Characterization of Bacillus cereus AFA01 Capable of Degrading Gluten and Celiac-Immunotoxic Peptides
Abstract
:1. Introduction
2. Materials and Methods
2.1. Synthetic Peptides
2.2. Strains and Culture Media
2.3. Degradation of Gluten
2.4. Degradation of Mixed Gliadins in Solution
2.5. Degradation of Gliadin in Gel (Gliadin Zymography)
2.6. Degradation of Immunotoxic Peptides
2.7. Growth and Gluten Degradation
2.8. Complete Genome Sequencing of B. cereus
2.9. RP-HPLC
2.10. Mass Spectrometry
2.11. Statistical Analysis
3. Results
3.1. Degradation of Gluten and Gliadin by B. cereus
3.2. Peptidasic Activity against Immunotoxic Peptides
3.3. Influence of Media Composition on Protease Activity
3.4. Whole Genome Sequencing and Bioinformatic Analysis of B. cereus
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Medium | Composition |
---|---|
BA | Peptone 10 g·L−1, Casein peptone 10 g·L−1, Yeast extract 2.0 g·L−1, Glucose 1.0 g·L−1, Sodium chloride 5.0 g·L−1, Agar 13 g·L−1, pH 7.0 ± 0.2 |
GA | Wheat gluten 23 g·L−1, Sodium chloride 5.0 g·L−1, Glucose 1.0 g·L−1, Sodium succinate 0.5 g·L−1, Soluble starch 1.0 g·L−1, Sodium pyruvate 1.0 g·L−1, Soluble pyrophosphate 0.25 g·L−1, L-Arginine 1.0 g·L−1, L-Cysteine 0.5 g·L−1, Haemin0.01 g·L−1, Vitamin K 0.001 g·L−1, Sodium bicarbonate 0.4 g·L−1, Agar 12 g·L−1 |
LB | Tryptone 10 g·L−1, Yeast extract 5.0 g·L−1, Sodium chloride 10 g·L−1 |
MCG-1 | Glucose 20 g·L−1, Gluten 30 g·L−1, CaCl2 0.05 g·L−1, ZnSO4 0.07 g·L−1, L-cysteine 0.05 g·L−1, Tween 80 0.1%, 60 mM Phosphate Buffer (pH 6.5), Agar 16 g·L−1 |
SCB | Starch 10 g·L−1, Casein 3.0 g·L−1, KNO3 2.0 g·L−1, NaCl 2.0 g·L−1, K2HPO4 2.0 g·L−1, MgSO4 0.05 g·L−1, CaCl2 0.02 g·L−1, FeSO4 0.01 g·L−1, pH 7.2 |
The Number of Genes | ||||
---|---|---|---|---|
KO ID | KO Description | CH | 21155 | AFA01 |
K01297 | muramoyltetrapeptide carboxypeptidase [EC:3.4.17.13] | 1 | 1 | 1 |
K07258 | serine-type D-Ala-D-Ala carboxypeptidase [EC:3.4.16.4] | 4 | 4 | 4 |
K01299 | carboxypeptidase Taq [EC:3.4.17.19] | 1 | 1 | 1 |
K08602 | oligoendopeptidase F [EC:3.4.24.-] | 3 | 3 | 3 |
K03798 | cell division protease FtsH [EC:3.4.24.-] | 1 | 1 | 1 |
K01265 | methionyl aminopeptidase [EC:3.4.11.18] | 3 | 3 | 3 |
K19689 | aminopeptidase [EC:3.4.11.-] | 3 | 3 | 3 |
K01258 | tripeptide aminopeptidase [EC:3.4.11.4] | 2 | 2 | 2 |
K01255 | leucyl aminopeptidase [EC:3.4.11.1] | 1 | 1 | 1 |
K03100, K12380 | signal peptidase I [EC:3.4.21.89] | 7 | 7 | 7 |
K03101 | signal peptidase II [EC:3.4.23.36] | 1 | 1 | 1 |
K08600 | sortase B [EC:3.4.22.71] | 1 | 1 | 1 |
K02236 | leader peptidase (prepilin peptidase)/N-methyltransferase [EC:3.4.23.43 2.1.1.-] | 1 | 1 | 1 |
K05995 | dipeptidase E [EC:3.4.13.21] | 1 | 1 | 1 |
K01270 | dipeptidase D [EC:3.4.13.-] | 1 | 1 | 1 |
K01273 | membrane dipeptidase [EC:3.4.13.19] | 1 | 1 | 1 |
K08651 | thermitase [EC:3.4.21.66] | 1 | 1 | 1 |
K17733 | peptidoglycan LD-endopeptidase CwlK [EC:3.4.-.-] | 1 | 1 | 1 |
K01419 | ATP-dependent HslUV protease, peptidase subunit HslV [EC:3.4.25.2] | 1 | 1 | 1 |
K01338 K04076 | ATP-dependent Lon protease [EC:3.4.21.53] | 1 | 1 | 1 |
K01358 | ATP-dependent Clp protease, protease subunit [EC:3.4.21.92] | 2 | 2 | 2 |
K20742 | gamma-D-glutamyl-L-lysine dipeptidyl-peptidase [EC:3.4.14.13] | 1 | 1 | 1 |
K01304 | pyroglutamyl-peptidase [EC:3.4.19.3] | 1 | 1 | 1 |
K08777 | neutral peptidase B [EC:3.4.24.-] | 1 | 1 | 1 |
K21472 | peptidoglycan LD-endopeptidase LytH [EC:3.4.-.-] | 1 | 1 | 1 |
K21471 | peptidoglycan DL-endopeptidase CwlO [EC:3.4.-.-] | 1 | 1 | 1 |
K11749 | regulator of sigma E protease [EC:3.4.24.-] | 1 | 1 | 1 |
K06383 | stage II sporulation protein GA [EC:3.4.23.-] | 1 | 1 | 1 |
K06402 | stage IV sporulation protein FB [EC:3.4.24.-] | 1 | 1 | 1 |
K06399 | stage IV sporulation protein B [EC:3.4.21.116] | 1 | 1 | 1 |
K06012 | spore protease [EC:3.4.24.78] | 1 | 1 | 1 |
K14647 | minor extracellular serine protease Vpr [EC:3.4.21.-] | 1 | 1 | 1 |
K03797 | carboxyl-terminal processing protease [EC:3.4.21.102] | 1 | 1 | 1 |
K08303 | putative protease [EC:3.4.-.-] | 2 | 2 | 2 |
K19701 | aminopeptidase YwaD [EC:3.4.11.6 3.4.11.10] | 1 | 1 | 1 |
K01271 | Xaa-Pro dipeptidase [EC:3.4.13.9] | 2 | 2 | 2 |
K01262 | Xaa-Pro aminopeptidase [EC:3.4.11.9] | 1 | 1 | 1 |
K01281 | X-Pro dipeptidyl-peptidase [EC:3.4.14.11] | 1 | 1 | 1 |
K01286 | D-alanyl-D-alanine carboxypeptidase [EC:3.4.16.4] | 12 | 12 | 10 |
K07260 | zinc D-Ala-D-Ala carboxypeptidase [EC:3.4.17.14] | 7 | 5 | 3 |
K19702 | aminopeptidase S [EC:3.4.11.24] | 1 | 0 | 0 |
K01387 | microbial collagenase [EC:3.4.24.3] | 5 | 4 | 5 |
K01400 | bacillolysin [EC:3.4.24.28] | 1 | 1 | 2 |
K09607 | immune inhibitor A [EC:3.4.24.-] | 4 | 3 | 4 |
K07284 | sortase A [EC:3.4.22.70] | 2 | 4 | 2 |
K13275 | major intracellular serine protease [EC:3.4.21.-] | 3 | 1 | 1 |
K20486 | lantibiotic leader peptide-processing serine protease [EC:3.4.21.-] | 2 | 0 | 0 |
K01356 | repressor LexA [EC:3.4.21.88] | 2 | 3 | 2 |
K01259 | proline iminopeptidase [EC:3.4.11.5] | 2 | 3 | 4 |
Total | 99 | 94 | 91 |
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Lu, J.; Wu, Y.; Yuan, J.; Yuan, J.; Wang, Z.; Gao, J.; Chen, H. Characterization of Bacillus cereus AFA01 Capable of Degrading Gluten and Celiac-Immunotoxic Peptides. Foods 2021, 10, 1725. https://doi.org/10.3390/foods10081725
Lu J, Wu Y, Yuan J, Yuan J, Wang Z, Gao J, Chen H. Characterization of Bacillus cereus AFA01 Capable of Degrading Gluten and Celiac-Immunotoxic Peptides. Foods. 2021; 10(8):1725. https://doi.org/10.3390/foods10081725
Chicago/Turabian StyleLu, Jun, Yong Wu, Juanli Yuan, Jin Yuan, Zhongliang Wang, Jinyan Gao, and Hongbing Chen. 2021. "Characterization of Bacillus cereus AFA01 Capable of Degrading Gluten and Celiac-Immunotoxic Peptides" Foods 10, no. 8: 1725. https://doi.org/10.3390/foods10081725