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A Detergent-Compatible Alkaline Metalloprotease from Bacillus pseudofirmus BBAU-19: Characterization and Application

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Proceedings of the National Academy of Sciences, India Section B: Biological Sciences Aims and scope Submit manuscript

Abstract

We isolated a bacterium (BBAU-19) producing alkaline protease from the saline soil of Uttar Pradesh, India. The isolate identified through 16S rRNA gene analysis was Bacillus pseudofirmus. The enzyme production of BBAU-19 was optimum at pH 9 and a temperature of 40 °C. The ammonium sulfate salt partially purified the crude enzyme. The filtered enzyme was analyzed under ion-exchange chromatography through a DEAE-cellulose column. The partially purified enzyme (PPE) activity increased up to ~ 1.92-fold compared to crude protease, and the recovery of protease enzyme was 54.60%. The molecular weight of the PPE was determined to be 42 kDa. The optimum temperature and pH of the PPE were 40 °C and 10.0, respectively. Ca++ and Mg++ ions enhanced the enzyme activity, while Cu++and Na+ ions suppressed it. The presence of ethylenediaminetetraacetic acid completely inhibited the enzyme activity. The findings of this study suggest that the enzyme is a Metallo-type protease in nature. We evaluated the efficiency of alkaline protease by testing its ability to remove stains of the cloth stained with Bradford reagent. The compatibility test with detergents enhanced the commercial use of this enzyme. The PPE (partially purified enzyme) was compatible with Ghadi and Nirma detergents, and the de-staining was maximum with 500 ml of the PPE.

Graphical Abstract

A Screening of the bacterial isolate BBAU-19, B Enzyme production, C Biochemical assay and enzyme activity determination of the bacterial isolate BBAU-19; D Purification and detergent formulation; BSA-soaked cloths were washed with—(DA) only water, (DB) local laundry detergents, (DC) local laundry detergents containing 250 ml of alkaline protease and (DD) local laundry detergents containing 500-ml alkaline protease followed by Bradford staining [Cloths washed with only water (DA) displayed maximum blue color, while cloths washed with detergent containing 500 ml of the enzyme (DD) displayed minimum blue color after Bradford staining]

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References

  1. Razzaq A, Shamsi S, Ali A, Ali Q, Sajjad M, Malik A, Ashraf M (2019) Microbial proteases applications. Front Bioeng Biotechnol 7:110. https://doi.org/10.3389/fbioe.2019.00110

    Article  PubMed  PubMed Central  Google Scholar 

  2. Sharma M, Gat Y, Arya S, Kumar V, Kumar A (2019) Review on microbial alkaline protease: an essential tool for various industrial approaches. Ind Biotechnol 15(2):69–78. https://doi.org/10.1089/ind.2018.0032

    Article  CAS  Google Scholar 

  3. Dorra G, Ines K, Imen SB, Laurent C, Sana A, Olfa T, Pascal C, Thierry J, Ferid L (2018) Purification and characterization of a novel high molecular weight alkaline protease produced by an endophytic Bacillus halotolerans strain CT2. Int J Biol Macromol 111:342–351. https://doi.org/10.1016/j.ijbiomac.2018.01.024

    Article  CAS  PubMed  Google Scholar 

  4. Cavello I, Bezus B, Cavalitto S (2021) The keratinolytic bacteria Bacillus cytotoxicus as a source of novel proteases and feather protein hydrolysates with antioxidant activities. J Genet Eng Biotechnol 19:107. https://doi.org/10.1186/s43141-021-00207-1

    Article  PubMed  PubMed Central  Google Scholar 

  5. Sundus H, Mukhtar H, Nawaz A (2016) Industrial applications and production sources of serine alkaline proteases: a review. J Bacteriol Mycol 3(1):1–4. https://doi.org/10.15406/jbmoa.2016.03.00051

    Article  Google Scholar 

  6. Cui H, Wang L, Yu Y (2015) Production and characterization of alkaline protease from a high yielding and moderately halophilic strain of SD11 MARINE BACteria. J Chem. https://doi.org/10.1155/2015/798304

    Article  Google Scholar 

  7. Chatterjee J, Giri S, Maity S, Sinha A, Ranjan A, Rajshekhar et al (2015) Production and characterization of thermostable alkaline protease of Bacillus subtilis (ATCC 6633) from optimized solid-state fermentation. Biotechnol Appl Biochem 62:709–718. https://doi.org/10.1002/bab.1309

    Article  CAS  PubMed  Google Scholar 

  8. Ibrahim ASS, Al-Salamah AA, Elbadawi YB, El-Tayab MA, Ibrahim SSS (2015) Production of extracellular alkaline protease halotolerentalkaliphilic Bacillus sp. NPST-AK15 isolated from hyper saline soda lakes. Electron J Biotechnol 18:236–243. https://doi.org/10.1016/j.ejbt.2015.04.001

    Article  Google Scholar 

  9. Verma J, Pandey S (2019) Characterization of partially purified alkaline protease secreted by halophilic bacterium Citricoccus sp. isolated from agricultural soil of northern India. Biocatal Agric Biotechnol 17:605–612. https://doi.org/10.1016/j.bcab.2019.01.020

    Article  Google Scholar 

  10. Cheng K, Lu FP, Li M, Liu LL, Liang XM (2010) Purification and biochemical characterization of a serine alkaline protease TC4 from a new isolated Bacillus alcalophilus TCCC11004 in detergent formulations. Afr J Biotechnol 9:4942–4953

    CAS  Google Scholar 

  11. Tsuchida O, Yamagata Y, Ishizuka J (1986) An Alkaline proteinase an alkalophilic Bacillus sp. Curr Microbiol 14:7–12. https://doi.org/10.1007/BF01568094

    Article  CAS  Google Scholar 

  12. Lowry OH, Rosebrough NI, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    Article  CAS  PubMed  Google Scholar 

  13. Upadhyay SK, Chandrashekar K (2012) Interaction of salivary and Midgut Proteins of Helicoverpa armigera with soybean Trypsin Inhibitors. Protein J 31:259–115. https://doi.org/10.1007/s10930-012-9402-0

    Article  CAS  PubMed  Google Scholar 

  14. Rao CS, Sathish T, Ravichandra P, Prakasham R (2009) Characterization of thermo-and detergent stable serine protease from isolated Bacillus circulans and evaluation of eco-friendly applications. Process Biochem 44(3):262–268. https://doi.org/10.1016/j.procbio.2008.10.022

    Article  CAS  Google Scholar 

  15. Mothe T, Sultanpuram VR (2016) Production, purification and characterization of a thermotolerant alkaline serine protease from a novel species Bacillus caseinilyticus. 3 Biotech 6:53. https://doi.org/10.1007/s13205-016-0377-y

    Article  PubMed  PubMed Central  Google Scholar 

  16. Bradford MM (1976) A rapid and sensitive method for the quantitation of microorganism quantities of protein utilizing the principle of protein–dye binding. Ann Biochem 72(1–2):248–254. https://doi.org/10.1016/0003-2697(76)90527-3

    Article  CAS  Google Scholar 

  17. Sen S, Veeranki VD, Mandal B (2009) Effect of physical parameters, carbon and nitrogen sources onthe production of alkaline protease from a newly isolated Bacillus pseudofirmus SVB1. Ann Microbiol 59(3):531–538

    Article  CAS  Google Scholar 

  18. Gupta R, Beg OK, Lorenz P (2002) Bacterial alkaline proteases: molecular approaches and industrial application. Appl Microbiol Biotechnol 59:15–32. https://doi.org/10.1007/s00253-002-1142-1

    Article  CAS  PubMed  Google Scholar 

  19. Saggu SK, Mishra PC (2017) Characterization of thermostable alkaline proteases from Bacillus infantis SKS1 isolated from garden soil. PLoS ONE 12(11):e0188724. https://doi.org/10.1371/journal.pone.0188724

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Sepahy AA, Jabalameli L (2011) Effect of culture conditions on the production of an extracellular protease by Bacillus sp. Isolated from soil sample of Lavizan Jungle park. Enzyme Res 2011:1–7. https://doi.org/10.4061/2011/219628

    Article  CAS  Google Scholar 

  21. Ahmetoglu N, Bekler FM, Acer O, Guven RG, Guven K (2015) Production, purification and characterization of thermostable metallo-protease from newly isolated Bacillus sp. KG5. Eurasia J Biosci 9:1–11. https://doi.org/10.5053/ejobios.2015.9.0.l

    Article  CAS  Google Scholar 

  22. Vijayraghavan P, Lazarus S, Vincent SGP (2014) De-hairing protease production by an isolated Bacillus cereus strain AT under solid-state fermentation using cow dung: Biosynthesis and properties. Saudi J Biol Sci 21(1):27–34. https://doi.org/10.1016/j.sjbs.2013.04.010

    Article  CAS  Google Scholar 

  23. Iqbal A, Hakim A, Hossain MS, Rahman MR, Islam K, Azim MF, Ahmed J, Assaduzzaman M, Hoq MM, Azad AK (2018) Partial purification and characterization of serine protease produced through fermentation of organic municipal solid wastes by Serratia marcescens A3 and Pseudomonas putida A2. J Genet Eng Biotechnol 16:29–37. https://doi.org/10.1016/j.jgeb.2017.10.011

    Article  PubMed  Google Scholar 

  24. Waghmore S, Gurav AA, Mali SA, Nadaf NH, Jadhav DB, Sonawane KD (2015) Purification and characterization of novel organic solvent tolerant 98 k Da alkaline protease isolated from Stenotrophomonas maltophilia strain SK. Protein Expr Purif 107:1–6. https://doi.org/10.1016/j.pep.2014.11.002

    Article  CAS  Google Scholar 

  25. Manavalan T, Manavalan A, Ramachandran S, Heese K (2020) Protease from Bacillus megaterium -TK1 for the detergent and leather industry. Biology 9:472. https://doi.org/10.3390/biology9120472

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Saxena S, Verma J, Shikha MDR (2014) RAPD-PCR and 16S rDNA phylogenetic analysis of alkaline protease producing bacteria isolated from soil of India: identification and detection of genetic variability. J Genet Eng Biotechnol 12:27–35. https://doi.org/10.1016/j.jgeb.2014.03.001

    Article  Google Scholar 

  27. Saggu SK, Gopaljee J, Mishra PC (2019) Enzymatic degradation of biofilm by metalloprotease from Microbacterium sp. Sks 10. Front Bioeng Biotechnol 7:192. https://doi.org/10.3389/fbioe.2019.00192

    Article  PubMed  PubMed Central  Google Scholar 

  28. Harwood CR, Kikuchi Y (2022) The ins and outs of Bacillus proteases: activities, functions and commercial significance. FEMS Microbiol Rev FUAB046 46:1–20. https://doi.org/10.1093/femsre/fuab046

    Article  CAS  Google Scholar 

  29. Arnaouteli S, Bamford NC, Stanley-Wall NR et al (2021) Bacillus subtilis biofilm formation and social interactions. Nat Rev Microbiol 19:600–614

    Article  CAS  PubMed  Google Scholar 

  30. Inbaraj S, Agrawal RK, Thomas P, Chaudhuri P, Verma A, Chaturvedi VK (2022) Isolation and characterization of vB_SenS_Ib_psk2 bacteriophage against drug resistant Salmonella enterica serovar Kentucky. Res Sq. https://doi.org/10.21203/rs.3.rs-1900211/v1

    Article  Google Scholar 

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Acknowledgements

This work was facilitated by the Department of Biotechnology from Babasaheb Bhimrao Ambedkar University, Lucknow 226025 (India), and the authors are thankful to execute the research work. The authors have no relevant financial or non-financial interests to disclose. Thus, there is no conflict of interest among the authors.

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Authors and Affiliations

  1. Department of Biotechnology, Babasaheb Bhimarao Ambedkar University, Vidya Vihar, Raebareli road, Lucknow, India

    Jyoti Verma & Sangeeta Saxena

  2. Amity Institute of Organic Agriculture, Amity University Uttar Pradesh, Noida, Uttar Pradesh, India

    Sangeeta Pandey, Chitranjan Kumar & Sangeeta Saxena

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  1. Jyoti Verma
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Correspondence to Sangeeta Pandey.

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We partially purified and characterized the bacteria Bacillus pseudofirmus (BBAU-19) from saline soil that produced alkaline protease. The optimum conditions for temperature and pH were 40 °C and 10, respectively. Ca++ and Mg++ ions activate the Metallo-type enzyme activity, whereas Cu ++and Na+ ions inhibit the enzyme activity. The enzyme compatibility was evaluated with two detergents (Ghadi and Nirma), and the identified enzyme was efficient in removing cloth stains.

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Verma, J., Pandey, S., Kumar, C. et al. A Detergent-Compatible Alkaline Metalloprotease from Bacillus pseudofirmus BBAU-19: Characterization and Application. Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. 93, 499–510 (2023). https://doi.org/10.1007/s40011-022-01436-1

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