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Molecular Cloning and Heterologous Expression of Laccase from Aeromonas hydrophila NIU01 in Escherichia coli with Parameters Optimization in Production

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Abstract

Prior studies disclosed that Aeromonas hydrophila NIU01 was a biodecolorization and bioelectricity bacterium which was isolated from a cross-strait of Taiwan. However, enzymatic function, laccase, involved in this strain had never been reported. This first attempt is to explore its laccase activity, the molecular cloning and heterologous recombinant expression in Escherichia coli. A full-length novel gene of 1,647 bp, LacA, encoding of 549 amino acids was successfully cloned by polymerase chain reaction. The recombinant pET-15b(+)-NIU-LacA expression was compared in different E. coli strains. By applying Taguchi’s L9 in culture optimization, the soluble laccase increased to 22.7 %, in which the conditions were obtained at 22 °C with initial shaking speed at 200 rpm, addition of lactose of 0.2 mM and CuSO4 of 0.5 mM to the medium, and shaking off while cell mass reached to OD600nm of 1.5. NIU-LacA was strongly inhibited by chloride ion. The optimal temperature was 60 °C and the optimum pH for ABTS (2,2′-azino-bis (3-ethylbenzthiazolinesulfonic acid) and 2,6-DMP (2,6-dimethoxyphenol) were pH 2.1 and pH 7.5 which enzymatic activity was 274.6 and 44.8 U/L, respectively. Further study in structural modeling of NIU-LacA showed the C terminal domain was the major variance in the three most closely A. hydrophila strains.

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Acknowledgments

The authors are grateful to the financial support by the Fundamental Research Funds for the Central Universities (2011121017), the Chinese National Natural Science Foundation (21206141), and the Fujian Provincial Department of Science and Technology (2012I0009). The authors also sincerely appreciate Professor Bor-Yann Chen who supports the bacterium and initiates the academic connection program between Xiamen University (China) and National I-Lan University (Taiwan).

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Authors and Affiliations

  1. Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China

    I-Son Ng, Xia Zhang, Yu Zhang & Yinghua Lu

  2. The Key Laboratory for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, 361005, China

    I-Son Ng & Yinghua Lu

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  1. I-Son Ng
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  2. Xia Zhang
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  3. Yu Zhang
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Correspondence to I-Son Ng.

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Ng, IS., Zhang, X., Zhang, Y. et al. Molecular Cloning and Heterologous Expression of Laccase from Aeromonas hydrophila NIU01 in Escherichia coli with Parameters Optimization in Production. Appl Biochem Biotechnol 169, 2223–2235 (2013). https://doi.org/10.1007/s12010-013-0128-z

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  • DOI: https://doi.org/10.1007/s12010-013-0128-z

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