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Optimizing Protein-Glutaminase Expression in Bacillus subtilis

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Abstract

Protein-glutaminase (PG) is a promising protein deaminase. It only hydrolyzes the side chain amido groups of protein-bound to generate ammonia and protein-L-glutamic acid and does not catalyze any other undesirable changes in protein structures. Deamidation of proteins via PG can influence the solubility, emulsification, foaming, and gelation properties of proteins, which are important properties for some food proteins. Therefore, there is great potential for the application of PG in the food industry. PG is derived from Chryseobacterium proteolyticum (C. proteolyticum); however, wild strains are difficult to industrialize because of their low levels of enzyme production. In this article, we studied different strategies for PG expression in B. subtilis. Results showed that PG produced from C. proteolyticum could be successfully secreted in B. subtilis WB800N, and actively secreted in B. subtilis 168(BS168) or DB403 containing a pro-peptide (pro-PG). The secreted PG from B. subtilis WB800N was inactive unless digested by exogenous proteases, such as trypsin, alkaline protease, and neutral protease. However, active PG was secreted by the self-processing of BS168 and DB403. The specific activity of purified PG reached 20.9 U/mg. PG showed maximum activity at pH 5.5, 55 °C and more than 80% of PG activity was retained within a range of pH 3.5–6.5. When Cbz-Gln-Gly was used as the substrate, PG activity was 31.1 ± 0.9 μM min−1 mg−1. Mg2+, Ca2+, and Zn2+ stabilized and even activated PG activity. These strategies concerning PG expression in B. subtilis and the enzymatic properties of PG provide efficient alternatives for PG research and contribute to the industrial-scale production of PG.

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Data Availability

All data generated or analyzed in this study are included in this published article and its additional files.

Abbreviations

pro-PG:

Protein-glutaminase with pro-peptide;

mPG:

Protein-glutaminase with mature peptide

pph:

Protein-glutaminase with pro-peptide and His-tag

GRAS:

Generally recognized as safe

LB:

Luria–Bertani

SDS-PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

PCR:

Polymerase chain reaction

IPTG:

Isopropyl-β-d-thiogalactoside

References

  1. Chobert JM, Briand L, Guéguen J, Popineau Y, Larré C, Haertlé T (1996) Recent advances in enzymatic modifications of food proteins for improving their functional properties. Nahrung-Food 40(4):177–182

    Article  CAS  Google Scholar 

  2. Zhao J, Tian Z, Chen L (2010) Effects of deamidation on structure and functional properties of Barley Hordein. J Agric Food Chem 58(21):11448–11455

    Article  CAS  Google Scholar 

  3. Riha WE, Izzo HV, Zhang J, Ho CT (1996) Nonenzymatic deamidation of food proteins. Crit Rev Food Sci Nutr 36(3):225–255

    Article  CAS  Google Scholar 

  4. Dong X, Zhao M, Jiang Y (2011) Advances in peanut protein modification. J Chin Cereals Oils Assoc 12:109–117

    Google Scholar 

  5. Xiao L, Ren G (2012) Research progress on rice protein modification. Food Ferment Ind 02:151–156

    Google Scholar 

  6. Li C, Li Y, Liu H, Jiang H, Chen Z (2000) Soybean protein modification. Food Ind Technol 03:75–76

    Google Scholar 

  7. Elnaggar SALM. Enzyme and chemical modification of lupin seed proteins. 1997.

  8. Larre C, Kedzior ZM, Chenu MG, Viroben G, Gueguen J (1992) Action of transglutaminase on an 11S seed protein (Pea legumin): influence of the substrate conformation. J Agric Food Chem 40(7):258–267

    Article  Google Scholar 

  9. Larre C, Chiarello M, Blanloeil Y, Chenu M, Gueguen J (1993) Gliadin modifications catalyzed by guinea pig liver transglutaminase. J Food Biochem 17(4):267–282

    Article  Google Scholar 

  10. Kato A, Tanaka A, Lee Y, Matsudomi N, Kobayashi K (1987) Effects of deamidation with chymotrypsin at pH 10 on the functional properties of proteins. J Agric Food Chem 35(2):285–288

    Article  CAS  Google Scholar 

  11. Kato A, Tanaka A, Matsudomi N, Kobayashi K (1987) Deamidation of food proteins by protease in alkaline pH. J Agric Food Chem 35:224–227

    Article  CAS  Google Scholar 

  12. Gill BP, O’Shaughnessey AJ, Henderson P, Headon DR (1985) An assessment of the potential of peptide glutaminases I and II in modifying the charge characteristics of casein and whey proteins. Irish J Food Sci Technol 9(1):33–41

    CAS  Google Scholar 

  13. Hamada JAS, Shih FRF, Frank ARW, Marshall WAE (1988) Deamidation of soy peptides and proteins by Bacillus circulans peptidoglutaminase. J Food Sci 53(2):671–672

    Article  CAS  Google Scholar 

  14. Hamada JS (1992) Effects of heat and proteolysis on deamidation of food proteins using peptidoglutaminase. J Agric Food Chem 40(5):719–723

    Article  CAS  Google Scholar 

  15. Yamaguchi S, Yokoe M (2000) A novel protein-deamidating enzyme from Chryseobacterium proteolyticum sp. Nov., a newly isolated bacterium from soil. Appl Environ Microbiol. 66(8):3337–3343

    Article  CAS  Google Scholar 

  16. Yamaguchi S, Jeenes DJ, Archer DB (2001) Protein-glutaminase from Chryseobacterium proteolyticum, an enzyme that deamidates glutaminyl residues in proteins. FEBS J 268(5):1410–1421

    CAS  Google Scholar 

  17. Zhang YF (2014) Screening of strains producing protein-glutaminase and identification of the enzyme. [Master]: East China Normal University

  18. Qu R (2016) Screening identification and optimization of culture medium of protein glutaminase producing strain. [Master]: East China Normal University

  19. He C (2016) Mutation breeding of Chryseobacterium proteolyticum for increasing protein glutaminase-production. [Master]: East China Normal University

  20. Kikuchi Y, Itaya H, Date M, Matsui K, Wu LF (2008) Production of Chryseobacterium proteolyticum protein-glutaminase using the twin-arginine translocation pathway in Corynebacterium glutamicum. Appl Microbiol Biotechnol 78(1):67–74

    Article  CAS  Google Scholar 

  21. Wang ZH (2013) Gene synthesis, expression and property research of protein-gutaminase. [Master]: East China Normal University

  22. Kiers JL, Van Laeken AEA, Rombouts FM, Nout MJR (2000) In vitro digestibility of Bacillus fermented soya bean. Int J Food Microbiol 60(2–3):163–169

    Article  CAS  Google Scholar 

  23. Boer AS, Diderichsen B (1991) On the safety of Bacillus subtilis and B. amyloliquefaciens: a review. Appl Microbiol Biotechnol 36(1):1–4

    Article  Google Scholar 

  24. Schallmey M, Singh A, Ward OP (2004) Developments in the use of Bacillus species for industrial production. Can J Microbiol 50(1):1–17

    Article  CAS  Google Scholar 

  25. Westers L, Westers H, Quax WJ (2004) Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochim Biophys Acta-Mol Cell Res 1694(1–3):299–310

    Article  CAS  Google Scholar 

  26. Meijer WJJ, Boer AJD, Tongeren SV, Venema G, Bron S (1995) Characterization of the replication region of the Bacillus subtilis plasmid pLS20: a noval type to replicon. Nucleic Acids Res 23(16):3214–3223

    Article  CAS  Google Scholar 

  27. Zhao X (2010) Study on the improvement of electrotransformation of Bacillus subtilis by trehalose. [Master]: Nanjing Agriculture University

  28. Kang L (2014) Preliminary study on fermentation and purification of protein glutaminase and its application. [Master]: East China Normal University

  29. Belokrylov GA, Ostrovskii AD (1968) On the effect of adrenalectomy, adrenal hormones and ACTH on the resistance of developing rats to staphylococcal exotoxin and the endotoxin of Escherichia coli. Zh Mikrobiol Epidemiol Immunobiol 45(5):33–37

    CAS  PubMed  Google Scholar 

  30. Wandersman C (1989) Secretion, processing and activation of bacterial extracellular proteases. Mol Microbiol 3(12):1825–1831

    Article  CAS  Google Scholar 

  31. Kikuchi Y, Date M, Yokoyama K, Umezawa Y, Matsui H (2003) Secretion of active-form Streptoverticillium mobaraense transglutaminase by Corynebacterium glutamicum: processing of the pro-transglutaminase by a cosecreted subtilisin-like protease from Streptomyces albogriseolus. Appl Environ Microbiol 69(1):358–366

    Article  CAS  Google Scholar 

  32. Marx CK, Hertel TC, Pietzsch M (2008) Purification and activation of a recombinant histidine-tagged pro-transglutaminase after soluble expression in Escherichia coli and partial characterization of the active enzyme. Enzyme Microb Technol 42(7):568–575

    Article  CAS  Google Scholar 

  33. Shen X (2011) Expression of transglutaminase proenzyme in Bacillus subtilis. [Master]: South China University of Technology

Download references

Acknowledgments

The authors wish to thank all members of the 344 Lab from the School of Life Science, East China Normal University, and we thank the Dongsheng Biotechnology Co., Ltd for financial support.

Funding

This work was supported by Dongsheng Biotechnology Co., Ltd for financial support.

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Author notes

  1. Xiaoying Ouyang and Yingjie Liu contributed equally to this work.

Authors and Affiliations

  1. School of Life Science, East China Normal University, 500 Dongchuan Road, Shanghai, 200241, People’s Republic of China

    Xiaoying Ouyang, Yingjie Liu, Ruidan Qu, Min Tian, Ting Yang, Rui Zhu, Hongliang Gao, Mingfei Jin & Jing Huang

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  1. Xiaoying Ouyang
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Contributions

MF.J and J.H designed the experiments; YJ.L and XY.OY performed most of the experiment; RD.Q and MT purified the PG; T.Y and R.Z analyzed the properties of PG; XY.OY and J.H wrote the manuscript, and MF.J and HL.G revised the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Hongliang Gao, Mingfei Jin or Jing Huang.

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Ouyang, X., Liu, Y., Qu, R. et al. Optimizing Protein-Glutaminase Expression in Bacillus subtilis. Curr Microbiol 78, 1752–1762 (2021). https://doi.org/10.1007/s00284-021-02404-0

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