Today's article was published in Food Chemistry, "Purification and characterization of the enzymes from Brevundimonas naejangsanensis that degrade ochratoxin A and B," by Associate Professor Liang Zhihong of China Agricultural University.
Mycotoxins are secondary metabolites produced primarily by certain species of Aspergillus, Penicillium and Fusarium species. They widely contaminate food and feed, are highly toxic to humans and animals, and cause huge economic losses to global agriculture. Ochratoxin (OTs) is one of the important mycotoxin classes. More than 20 OTs have been identified, of which ochratoxin A (OTA) is the main ochratoxin detected in agricultural products. Multiple OTs are often present in food, and OTAs and ochratoxin B (OTB) have been found to be co-contaminated in wheat, red wine, and dried fruits. OTAs are nephrotoxic, hepatotoxic, immunotoxic and carcinogenic to animals and are classified as Class 2B carcinogens by the International Agency for Research on Cancer (IARC). OTBs have fewer toxicological studies than OTAs. OTBs are less toxic than OTAs. Cell line studies have found that OTB is potentially immunotoxic and teratogenic.
Detoxification strategies for mycotoxins typically include physical, chemical, and biological methods. Biological detoxification, in short, is the use of microorganisms or enzymes to degrade toxins, with high efficiency, strong specificity, and no destruction of nutrients. There are many microorganisms that degrade OTAs. Although abundant microbial resources for degrading OTA have been discovered, most of the degrading enzyme genes have not yet been mined. Only a few carboxypeptidases and amides degrade OTA in microorganisms. The discovery of these degradation genes has great application prospects for the biological detoxification of OTAs. There are two main types of enzymes that degrade OTA, namely carboxypeptidase and amide enzymes. Carboxypeptidase A (CPA) derived from cattle was the first enzyme to be found to hydrolyze OTA. It was then also found that carboxypeptidase B (CPB) and carboxypeptidase Y (CPY) could degrade OTA. Another important group of OTA-degrading enzymes is amide enzymes, first identified in A. niger. Dobritzsch et al. purified an OTA-degrading amide enzyme from A. niger's commercial lipase A with a degradation rate of 50% (50 ng/mL) within 60 minutes. Amidases that degrade OTAs have also been found in Alc. faecalis and Stenotrophomonas acidaminiphila. The mechanism of degradation of OTA by these enzymes is the hydrolysis of the amide bonds of OTA to produce OTα and phenylalanine.
The authors' team previously obtained the Brevundimonas naejangsanensis strain ML17 with OTA and OTB degradation capabilities. The purpose of this study is to extract OTA and OTB detoxification enzymes from this strain. OTA-degrading enzymes were continuously purified by (NH4)2SO4 salting, hydrophobic interaction chromatography (HIC), and high-resolution transparent natural polyacrylamide gel electrophoresis (hrCNE). The enzymes in the active ingredient were identified by LC-MS/MS. Genes for potential degradation enzymes were then recombinantly expressed in E. coli BL21 (DE3) strains to verify OTA and OTB degradation functions. Finally, the cytotoxicity of OTA and OTB degradation products was evaluated in the human embryonic kidney cell line (HEK293). In this study, four novel OTA-degrading enzymes were found to degrade OTA and OTB into low-toxicity products, providing basic research content for the biological detoxification of ochratoxin.
Purified with (NH4)2SO4 salting out
Salting out proteins with (NH4)2SO4 greatly reduces solution volume while preserving the biological activity of the protein, and is therefore often used as a first step in protein purification. First, OTA-degrading enzymes in the extracellular metabolites of ML17 strains were extracted by salting out method. Proteins with (NH4)2SO4 salted out at saturations of 20%, 40%, 60%, and 80% have OTA degradation activity (Figure 1). The main degrading enzymes can be salted out with 80% saturation (NH4)2SO4. To obtain more active ingredients, subsequent purification uses (NH4)2SO4 with 80% saturation to concentrate the OTA-degrading enzyme. This step reduced the volume of crude enzyme by approximately 16-fold, and the degrading enzyme was purified 5.1-fold with a yield of 18.9%.
HIC purification
HIC separates different proteins based on hydrophobic coefficient under specific conditions. Most proteins are adsorbed by hydrophobic ligands of HIC at high ionic concentrations and eluted in low-ionic solutions. The second step of purification is the purification of OTA-degrading enzymes using HIC. As shown in Figures 2A and B, OTA degrading enzymes are concentrated at the elution peak of 100 mmol/L (NH4)2SO4. Proteins with different eluting components vary widely, indicating that they are well separated (Figure 2C). Components of 3-7 have the strongest OTA degradation activity and share the same band around 50 and 70 kDa, which may be OTA degrading enzymes (Figures 2B and C). The HIC purification step reduced the volume of the crude enzyme solution by approximately 3.4-fold with a purification factor of 7.2 and a yield of 9.2%.
Two hrCNE purifications
hrCNE separates proteins based on their charge and molecular weight, while preserving their biological activity. The next purification step is to use two hrCNEs to purify the OTA-degrading enzyme. At the first hrCNE, the gel labeled lane and one of the sample lanes is cut off and stained for observation (Figure 3A). The proteins are well separated in the gel of hrCNE. The gels of the sample are then cut into 6 equal parts from top to bottom and the proteins in the gel are recovered by electrophoresis. As shown in Figures 3B and C, the proteins recovered from the first three gels have OTA degrading activity, and these components have a common band around the 440 kDa label, which may be the location of the OTA-degrading enzyme. Proteins recovered from band 2 of the first hrCNE are subjected to a second hrCNE. Similar to the first hrCNE, divide the gel of the second hrCNE into 5 equal parts and then recover the protein. Proteins recovered from bands 1 and 2 of the second hrCNE have better OTA degradation activity (Figure 3F). The electrophoresis of hrCNE (Figure 3D) and SDS-PAGE (Figure 3E) showed that after the second hrCNE, the degrading enzyme component was relatively pure.
Proteins recovered from bands 1 and 2 of the 2nd hrCNE were identified by mass spectrometry. There are four potential OTA-degrading enzymes in the recovered proteins, which belong to the M3 family of peptidases, serine carboxypeptidases, M14 metallopeptidases, and amide hydrolase family proteins. The four enzymes were named BnOTAase1, BnOTAase2, BnOTAase3 and BnOTAase4, with molecular weights of 79.7, 58.4, 67.7 and 45.8 kDa, respectively. The amino acid sequences of these four enzymes were BLAST compared to reported OTA-degrading enzymes, including carboxypeptidases from bovine, Bacillus amylis, Bacillus subtilis, and lysosis, and amides from Aspergillus niger, Lysobacterium faecalis, and Narrowimomonas species. The amino acid sequences of the four enzymes were less than 25% identical to the found OTA-degrading enzymes, indicating that these enzymes are novel potential OTA-degrading enzymes.
Recombinant expression of degrading enzymes
Genes for four potential OTA-degrading enzymes were expressed in E. coli BL21 (DE3) strains to validate their OTA and OTB degradation activities. As shown in Figure 4, SDS-PAGE (Figure 4A) and Westernblot (Figure 4B) detected successful expression of the gene of interest. BnOTAase1, BnOTAase2 and BnOTAase3 are mainly solubilically expressed in cells. BnOTAase4 is expressed in both intracellular solubility and inclusion bodies. These enzymes are purified by nickel affinity chromatography to obtain products with approximate theoretical molecular weights (Figure 4C).
OTA and OTB degradation activities of recombinant enzymes
The degradation rates of recombinant enzymes BnOTAase1, BnOTAase2, BnOTAase3 and BnOTAase4 to OTA and OTB were determined by HPLC. The retention times (RT) for the OTA, OTα, OTB, and OTβ standards were 4.270, 3.427, 3.443, and 2.899 minutes, respectively (Figures 5A and B). In a reaction system between recombinase and OTA or OTB, the respective products are detected with chromatograms similar or identical to the standards. These four recombinase degrade OTA and OTB to OTα and OTβ, respectively, with a degradation rate of 100%. The RT of OTA and OTB degradation products hydrolyzed by recombinase is the same as that of CPA, indicating that recombinase and CPA have a similar degradation mechanism as OTA and OTB, i.e., hydrolyzing the amide bonds of OTA and OTB (Figure 5B).
The Mie's constant Km for recombinase used to hydrolyze OTA or OTB is determined by double-reciprocal mapping (Lineweaver-Burk mapping). Using OTA as the substrate, the Km values of BnOTAase1, BnOTAase2, BnOTAase3 and BnOTAase4 were 19.38, 0.92, 12.11 and 1.09 μmol/L, respectively. With OTB as the substrate, the Km values of these four recombinases were 0.76, 2.43, 0.60 and 0.64 μmol/L, respectively.
Cytotoxicity of OTA and OTB degradation products
To determine whether the toxicity of the degradation products (OTα and OTβ) of OTA and OTB is reduced, the effects of OTA, OTB and their degradation products on the HEK293 cell line induced cell proliferation toxicity, apoptosis rate, and cell cycle arrest were investigated. Exposure to OTA and/or OTB (0–30 μmol/L) results in a concentration-dependent decrease in cell viability, and their degradation products are not proliferatively toxic to HEK293 cell lines within the experimental concentration range (Figure 6A). The apoptosis rates in the media control (CK), OTA, OTB, OTA, OTA, and OTB groups were 4.21±0.06%, 20.66±0.50%, 17.44±0.21%, and 20.14±0.06%, respectively (Figure 6B). The degradation product treatment group had no significant effect on apoptosis rate (p greater than 0.05). The combined treatment of OTA and OTB significantly alters the cell cycle. The proportion of cells in the G0/G1 phase decreased to 77.08% of the control group, and the proportion of cells in the S phase and G2/M phase increased by 1.15-fold and 3.48-fold, respectively, indicating that the cell cycle was stagnant in the S phase and the G2/M phase (Figure 6C). The degradation product treatment group did not significantly alter the cell cycle progression of HEK293. The above results show that the degradation products of OTA and OTB are less toxic to the HEK293 cell line.
DOI:10.1016/j.foodchem.2023.135926
Article link: https://doi.org/10.1016/j.foodchem.2023.135926