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Cloning and expression of a thermostable keratinase gene from Thermoactinomyces sp. YT06 in Escherichia coli and characterization of purified recombinant enzymes

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Abstract

The feather-degrading strain Thermoactinomyces sp. YT06 secretes an extracellular keratinolytic protease (KERTYT); however, the gene encoding this protease remains unknown. The kerT1 gene (1170 bp) encoding keratinase was cloned and expressed in Escherichia coli BL21(DE3). Purified recombinant keratinase (rKERTYT) was achieved at a yield of 39.16% and 65.27-fold purification with a specific activity of 1325 U/mg. It was shown that rKERTYT has many similarities to the native enzyme (KERTYT) by characterization of rKERTYT. The molecular weight of rKERTYT secreted by recombinant E. coli was approximately 28 kDa. The optimal temperature and the pH values of rKERTYT were 65 °C and 8.5, respectively, and the protein remained stable from 50 to 60 °C and pH 6–11. The keratinase was strongly inhibited by phenyl methane sulfonyl fluoride (PMSF), suggesting that it belongs to the serine protease family. It was significantly activated by Mn2+ and β-mercaptoethanol (β-Me). rKERTYT showed stability and retained over 80% activity with the existence of organic solvents such as acetone, methylbenzene and dimethyl sulfoxide. These findings indicated that rKERTYT will be a promising candidate for the enzymatic processing of keratinous wastes.

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Acknowledgements

This research was funded by the Natural Science Foundation of Jiangsu Province (Grants No. BK20170113) and the National Natural Science Foundation of China (Grants No. 31700099).

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Authors and Affiliations

  1. Nanjing Institute of Agricultural Sciences in Jiangsu Hilly Area, Nanjing, 210046, People’s Republic of China

    Lin Wang, Ying Zhou, Ying Huang, Qishun Wei & Chengbao Guo

  2. Circular Agriculture Research Center, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, People’s Republic of China

    Hongying Huang

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  1. Lin Wang
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  2. Ying Zhou
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  3. Ying Huang
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  4. Qishun Wei
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Correspondence to Chengbao Guo.

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Wang, L., Zhou, Y., Huang, Y. et al. Cloning and expression of a thermostable keratinase gene from Thermoactinomyces sp. YT06 in Escherichia coli and characterization of purified recombinant enzymes. World J Microbiol Biotechnol 35, 135 (2019). https://doi.org/10.1007/s11274-019-2710-1

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