The aim of this study
The aim of this study was to discover and characterize a novel thermostable gluten-specific prolyl endopeptidase suitable for use during the mashing step of beer production. Subsequently to express the recombinant enzyme in E.coli and to demonstrate degradation of immunogenic gluten derived peptides.
Materials and methods
Results and discussion
Conclusions The present study reveals an open reading frame composed of 2064bp, (UniProtKB − D1C7Y4) from Sphaerobacter thermophiles encoding a gene with proline specific prolyl endopeptidase activity. When expressed in Escherichia coli and isolated, it was found to have a broad range of pH stability and high temperature activity and stability. The enzyme degrades the celiac-active peptides we tested, i.e. α-gliadin LGQQQPFPPQQPY, PQPQLPYPQPQLPY and γ-hordein SQQQFPQPQQPFPQQP by cleaving at internal proline residues. It is one of the few PEP enzymes which have been confirmed to have such activity. It is thus a promising enzyme for applications where gluten degradation at high temperatures offers processing advantages, for example to degrade and reduce gluten during the high temperature mashing process of beer brewing.
Conflict of interest
Funding This work was supported by the Novozymes and Henning Holck Larsen foundation. The Centre for Advanced Food Studies is acknowledged for funding the Ultraflex TOF/TOF mass spectrometer.
Introduction Botulinum neurotoxin (BoNT) is a uniquely potent protein synthesized by Clostridium botulinum, C. baratii and C. butyricum, (Rosetto et al., 2014, Kukreja and Singh, 2014). It acts on cholinergic nerve endings and blocks the release of Oxaliplatin causing flaccid paralysis. It is responsible for the disease botulism, which results in flaccid muscle paralysis and leads to death. Botulism can be spread by accidental food poisoning, wound, air, and liquid. For nearly three decades now it is also being used as a therapeutic agent against disorders of uncontrolled peripheral cholinergic nerve activity, such as dystonia (Kukreja and Singh, 2015). In the disease form, it is considered as the most poisonous of all poisons (Lamanna, 1959, Singh, 2000, Kumar et al., 2014). BoNT, an AB type toxin, is a 150 kDa protein composed of two functional domains: a 100 kDa heavy chain (HC) and a 50 kDa light chain (LC), linked through a disulfide bond. The C-terminal of heavy chain specifically target the neuronal cells and facilitate receptor-mediated endocytosis. Whereas, N-terminal (50 kDa) of heavy chain facilitates translocation of light chain to the cytosol inside the neuronal cells. The LC is a zinc-endopeptidase, and is associated with the intracellular activity of blocking acetylcholine release (Sharma and Singh, 2004, Rosetto et al., 2014). In bacterial culture, BoNT/A is produced in three progenitor toxin forms: M, L, and LL forms. The M form consists of neurotoxin (150 kDa) and a nontoxic protein component (140 kDa) which is called nontoxic non-hemagglutinin component, NTNH (Gu et al., 2012) with 12S molecular size (the molecular size of complex forms is expressed as sedimentation equilibrium values) (East and Collins, 1994). The L form has a molecular weight of about 500 kDa and a molecular size of 16S. The LL form is about 900 kDa and 19S. The L and LL complexes consist of several hemagglutinin components besides the BoNT and NBP, and exhibit hemagglutinin activity (Somers and DasGupta, 1991, Fujii, 1995, Singh et al., 2014). These are referred to as neurotoxin associated proteins (NAPs), and also as complexing, ancillary, or accessory proteins (Singh et al., 2014). Stabilized through non-covalent interactions, NAPs account for up to 80% of the total mass of BoNT complex. In case of natural botulism, it is the complex form of BoNT that is encountered. In general, BoNT in the complex form is resistant to environmental stress, such as pH, temperature, and proteases. L form of the complex is used in this study.