Detoxification of aflatoxin B1 by a Bacillus subtilis spore coat protein through formation of the main metabolites AFQ1 and epi-AFQ1

Abstract

A variety of important agricultural crops host fungi from the Aspergillus genus can produce cancerogenic secondary metabolites such as aflatoxins. Consequently, novel strategies for detoxification and their removal from food and feed chains are required. Here, detoxification of Aflatoxin B1 (AFB1) by the Bacillus subtilis multi-copper oxidase CotA (BsCotA) was investigated. This laccase was recombinantly produced in E. coli while codon optimization led to duplication of the amount of active protein obtained. CuCl₂ was added to the cultivation medium leading to a 25-fold increase of V _max corresponding to improved incorporation of Cu²⁺ into the enzyme protein which is essential for the catalytic reaction. To avoid potential cytotoxicity of Cu²⁺, cultivation was performed at microaerobic conditions indeed leading to 100x more functional protein when compared to standard aerobic conditions. This was indicated by an increase of V _max from 0.30 ± 0.02 to 33.56 ± 2.02 U/mg. Degradation kinetics of AFB1 using HPLC with fluorescence detection (HPLC-FLD) analysis indicated a theoretical substrate saturation above solubility in water. At a relatively high concentration of 500 μg/L, AFB1 was decomposed at 10.75 μg/Lh (0.17 nmol^*min^-1*mg^-1) at a dosage of 0.2 μM BsCotA. AFQ1 and epi-AFQ1 were identified as the initial oxidation products according to mass spectrometry (i.e., HPLC-MS, HPLC-QTOF). None of these molecules were substrates for laccase but both decomposed in buffer. However, decomposition does not seem to be due to hydration of the vinyl ether in the terminal furan ring. Genotoxicity of the formed AFB1 was assessed in several dilutions based on the de-repression of the bacterial SOS response to DNA damage indicating about 80-times reduction in toxicity when compared to AFQ1. The results of this study indicate that BsCotA has high potential for the biological detoxification of aflatoxin B1.

Keywords: copper incorporation; enzymatic degradation; genotoxicity; laccase; mycotoxins.

Figure 1

Determination of pH optima for different variants of the BsCotA laccase with ABTS at 37°C. Experiments were performed in duplicates using 0.01 μM BsCotA_Strep. BsCotA: laccase CotA from Bacillus subtilis.

Figure 2

Degradation of AFB1 by the BsCotA_Strep laccase monitored using HPLC/MS. 5 ppm of (A) AFB1(circle symbol), (B) AFQ1 (square), (C) epi-AFQ1 (triangle) were each incubated with enzyme (full line) or without (dashed). Increase of instability products of AFQ1 (inverted triangle) and epi-AFQ1 (diamond) are shown as increases in peak areas (second y-axis).

Figure 3

AFB1 degradation products after enzymatic treatment. Total ion chromatogram (TIC) of the Q1 scan in positive ionization mode after 48 (blue) hours of incubation with BsCotA_Strep. The blue curve displays the sample of AFB1 after enzymatic treatment and the red curve the sample of the laccase in buffer without AFB1 after 72 of incubation. The peaks 14.35, 14.90, and 17.50 have their origin to epi-AFQ1, AFQ1, and AFB1. The peaks at 10.23 min and 10.01 min have their origin to the instability of AFQ1 in the buffer used for incubation.

Figure 4

Assessment of the genotoxic potential of AFB1 and AFQ1 by the SOS Chromotest based on the measurement of the primary response of E.coli cells to genetic damage. Estimated SOSIF values for AFB1 against AfQ1 resulting after decomposition by laccase. SOSIF, SOS induction factor.