標題: | Cloning and Expression of Chitinase A from Serratia marcescens for Large-scale Preparation of N,N-Diacetyl Chitobiose |
作者: | Wu, Yue-Jin Cheng, Chih-Yu Li, Yaw-Kuen 應用化學系 Department of Applied Chemistry |
關鍵字: | Serratia marcescens;Chitinase A;Colloidal chitin;N,N-Diacetyl chitobiose |
公開日期: | 1-八月-2009 |
摘要: | The gene of Serratia marcescens chitinase A (chiA) was cloned by PCR. The complete gene was constructed into a pRSET vector and expressed in Escherichia coli. The recombinant enzyme was purified to > 90% homogeneity by hydrophobic interaction chromatography followed by ion-exchange separation. Measured with an electrospray-ionization mass spectrometer, the molecular mass of the protein was 58,607 Da, consistent with a theoretical calculation of the deduced protein without the signal peptide. The recombinant enzyme was characterized and tested for the preparation of chitobiose. In general, the recombinant Chtinase A exhibited an exo-type catalytic activity toward colloidal chitin and released both N-acetylglucosamine and N,N-diacetyl chitobiose as products. After extensive testing, we produced N,N-diacetyl chitobiose as the predominant product when the enzymatic reaction was performed in sodium acetate buffer at pH 5.5; under such conditions, an enzymatic process is established for the production of the disaccharide on a 100-g scale. |
URI: | http://hdl.handle.net/11536/6904 |
ISSN: | 0009-4536 |
期刊: | JOURNAL OF THE CHINESE CHEMICAL SOCIETY |
Volume: | 56 |
Issue: | 4 |
起始頁: | 688 |
結束頁: | 695 |
顯示於類別: | 期刊論文 |