Overproduction of d-hydantoinase and carbamoylase in a soluble form in Escherichia coli |
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| Authors: | Y-P Chao C-J Chiang T-E Lo H Fu |
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| Institution: | (1) Department of Chemical Engineering, Feng Chia University, 100 Wenhwa Road, Taichung, Taiwan e-mail: ypchao@fcu.edu.tw Fax: +886-4-4510890, TW;(2) Widetex Chemical Corporation Limited, 160 Nanking E. Road, Taipei, Taiwan, TW;(3) Tungfang Junior College of Technology and Commence, Kaoshung, Taiwan, TW;(4) Institute of Botany, Academia Sinica, Nankang, Taipei, Taiwan, TW |
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| Abstract: | The production of d-hydantoinase and carbamoylase from Agrobacterium radiobacter NRRL B11291 using T7 and trc promoters, respectively, was found to cause protein aggregates in Escherichia coli. We initiated a systematic study aimed at overproducting these two proteins in a soluble form. As a result, the protein aggregate
from carbamoylase overproduction could be alleviated with the aid of GroEL/GroES. In contrast, the production of a high level
of d-hydantoinase in an active form can be achieved at low temperature (25 °C) or by the coproduction of DnaJ/DnaK. Overall, with
such approaches both recombinant proteins gain more than a four-fold increase in enzyme activity. In addition, by fusion with
thioredoxin, d-hydantoinase activity can be increased 25% more than the unfused counterpart in the presence of DnaJ/DnaK. These results
indicate the success of our approaches to overproducing d-hydantoinase and carbamoylase in a soluble form in E. coli.
Received: 26 November 1999 / Received revision: 28 February 2000 / Accepted: 10 March 2000 |
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