Benoit Granier

Multiplex dipstick immunoassay for semi-quantitative determination of Fusarium mycotoxins in cereals.

A multiplex dipstick immunoassay based method for the simultaneous determination of major Fusarium toxins, namely zearalenone, T-2 and HT-2 toxins, deoxynivalenol and fumonisins in wheat, oats and maize has been developed. The dipstick format was based on an indirect competitive approach. Four test lines (mycotoxin-BSA conjugates) and one control line were located on the strip membrane. Labelled antibodies were freeze-dried within the microwell. Two matrix-related sample preparation protocols have been developed for wheat/oats (not containing fumonisins) and maize (containing fumonisins) respectively. The use of a methanol/water mixture for sample preparation allowed recoveries in the range 73-109% for all mycotoxins in all tested cereals, with relative standard deviation less than 10%. The optimized immunoassay was able to detect target mycotoxins at cut off levels equal to 80% of EU maximum permitted levels, i.e. 280, 400, 1400 and 3200 μg kg(-1), respectively, for zearalenone, T-2/HT-2 toxins, deoxynivalenol and fumonisins in maize, and 80, 400 and 1400 μg kg(-1), respectively, for zearalenone, T-2/HT-2 toxins and deoxynivalenol in wheat and oats. Analysis of naturally contaminated samples resulted in a good agreement between multiplex dipstick and validated confirmatory LC-MS/MS. The percentage of false positive results was less than or equal to 13%, whereas no false negative results were obtained. Data on the presence/absence of 6 mycotoxins at levels close to EU regulatory levels were obtained within 30 min. The proposed immunoassay protocol is rapid, inexpensive, easy-to-use and fit for purpose of rapid screening of mycotoxins in cereals.

Serine-Type D-Ala-D-Ala Peptidases and Penicillin-Binding Proteins

Publisher Summary This chapter describes serine-type D-Ala-D-Ala peptidases and penicillin-binding proteins. Penicillin is a suicide substrate. Because of the endocyclic nature of the scissile β-lactam amide bond, the leaving group of the enzyme acylation step remains part of the acyl enzyme. The first part only of the transfer cycle is achieved, leading to a long-lived; serine ester-linked acyl(penicilloyl)-enzyme and the enzyme behaves as a penicillin-binding protein (PBP). All bacteria possess an assortment of low and high molecular weight membrane-bound PBPs. The low molecular weight PBPs are single catalytic entities. The bulk of the protein is on the outer face of the plasma membrane and bears a carboxy-terminal extension, the end of which serves as membrane anchor. The low molecular weight PBPs helps to control the extent of wall peptidoglycan cross-linking throughout the life cycle of the cells. The high molecular weight PBPs involved in wall peptidoglycan assembly and cell morphogenesis are multimodule proteins. The bulk of the protein is on the outer face of the membrane and consists of an N-terminal module, fused to a C-terminal, penicillin-binding module.

The precursor of the Streptomyces R61 DD‐peptidase containing a C‐terminal extension is inactive

The Streptomyces R61 DD‐peptidase gene encodes a 26‐residue C‐terminal extension which is not found in the mature protein. When the gene was expressed in Escherichia coli, the extension was not cleaved and the precursor protein was not enzymatically active. It also reacted with penicillins significantly more slowly than the mature protein. The introduction of a ‘stop’ codon after that corresponding to the C‐terminal residue of the mature protein resulted in the production of an active protein in the periplasm of E. coli.