Production and characterization of two major Aspergillus oryzae secreted prolyl endopeptidases able to efficiently digest proline-rich peptides of gliadin

Abstract

Prolyl endopeptidases are key enzymes in the digestion of proline-rich proteins. Fungal extracts rich in prolyl endopeptidases produced by a species such as Aspergillus oryzae used in food fermentation would be of particular interest for the development of an oral enzyme therapy product in patients affected by intolerance to gluten. Two major A. oryzae secreted prolyl endopeptidases of the MEROPS S28 peptidase family, AoS28A and AoS28B, were identified when this fungus was grown at acidic pH in a medium containing soy meal protein or wheat gliadin as the sole source of nitrogen. AoS28B was produced by 12 reference A. oryzae strains used in food fermentation. AoS28A was secreted by six of these 12 strains. This protease is the orthologue of the previously characterized Aspergillus fumigatus (AfuS28) and Aspergillus niger (AN-PEP) prolyl endopeptidases which are encoded by genes with a similar intron-exon structure. Large amounts of secreted AoS28A and AoS28B were obtained by gene overexpression in A. oryzae. AoS28A and AoS28B are endoproteases able to cleave Nterminally blocked proline substrates. Both enzymes very efficiently digested the proline-rich 33-mer of gliadin, the most representative immunotoxic peptide deriving from gliadin, with some differences in terms of specificity and optimal pH. Digestion of the gliadin peptide in short peptides with both enzymes was found to occur from its N terminus.

Comparative systems analysis of the secretome of the opportunistic pathogen Aspergillus fumigatus and other Aspergillus species

“…An extensive literature search was undertaken to compile high-throughput experimental studies that have characterized the secretome of Aspergillus species. A systematic search led to a comprehensive list of 46 high-throughput proteomic studies 27 – 73 on the secretome of 6 Aspergillus species analyzed here. Note that many high-throughput studies report proteins with transmembrane (TM) domains as part of the secretome but a few studies have specifically identified cell membrane and cell wall associated proteins along with the secretome 30 , 36 .…”

“…Note that many high-throughput studies report proteins with transmembrane (TM) domains as part of the secretome but a few studies have specifically identified cell membrane and cell wall associated proteins along with the secretome 30 , 36 . Our compilation of high-throughput proteomic studies led to experimentally identified lists of 437, 364, 454, 96, 469 and 171 secreted proteins in A. fumigatus 27 – 41 , A. flavus 30 , 42 – 45 , A. niger 30 , 46 – 58 , A. terreus 30 , 59 , 60 , A. nidulans 58 , 61 – 65 and A. oryzae 57 , 66 – 73 , respectively (Methods; Supplementary Table S1 ).…”

“…We performed an extensive literature search to compile 46 published high-throughput experimental studies 27 – 73 which have characterized the secretome of 6 Aspergillus species studied here (Supplementary Table S1 ). Note that protein identifiers in these experimental studies for the reference strain or other strains of the same Aspergillus species were mapped to standard identifiers in the reference sequence using OrthoMCL 100 ( http://orthomcl.org/orthomcl/ ) with inflation option set at 1.5.…”

A fast and novel approach to evaluate technical enzyme preparations for an efficient protein hydrolysis

“…The TEPs currently used in the food industry are derived mainly from microorganisms, such as Aspergillus spp. [6][7][8][9] and Bacillus spp. [10][11][12][13].…”

Comparative systems analysis of the secretome of the opportunistic pathogen Aspergillus fumigatus and other Aspergillus species

“…terreus 30,59,60 , A. nidulans 58,[61][62][63][64][65] and A. oryzae 57,[66][67][68][69][70][71][72][73] , respectively (Methods; Supplementary Table S1).…”