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Production of D-hydantoinase via surface display and self-cleavage system.

Published on Nov 1, 2013in Journal of Bioscience and Bioengineering2.03
· DOI :10.1016/j.jbiosc.2013.05.002
Chia-Chi Lin5
Estimated H-index: 5
(NCHU: National Chung Hsing University),
Tzu-Tsen Liu2
Estimated H-index: 2
(NCHU: National Chung Hsing University)
+ 6 AuthorsYung-Chuan Liu24
Estimated H-index: 24
(NCHU: National Chung Hsing University)
Abstract
In this study, the cell surface expression system was the first time used to directly produce extracellular enzyme. In the plasmid construction, the truncated ice nucleation protein (INP) was fused with intein (INT) and target protein, d -hydantoinase ( DHT ase), to form the INP-INT-DHTase gene. The plasmid containing this gene was transformed into Escherichia coli ER2566 cells. The gene construct enables the expression of INP-INT-DHTase fusion protein, which might anchor on cell membrane surface. The induction conditions were studied and optimal conditions were as follows: E. coli ER2566 was incubated at 37°C and 200 rpm till OD 600 reached 0.6. Then, 0.05 mM IPTG was added and the induction was conducted at 15°C for 24 h. The cell was harvested and resuspended in the cleavage buffer (50 mM Tris-HCl buffer, pH 6). The cleavage reaction was carried out at 25°C, and 100 rpm for 24 h. The DHT ase with an activity of 0.225 U/ml and a purity of 63.2% was obtained via centrifugation. This study demonstrated the feasibility of direct extracellular enzyme production using E. coli via only two steps of centrifugation.
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  • Citations (2)
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