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Isolation, Purification and Characterization of Two Laccases from Carrot (Daucus carota L.) and Their Response to Abiotic and Metal Ions Stresses

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Abstract

Laccases, which belong to the blue copper oxidase enzyme family, oxidize many organic and inorganic compounds. The laccase-encoding genes DcLac1 and DcLac2 were isolated from the economically important tuberous root carrot, and their proteins were successfully expressed and purified using the Escherichia coli expression system BL21(DE3). DcLac1 and DcLac2 had molecular masses of approximately 64 and 61.9 kDa, respectively. With 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate acid) as the substrate, DcLac1 and DcLac2 had K m values of 3.9043 and 1.255 mM, respectively, and V max values of 54.0832 and 81.7996 μM mg−1 min−1, respectively. Moreover, DcLac1 and DcLac2 had optimal pH values of 2.8 and 2.6, respectively, and optimal temperatures of 45 and 40 °C, respectively. The activities of the two enzymes were promoted by Ca2+, Mg2+, Cu2+, and Na+ but inhibited by Fe2+, Zn2+, Mn2+, K+, SDS, and EDTA. Expression profiles showed that the two DcLac genes had almost identical responses to high and low temperature stresses but different responses to salt, drought, and metal stresses. This study provided insights into the characteristics and tolerance response mechanisms of laccase in carrot.

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Abbreviations

ABTS:

2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulfonate acid)

SDS:

Sodium dodecyl sulfate, sodium salt

EDTA:

Ethylene diamine tetraacetic acid

qRT-PCR:

Quantitative real-time PCR

SDS-PAGE:

Sodium dodecyl sulfate, sodium salt-polyacrylamide gel electrophoresis

McIlvaine buffer:

Citric acid–Na2HPO4 buffer

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Acknowledgments

The research was supported by New Century Excellent Talents in University (NCET-11-0670), Jiangsu Natural Science Foundation (BK20130027), Priority Academic Program Development of Jiangsu Higher Education Institutions.

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  1. State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, 1 Weigang, Nanjing, 210095, China

    Jing Ma, Zhi-Sheng Xu, Feng Wang & Ai-Sheng Xiong

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Ma, J., Xu, ZS., Wang, F. et al. Isolation, Purification and Characterization of Two Laccases from Carrot (Daucus carota L.) and Their Response to Abiotic and Metal Ions Stresses. Protein J 34, 444–452 (2015). https://doi.org/10.1007/s10930-015-9639-5

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