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A novel β-mannanase from Pantoea agglomerans A021: gene cloning, expression, purification and characterization

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Abstract

This paper describes the construction of a genomic library from the bacterium Pantoea agglomerans A021 and the subsequent cloning and expression of a novel mannanase gene (man26P). The gene consists of 1,047 bp and encodes a peptide (Man26P) of 348 amino acids with a calculated molecular mass of 38.5 kDa. Man26P is 63% identical with mannanase from Pectobacterium carotovorum at protein level and considered to be a member of the glycoside hydrolase family 26 (GH26). Man26P was expressed efficiently in E.coli BL21 (DE3) after induction with isopropylthiogalactoside (IPTG) and purified with a GST Bind Purification Kit. Maximum activity of purified Man26P was 514 U mg−1, which was seen at pH 6.0 and a temperature of 55°C. Man26P was stable on exposure to buffers ranging from pH 4.0–10.0, and tolerant of temperature below 60°C. Zn2+, Mg2+ and Co2+ enhanced the activity, while Mn2+, Cu2+ and Hg+ had a negative effect. β-mercaptoethanol (1%) increased the activity twofold, while SDS (1%) inhibited it significantly. The enzyme showed optimal activity in a NaCl solution. The properties make it a candidate for various industrial applications.

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Acknowledgments

We would like to thank Dr. Qifa Zhang for many valuable suggestions. This study was supported by grants from the National Natural Science Foundation of China (30770021 and 30570057) and the 111 Project (B07041).

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Authors and Affiliations

  1. State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, 430070, Wuhan, People’s Republic of China

    Jing Wang, Chanjuan Li, Xiaoyu Fu & Ziduo Liu

  2. College of Plant Science and Technology, Huazhong Agricultural University, 430070, Wuhan, People’s Republic of China

    Yuzhi Hong

  3. Key Laboratory of Marine Biogenetic Resources, The Third Institute of Oceanography, State of Oceanic Administration, 361005, Xiamen, People’s Republic of China

    Zongze Shao

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  1. Jing Wang
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  2. Zongze Shao
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  3. Yuzhi Hong
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  4. Chanjuan Li
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  5. Xiaoyu Fu
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  6. Ziduo Liu
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Correspondence to Ziduo Liu.

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Wang, J., Shao, Z., Hong, Y. et al. A novel β-mannanase from Pantoea agglomerans A021: gene cloning, expression, purification and characterization. World J Microbiol Biotechnol 26, 1777–1784 (2010). https://doi.org/10.1007/s11274-010-0358-y

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  • DOI: https://doi.org/10.1007/s11274-010-0358-y

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