Enzymatic Production of Chitooligosaccharides throgh the Cooperative Reaction from Chitinase and Lysozyme and Their Protein Engineered Chimera Enzymes

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Project title

Code/計畫編號

NSC93-2313-B019-026

Translated Name/計畫中文名

以幾丁脢與溶菌脢及其蛋白質工程產物複合酵素進行幾丁寡醣的酵素生產

Project Coordinator/計畫主持人

Fu-Pang Lin

Funding Organization/主管機關

National Science and Technology Council

Co-Investigator(s)/共同執行人

陳幸臣

Department/Unit

Department of Bioscience and Biotechnology

Website

https://www.grb.gov.tw/search/planDetail?id=990816

Year

2004

Start date/計畫起

01-08-2004

Expected Completion/計畫迄

31-07-2005

Bugetid/研究經費

846千元

ResearchField/研究領域

食品科技(農)

" 將文獻中已知的數個具有轉糖基功能的幾丁質.基因，先以PCR 的方式選殖出來，再由大腸桿菌的表現系統－載體pET-20b(+)配合最適合的表現宿主，取得具酵素活性的重組幾丁質.，測試其對幾丁質水解的能力及產物分析後，再將其與市售之溶菌.（Lysozyme）搭配，觀察其是否能提高轉糖反應的效率？找出其最適的反應條件。並將以基因工程的技術找出溶菌.的轉糖功能之部位，再以基因融合的方式與具轉糖功能之幾丁質.做複合酵素（Chimera Enzyme），測試其是否具雙功能－即水解幾丁質後，又能夠同時進行轉糖來提高幾丁寡糖的酵素生產率。若能得到具雙功能活性的複合酵素，則將對其基因進行人工演化（Molecular Evolution），並將對其結構與功能的關係以X-光結晶繞射或核磁共振的方式做深度剖析。 " " Genes encoding chitinase with putative transglycosylation activity from either prokaryotic or eukaryotic organisms will be cloned by PCR techniques. The cloned chitinase genes will be overexpressed in E. coli expression system using the expression vector pET-20b(+). Hydrolysis of chitin and their transglycosylation activity would be analyzed in these recombinant chitinases and their optimized reaction conditions for transglycosylation in conjunction with the commercially available lysozyme are to be performed. The region responsible for transglycosylation activity of the lysozyme molecule will be investigated by genetic manipulation techniques. The chimera enzymes between chitinase and lysozyme』s region with transglycosylation activity will be constructed and further molecular evolution to increase the efficiency of chitooligosaccharides production is also planned to carry out. Finally, the function and structure relationships of this bifunctional chimera enzyme will be thoroughly analyzed by modern techniques such as X-ray crystallography or NMR methods. "

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Enzymatic Production of Chitooligosaccharides throgh the Cooperative Reaction from Chitinase and Lysozyme and Their Protein Engineered Chimera Enzymes

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