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A new esterase EstD2 isolated from plant rhizosphere soil metagenome

  • Biotechnologically Relevant Enzymes and Proteins
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Abstract

Soil metagenome constitutes a reservoir for discovering novel enzymes from the unculturable microbial diversity. From three plant rhizosphere metagenomic libraries comprising a total of 142,900 members of recombinant plasmids, we obtained 14 recombinant fosmids that exhibited lipolytic activity. A selected recombinant plasmid, pFLP-2, which showed maximum lipolytic activity, was further analyzed. DNA sequence analysis of the subclone in pUC119, pELP-2, revealed an open reading frame of 1,191 bp encoding a 397-amino-acid protein. Purified EstD2 exhibited maximum enzymatic activity towards p-nitrophenyl butyrate, indicating that it is an esterase. Purified EstD2 showed optimal activity at 35 °C and at pH 8.0. The K m and K cat values were determined to be 79.4 μM and 120.5/s, respectively. The esterase exhibited an increase in enzymatic activity in the presence of 15% butanol and 15% methanol. Phylogenetic analysis revealed that the lipolytic protein EstD2 may be a member of a novel family of lipolytic enzymes. Several hypothetical protein homologs of EstD2 were found in the database. A hypothetical protein from Phenylobacterium zucineum HLK1, a close homolog of EstD2, displayed lipolytic activity when the corresponding gene was expressed in Escherichia coli. Our results suggest that the other hypothetical protein homologs of EstD2 might also be members of this novel family.

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Acknowledgments

This research was supported by a grant (MG 08-0104-1-0) from the Microbial Genomics and Application Center of the 21st Century Frontier Research Program funded by the Ministry of Education, Science and Technology of the Republic of Korea.

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Authors and Affiliations

  1. Department of Applied Biology, College of Natural Resources and Life Science, Dong-A University, Busan, 604-714, Republic of Korea

    Myung Hwan Lee, Shweta Malhotra, Ji-Hye Park, Eul Chul Hwang & Seon-Woo Lee

  2. Department of Medical Bioscience, Dong-A University, Busan, 604-714, Republic of Korea

    Myung Hwan Lee, Weixin Tao & Seon-Woo Lee

  3. Biomaterial Research Center, Korea Research Institute of Chemistry Technology, Daejeon, 305-600, Republic of Korea

    Kyung Sik Hong & Young Sup Kim

  4. Department of Genetic Engineering, Dong-A University, Busan, 604-714, Republic of Korea

    Hong Kyu Choi

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  1. Myung Hwan Lee
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  9. Seon-Woo Lee
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Correspondence to Seon-Woo Lee.

Additional information

Myung Hwan Lee and Kyung Sik Hong contributed equally to this study.

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Fig. S1

SDS-PAGE gel showing the expressed EstD2 protein produced in E. coli and purified EstD2. a Expression of EstD2 in E. coli BL21(DE3)/pLysS carrying pELP28. To induce his-tag fusion protein in E. coli, 1 mM of IPTG was added to the bacterial culture from time to time. Lanes: S molecular weight standards with sizes written on the left; 1 before IPTG addition; 2, 4, 6, 8 uninduced culture (1, 2, 3, and 4 h without IPTG addition); 3, 5, 7, 9 induced culture (1, 2, 3, and 4 h after IPTG addition). Arrow indicates the expressed protein. b Purified EstD2 protein by affinity purification. Lanes: S molecular weight standards with sizes noted on the left; 1 purified protein (arrow). (PPT 255 kb)

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Lee, M.H., Hong, K.S., Malhotra, S. et al. A new esterase EstD2 isolated from plant rhizosphere soil metagenome. Appl Microbiol Biotechnol 88, 1125–1134 (2010). https://doi.org/10.1007/s00253-010-2729-6

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