Skip to main content

Advertisement

SpringerLink
Log in

Proteomic analysis and optimized production of Alkalihalobacillus patagoniensis PAT 05T extracellular proteases

  • Research Paper
  • Published:
Bioprocess and Biosystems Engineering Aims and scope Submit manuscript

Abstract

Extracellular proteolytic extracts from the haloalkalitolerant strain Alkalihalobacillus patagoniensis PAT 05T have proved highly efficient to reduce wool felting, as part of an ecofriendly treatment suitable for organic wool. In the present study, we identified the extracellular proteases produced by PAT 05T and we optimized its growth conditions for protease production through statistical methods. A total of 191 proteins were identified in PAT 05T culture supernatants through mass spectrometry analysis. Three of the 6 detected extracellular proteases belonged to the serine-endopeptidase family S8 (EC 3.4.21); two of them showed 86.3 and 67.9% identity with an alkaline protease from Bacillus alcalophilus and another one showed 50.4% identity with Bacillopeptidase F. The other 3 proteases exhibited 55.3, 49.4 and 61.1% identity with D-alanyl-D-alanine carboxypeptidase DacF, D-alanyl-D-alanine carboxypeptidase DacC and endopeptidase LytE, respectively. Using a Fractional Factorial Design followed by a Central Composite Design optimization, a twofold increase in protease production was reached. NaCl concentration was the most influential factor on protease production. The usefulness of PAT 05T extracellular proteolytic extracts to reduce wool felting was possible associated with the activity of the serine-endopeptidases closely related to highly alkaline keratinolytic proteases. The other identified proteases could cooperate, improving protein hydrolysis. This study provided valuable information for the exploitation of PAT 05T proteases which have potential for the valorization of organic wool as well as for other industrial applications.

Graphic abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Olivera N, Siñeriz F, Breccia JD (2005) Bacillus patagoniensis sp. nov., a novel alkalitolerant bacterium from the rhizosphere of Atriplex lampa in Patagonia Argentina. Int J Syst Evol Microbiol. https://doi.org/10.1099/ijs.0.63348-0

    Article  PubMed  Google Scholar 

  2. Patel S, Gupta RS (2020) A phylogenomic and comparative genomic framework for resolving the polyphyly of the genus Bacillus: proposal for six new genera of Bacillus species, Peribacillus gen. nov., Cytobacillus gen. nov., Mesobacillus gen. nov., Neobacillus gen. nov., Metabacillus gen. nov. and Alkalihalobacillus gen. nov. Int J Syst Evol Microbiol. https://doi.org/10.1099/ijsem.0.003775

    Article  PubMed  Google Scholar 

  3. Olivera N, Sequeiros C, Siñeriz F, Breccia JD (2006) Characterization of alkaline proteases from a novel alkalitolerant bacterium Bacillus patagoniensis. World J Microbiol Biotechnol 22:737–743. https://doi.org/10.1007/s11274-005-9099-8

    Article  CAS  Google Scholar 

  4. Iglesias MS, Sequeiros C, García S, Olivera NL (2019) Eco-friendly anti-felting treatment of wool top based on biosurfactant and enzymes. J Clean Prod. https://doi.org/10.1016/j.jclepro.2019.02.165

    Article  Google Scholar 

  5. Hassan MM, Carr CM (2019) A review of the sustainable methods in imparting shrink resistance to wool fabrics. J Adv Res. https://doi.org/10.1016/j.jare.2019.01.014

    Article  PubMed  PubMed Central  Google Scholar 

  6. Shen J (2009) Wool finishing and the development of novel finishes. In: Johnson NAG, Russell IM (eds) Advances in wool technology. Woodhead Publishing Series in Textiles, Cambridge, pp 147–182

    Chapter  Google Scholar 

  7. Kettlewell R, De Boos A, Jackson J (2015) Commercial shrink-resist finishes for wool. In: Paul R (ed) Functional finishes for textiles. Improving comfort, performance and protection. Woodhead Publishing Series in Textiles, Cambridge. https://doi.org/10.1016/C2013-0-16373-8

  8. GOTS. Version 5.0 Global Organic Textile Standard International Working Group (2017) https://global-standard.org/the-standard.html. Accessed 20 March 2019

  9. Sharma KM, Kumar R, Panwar S, Kumar A (2017) Microbial alkaline proteases: optimization of production parameters and their properties. J Genet Eng Biotechnol. https://doi.org/10.1016/j.jgeb.2017.02.001

    Article  PubMed  PubMed Central  Google Scholar 

  10. Conesa A, Götz S, Garcia-Gomez JM, Terol J, Talon M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics. https://doi.org/10.1093/bioinformatics/bti610

    Article  PubMed  Google Scholar 

  11. Ye J, Zhang Y, Cui H et al (2018) WEGO 2.0: a web tool for analyzing and plotting GO annotations. Nucleic Acids Res. https://doi.org/10.1093/nar/gky400

    Article  PubMed  PubMed Central  Google Scholar 

  12. Petersen TN, Brunak S, Heijne GV, Nielsen H (2010) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods. https://doi.org/10.1038/nmeth.1701

    Article  Google Scholar 

  13. Cupp-Enyard C (2008) Sigma’s non-specific protease activity assay-casein as a substrate. J Vis Exp. https://doi.org/10.3791/899

    Article  PubMed  PubMed Central  Google Scholar 

  14. Fitzhenry K, Rowan N, Val del Rio A, Cremillieux A, Clifford E (2019) Inactivation efficiency of Bacillus endospores via modified flow-through PUV treatment with comparison to conventional LPUV treatment. J Water Process Eng. https://doi.org/10.1016/j.jwpe.2018.11.009

    Article  Google Scholar 

  15. Haaland PD (1989) Experimental Design in Biotechnology. CRC Press, New York

    Google Scholar 

  16. Wang C, Yu S, Song T, He T, Shao H, Wang H (2016) Extracellular proteome profiling of Bacillus pumilus SCU11 producing alkaline protease for dehairing. J Microbiol Biotechnol. https://doi.org/10.4014/jmb.1602.02042

    Article  PubMed  PubMed Central  Google Scholar 

  17. Madeira J-P, Alpha-Bazin B, Armengaud J, Duport C (2015) Time dynamics of Bacillus cereus exoproteome are shaped by cellular oxidation. Front Microbiol. https://doi.org/10.3389/fmicb.2015.00342

    Article  PubMed  PubMed Central  Google Scholar 

  18. Vazquez-Gutierrez P, Stevens MJ, Gehrig P, Barkow-Oesterreicher S, Lacroix C, Chassard C (2017) The extracellular proteome of two Bifidobacterium species reveals different adaptation strategies to low iron conditions. BMC Genomics. https://doi.org/10.1186/s12864-016-3472-x

    Article  PubMed  PubMed Central  Google Scholar 

  19. Peterson BW, Sharma PK, van der Mei HC, Busscher HJ (2012) Bacterial cell surface damage due to centrifugal compaction. Appl Environ Microbiol. https://doi.org/10.1128/AEM.06780-11

    Article  PubMed  PubMed Central  Google Scholar 

  20. Vidmar B, Vodovnik M (2018) Microbial keratinases: enzymes with promising biotechnological applications. Food Technol Biotechnol. https://doi.org/10.17113/ftb.56.03.18.5658

    Article  PubMed  PubMed Central  Google Scholar 

  21. Rawlings ND, Barrett AJ, Thomas PD, Huang X, Bateman A, Finn RD (2018) The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Res. https://doi.org/10.1093/nar/gkx1134

    Article  PubMed  PubMed Central  Google Scholar 

  22. Kamal S, Rehman S, Iqbal HM (2016) Biotechnological valorization of proteases: from hyperproduction to industrial exploitation-a review. Environ Prog Sustain Energy. https://doi.org/10.1002/ep.12447

    Article  Google Scholar 

  23. Liu B, Zhang J, Li B, Liao X, Du G, Chen J (2013) Expression and characterization of extreme alkaline, oxidation-resistant keratinase from Bacillus licheniformis in recombinant Bacillus subtilis WB600 expression system and its application in wool fiber processing. World J Microbiol Biotechnol. https://doi.org/10.1007/s11274-012-1237-5

    Article  PubMed  Google Scholar 

  24. Zhang R, Wang A (2015) Modification of wool by air plasma and enzymes as a cleaner and environmentally friendly process. J Clean Prod. https://doi.org/10.1016/j.jclepro.2014.10.004

    Article  Google Scholar 

  25. Hageman JH (2013) Bacillopeptidase F. In: Rawlings ND, Salvesen G (eds) Handbook of Proteolytic Enzymes. Academic Press, London, pp 3170–3171

    Chapter  Google Scholar 

  26. Meng D, Dai M, Xu BL, Zhao ZS, Liang X, Wang M, Tang XF, Tang B (2016) Maturation of fibrinolytic Bacillopeptidase F involves both hetero-and autocatalytic processes. Appl Environ Microbiol. https://doi.org/10.1128/AEM.02673-15

    Article  PubMed  PubMed Central  Google Scholar 

  27. Sauvage E, Duez C, Herman R, Kerff F, Petrella S, Anderson JW, Adediran SA, Pratt RF, Frère JM, Charlier P (2007) Crystal structure of the Bacillus subtilis penicillin-binding protein 4a, and its complex with a peptidoglycan mimetic peptide. J Mol Biol. https://doi.org/10.1016/j.jmb.2007.05.071

    Article  PubMed  Google Scholar 

  28. Wu JJ, Schuch R, Piggot PJ (1992) Characterization of a Bacillus subtilis sporulation operon that includes genes for an RNA polymerase σ factor and for a putative DD-carboxypeptidase. J Bacteriol. https://doi.org/10.1128/jb.174.15.4885-4892.1992

    Article  PubMed  PubMed Central  Google Scholar 

  29. Damblon C, Zhao GH, Jamin M, Ledent P, Dubus A, Vanhove M, Raquet X, Christiaens L, Frère JM (1995) Breakdown of the stereospecificity of DD-peptidases and beta-lactamases with thiolester substrates. Biochem J pt2:431–436. https://doi.org/10.1042/bj3090431

    Article  Google Scholar 

  30. Kasahara J, Kiriyama Y, Miyashita M, Kondo T, Yamada T, Yazawa K, Yoshikawa R, Yamamoto H (2016) Teichoic acid polymers affect expression and localization of DL-endopeptidase LytE required for lateral cell wall hydrolysis in Bacillus subtilis. J Bacteriol. https://doi.org/10.1128/JB.00003-16

    Article  PubMed  PubMed Central  Google Scholar 

  31. Noor YM, Samsulrizal NH, Jema’on NA et al (2014) A comparative genomic analysis of the alkalitolerant soil bacterium Bacillus lehensis G1. Gene. https://doi.org/10.1016/j.gene.2014.05.012

    Article  PubMed  Google Scholar 

  32. Srivastava B, Khatri M, Singh G, Kumar Arya S (2000) Microbial keratinases: an overview of biochemical characterization and its eco-friendly approach for industrial applications. J Clean Prod. https://doi.org/10.1016/j.jclepro.2019.119847

    Article  Google Scholar 

  33. Reddy LVA, Wee YJ, Yun JS, Ryu HW (2008) Optimization of alkaline protease production by batch culture of Bacillus sp. RKY3 through Plackett-Burman and response surface methodological approaches. Bioresour Technol. https://doi.org/10.1016/j.biortech.2007.05.006

    Article  PubMed  Google Scholar 

  34. Joo AS, Kumar CG, Park GC, Kim KT, Paik SR, Chang CS (2002) Optimization of the production of an extracellular alkaline protease from Bacillus horikoshii. Process Biochem. https://doi.org/10.1016/S0032-9592(02)00061-4

    Article  Google Scholar 

  35. Patel A, Dodia M, Singh SP (2005) Extracellular alkaline protease from a newly isolated haloalkaliphilic Bacillus sp.: production and optimization. Process Biochem. https://doi.org/10.1016/j.procbio.2005.03.049

    Article  Google Scholar 

  36. Bhunia B, Dey A (2012) Statistical approach for optimization of physiochemical requirements on alkaline protease production from Bacillus licheniformis NCIM 2042. Enzyme Res. https://doi.org/10.1155/2012/905804

    Article  PubMed  PubMed Central  Google Scholar 

  37. Queiroga AC, Pintado ME, Malcata FX (2013) Medium factors affecting extracellular protease activity by Bacillus sp. HTS 102-a novel wild strain isolated from Portuguese merino wool. Nat Sci. https://doi.org/10.4236/ns.2013.56A007

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the Agencia Nacional de Promoción Científica y Tecnológica [PICT Start Up 2012-2004; PICT 2015-1689] and the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) [PUE-IPEEC 22920160100044], from Argentina. Martín Iglesias is grateful to CONICET for his Ph.D. grant. We acknowledge Dr. Magalí Marcos for her comments about this manuscript.

Author information

Authors and Affiliations

  1. Instituto Patagónico Para el Estudio de los Ecosistemas Continentales (IPEEC-CCT CONICET-CENPAT), Blvd. Brown 2915, U9120ACD, Puerto Madryn, Chubut, Argentina

    Nelda L. Olivera & Martín Iglesias

  2. Centro Para el Estudio de Sistemas Marinos (CESIMAR-CCT CONICET-CENPAT), Blvd. Brown 2915, U9120ACD, Puerto Madryn, Chubut, Argentina

    Cynthia Sequeiros & Marina Nievas

Authors
  1. Nelda L. Olivera
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cynthia Sequeiros
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Martín Iglesias
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marina Nievas
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nelda L. Olivera.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 510 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Olivera, N.L., Sequeiros, C., Iglesias, M. et al. Proteomic analysis and optimized production of Alkalihalobacillus patagoniensis PAT 05T extracellular proteases. Bioprocess Biosyst Eng 44, 225–234 (2021). https://doi.org/10.1007/s00449-020-02436-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00449-020-02436-z

Keywords

Search

Navigation