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標題: Production of D-hydantoinase via surface display and self-cleavage system
關鍵字: Biocatalysis;Enzyme production;Ice nucleation protein-intein surface display system;Protein recovery;Self-cleavage;D-Hydantoinase
Project: Journal of Bioscience and Bioengineering, Volume 116, Issue 5, Page(s) 562-566.
In this study, the cell surface expression system was the first time used to directly produce extracellular enzyme. In theplasmid construction, the truncated ice nucleation protein (INP) was fused with intein (INT) and target protein,D-hydantoinase (DHTase), to form the INP-INT-DHTase gene. The plasmid containing this gene was transformed intoEscherichia coli ER2566 cells. The gene construct enables the expression of INP-INT-DHTase fusion protein, which mightanchor on cell membrane surface. The induction conditions were studied and optimal conditions were as follows: E. coliER2566 was incubated at 37 C and 200 rpm till OD600 reached 0.6. Then, 0.05 mM IPTG was added and the induction wasconducted at 15 C for 24 h. The cell was harvested and resuspended in the cleavage buffer (50 mM TriseHCl buffer, pH 6).The cleavage reaction was carried out at 25 C, and 100 rpm for 24 h. The DHTase with an activity of 0.225 U/ml and apurity of 63.2% was obtained via centrifugation. This study demonstrated the feasibility of direct extracellular enzymeproduction using E. coli via only two steps of centrifugation.
Appears in Collections:化學工程學系所

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