Today's article is "Purification and characterization of the enzymes from Brevundimonas naejangsanensis that degrade ochratoxin A and B" published in Food Chemistry, and the corresponding author is Associate Professor Liang Zhihong from the College of Food Science and Nutritional Engineering, China Agricultural University.
1
(NH4)2SO4 salting out purification
Salting out proteins with (NH4)2SO4 greatly reduces solution volume while preserving the biological activity of proteins, and is therefore often used as the first step in protein purification. First, OTA-degrading enzymes in the extracellular metabolites of ML17 strains were extracted by salting out. (NH4)2SO4 proteins saturated with 20%, 40%, 60%, and 80% salts have OTA degradation activity (Figure 1). The main degrading enzyme can be precipitated by 80% saturated (NH4)2SO4 salt. To obtain more active ingredients, subsequent purification uses (NH4)2SO4 with 80% saturation to concentrate the OTA-degrading enzyme. This step reduces the crude enzyme volume by about 16-fold, purifies the degrading enzyme by 5.1-fold, and yields 18.9%.
Figure 1
2
HIC purification
HIC separates different proteins under specific conditions based on their hydrophobic coefficient. Most proteins are adsorbed by hydrophobic ligands of HIC at high ionic concentrations and eluted in low-ionic solutions. The second step in purification is to purify the OTA-degrading enzyme using HIC. As shown in Figures 2A and B, the OTA degrading enzyme is concentrated on the elution peak of 100 mmol/L (NH4)2SO4. Proteins with different eluting components vary greatly, indicating that they are well separated (Figure 2C). Fractions 3-7 have the strongest OTA-degrading activity and have the same bands around 50 and 70 kDa, which may be OTA-degrading enzymes (Figures 2B and C). The HIC purification step resulted in a approximately 3.4-fold reduction in the size of the crude enzyme solution with a purification factor of 7.2 and a yield of 9.2%.
Figure 2
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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. In the 1st hrCNE, the gel labeled lane and one of the sample lanes is cut off and stained for observation (Figure 3A). Proteins are well separated in hrCNE gels. The sample gel is then cut into 6 aliquots from top to bottom and the proteins in the gel are recovered by electrophoresis elute. As shown in Figures 3B and C, proteins recovered from the first three gels have OTA degrading activity, and these components have a common band around the 440 kDa mark, which may be the location of the OTA-degrading enzyme. Proteins recovered from band 2 of the first hrCNE proceed to the second hrCNE. Similar to the 1st hrCNE, the gel of the 2nd hrCNE is divided into 5 aliquots and the proteins are then recovered. Proteins recovered from bands 1 and 2 of the second hrCNE have better OTA degradation activity (Figure 3F). From the electrophoresis maps of hrCNE (Figure 3D) and SDS-PAGE (Figure 3E), it can be found that after the second hrCNE, the degrading enzyme components are relatively pure.
Identify proteins recovered from bands 1 and 2 of the second hrCNE by mass spectrometry. Enzymes with OTA degrading activity have been reported to include carboxypeptidase and amide hydrolase. Among the recovered proteins are four potential OTA-degrading enzymes, which belong to the M3 family peptidases (gene 0095 code), serine carboxypeptidase (gene 1826 code), M14 family metallopeptidases (gene 2253 code), and amide hydrolase family proteins (gene 0484 code). 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 bovines, Bacillus amylis, Bacillus subtilis, and lysophilus, and amides from Aspergillus niger, Lysobacterium faecaliensis, and Oligomonas. The amino acid sequences of all four enzymes are less than 25% identical to the discovered OTA-degrading enzymes, indicating that these enzymes are novel potential OTA-degrading enzymes.
Figure 3
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Recombinant expression of degrading enzymes
Genes for four potential OTA-degrading enzymes were expressed in the E. coli BL21 (DE3) strain to validate their OTA and OTB degrading activities. As shown in Figure 4, the gene of interest was successfully expressed by SDS-PAGE (Figure 4A) and Western blot (Figure 4B). BnOTAase1, BnOTAase2 and BnOTAase3 are mainly expressed in cells as solubility. BnOTAase4 is expressed in intracellular solvable and inclusion bodies. These enzymes are purified by nickel affinity chromatography to obtain products with approximate theoretical molecular weights (Figure 4C).
Figure 4
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OTA and OTB degradation activity of recombinases
The degradation rates of recombinase to OTA and OTB BnOTAase1, BnOTAase2, BnOTAase3 and BnOTAase4 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 (Figure 5A and B). The respective products were detected in the reaction system of the recombinase with OTA or OTB with a chromatogram similar or identical to the standard. These four recombinase degrade OTA and OTB to OTα and OTβ, respectively, with a degradation rate of 100%. The RT of the recombinase hydrolysis of OTA and OTB degradation products is the same as that of CPA, indicating that recombinase and CPA have similar degradation mechanisms to OTA and OTB. That is, the amide bonds of OTA and OTB are hydrolyzed (Figure 5B).
The Michael's constant Km of recombinase that hydrolyzes 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. Using OTB as substrate, the Km values of these four recombinases were 0.76, 2.43, 0.60, and 0.64 μmol/L, respectively.
Figure 5
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Cytotoxicity of OTA and OTB degradation products
To determine whether the toxicity of the degradation products (OTα and OTβ) of OTA and OTB is decreasing, this study examined the cytoproliferative toxicity, apoptosis rate and cell cycle arrest effects of OTA, OTB and their degradation products on HEK293. Exposure of cells to OTA and/or OTB (0-30 μmol/L) results in a concentration-dependent decrease in cell viability, and their degradation products have no proliferative toxicity to the HEK293 cell line within the experimental concentration (Figure 6A). The apoptosis rates of the media control (CK), OTA, OTB, and OTA and OTB combined treatment groups were 4.21±0.06%, 20.66±0.50%, 17.44±0.21%, and 20.14±0.06%, respectively (Figure 6B). The treated group of degradation products had no significant effect on the apoptosis rate. The combination 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 treated group of degradation products 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.
Figure 6
Enzymes that degrade OTAs, including carboxypeptidase, amide enzymes, peroxidases, protease A, etc., have been found in animals, plants, bacteria, and fungi, indicating that enzymes that degrade OTAs are widely present in organisms. Although many bacteria have been found to have OTA degrading activity, only a few bacteria-derived degrading enzymes have been identified. This limits the utilization of microbial resources. A bacterial strain ML17 that can hydrolyze OTA and OTB was previously discovered. In this study, potential OTA and OTB degrading enzymes were purified from extracellular metabolites of ML17 strains, and the degradation activity of these enzymes was verified by recombinant expression in E. coli BL21(DE3) strains, and the cytotoxicity of degradation products of ML17 strains was evaluated. Multiple purification methods based on protein properties are combined with effective strategies for discovering novel mycotoxin detoxification enzymes, such as the discovery of aflatoxin B1 detoxification enzyme FAO and the discovery of zearalenone detoxification enzyme ZHD101. Salinization is a commonly used method in the coarse separation stage. The OTA-degrading components are dispersed in 20-80% (NH4)2SO4 saturation (Figure 1), indicating that the OTA-degrading enzyme may be multicomponent. In the medium purification stage, hydrophobic or ion exchange chromatography is often used. However, ion exchange chromatography requires the sample to maintain a low initial ion concentration (typically less than 20 mmol/L), so the sample needs to be dialyzed or otherwise desalted. If the protein is purified by HIC at this stage, no desalting is required. The purification factor of OTA-degrading components in this process was 7.2-fold, indicating that HIC is suitable for purification of OTA-degrading enzymes. During the fine purification phase, the authors purified OTA-degrading enzymes by hrCNE and identified four potential OTA-degrading enzymes in the recovered active ingredient.
By recombinant expression in the E. coli BL21 (DE3) strain, the authors verified that the four enzymes had OTA and OTB degrading activity. These enzymes have molecular weights of 79.7, 58.4, 67.7, and 45.8 kDa, respectively, and are named BnOTase1, BnOTase2, BnOTase3, and BnOTase4, respectively. BnOTase1, BnOTase2, and BnOTase3 belong to peptidases, and BnOTase4 belongs to amides. These four enzymes have less than 25% agreement with the amino acid sequences of the discovered OTA-degrading enzymes, indicating that these enzymes are novel OTA-degrading enzymes. The OTA degradation rate of 100% for the four degrading enzymes was higher than the reported bacterial carboxypeptidases DacA, DacB, and DacC. It has been reported that an amide hydrolase, ADH3, can completely degrade 50 ng/mL OTA in 90 seconds. This is the most active enzyme for OTA degradation that has been reported so far. Although the OTA-degrading enzyme activity reported in this paper is lower than that of ADH3, it provides materials for further exploration of the OTA degradation mechanism.
Relatively few enzymes have been reported to be able to degrade OTB. CPAs have been reported to degrade the performance of OTAs and OTBs. Both OTA and OTB can be completely degraded by BnOTase1, BnOTase2, BnOTase3, BnOTase4, and the degradation mechanism is the same as CPA, that is, the hydrolysis of amide bonds in molecules. This suggests that due to the structural similarity between OTA and OTB, enzymes that degrade OTA may also degrade OTB.
Most ochratoxins such as OTA, OTB, OTC, 4-OH OTA, 4-OH OTB, and 10-OH OTA contain amide bonds in molecules, which are likely to be found in amide bond hydrolysis. OTA degrading enzymes may help mitigate co-contamination of multiple ochratoxins in food and feed. OTA causes oxidative stress, inhibits cell proliferation, induces apoptosis, and blocks the normal processes of the cell or animal cell cycle. The main mechanism of toxicity lies in the phenylalanine residue in the molecule.
Therefore, the hydrolysis of amide bonds of OTA to generate OTα and phe is an important detoxification pathway. Early toxicological studies have shown that OTα is less toxic than OTA. For example, BALB/c mice did not exhibit immunotoxicity after intraperitoneal injection of a 1 μg/kg dose. The elimination half-life of OTα, which is 9.6 hours in rats, is about one-tenth of OTA (103 hours). There are no toxicological data for OTβ. The amide bonds of OTA or OTB can be hydrolyzed to OTα or OTβ by ML17 strains and their metabolites. OTA and OTB have significant cytotoxicity against HEK293 cells, i.e., inhibit cell proliferation and induce apoptosis.
Apoptosis and blocks the cell cycle (Figure 6). Their degradation products OTα and OTβ did not exhibit significant cytotoxicity within the test concentrations. This suggests that the four degradation enzymes identified in this study can mitigate the cytotoxicity of OTA and OTB. However, this study only preliminarily evaluated the cytotoxicity of OTα and OTβ, and more extensive safety evaluation at the animal level is still needed.
In summary, the authors identified four novel ochratoxin-degrading enzymes with detoxification mechanisms similar to CPAs, i.e., they can hydrolyze OTA and OTB into low-toxicity products OTα and OTβ, respectively. The amino acid sequences of these enzymes are less than 25% consistent with the reported OTA detoxification enzymes, indicating novelty. Although the detoxification activity is not as good as that of ADH3, its degradation rate for both OTA and OTB reaches 100%. Since OTA-degrading enzymes also have the function of degrading OTBs, based on the structural similarity of ochratoxin, it is possible to combine multiple ochratoxin detoxification enzymes for the removal of multiple ochratoxin from food and feed.
Article Information:
DOI:https://doi.org/10.1016/j.foodchem.2023.135926