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Molecular characterization of gene encoding an extracellular alkaline protease in Vibrio metschnikovii

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Abstract

The gene, vapK, encoding an extracellular alkaline protease, VapK, in Vibrio metschnikovii strain RH530 was cloned and expressed in Escherichia coli. The nucleotide sequence of the vapK revealed a single open reading frame of 1266 bp encoding 422 amino acids. The biochemical characteristics of the expressed protease were indistinguishable from those of VapK from the wild type strain. The recombinant VapK showed resistant activity to surfactants such as sodium α-olefin sulfonate, polyoxyethylene oxide, SDS and urea. The surfactant, sodium alkylbenzenesulfonate, strongly inhibited the activity of re-VapK. However in the presence of POE or Na2SO4, its activity was completely restored. By N-terminal sequencing of VapK, the cleavage site for the mature protease could be identified. The cleavage site was Ala140 and the calculated molecular mass of the mature enzyme was 29 609 Da, corresponding to the purified VapK. The active sites and putative calcium binding sites of recombinant VapK (re-VapK) were highly conserved when compared to subtilisin Carlsberg.

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Authors and Affiliations

  1. Department of Bioscience and Biotechnology, College of Natural Sciences, Hankuk University of Foreign Studies, Yongin-Si, Kyunggi-Do, 449-791, Korea

    So Sun Chung, Hee Jin Kim, Yong Uk Shin & Hyune Hwan Lee

  2. R & D Center, Household Division, Cheiljedang Corporation, Inchon, 400-103, Korea

    Ghee Hong Jin

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  1. So Sun Chung
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  2. Hee Jin Kim
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  3. Yong Uk Shin
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  4. Ghee Hong Jin
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  5. Hyune Hwan Lee
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Correspondence to Hyune Hwan Lee.

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Chung, S.S., Kim, H.J., Shin, Y.U. et al. Molecular characterization of gene encoding an extracellular alkaline protease in Vibrio metschnikovii. Biotechnology Letters 23, 1175–1182 (2001). https://doi.org/10.1023/A:1010525428239

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