Skip to main content
SpringerLink
Log in

Combinatorial mutagenesis of Bacillus amyloliquefaciens for efficient production of protease

  • Original Article
  • Published:
Systems Microbiology and Biomanufacturing Aims and scope Submit manuscript

Abstract

As an important industrial enzyme, protease is widely used in feed, food and other fields. At present, the insufficient protease activity obtained from microorganisms cannot meet the purpose of industrial production. In this study, Bacillus amyloliquefaciens with high protease production was screened from animal feces by plate transparent circle method. To improve the production of protease, atmospheric room temperature plasma (ARTP) mutagenesis was used in the first round, protease activity reached 315.0 U/mL. Then, to enhance production of protease, 60Co-γ irradiation was used for combined mutagenesis, leading to protease activity of B. amyloliquefaciens FMME ZK003 up to 355.0 U/mL. Furthermore, to realize the efficient production of protease, after optimization of fermentation conditions, protease activity was increased to 456.9 U/mL. Finally, protease activity of B. amyloliquefaciens FMME ZK003 reached 823.0 U/mL in a 5 L fermenter. These results indicate that B. amyloliquefaciens can efficiently produce protease, which provides a good foundation for the industrial production of protease.

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
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Data availability

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

References

  1. Razzaq A, Shamsi S, Ali A, Ali Q, Sajjad M, Malik A, Ashraf M. Microbial proteases applications. front bioeng. Biotechnol. 2019;7:110.

    Google Scholar 

  2. Contesini FJ, de Melo RR, Sato HH. An overview of Bacillus proteases: from production to application. Crit Rev Biotechnol. 2018;38:321–34.

    Article  CAS  PubMed  Google Scholar 

  3. Wang L, Wang Y-J. Rice starch isolation by neutral protease and high-intensity ultrasound. J Cereal Sci. 2004;39:291–6.

    Article  CAS  Google Scholar 

  4. Nalinanon S, Benjakul S, Kishimura H, Shahidi F. Functionalities and antioxidant properties of protein hydrolysates from the muscle of ornate threadfin bream treated with pepsin from skipjack tuna. Food Chem. 2011;124:1354–62.

    Article  CAS  Google Scholar 

  5. Hejdysz M, Kaczmarek SA, Kubiś M, Wiśniewska Z, Peris S, Budnik S, Rutkowski A. The effect of protease and Bacillus licheniformis on nutritional value of pea, faba bean, yellow lupin and narrow-leaved lupin in broiler chicken diets. Br Poult Sci. 2020;61:287–93.

    Article  CAS  PubMed  Google Scholar 

  6. Naveed M, Nadeem F, Mehmood T, Bilal M, Anwar Z, Amjad F. Protease—a versatile and ecofriendly biocatalyst with multi-industrial applications: an updated review. Catal Lett. 2021;151:307–23.

    Article  CAS  Google Scholar 

  7. Vojcic L, Pitzler C, Körfer G, Jakob F, Ronny M, Maurer K-H, Schwaneberg U. Advances in protease engineering for laundry detergents. New Biotechnol. 2015;32:629–34.

    Article  CAS  Google Scholar 

  8. Guleria S, Walia A, Chauhan A, Shirkot CK. Purification and characterization of detergent stable alkaline protease from Bacillus amyloliquefaciens SP1 isolated from apple rhizosphere. J Basic Microbiol. 2016;56:138–52.

    Article  CAS  PubMed  Google Scholar 

  9. Dayanandan A, Kanagaraj J, Sounderraj L, Govindaraju R, Rajkumar GS. Application of an alkaline protease in leather processing: an ecofriendly approach. J Cleaner Prod. 2003;11:533–6.

    Article  Google Scholar 

  10. Rehman R, Ahmed M, Siddique A, Hasan F, Hameed A, Jamal A. Catalytic role of thermostable metalloproteases from bacillus subtilis KT004404 as dehairing and destaining agent. Appl Biochem Biotechnol. 2017;181:434–50.

    Article  CAS  PubMed  Google Scholar 

  11. Gupta R, Beg QK, Lorenz P. Bacterial alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotechnol. 2002;59:15–32.

    Article  CAS  PubMed  Google Scholar 

  12. Calik P, Ozdamar TH. Carbon sources affect metabolic capacities of Bacillus species for the production of industrial enzymes: theoretical analyses for serine and neutral proteases and alpha-amylase. Biochem Eng J. 2001;8:61–81.

    Article  CAS  PubMed  Google Scholar 

  13. Ahmed Z, Donkor O, Street WA, Vasiljevic T. Proteolytic activities in fillets of selected underutilized Australian fish species. Food Chem. 2013;140:238–44.

    Article  CAS  PubMed  Google Scholar 

  14. Chalamaiah M, Dineshkumar B, Hemalatha R, Jyothirmayi T. Fish protein hydrolysates: proximate composition, amino acid composition, antioxidant activities and applications: a review. Food Chem. 2012;135:3020–38.

    Article  CAS  PubMed  Google Scholar 

  15. Mala BR, Aparna MT, Mohini SG, Vasanti VD. Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev. 1998;62:597–635.

    Article  Google Scholar 

  16. Tufvesson P, Lima-Ramos J, Nordblad M, Woodley JM. Guidelines and cost analysis for catalyst production in biocatalytic processes. Org Process Res Dev. 2011;15:266–74.

    Article  CAS  Google Scholar 

  17. Sn N, D J. Optimization of alkaline protease production from Bacillus subtilis NS isolated from sea water. Afr J Biotechnol. 2014;13:1707–13.

    Article  Google Scholar 

  18. Ali N, Ullah N, Qasim M, Rahman H, Khan SN, Sadiq A, Adnan M. Molecular characterization and growth optimization of halo-tolerant protease producing Bacillus Subtilis Strain BLK-1.5 isolated from salt mines of Karak. Pakistan Extremophiles. 2016;20:395–402.

    Article  PubMed  Google Scholar 

  19. Karray A, Alonazi M, Horchani H, Ben BA. A novel thermostable and alkaline protease produced from Bacillus stearothermophilus isolated from olive oil mill sols suitable to industrial biotechnology. Molecules. 2021. https://doi.org/10.3390/molecules26041139.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Elshaghabee FMF, Rokana N, Gulhane RD, Sharma C, Panwar H. Bacillus as potential probiotics: status, concerns, and future perspectives. Front Microbiol. 2017. https://doi.org/10.3389/fmicb.2017.01490.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Soares VF, Castilho LR, Bon EPS, Freire DMG. High-yield bacillus subtilis protease production by solid-state fermentation. Appl Biochem Biotechnol. 2005;121:311–9.

    Article  PubMed  Google Scholar 

  22. Elyasi Far B, Yari Khosroushahi A, Dilmaghani A. In silico study of alkaline serine protease and production optimization in Bacillus sp. Khoz1 closed bacillus safensis isolated from honey. Int J Pept Res Ther. 2020;26:2241–51.

    Article  CAS  Google Scholar 

  23. Lario LD, Chaud L, Almeida MdG, Converti A, Durães Sette L, Pessoa A. Production, purification, and characterization of an extracellular acid protease from the marine Antarctic yeast Rhodotorula mucilaginosa L7. Fungal Biol. 2015;119:1129–36.

    Article  CAS  PubMed  Google Scholar 

  24. Yegin S, Fernandez-Lahore M, JoseGamaSalgado A, Guvenc U, Goksungur Y, Tari C. Aspartic proteinases from Mucor spp in cheese manufacturing. Appl Microbiol Biotechnol. 2011;89:949–60.

    Article  CAS  PubMed  Google Scholar 

  25. Kalahroudi RJ, Valizadeh V, Atyabi SM, Keramati M, Cohan RA, Aghai A, Norouzian D. Increment in protease activity of Lysobacter enzymogenes strain by ultra violet radiation. Iran J Microbiol. 2020;12:601–6.

    PubMed  PubMed Central  Google Scholar 

  26. Tuly JA, Ma H, Zabed HM, Dong Y, Janet Q, Golly MK, Feng L, Li T, Chen G. Harnessing the keratinolytic activity of bacillus licheniformis through random mutagenesis using ultraviolet and laser irradiations. Appl Biochem Biotechnol. 2022;194:1546–65.

    Article  CAS  PubMed  Google Scholar 

  27. Wang Y, Wang J, Zhang X, Tong Y, Yang R. Genomic and transcriptomic analysis of Bacillus subtilis JNFE1126 with higher nattokinase production through ultraviolet combined 60Co-γ ray mutagenesis. LWT. 2021;147:111652.

    Article  CAS  Google Scholar 

  28. Yadav SK, Singh P, Dubey KK, Singh BP. Combined mutagenic improvement of Bacillus licheniformis SK7 for cost-effective protease production. Asian J Bio Sci. 2016;11:91–4.

    Article  Google Scholar 

  29. Zhang X. Applying the mutation of Bacillus subtilis and the optimization of feather fermentation medium to improve Keratinase activity. Adv Biol Chem. 2012;02:64–9.

    Article  CAS  Google Scholar 

  30. Zhang JF, Zhu BY, Li XY, Xu XJ, Li DK, Zeng F, Zhou CX, Liu YH, Li Y, Lu FP. Multiple modular engineering of Bacillus Amyloliquefaciens cell factories for enhanced production of alkaline proteases from B. Clausii Front Bioeng Biotechnol. 2022;10:866066.

    Article  PubMed  Google Scholar 

  31. Zhou CX, Yang GC, Zhang L, Zhang HT, Zhou HY, Lu FP. Construction of an alkaline protease overproducer strain based on Bacillus licheniformis 2709 using an integrative approach. Int J Biol Macromol. 2021;193:1449–56.

    Article  CAS  PubMed  Google Scholar 

  32. Zhou C, Zhou H, Li D, Zhang H, Wang H, Lu F. Optimized expression and enhanced production of alkaline protease by genetically modified Bacillus licheniformis 2709. Microb Cell Fact. 2020;19:45.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Mo QS, Tian Y, Zhang HT, Bu LJ, Lu FP. Using 16S rDNA as target site for homologous recombination to improve the alkaline protease production of Bacillus alcalophilus. Adv Mater Res. 2014;886:349–54.

    Article  Google Scholar 

  34. Suberu Y, Akande I, Samuel T, Lawal A, Olaniran A. Optimization of protease production in indigenous Bacillus species isolated from soil samples in Lagos. Nigeria using response surface methodology: Biocatal Agric Biotechnol; 2019. https://doi.org/10.1016/j.bcab.2019.01.049.

    Book  Google Scholar 

  35. Cai CG, Zheng XD. Medium optimization for keratinase production in hair substrate by a new Bacillus subtilis KD-N2 using response surface methodology. J Ind Microbiol Biotechnol. 2009;36:875–83.

    Article  CAS  PubMed  Google Scholar 

  36. He F, Chao J, Yang D, Zhang X, Yang C, Xu Z, Jiewei T, Yongqiang T. Optimization of fermentation conditions for production of neutral metalloprotease by Bacillus subtilis SCK6 and its application in goatskin-dehairing. Prep Biochem Biotechnol. 2021;52:789–99.

    Article  PubMed  Google Scholar 

  37. Ding Q, Luo QL, Zhou J, Chen XL, Liu LM. Enhancing l-malate production of Aspergillus oryzae FMME218-37 by improving inorganic nitrogen utilization. Appl Microbiol Biotechnol. 2018;102:8739–51.

    Article  CAS  PubMed  Google Scholar 

  38. Liu H, Zhang Z, Hong P, Zhou C, Yang P, Chen K, Wu J. Isolation of microbial strain producing thermostable protease and characterization of the enzyme. Sci Technol Food Ind. 2017;38:133.

    CAS  Google Scholar 

  39. Shu L, Si XG, Yang XD, Ma WY, Sun JL, Zhang J, Xue XL, Wang DP, Gao Q. Enhancement of acid protease activity ofaspergillus oryzaeusing atmospheric and room temperature plasma. Front Microbiol. 2020. https://doi.org/10.3389/fmicb.2020.01418.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Gao X, Liu E, Yin Y, Yang L, Huang Q, Chen S, Ho C-T. Enhancing activities of salt-tolerant proteases secreted by aspergillus oryzae using atmospheric and room-temperature plasma mutagenesis. J Agric Food Chem. 2020;68:2757–64.

    Article  CAS  PubMed  Google Scholar 

  41. Xue G, Chen L, Wu B, He B. Selection of high-yield thermostable protease producing strain by ARTP and the study on its enzymological properties. Sci Technol Food Ind. 2015;36(177–180):206.

    CAS  Google Scholar 

  42. Wang S, Luo Q, Liu J, Liu L, Chen X. Mutation and fermentation optimization of Bacillus amyloliquefaciens for acetoin production. Chin J Biotechnol. 2018;34:803–11.

    CAS  Google Scholar 

  43. Liu S, Fang Y, Lv M, Wang S, Chen L. Optimization of the production of organic solvent-stable protease by Bacillus sphaericus DS11 with response surface methodology. Bioresour Technol. 2010;101:7924–9.

    Article  CAS  PubMed  Google Scholar 

  44. Singh S, Bajaj BK. Medium optimization for enhanced production of protease with industrially desirable attributes from Bacillus subtilis K-1. Chem Eng Commun. 2015;202:1051–60.

    Article  CAS  Google Scholar 

  45. Keshavamurthy M, Vishwanatha T, Kumar SM, Gaddad SM. Enhanced production of alkaline protease from novel bacterium Bacillus cereus GVK21 under submerged fermentation. Biosci Biotechnol Res Commun. 2018;11:416–25.

    Article  Google Scholar 

Download references

Funding

This study was supported by the Provincal Outstanding Youth Foundation of Jiangsu Province (BK20211529) and the National Science Fund for Excellent Young Scholars (22122806).

Author information

Authors and Affiliations

  1. State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, China

    Kuo Zhao, Haoyu Liu, Cong Gao, Liang Guo & Xiulai Chen

  2. International Joint Laboratory On Food Safety, Jiangnan University, Wuxi, 214122, China

    Kuo Zhao, Haoyu Liu, Cong Gao, Liang Guo & Xiulai Chen

  3. School of Life Sciences and Health Engineering, Jiangnan University, Wuxi, 214122, China

    Wei Song & Jing Wu

Authors
  1. Kuo Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Haoyu Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wei Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jing Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Cong Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Liang Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xiulai Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xiulai Chen.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Rights and permissions

Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhao, K., Liu, H., Song, W. et al. Combinatorial mutagenesis of Bacillus amyloliquefaciens for efficient production of protease. Syst Microbiol and Biomanuf 3, 457–468 (2023). https://doi.org/10.1007/s43393-022-00130-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s43393-022-00130-7

Keywords

Search

Navigation