A Superefficient Ochratoxin A Hydrolase with Promising Potential for Industrial Applications

Abstract

As the most seriously controlled mycotoxin produced by Aspergillus spp. and Penicillium spp., ochratoxin A (OTA) results in various toxicological effects and widely contaminates agro-products. Biological detoxification of OTA is the most priority in food and feed industry, but currently available detoxification enzymes are relatively low effectiveness in time and cost. Here we show a superefficient enzyme ADH3 identified from Stenotrophomonas acidaminiphila with a strong ability to transform OTA into non-toxic ochratoxin-α by acting as an amidohydrolase. Recombinant ADH3 (1.2 μg/mL) completely degrades 50 μg/L OTA within 90 seconds, while the availably most efficient OTA hydrolases takes several hours. The kinetic constant showed that rADH3 ( Kcat/Km ) catalytic efficiency was 56.7-35000 times higher than those of previous hydrolases rAfOTase, rOTase and commercial carboxypeptidase A (CPA). Protein structure-based assay suggested that ADH3 has a preference for hydrophobic residues to form a larger hydrophobic area than other detoxifying enzymes at the cavity of the catalytic sites, and this structure makes the OTA easier to access to catalytic sites. In addition, ADH3 shows considerable temperature adaptability to exert hydrolytic function at the temperature down to 0°C or up to 70°C. Collectively, we report a superefficient OTA detoxifying enzyme with promising potential for industrial applications. IMPORTANCE Ochratoxin A (OTA) can result in various toxicological effects and widely contaminates agro-products and feedstuffs. OTA detoxifications by microbial strains and bio-enzymes are significant to food safety. Although previous studies showed OTA could be transformed through several pathways, the ochratoxin-α pathway is recognized as the most effective one. However, the most currently available enzymes are not efficient enough. Here, a superefficient hydrolase ADH3 which can completely transform 50 μg/L OTA into ochratoxin-α within 90 seconds was screened and characterized. The hydrolase ADH3 shows considerable temperature adaptability (0-70°C) to exert the hydrolytic function. Findings of this study supplied an efficient OTA detoxifying enzyme and predicted the superefficient degradation mechanism which lay a foundation for future industrial applications.

Isoenzyme N -Acyl- l -Amino Acid Amidohydrolase NA Increases Ochratoxin A Degradation Efficacy of Stenotrophomonas sp. CW117 by Enhancing Amidohydrolase ADH3 Stability

“…Open reading frames (ORFs) of two genes that encode enzymes NA and ADH3 were amplified from the CW117 genome, ligated to vector pGEX-4T-1, and transferred into E. coli BL21 for protein expression. Gene adh3 has been fully characterized in Luo et al ( 11 ), the repeated ADH3 results were not shown (or only as a parallel comparison) in this study. As shown in Fig.…”

“…As described in a previous publication, 53 hydrolase genes, which may potentially be used for OTA detoxification, were selected for degradation testing from the CW117 genome (GenBank accession no. CP062156.1 ) ( 11 ). Two genes, adh3 (protein, ADH3) and na (protein, NA), were screened and characterized for OTA degradation activity by heterologous protein expression in Escherichia coli BL21 and enzymatic degradation test in vitro .…”

“…Degradation dynamics and product identification. OTA degradation product from rNA was purified using an OchraTest column (Vicam, Milford, MA, USA) and identified by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) using the same procedures as Luo et al ( 11 ). The OTA degradation product from rNA produced [M + H + ] at m/z 257 as a parent ion from the full scan MS spectrum, and the parent ion produced [M + H + ] at m/z 167 and 211 as daughter ions in MS/MS spectrum, which was consistent with the rADH3-degraded product OTα discussed previously ( Fig.…”

“…Development of detoxification methods on mycotoxin-contaminated foods and feedstuffs receives great interest in the food and agriculture fields ( 6 –7 ). Detoxification studies have shown that OTA is mainly transferred by three possible pathways (i.e., lactone ring opening, producing OP-OTA; isocoumarin ring dechlorinating, producing OTB; or peptidic bond cleaving, producing OTα and l -β-phenylalanine) ( 8 – 11 ). Toxicological evaluation showed that OTα is a much less toxic metabolite compared to the parent chemical OTA, followed by OTB and OTA lactone-opened product (OP-OTA) ( 10 –11 ).…”

“…CW117 and ochratoxinase (i.e., OTase) from Aspergillus niger were the most efficient and investigated hydrolases, and OTase was the first crystal structure characterized detoxifying enzyme that was highly significant to understanding the detoxifying mechanism ( 11 , 15 ). Moreover, the activities of amidohydrolase ADH3, OTase, and the N -acyl- l -amino acid amidohydrolase (i.e., AfOTase) from Alcaligenes faecalis , were much more efficient than bovine pancreatic carboxypeptidase (CPA) (the first OTA-detoxifying enzyme) and other characterized hydrolases ( 11 , 16 ). Other recently isolated carboxypeptidases, such as CP from Bacillus amyloliquefaciens , PJ_1540 from Acinetobacter sp., and CP4 from Lysobacter sp.…”