Immunochemical evaluation of proteolysis of cereal proteins causing celiac disease by microbial proteases

Figures & data

Table 1. Summary of proteolytic conditions.

Protease	Molecular weight	Reaction buffer	Temperature (°C)	Cofactor	Inhibitor
B. licheniformis (EC 3.4.21.62)	27,300	0.1 mol/l Tris-HCl pH 9	70	–	Pefabloc^a
B. stearothermophilus (EC 3.4.21.62)	27,500	0.1 mol/l Tris-HCl pH 8	70	–	Pefabloc^a
B. thermoproteolyticus (EC 3.4.24.27)	34,000	0.1 mol/l Tris-HCl pH 8	70	Zn^2+	EDTA
S. griseus^b	16–27,000	0.1 mol/l Tris-HCl pH 8	37	Zn^2+	EDTA

^a4-(2-Aminoethyl)-benzenesulfonyl-fluoride, hydrochloride (AEBSF).

^bGroup of at least 10 different proteases, including serine- and zinc-proteases.

Figure 1. Proteolysis of prolamins detected by SDS-PAGE (a) and Western blot (b) after 60 min of hydrolysis using protease from Bacillus licheniformis: CW1 – common wheat, cultivar Saxana, spring form; CW2 – common wheat, cultivar Blava, winter form; DW – durum wheat, cultivar Soldur; SW – spelt wheat, cultivar Rubiota; R – rye, cultivar Dankowskie Nowe; T – triticale, cultivar Wanad; B – barley, cultivar Ludan; O – oat, cultivar Detvan; lane 1 – prolamins treated using protease; lane 2 – untreated prolamins; M – molecular marker; arrows indicate fragments after proteolysis.

Figure 2. Proteolysis of prolamins detected by SDS-PAGE (a) and Western blot (b) after 60 min of hydrolysis using protease from B. stearothermophilus: CW1 – common wheat, cultivar Saxana, spring form; CW2 – common wheat, cultivar Blava, winter form; DW – durum wheat, cultivar Soldur; SW – spelt wheat, cultivar Rubiota; R – rye, cultivar Dankowskie Nowe; T – triticale, cultivar Wanad; B – barley, cultivar Ludan; O – oat, cultivar Detvan; lane 1 – prolamins treated using protease; lane 2 – untreated prolamins; M – molecular marker; arrows indicate fragments after proteolysis.

Figure 3. Proteolysis of prolamins detected by SDS-PAGE (a) and Western blot (b) after 60 min of hydrolysis using protease from Bacillus thermoproteolyticus: CW1 – common wheat, cultivar Saxana, spring form; CW2 – common wheat, cultivar Blava, winter form; DW – durum wheat, cultivar Soldur; SW – spelt wheat, cultivar Rubiota; R – rye, cultivar Dankowskie Nowe; T – triticale, cultivar Wanad; B – barley, cultivar Ludan; O – oat, cultivar Detvan; lane 1 – prolamins treated using protease; lane 2 – untreated prolamins; M – molecular marker; arrows indicate fragments after proteolysis.

Figure 4. Proteolysis of prolamins detected by SDS-PAGE (a) and Western blot (b) after 60 min of hydrolysis using protease from Streptomyces griseus: CW1 – common wheat, cultivar Saxana, spring form; CW2 – common wheat, cultivar Blava, winter form; DW – durum wheat, cultivar Soldur; SW – spelt wheat, cultivar Rubiota; R – rye, cultivar Dankowskie Nowe; T – triticale, cultivar Wanad; B – barley, cultivar Ludan; O – oat, cultivar Detvan; lane 1 – prolamins treated using protease; lane 2 – untreated prolamins; M – molecular marker; arrows indicate fragments after proteolysis.

Figure 5. Proteolysis of prolamins detected by R5 ELISA after 60 min of hydrolysis using different microbial proteases; *ppm values lower than 20 mg/kg (Codex Alimentarius limit); ^†inconclusive results; ^‡<LOD (limit of detection).

Table 2. Relative residual immunoreactivity of extracted prolamins after proteolysis (R5 ELISA).

	Relative residual immunochemical activity (%)
Cereal species/protease used	B. licheniformis	B. stearothermophilus	B. thermoproteolyticus	S. griseus
Common wheat, Saxana, spring form	3.1	4.5	0.7	0.2
Common wheat, Blava, winter form	0.5	2.4	0.2	0.3
Durum wheat, Soldur	0.8	6.9	1.9	1.2
Spelt wheat, Rubiota	0.5	5.1	1.4	0.8
Rye, Dankowskie Nowe	9.7	6.6	1.7	0.9
Triticale, Wanad	4.5	5.1	0.3	2.6
Barley, Ludan	80.5	–^a	38.0	22.0
Oat, Detvan	26.5	2.9	10.7	<LOD

Note: LOD limit of detection (1.5 mg/kg gliadin).

^aInconclusive results.

Table 3. Relative residual immunoreactivity of extracted prolamins after proteolysis (G12 ELISA).

	Relative residual immunochemical activity (%)
Cereal species/protease used	B. licheniformis	B. stearothermophilus	B. thermoproteolyticus	S. griseus
Common wheat, Saxana, spring form	16.0	15.1	10.3	6.4
Common wheat, Blava, winter form	10.9	10.8	9.4	6.8
Durum wheat, Soldur	1.5	8.9	2.2	0.7
Spelt wheat, Rubiota	10.0	7.6	6.0	2.3
Rye, Dankowskie Nowe	5.0	33.5	11.0	0.9
triticale, Wanad	4.3	6.4	2.0	1.3
Barley, Ludan	34.9	41.9	18.1	33.8
Oat, Detvan	8.6	55.7	13.5	5.3