Fig 1.
Comparison of glutenase activity of the 2RA3 strain with a reference gluten degrader in TSA medium with gliadin (0.1% w/v).
A. 1, strain 2RA3; 2, Flavobacterium meningosepticum; 3 and 4, Escherichia coli (negative control). B. 1, strain 2RA3; 2, Myxococcus xanthus; 3, Sphingomonas capsulata.
Fig 2.
Detection of glutenase activity in microencapsulated bacteria.
Fluorescence intensity of each individual alginate particle was detected and quantified with the moAb G12-FITC at different incubation times. Micrographs corresponding to the most representative samples are shown. PEP (+) show the fluorescence of microencapsulated bacteria hydrolyzing the gliadin, and PEP (-), containing microparticles without bacteria.
Fig 3.
Reduction of gluten immunogenic peptides by the glutenase activity in 2RA3 cell fractions.
Example of G12 immunochromatographic test containing a mixture of gliadin solution at a concentration of 90 ppm: 1, without cellular fraction; 2, intracellular fraction of strain 2RA3; and 3, with extracellular fraction of strain 2RA3.
Fig 4.
Dendrogram showing the phylogenetic relationship between strain 2RA3 and a group of related Chryseobacterium species.
Table 1.
Distinctive phenotypic characteristics of the strain 2RA3 and related species of the genus Chryseobacterium.
Taxa: 1, Chryseobacterium sp. 2RA3; 2, C. taeanense DSM 17071T; 3, C. taichungense DSM 17453T. Except for the hydrolysis data of the various substrates, all data for reference species was taken from Shen [40] and Park [4,1]. ND, no data available.
Table 2.
Chryseobacterium taeanense 2RA3 gene-encoding proteases of the S9 family and main characteristics of deduced translation products.
Fig 5.
Strategy for the identification of protein responsible for the glutenase activity in C. taeanense strain 2RA3.
A, Gliadin zymography of culture supernatant. Lane 1, molecular weight marker, lane 2, strain 2RA3. B, Nano-ESI-Q-TOF mass-spectrometer analysis of the potential active band after trypsin treatment; C, Amino acid sequence of annotated protease in the genome of C. taeanense strain 2RA3 responsible for the activity detected in the excised band.
Fig 6.
Purification of PEP 2RA3 from E. coli REG-1 [pALEX2-HCa].
SDS-PAGE was Coomassie stained. M, Molecular weight marker; I, Fraction of E. coli induced culture before purifying; FS, Solubilized fraction; FT, Flow-through fraction from Ni-NTA resin; E1-E10, Elution fractions of desired protein at different concentrations of imidazole (25 mM -500 mM).
Fig 7.
Characterization of the recombinant PEP.
A, Hydrolysis of Suc-Ala-Pro-pNA and Z-Gly-Pro-pNA by recombinant PEP. B, Activity profiles measured using Suc-Ala-Pro-pNA at various concentrations. C, The effect of pH was determined by incubating the enzyme in citrate/disodium phosphate buffer (pH 2–8), Tris-HCl (9–10), and glycine/NaOH (pH 11–12) for 30 min with 1.5 mM Suc-Ala-Pro-pNA. D, Hydrolysis of 1.5 mM Suc-Ala-Pro-pNA at various temperatures. The activity for optimum temperature was determined under conditions at 20–70°C and optimum pH for 30 min.
Fig 8.
Effect of PEP in beer gluten peptides.
A. Coomassie stained protein gel. M, Molecular weight marker; 1, Beer fraction after addition of PEP showing the hydrolysis of gluten beer peptides by the purified recombinant PEP. 2, Beer fraction. B. Western blots using the detector antibody G12. M, Molecular weight marker; 1, Beer fraction; 2, Beer fraction after addition of PEP.