Identification of a novel and thermostable laccase from Bacillus velezensis GLY4 and its application in the biotransformation of aflatoxins

Abstract

Recently, enzyme-based transformation of aflatoxins (AFs) has drawn great attention. The microbial laccases are most intensively studied. This study identified a thermostable laccase (designated LacGLY4) from Bacillus velezensis GLY4. The recombinant enzyme could be smoothly expressed in Escherichia coli and purified by Ni²⁺-affinity chromatography. The LacGLY4 displayed the optimal pH and temperature at pH 8.0 and 85 °C, and maintained a relatively high activity at 75–95 °C against AFB₁. At the optimized condition, over 95 % of AFB₁ (10 μg/mL) would be transformed by 20 μg/mL of LacGLY4 after 30 min. The half-life (t_1/2) of LacGLY4 at 40, 50 and 60 °C was calculated to be 77, 63 and 53 min, respectively. LacGLY4 could extensively transform a variety of AFB₁ analogues that showed the specific activity of 0.132, 0.077, 0.071, and 0.107 U/mg for AFB₂, AFM₁, AFG₁ and AFG₂, respectively. In addition, the LacGLY4 exhibited about 57 % relative activity towards ZEN, but only showed less than 10 % of activity against DON and PAT compared to that of AFB₁. A significant transformation product from AFB₁ by LacGLY4 was identified as C₂₄H₃₀O₆ by liquid chromatography–tandem mass spectrometry. The generated product is speculated to be formed by a complex addition reduction rather than a simple hydrolase reaction from AFB₁. This bacterial laccase combines good thermostability and efficient transformation, providing a novel enzymatic tool for the biocontrol of AFB₁ contamination such as in the oil refinement degumming step in which the temperature is around 80 °C and the processing time is about 30 min.