Heterologous production of basidiomycetous peptidases to decrease sodium chloride in food

Abstract

In the past, the topic of excessive salt consumption associated with various chronic diseases became a focus of nutritional science. Enzymes such as hydrolases and specifically peptidases are widely used in industry since they do not require cofactors, are often stable at high pH values, and have a broad cleavage specificity. Thus, a new approach to use peptidases would be the generation of L‐arginyl dipeptides, which do not have a taste on their own, but are able to enhance the salty taste. This thesis should focus on the isolation and characterisation of arginyl‐specific peptidase genes from basidiomycetes to generate these dipeptides.First, various peptidase genes from Trametes versicolor, Phanerochaete chrysosporium and Schizophyllum commune were isolated and expressed in Escherichia coli or Komagataella phaffii. The serine (ABB73029) peptidase from P. chrysosporium and the aspartate peptidase from T. versicolor (EIW62808) were successfully purified. Activity assays showed that the aspartate peptidase from T. versicolor is not an arginyl‐specific peptidase, while the analysis of cleavage sites indicates that the serine peptidase from P. chrysosporium is an exopeptidase. Next, 29 basidiomycetes were cultivated over 24 days in minimal medium with 1 % gluten and analysed regarding their endo‐ and exopeptidase activity. Out of 29 fungi, five interesting species with arginyl‐specific peptidase activity were chosen, namely Agrocybe aegerita, Fomitop‐ sis pinicola, Flammulina velutipes, Hypholoma sublateritium and Pleurotus eryngii. After purification of the culture supernatant using size exclusion chromatography, active fractions were used to hydrolyse the substrate casein, and cleavage products were analysed. Cleavage sites were only detected for A. aegerita, F. velutipes and P. eryngii. Beside arginyl‐specific cleavage sites also a high amount of undesired exopeptidase cleavage sites was determined assuming that these peptidases are probably exopeptidases rather than arginyl‐specific peptidases.Generation of salt taste enhancing dipeptides should finally be achieved with a dipeptidyl‐ peptidase V (DPPV) from Pleurotusfloridanus. PflDPPV was heterologously produced in E. coli, had optima at pH 8.5 and 60 °C and was not completely inhibited by the tested peptidase inhibitors.