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KMID : 0380020190340040258
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Korean Journal of Biotechnology and Bioengineering
2019 Volume.34 No. 4 p.258 ~ p.266
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Secretional Optimization of Ferritin Protein in Bacillus subtilis and Its Biological Activity in Laying Hen
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Lee Seung-Woo
Kim Min-Ju
Choi Jang-Won
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Abstract
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In order to maximize the production and secretion of ferritin protein, the replication origin (0.5 kb) of the pRBASFer (6.93 kb) which was already constructed was amplified using PCR and introduced into the pRBASFer vector with tandem arrangement. Finally, the recombinant pRBASFer-ori (7.43 kb) vector with two replication origins was newly constructed. And then, two type of the recombinant vectors (pRBASFer and pRBASFer-ori) containing aprE promoter, aprE signal sequence and ferritin gene were transferred to Bacillus subtilis LKS87, respectively and the right transformant were confirmed by restriction enzyme digestion, PCR amplification and DNA sequencing. The effect of additional replication origin on the secretion of ferritin protein was investigated according to various culture conditions and the secretion levels of ferritin were analyzed in SDS-PAGE and western-blot. As a result, the secretion level of ferritin protein in B. subtilis LKS87 harboring the pRBASFer-ori vector was increased to approximately 20% of total secreted proteins (165 μg/mL) more than that of pRBASFer vector. Maximum level of the ferritin secretion was accomplished in batch culture as followings: 2% soy peptone as nitrogen source, 2% barley as carbon source, and 48 h incubation time. Mass production of the ferritin was carried out using 50 L fermenter under the optimal conditions established in batch culture. The liquid type of feed additive containing the ferritin was formulated and the biological activity was tested by its administration to drinking water in laying hens, resulting in the improvement (10%) of all index related to eggshell color, egg weight and Haugh unit, indicating that the liquid type feed show the possibility as new feed additive in laying hen to increase egg quality.
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KEYWORD
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Bacillus subtilis, egg quality, ferritin, feed additive, replication origin
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