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Characterization of alginate lyase gene using a metagenomic library constructed from the gut microflora of abalone

  • Biotechnology Methods
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

A metagenomic fosmid library was constructed using a genomic DNA mixture extracted from the gut microflora of abalone. The library gave an alginate lyase positive clone (AlyDW) harboring a 31.7-kbp insert. The AlyDW insert consisted of 22 open reading frames (ORFs). The deduced amino acid sequences of ORFs 11–13 were similar to those of known alginate lyase genes, which are found adjacent in the genome of Klebsiella pneumoniae subsp. aerogenes, Vibrio splendidus, and Vibrio sp. belonging to the phylum Gammaproteobacteria. Among the three recombinant proteins expressed from the three ORFs, alginate lyase activity was only observed in the recombinant protein (AlyDW11) coded by ORF 11. The expressed protein (AlyDW11) had the highest alginate lyase activity at pH 7.0 and 45°C in the presence of 1 mM AgNO3. The alginate lyase activity of ORF 11 was confirmed to be endolytic by thin-layer chromatography. AlyDW11 preferred poly(β-d-mannuronate) as a substrate over poly(α-l-guluronate). AlyDW11 contained three highly conserved regions, RSEL, QIH, and YFKAGVYNQ, which may act to stabilize the three-dimensional conformation and function of the alginate lyase.

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Acknowledgments

This study received financial support from the Ministry for Food, Agriculture, Forestry and Fisheries and the 21C Frontier Microbial Genomics and Applications Center Program, Ministry of Education, Science & Technology, Republic of Korea.

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Authors and Affiliations

  1. Department of Food Science and Technology and Functional Food Research Center, Chonnam National University, Gwangju, 500-757, Republic of Korea

    Su-Jung Sim, Jeong-Yong Cho & Duwoon Kim

  2. Department of Biology, Sunchon National University, Suncheon, 540-742, Jeonnam, Republic of Korea

    Keun Sik Baik, Seong Chan Park, Han Na Choe & Chi Nam Seong

  3. Department of Food Science and Nutrition, Chonnam National University, Yeosu, 550-757, Republic of Korea

    Tai-Sun Shin

  4. Department of Chemical Engineering, Pukyung National University, Busan, 608-739, Republic of Korea

    Hee Chul Woo

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  1. Su-Jung Sim
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Correspondence to Duwoon Kim.

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Su-Jung Sim and Keun Sik Baik contributed equally to this study.

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Sim, SJ., Baik, K.S., Park, S.C. et al. Characterization of alginate lyase gene using a metagenomic library constructed from the gut microflora of abalone. J Ind Microbiol Biotechnol 39, 585–593 (2012). https://doi.org/10.1007/s10295-011-1054-0

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  • DOI: https://doi.org/10.1007/s10295-011-1054-0

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