A superefficient ochratoxin A hydrolase with promising potential for industrial applications

2021 ◽  
Author(s):  
Han Luo ◽  
Gan wang ◽  
Nan Chen ◽  
Zemin Fang ◽  
Yazhong Xiao ◽  
...  
Keyword(s):  
Ochratoxin A ◽  
Degradation Mechanism ◽  
Kinetic Constant ◽  
Industrial Applications ◽  
Detoxification Enzymes ◽  
Toxicological Effects ◽  
Catalytic Sites ◽  
Ochratoxin Α ◽  
Detoxifying Enzyme ◽  
Temperature Adaptability

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.

scholarly journals Truncated Expression of a Carboxypeptidase A from Bovine Improves Its Enzymatic Properties and Detoxification Efficiency of Ochratoxin A

Toxins ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 680
Author(s):  
Lu Xiong ◽  
Mengxue Peng ◽  
Meng Zhao ◽  
Zhihong Liang
Keyword(s):  
Ochratoxin A ◽  
Industrial Applications ◽  
Optimum Temperature ◽  
Carboxypeptidase A ◽  
Maximum Reaction Rate ◽  
Maximum Reaction ◽  
Toxic Product ◽  
Wide Range ◽  
Ochratoxin Α ◽  
Penicillium Spp

Ochratoxin A (OTA) is a toxic secondary metabolite produced mainly by Penicillium spp. and Aspergillus spp. and commonly found in foodstuffs and feedstuffs. Carboxypeptidase A (CPA) can hydrolyze OTA into the non-toxic product ochratoxin α, with great potential to realize industrialized production and detoxify OTA in contaminated foods and feeds. This study constructed a P. pastoris expression vector of mature CPA (M-CPA) without propeptide and signal peptide. The results showed that the degradation rate of OTA by M-CPA was up to 93.36%. Its optimum pH was 8, the optimum temperature was 40 °C, the value of Km was 0.126 mmol/L, and the maximum reaction rate was 0.0219 mol/min. Compared with commercial CPA (S-CPA), the recombinant M-CPA had an improve stability, for which its optimum temperature increased by 10 °C and stability at a wide range pH, especially at pH 3–4 and pH 11. M-CPA could effectively degrade OTA in red wine. M-CPA has the potential for industrial applications, such as can be used as a detoxification additive for foods and feeds.

scholarly journals Heterologous Expression and Characterization of A Novel Ochratoxin A Degrading Enzyme, N-acyl-L-amino Acid Amidohydrolase, from Alcaligenes faecalis

Toxins ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 518
Author(s):  
Honghai Zhang ◽  
Yunpeng Zhang ◽  
Tie Yin ◽  
Jing Wang ◽  
Xiaolin Zhang
Keyword(s):  
Amino Acid ◽  
Ochratoxin A ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Alcaligenes Faecalis ◽  
Degrading Enzyme ◽  
Sds Page ◽  
Ochratoxin Α ◽  
Acid Amidohydrolase

Ochratoxin A (OTA) is a well-known, natural contaminant in foods and feeds because of its toxic effects, such as nephrotoxicity in various animals. Recent studies have revealed that Alcaligenes faecalis could generate enzymes to efficiently degrade OTA to ochratoxin α (OTα) in vitro. In an effort to obtain the OTA degrading mechanism, we purified and identified a novel degrading enzyme, N-acyl-L-amino acid amidohydrolase (AfOTase), from A. faecalis DSM 16503 via mass spectrometry. The same gene of the enzyme was also encountered in other A. faecalis strains. AfOTase belongs to peptidase family M20 and contains metal ions at the active site. In this study, recombination AfOTase was expressed and characterized in Escherichia coli. The molecular mass of recombinant rAfOTase was approximately 47.0 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited a wide temperature range (30–70 °C) and pH adaptation (4.5–9.0) and the optimal temperature and pH were 50 °C and 6.5, respectively.

scholarly journals Analysis of the Carry-Over of Ochratoxin A from Feed to Milk, Blood, Urine, and Different Tissues of Dairy Cows Based on the Establishment of a Reliable LC-MS/MS Method

Molecules ◽  
2019 ◽  
Vol 24 (15) ◽  
pp. 2823 ◽  
Author(s):  
Zhiqi Zhang ◽  
Zhichen Fan ◽  
Dongxia Nie ◽  
Zhihui Zhao ◽  
Zheng Han
Keyword(s):  
Dairy Cows ◽  
Ochratoxin A ◽  
Limit Of Quantification ◽  
Fresh Milk ◽  
Liquid Chromatography Tandem Mass ◽  
Ochratoxin Α ◽  
Carry Over ◽  
First Time ◽  
Different Tissues

A rapid and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous determination of ochratoxin A (OTA) and its metabolite ochratoxin α (OTα), for the first time, in dairy cow plasma, milk, urine, heart, liver, spleen, lung, and kidney. The established method was extensively validated by determining the linearity (R2 ≥ 0.990), sensitivity (lower limit of quantification, 0.1–0.2 ng mL−1), recovery (75.3–114.1%), precision (RSD ≤ 13.6%), and stability (≥83.0%). Based on the methodological advances, the carry-over of OTA was subsequently studied after oral administration of 30 μg/kg body weight OTA to dairy cows. As revealed, OTA and OTα were detected in urine, with maximal concentrations of 1.8 ng mL−1 and 324.6 ng mL−1, respectively, but not in milk, plasma, or different tissues, verifying the protection effects of rumen flora against OTA exposure for dairy cows. Moreover, 100 fresh milk samples randomly collected from different supermarkets in Shanghai were also analyzed, and no positive samples were found, further proving the correctness of the in vivo biotransformation results. Thus, from the currently available data, regarding OTA contamination issues on dairy cows, no significant health risks were related to OTA exposure due to the consumption of these products.

Ameliorative role of dietary activated carbon against ochratoxin-A induced oxidative damage, suppressed performance and toxicological effects

Toxin Reviews ◽  
2020 ◽  
pp. 1-11
Author(s):  
Sheraz Ahmed Bhatti ◽  
Muhammad Zargham Khan ◽  
Muhammad Kashif Saleemi ◽  
Zahoor Ul Hassan ◽  
Ahrar Khan
Keyword(s):  
Activated Carbon ◽  
Oxidative Damage ◽  
Ochratoxin A ◽  
Toxicological Effects

INFLUENCE OF NANOFILLER ON THERMAL DEGRADATION RESISTANCE OF HYDROGENATED NITRILE BUTADIENE RUBBER

2018 ◽  
Vol 92 (2) ◽  
pp. 263-285 ◽  
Author(s):  
Tuhin Saha ◽  
Anil K. Bhowmick
Keyword(s):  
Atomistic Simulation ◽  
Model Compound ◽  
Degradation Mechanism ◽  
Decomposition Temperature ◽  
Industrial Applications ◽  
Gas Chromatography Mass Spectrometry ◽  
Butadiene Rubber ◽  
Pyrolysis Gas ◽  
Initial Decomposition Temperature ◽  
Acrylonitrile Butadiene Rubber

ABSTRACT Studies on the degradation of elastomers and their prevention have become increasingly important in recent years because of stringent environmental conditions in many industrial applications. The reactive atomistic simulation was executed on a hydrogenated acrylonitrile-butadiene rubber (HNBR40) model compound composed of 40 monomer units. The reactive simulation was used to study the decomposition behavior of HNBR40, to visualize different pyrolysis products, and also to analyze the degradation mechanism of HNBR40. Ethylene, propylene, and acrylonitrile were observed as dominant products at lower temperature, and 1-butene was found at higher temperature. Pyrolysis–gas chromatography–mass spectrometry was used to verify the decomposition products obtained from the prediction of atomistic simulation. In this study, nanofillers, especially nanoclays and nanosilicas, were used to prevent degradation significantly. Restricted degradation by the nanofiller-reinforced rubber prolonged the durability. Furthermore, the reactive simulation was performed to understand thermal decomposition characteristics of the model compound in the presence of the nanofiller. The initial decomposition temperature, the final degradation temperature, and the rate of degradation improved to a great extent on the addition of the model nanosilica compound as obtained from the simulation studies. Moreover, the lifetime of nanoclay- and nanosilica-reinforced hydrogenated acrylonitrile–butadiene rubber was calculated by using thermogravimetric analysis, and its useful lifetime was compared with that of the pristine polymer in the application temperature range of 150 °C.

A Spectrophotometric Procedure, Using Carboxypeptidase A, for the Quantitative Measurement of Ochratoxin A

1976 ◽  
Vol 59 (1) ◽  
pp. 128-129
Author(s):  
Karl Hult ◽  
Sten Gatenbeck
Keyword(s):  
Quantitative Determination ◽  
Ochratoxin A ◽  
Quantitative Measurement ◽  
Carboxypeptidase A ◽  
Excitation Spectra ◽  
Fluorescence Excitation ◽  
Fluorescence Excitation Spectra ◽  
Ochratoxin Α ◽  
The Difference

Abstract In the method described, ochratoxin A is cleaved into ochratoxin α (free isocoumarin chromophore) and phenylalanine, using carboxypeptidase. Detection is based on the difference in fluorescence excitation spectra of ochratoxin A (380 nm, maximum) and ochratoxin α (340 nm, maximum). The quantitation of ochratoxin A is based on the loss of fluorescence intensity at 380 nm. The method has been used for the quantitative determination of as little as 4 μg ochratoxin A/kg barley and barley meal but it could be extended to other products.

Mycotoxins in grapes and wine in Europe: occurrence, factors affecting the occurrence and related toxicological effects

2010 ◽  
Vol 3 (3) ◽  
pp. 283-300 ◽  
Author(s):  
I. Stratakou ◽  
H. van der Fels-Klerx
Keyword(s):  
Ochratoxin A ◽  
Agricultural Practices ◽  
Natural Occurrence ◽  
Factors Affecting ◽  
Toxicological Effects ◽  
Occurrence Data ◽  
Alternaria Toxins ◽  
Low Concentrations ◽  
Sweet Wines ◽  
Southern European Countries

In 2006, the European Commission has established maximum levels for ochratoxin A in wine and grape products, using occurrence data up to 2001 and toxicity data up to 2006. This paper presents an up-to-date overview of the occurrence of mycotoxins in grapes and wine produced in Europe in the period 1995-2010. In addition, for the most frequently occurring mycotoxins, factors influencing the occurrence, and the toxicological effects are presented. To evaluate possible trends in occurrence, contamination data were grouped into three periods of time, i.e. 1995-1999, 2000-2006 and 2007-2010. Most of the available contamination data on mycotoxins in grapes and wine refer to ochratoxin A, but occurrence data on this toxin from 2006 onwards are very limited. The occurrence of ochratoxin A is higher in the southern European countries than in the northern countries, and higher in red and sweet wines as compared to white wines. Fumonisins occur frequently, but in low concentrations. Data on the natural occurrence of Alternaria toxins are not available. The most important factors that influence ochratoxin A contamination of grapes and wine include: temperature and relative humidity in the month before harvesting the berries, the type of wine (maceration), and the percentage of damaged berries before vinification. Applying good agricultural practices in the vineyard, including minimizing damaged berries and chemical or biological control of the fungi, are the best methods to limit mycotoxin formation in grapes and wine. Ochratoxin A, Alternaria toxins and fumonisins are toxic to animals. These toxins are of concern to human health, but clear evidence on their relationship with human disease is not available yet. Therefore, more research in this area would be desirable.

scholarly journals New Insight into the Ochratoxin A Biosynthetic Pathway through Deletion of a Nonribosomal Peptide Synthetase Gene in Aspergillus carbonarius

2012 ◽  
Vol 78 (23) ◽  
pp. 8208-8218 ◽  
Author(s):  
Antonia Gallo ◽  
Kenneth S. Bruno ◽  
Michele Solfrizzo ◽  
Giancarlo Perrone ◽  
Giuseppina Mulè ◽  
...  
Keyword(s):  
Ochratoxin A ◽  
Biosynthetic Pathway ◽  
Nonribosomal Peptide Synthetase ◽  
Gene Encoding ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Peptide Synthetase ◽  
Ochratoxin Α ◽  
Hydrolysis Of ◽  
Insight Into

ABSTRACTOchratoxin A (OTA), a mycotoxin produced byAspergillusandPenicilliumspecies, is composed of a dihydroisocoumarin ring linked to phenylalanine, and its biosynthetic pathway has not yet been completely elucidated. Most of the knowledge regarding the genetic and enzymatic aspects of OTA biosynthesis has been elucidated inPenicilliumspecies. InAspergillusspecies, onlypksgenes involved in the initial steps of the pathway have been partially characterized. In our study, the inactivation of a gene encoding a nonribosomal peptide synthetase (NRPS) in OTA-producingA. carbonariusITEM 5010 has eliminated the ability of this fungus to produce OTA. This is the first report on the involvement of annrpsgene product in OTA biosynthetic pathway in anAspergillusspecies. The absence of OTA and ochratoxin α, the isocoumaric derivative of OTA, and the concomitant increase of ochratoxin β, the dechloro analog of ochratoxin α, were observed in the liquid culture of transformed strain. The data provide the first evidence that the enzymatic step adding phenylalanine to polyketide dihydroisocoumarin precedes the chlorination step to form OTA inA. carbonariusand that ochratoxin α is a product of hydrolysis of OTA, giving an interesting new insight into the biosynthetic pathway of the toxin.

Removal of ochratoxin A by a carboxypeptidase and peptides present in liquid cultures of Bacillus subtilis CW14

2018 ◽  
Vol 11 (4) ◽  
pp. 559-570 ◽  
Author(s):  
H.N. Hu ◽  
X. Jia ◽  
Y.P. Wang ◽  
Z.H. Liang
Keyword(s):  
Bacillus Subtilis ◽  
Ochratoxin A ◽  
Culture Supernatant ◽  
Physical Adsorption ◽  
Proteinase K ◽  
Small Peptides ◽  
Liquid Cultures ◽  
Ochratoxin Α ◽  
Peptide Complexes

Ochratoxin A (OTA) is an important mycotoxin that contaminates a variety of agricultural products. The cell-free supernatant of Bacillus subtilis CW14 liquid cultures were reported previously to be capable of removing OTA efficiently. In this work, we examined several substances that are probably involved in this removal of OTA using in vitro experiments. The strain CW14 culture supernatant that was separated by ultrafiltration showed that the fractions collected at >10 kDa and <3 kDa had a significant ability to reduce OTA (84.9 and 74.8%, respectively) when incubated with 6 μg/ml OTA at 37 °C for 24 h. A putative metalloenzyme was responsible for the activity of the >10-kDa fraction, which was confirmed by the detrimental effects of heat treatments or addition of SDS, proteinase K, or EDTA. Subsequently, a carboxypeptidase (CP) gene that was likely related to the enzymatic conversion of OTA by the >10-kDa fraction was cloned from the B. subtilis CW14 genome, and over-expressed in Escherichia coli. The recombinant CP degraded 71.3% of OTA at 37 °C for 24 h, and ochratoxin α (OTα) was confirmed as a degradation product. From the <3-kDa fraction, some small peptides (1.7 kDa >Mw >0.7 kDa) were purified and decreased OTA by 45.0% under the same conditions, but no product was detected. These peptides were presumed to be capable of binding OTA due to their affinity with the OTA molecule, and the OTA-peptide complexes escaped from the extraction procedures for OTA quantification. These results indicated there was a probable synergistic effect that was involved in removal of OTA by the strain CW14 culture supernatant, which included enzymatic degradation by a CP and physical adsorption by some small peptides.

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