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J Agric Food Chem. 2013 Jun 12;61(23):5468-73. doi: 10.1021/jf4010498. Epub 2013 Jun 3.

Purification and characterization of a temperature- and pH-stable laccase from the spores of Bacillus vallismortis fmb-103 and its application in the degradation of malachite green.

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Laboratory of Enzyme Engineering, College of Food Science and Technology, Nanjing Agriculture University, Nanjing 210095, PR China.


Malachite green residue can affect aquaculture food safety. Bioremediation of contaminated water by enzyme treatment is an environmentally friendly and economical way to remove contaminating substances. In the present study, a temperature- and pH-stable laccase was purified from the spores of Bacillus ballismortis fmb-103 and was used to degrade malachite green. The laccase from fmb-103 (fmb-L103) was purified 15.2-fold to homogeneity (389.9 mU/mg protein with respect to ABTS as a substrate) by precipitation with 30-80% (NH4)2SO4, DEAE-Sephadex A-50 ion exchange chromatography, and Sephadex G-100 chromatography. fmb-L103 is a nonblue laccase with a molecular weight of 55.0 kDa and Cu content of 2.5 (mol:mol). fmb-L103 retained more than 50% activity after 10 h at 70 °C and demonstrated broad pH stability in both acidic and alkaline conditions. The effects of inhibitors and metal ions on fmb-L103 activity were also examined. A kinetic study revealed that ABTS was a suitable substrate with a Km of 22.7 μmol and a Vmax of 3.32 μmol/mL/min. fmb-L103 can efficiently degrade malachite green after a 48 h treatment period in combination with a mediator, without the appearance of leucomalachite green.

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