Comparison of carboxypeptidase Y and thermolysin for ochratoxin A electrochemical biosensing

Abstract

International audienceHerein, carboxypeptidase Y (CPY) and thermolysin (TLN) were compared as sensing elements to develop an original biosensor for the direct detection of ochratoxin A (OTA) in olive oil. Electrochemical detection of OTA was performed using biosensors containing either CPY or TLN immobilized through glutaraldehyde vapor cross-linking onto gold interdigitated microelectrodes. Different parameters affecting biosensor sensitivity towards OTA were first optimized. TLN and CPY exhibited an optimal activity under the same conditions: 35 min cross-linking time, working pH of 7 and temperature of 25 degrees C. TLN and CPY biosensors exhibited comparable analytical performances. The conductometric response of both modified electrodes was linear up to 75 mu M. The limits of detection were also very similar (1 mu M for the CPY biosensor and 0.7 mu M for the TLN biosensor). For both enzymes, initial rate vs. OTA concentration plots could be described by hyperbolic curves consistent with the Michaelis-Menten model. K-M(app) and V-max(app), deduced from these curves, were 26 mu M and 3.3 mu S min(-1) respectively for the TLN biosensor. The biosensors signal was reproducible, relative standard deviations calculated from three consecutive measurements of standards in the 1-75 mu M range, being lower than 5%. The signals were also very stable over a 30 days period when biosensors were stored at 4 degrees C in 20 mM phosphate buffer between two measurements. The TLN biosensor was further evaluated for OTA determination in spiked olive oil samples. Recovery values were close to 100% demonstrating the suitability of this method for OTA screening in olive oil. Interestingly, OTA concentrations could be determined without pretreatment of the sample and no matrix effect was observed

An In Silico Target Fishing Approach to Identify Novel Ochratoxin A Hydrolyzing Enzyme

“…It is worth noticing that proteins/enzymes already known to bind/hydrolyze OTA were included in the list of entries identified (Table 1). In particular, bovine carboxypeptidase A (CPA) and thermolysin were already shown to hydrolyze OTA into OTα and phenylalanine [20,21,28], while serum albumin has been repeatedly described as OTA binder [29][30][31]. These results corroborate the efficacy of the in silico approach presented here to identify proteins that are able to interact/transform OTA.…”

Enzymes for Detoxification of Various Mycotoxins: Origins and Mechanisms of Catalytic Action

“…Although the presence of phenylalanine in ochratoxins makes these substrates acceptable for carboxypeptidase A, a halogen atom (Cl or Br) worsens the catalytic constant of the enzyme by an order of magnitude despite some improvement in K m (about 6 times) [115]. At the same time, carboxypeptidase A has a huge number of very close structural and functional analogues for screening of more active enzymes towards ochratoxin A, for example: thermolysin from bacteria Bacillus thermoproteolyticus rokko and carboxypeptidase Y from yeast Saccharomyces cerevisiae [116], or fungal homologues from Rhizopus oryzae and Trichoderma reesei [117], or bacterial homologues from Bacillus amyloliquefaciens [118], Acinetobacter sp. [119], etc.…”

Biodegradation of Mycotoxins: Tales from Known and Unexplored Worlds

“…The main detoxification pathway of OTA is the hydrolyzation of the amide bond between the isocoumarin residue and phenylalanine by a carboxypeptidase. Two classes of carboxypeptidases have been associated with degradation of OTA namely Carboxypeptidase A (CPA) (Stander et al, 2001 ; Chang et al, 2015 ) and Y (CPY) (Dridi et al, 2015 ). The main difference between both is the use of a zinc ion within the protein for hydrolysis of the peptide at the C-terminal of the amino acid.…”