Only NL-L shows activity in E. coli expression system. In addition, NL-B and NL-J show activity in Saccharomyces cerevisiae expression system, that modify these LIPs with glycosylation. As comparison of NL-J, NL-JF does not show enzyme activity and its sequence only has differences of three amino acids. Thermostability of NL-J is showed the more unstable than the other CRL. The difference of NL-J with LIP-4 has twenty-eight amino acids, and they demonstrate 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 acids differently, and they show ninety-five percent identity. However, they are different in the substrate specificity of lipase and esterase. To improve the thermostability of NL-J, site-directed mutagenesis was used to increase the glycosylation by replacing Phe-314 into Asn. Using error-prone PCR, NL-J was performed to improve its thermostability by 55 .degree.C treatment to screen the lipase activity.

DSpace CRIS

Candida rugosa Lipases Protein Engineering Using Directed Eevolution (II)

基本資料

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