Discovery, cloning and characterisation of proline specific prolyl endopeptidase, a gluten degrading thermo-stable enzyme from Sphaerobacter thermophiles

Abstract

Gluten free products have emerged during the last decades, as a result of a growing public concern and technological advancements allowing gluten reduction in food products. One approach is to use gluten degrading enzymes, typically at low or ambient temperatures, whereas many food production processes occur at elevated temperature. We present in this paper, the discovery, cloning and characterisation of a novel recombinant thermostable gluten degrading enzyme, a proline specific prolyl endoprotease (PEP) from Sphaerobacter thermophiles. The molecular mass of the prolyl endopeptidase was estimated to be 77kDa by using SDS-PAGE. Enzyme activity assays with a synthetic dipeptide Z-Gly-Pro-p-nitroanilide as the substrate revealed that the enzyme had optimal activity at pH 6.6 and was most active from pH 5.0-8.0. The optimum temperature was 63 °C and residual activity after one hour incubation at 63 °C was higher than 75 %. The enzyme was activated and stabilized by Co and inhibited by Mg, K and Ca followed by Zn, Na Mn, Al and Cu. The K and k values of the purified enzyme for different substrates were evaluated. The ability to degrade immunogenic gluten peptides (PQPQLPYPQPQLPY (a-gliadin) and SQQQFPQPQQPFPQQP (γ-hordein)) was also confirmed by enzymatic assays and mass spectrometric analysis of cleavage fragments. Addition of the enzyme during small scale mashing of barley malt reduced the gluten content. The findings here demonstrate the potential of enzyme use during mashing to produce gluten free beer, and provide new insights into the effects of proline specific proteases on gluten degradation.

A new microbial gluten-degrading prolyl endopeptidase: Potential application in celiac disease to reduce gluten immunogenic peptides

“…and Aeromonas hydrophila had optimal activity at pH 7.7 [50–51] and Flavobacterium meningosepticum had its highest activity at pH 7.0 [52]. Conversely, other PEPs as Aspergillus oryzae , Aspergillus niger and Sphaerobacter thermophiles achieved the maximum activity at the acidophilic pH values 4.0, 4.2 and 6.6, respectively [14, 53–54].…”

“…Although membrane bound form of PEP enzyme has been characterized, it is generally reported to be cytosolic [13]. PEPs are structurally and functionally closely related to that of the dipeptidyl peptidase IV (DPP-IV), oligo peptidase B and acyl-aminoacyl peptidase sub-families [14].…”

Gluten-degrading bacteria: availability and applications

“…However, POP from the thermophilic archaeon Pyrococcus furiosus has been shown to cleave larger substrates (Harwood et al 1997;Harris et al 2001). The Sphaerobacter thermophilus enzyme also seems to cleave full intact gluten molecules, although the enzymatic assay was performed in a complex mixture that also contains malt (Shetty et al 2017). The POP studied so far originate from a wide range of environmental bacteria and archaea from phyla/class: Actinobacteria (Cellulomonas sp.…”

Obtenção de extratos enzimáticos enriquecidos em proteases a partir de fungos associados a caules e folhas de mandioca visando a digestão do glúten

“…Devido a sua atuação no metabolismo humano e, principalmente em processos digestivos, problemas relacionados a produção de algumas enzimas específicas têm sido muito estudados. Como exemplo pode ser citada a deficiência na produção de enzimas específicas envolvidas na hidrólise das ligações peptídicas presentes no glúten, o que leva à desconfortos e sintomas característicos da intolerância ao glúten, 14), pertencente a subfamília S9A das serino endopeptidase, tem se mostrado promissora, uma vez que mostra capacidade de hidrolisar as sequências peptídicas específicas responsáveis pela intolerância ao glúten (YADAV et al, 2018;SHETTY et al, 2017;HAGER et al, 2014).…”

“…Figura 15 -Quebra do peptídeo 3-mer da gliadina pela prolil oligopeptidade Fonte: Shan et al, 2002Observa-se que essa enzima demonstra alta capacidade de hidrolisar o peptídeo em questão. Assim, é possível afirmar que as proteases possuem grande relevância no estudo de alternativas aos tratamentos atualmente aplicados em pacientes que sofrem de alguma alteração fisiológica pela presença de peptídeos imunogênicos, razão pela qual fontes dessas enzimas têm sido estudadas e alguns microrganismos, como os fungos, têm se mostrado promissores, uma vez que possuem alta capacidade de síntese dessas moléculas.Como destaque, podem ser reportados trabalhos envolvendo a aplicação deAspergillus niger na produção de cervejas visando a quebra da fração prolamina rica em prolina do glúten, além de outros trabalhos que demonstram a quebra do glúten da farinha de trigo utilizando extratos enzimáticos obtidos a partir de Flavobacterium meningosepticum, Xanthomonas sp., Aermonas hydrophilic, Sphingoonas capsulate, Halobacterium halobium S9, Lactobacillus helveticus, Myxococcus xanthus, Aspergillus oryzae e Aspergillus niger, que mostraram eficiência na degradação peptídeos imunogênicos de gliadina do trigo(SHETTY et al, 2017;HAGER et al, 2014).49…”