Advertisement

Agricultural waste materials enhance protease production by Bacillus subtilis B22 in submerged fermentation under blue light-emitting diodes

Abstract

Bacillus bacteria have major utility in large-scale production of industrial enzymes, among which proteases have particular importance. B. subtilis B22, an aerobic and chemotrophic strain, was isolated from kimchi and identified by 16S rRNA gene sequencing. Extracellular protease production was determined in basic medium, with 1% (w/v) casein as substrate, by submerged fermentation at 37 °C under blue, green, red and white light-emitting diodes (LEDs), white fluorescent light and darkness. Fermentation under blue LEDs maximized protease production (110.79 ± 1.8 U/mL at 24 h). Various agricultural waste products enhanced production and groundnut oil cake yielded the most protease (334 ± 1.8 U/mL at 72 h). Activity and stability of the purified protease were optimum at pH 7–10 and 20–60 °C. Activity increased in the presence of Ca2+, Mg2+ and Mn2+, while Fe2+, Zn2+, Co2+ and Cu2+ moderated activity, and Ni2+ and Hg2+ inhibited activity. Activity was high (98%) in the presence of ethylenediaminetetraacetic acid (EDTA) but inhibited by phenylmethanesulfonyl fluoride (PMSF). The protease was unaffected by nonionic surfactants, tolerated an anionic surfactant and oxidizing agents, and was compatible with multiple organic solvents. These properties suggest utility of protease produced by B. subtilis B22 under blue LEDs for industrial applications.

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

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 199

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. 1.

    Blanco AS, Durive OP, Pérez BS, Montesa DZ, Guerra PN (2016) Simultaneous production of amylases and proteases by Bacillus subtilis in brewery wastes. Braz J Microbiol 47:665–674. https://doi.org/10.1016/j.bjm.2016.04.019

  2. 2.

    Asoodeh A, Emtenani S, Emtenani S, Jalal R (2014) Enzymatic Molecular cloning and biochemical characterization of a thermoacidophilic, organic-solvent tolerant a-amylase from a Bacillus strain in Escherichia coli. J Mol Catal B Enzym 99:114–120. https://doi.org/10.1016/j.molcatb.2013.10.025

  3. 3.

    Farhadian S, Asoodeh A, Lagzian M (2015) Purification, biochemical characterization and structural modeling of a potential htrA-like serine protease from Bacillus subtilis DR8806. J Mol Catal B Enzym 115:51–58. https://doi.org/10.1016/j.molcatb.2015.02.001

  4. 4.

    Uttatree S, Charoenpanich J (2016) Isolation and characterization of a broad pH- and temperature-active, solvent and surfactant stable protease from a new strain of Bacillus subtilis. Biocatal Agric Biotechnol 8:32–38. https://doi.org/10.1016/j.bcab.2016.08.003

  5. 5.

    Lakshmi BKM, Muni Kumar D, Hemalatha KPJ (2018) Purification and characterization of alkaline protease with novel properties from Bacillus cereus strain S8. J Genet Eng Biotechnol 16:295–304. https://doi.org/10.1016/j.jgeb.2018.05.009

  6. 6.

    dos Santos Aguilar JG, Sato HH (2018) Microbial proteases: production and application in obtaining protein hydrolysates. Food Res Int 103:253–262. https://doi.org/10.1016/j.foodres.2017.10.044

  7. 7.

    Hmidet N, Ali NEH, Haddar A, Kanoun S, Alya KS, Nasri M (2009) Alkaline proteases and thermostable α-amylase co-produced by Bacillus licheniformis NH1: characterization and potential application as detergent additive. Biochem Eng J 47:71–79. https://doi.org/10.1016/j.bej.2009.07.005

  8. 8.

    Ward OP, Rao MB, Kulkarni A (2009) Proteases, production. Encycl Microbiol. https://doi.org/10.1016/B978-012373944-5.00172-3

  9. 9.

    Parrado J, Rodriguez-Morgado B, Tejada M, Hernandez T, Garcia C (2014) Proteomic analysis of enzyme production by Bacillus licheniformis using different feather wastes as the sole fermentation media. Enzyme Microb Technol 57:1–7. https://doi.org/10.1016/j.enzmictec.2014.01.001

  10. 10.

    Ramachandran S, Singh SK, Larroche C, Soccol CR, Pandey A (2007) Oil cakes and their biotechnological applications—a review. Bioresour Technol 98:2000–2009. https://doi.org/10.1016/j.biortech.2006.08.002

  11. 11.

    Hashemi M, Razavi SH, Shojaosadati SA, Mousavi SM, Khajeh K, Safari M (2010) Development of a solid-state fermentation process for production of an alpha amylase with potentially interesting properties. J Biosci Bioeng 110:333–337. https://doi.org/10.1016/j.jbiosc.2010.03.005

  12. 12.

    De Castro RJS, Sato HH (2013) Synergistic effects of agro industrial wastes on simultaneous production of protease and α-amylase under solid state fermentation using a simplex centroid mixture design. Ind Crops Prod 49:813–821. https://doi.org/10.1016/j.indcrop.2013.07.002

  13. 13.

    Karataş H, Uyar F, Tolan V, Baysal Z (2013) Optimization and enhanced production of α-amylase and protease by a newly isolated Bacillus licheniformis ZB-05 under solid-state fermentation. Ann Microbiol 63:45–52. https://doi.org/10.1007/s13213-012-0443-6

  14. 14.

    Sukumprasertsri M, Unrean P, Pimsamarn J, Kitsubun P, Tongta A (2013) Fuzzy logic control of rotating drum bioreactor for improved production of amylase and protease enzymes by Aspergillus oryzae in solid-state fermentation. J Microbiol Biotechnol 23:335–342. https://doi.org/10.4014/jmb.1204.04038

  15. 15.

    Dar GH, Kamili AN, Nazir R, Bandh SA, Jan TR, Chishti MZ (2015) Enhanced production of α-amylase by Penicillium chrysogenum in liquid culture by modifying the process parameters. Microb Pathog 88:10–15. https://doi.org/10.1016/j.micpath.2015.07.016

  16. 16.

    Robertson JB, Davis CR, Jhonson JH (2013) Visible light alters yeast metabolic rhythms by inhibiting respiration. PNAS 110:21130–21135. https://doi.org/10.1073/pnas.1313369110

  17. 17.

    Richert I (2014) The influence of light and water mass on bacterial population dynamics in the Amundsen Sea Polynya. Elem Sci Anth 3:000044. https://doi.org/10.12952/journal.elementa.000044

  18. 18.

    Davis SJ, Vener AV, Vierstra RD (1999) Bacteriophytochromes: Phytochrome-like photoreceptors from nonphotosynthetic eubacteria. Science 286:2517–2520. https://doi.org/10.1126/science.286.5449.2517

  19. 19.

    Losi A, Polverini E, Quest B, Gärtner W (2002) First evidence for phototropin-related blue-light receptors in prokaryotes. Biophys J 82:2627–2634. https://doi.org/10.1016/S0006-3495(02)75604-X

  20. 20.

    Gomelsky M, Hoff WD (2011) Light helps bacteria make important lifestyle decisions. Trends Microbiol 19:441–448. https://doi.org/10.1016/j.tim.2011.05.002

  21. 21.

    Elumalai P, Park YJ, Min C, Shea PJ, Oh BT (2019) Red yeast rice fermentation with Bacillus subtilis B2 under blue light emitting diodes increases antioxidant secondary products. Bioprocess Biosyst Eng 42:529–539. https://doi.org/10.1007/s00449-018-2056-3

  22. 22.

    Elumalai P, Lim JM, Park YJ, Min C, Shea PJ, Oh BT (2018) Enhanced amylase production by a Bacillus subtilis strain under blue light-emitting diodes. Prep Biochem Biotechnol 49:143–150. https://doi.org/10.1080/10826068.2018.1550656

  23. 23.

    Margit Olle AV (2013) The effect of light-emitting diode lighting on greenhouse plant growth and quality. Agric Food Sci 22:223–234. https://doi.org/10.1016/j.envexpbot.2009.06.011

  24. 24.

    Yi ZL, Huang WF, Ren Y, Onac E, Zhou GF, Pengb S, Wanga XJ, Li HH (2014) LED lights increase bioactive substances at low energy costs in culturing fruiting bodies of Cordyceps militaris. Sci Hortic 175:139–143. https://doi.org/10.1016/j.scienta.2014.06.006

  25. 25.

    Morrow RC (2008) LED lighting in horticulture. Hort Sci 43:1947–1950. https://doi.org/10.1007/978-981-10-5807-3_7

  26. 26.

    Elumalai P, Parthipan P, Narenkumar J, Anandakumar B, Madhavan J, Oh BT, Rajasekar A (2019) Role of thermophilic bacteria (Bacillus and Geobacillus) on crude oil degradation and biocorrosion in oil reservoir environment. 3 Biotech 9:79. https://doi.org/10.1007/s13205-019-1604-0

  27. 27.

    Qureshi AS, Khushk I, Ali CH, Chisti Y, Ahmad A, Majeed H (2016) Coproduction of protease and amylase by thermophilic Bacillus sp. BBXS-2 using open solid-state fermentation of lignocellulosic biomass. Biocatal Agric Biotechnol 8:146–151. https://doi.org/10.1016/j.bcab.2016.09.006

  28. 28.

    Association of Official Analytical Chemists (AOAC) (2010) In: Horwitz W (Ed) Official methods of analysis of the Association of Official Agriculture Chemistry, Washington

  29. 29.

    Salihi A, Asoodeh A, Aliabadian M (2017) Production and biochemical characterization of an alkaline protease from Aspergillus oryzae CH93. Int J Biol Macromol 94:827–835. https://doi.org/10.1016/j.ijbiomac.2016.06.023

  30. 30.

    Acebal C, Castillon MP, Estrada P, Mata I, Costa E, Aguado J, Romero D, Jimenez F (1986) Enhanced cellulase production from Trichoderma reesei QM 9414 on physically treated wheat straw. Appl Microbiol Biotechnol 24:218–233. https://doi.org/10.1007/BF00261540

  31. 31.

    Sathishkumar R, Ananthan G, Arun J (2015) Production, purification and characterization of alkaline protease by ascidian associated Bacillus subtilis GA CAS8 using agricultural wastes. Biocatal Agric Biotechnol 4:214–220. https://doi.org/10.1016/j.bcab.2014.12.003

  32. 32.

    Sankareswaran M, Anbalagan S, Prabhavathi P (2014) Optimization of production of an extracellular alkaline protease by soil isolated Bacillus species using submerged and solid-state fermentation with agricultural wastes. Afr J Microbiol Res 8:872–877. https://doi.org/10.5897/AJMR11.1273

  33. 33.

    Lee S, Lee J, Jin YI, Jeong JC, Chang YH, Lee Y, Jeong Y, Kima M (2017) Probiotic characteristics of Bacillus strains isolated from Korean traditional soy sauce. LWT Food Sci Technol 79:518–524. https://doi.org/10.1016/j.lwt.2016.08.040

  34. 34.

    Deng A, Wu J, Zhang Y, Zhang G, Wen T (2010) Purification and characterization of a surfactant-stable high-alkaline protease from Bacillus sp. B001. Bioresour Technol 101:7100–7106. https://doi.org/10.1016/j.biortech.2010.03.130

  35. 35.

    Jain D, Pancha I, Mishra SK, Shrivastav A, Mishra S (2012) Purification and characterization of haloalkaline thermoactive, solvent stable and SDS-induced protease from Bacillus sp.: a potential additive for laundry detergents. Bioresour Technol 115:228–236. https://doi.org/10.1016/j.biortech.2011.10.081

  36. 36.

    Annamalai N, Rajeswari MV, Sahu SK, Balasubramanian T (2014) Purification and characterization of solvent stable, alkaline protease from Bacillus firmus CAS 7 by microbial conversion of marine wastes and molecular mechanism underlying solvent stability. Process Biochem 49:1012–1019. https://doi.org/10.1016/j.procbio.2014.03.007

  37. 37.

    Hadjidj R, Badis A, Mechri S, Eddouaouda K, Khelouia L, Annane R, El Hattab M, Jaouadi B (2018) Purification, biochemical, and molecular characterization of novel protease from Bacillus licheniformis strain K7A. Int J Biol Macromol 114:1033–1048. https://doi.org/10.1016/j.ijbiomac.2018.03.167

  38. 38.

    Jaouadi B, Ellouz-Chaabouni S, Rhimi M, Bejar S (2008) Biochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS with high catalytic efficiency. Biochimie 90:1291–1305. https://doi.org/10.1016/j.biochi.2008.03.004

  39. 39.

    Demirkan E, Dincbas S, Sevinc N, Ertan F (2011) Immobilization of B. amyloliquefaciens α-amylase and comparison of some of its enzymatic properties with the free form. Rom Biotechnol Lett 16:6690–6701

  40. 40.

    Saggu SK, Mishra PC (2017) Characterization of thermostable alkaline proteases from Bacillus infantis SKS1 isolated from garden soil. PLoS ONE 12:e0188724. https://doi.org/10.1371/journal.pone.0188724

  41. 41.

    Mokashe N, Chaudhari A, Patila U (2015) Optimal production and characterization of alkaline protease from newly isolated halotolerant Jeotgalicoccus sp. Biocatal Agric Biotechnol 4:235–243. https://doi.org/10.1016/j.bcab.2015.01.003

  42. 42.

    Kecha M, Benallaoua S, Touzel JP, Bonaly R, Duchiron F (2007) Biochemical and phylogenetic characterization of a novel terrestrial hyperthermophilic archaeon pertaining to the genus Pyrococcus from an Algerian hydrothermal hot spring. Extremophiles 11:65–73. https://doi.org/10.1007/s00792-006-0010-9

  43. 43.

    Joo HS, Kumar CG, Park GC, Paik SR, Chang CS (2003) Oxidant and SDS-stable alkaline protease from Bacillus clausii I-52: production and some properties. J Appl Microbiol 95:267–272. https://doi.org/10.1046/j.1365-2672.2003.01982.x

  44. 44.

    Jellouli K, Ghorbel-Bellaaj O, Ayed HB, Manni L, Agrebi R, Nasri M (2011) Alkaline-protease from Bacillus licheniformis MP1: purification, characterization and potential application as a detergent additive and for shrimp waste deproteinization. Process Biochem 46:1248–1256. https://doi.org/10.1016/j.procbio.2011.02.012

  45. 45.

    Singh SK, Singh SK, Tripathi VR, Garg SK (2012) Purification, characterization and secondary structure elucidation of a detergent stable, halotolerant, thermoalkaline protease of Bacillus cereus SIU1. Process Biochem 47:1479–1487. https://doi.org/10.1016/j.procbio.2012.05.021

  46. 46.

    Shah K, Mody K, Keshri J, Jha B (2010) Purification and characterization of a solvent, detergent and oxidizing agent tolerant protease from Bacillus cereus isolated from the Gulf of Khambhat. J Mol Catal B Enzym 67:85–91. https://doi.org/10.1016/j.molcatb.2010.07.010

Download references

Acknowledgements

This work was supported by Korea Environment Industry & Technology Institute (KEITI) through Public Technology Program based on Environmental Policy, funded by Korea Ministry of Environment (MOE) (2018000200001).

Author information

Correspondence to Min Cho or Byung-Taek Oh.

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 (DOCX 231 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Elumalai, P., Lim, J., Park, Y. et al. Agricultural waste materials enhance protease production by Bacillus subtilis B22 in submerged fermentation under blue light-emitting diodes. Bioprocess Biosyst Eng (2020). https://doi.org/10.1007/s00449-019-02277-5

Download citation

Keywords

  • Agricultural waste products
  • Blue LEDs
  • B. subtilis
  • Protease
  • Submerged fermentation