Skip to main content
SpringerLink
Log in

Biochemical analysis and investigation on the prospective applications of alkaline protease from a Bacillus cereus strain

  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Proteases have prospective financial and environment-friendly applications; hence attention is focused currently on the finding of new protease producing microorganism so as to meet the requirements of industry. A thermophilic bacterial strain producing extracellular protease activity was isolated from soil and identified as Bacillus cereus by analysis of 16S rRNA. Protease production by the microorganism was improved by studying the impact of the type of nitrogen and carbon source, fermentation period, growth temperature and initial pH of the culture medium in cultivation optimization experiments. The enzyme was purified to homogeneity in two step procedure involving Sephadex G-75 and Q-Sepharose chromatography. The molecular weight of purified enzyme was found to be 58 kDa by SDS-PAGE. Protease exhibited a pH and temperature optima of 7.5 and 60°, respectively. The enzyme was active in the pH range of 6.0–9.0 and stable up to 70°C. Histological analysis of protease treated goat and cow skin pelts showed complete removal of non leather forming structures such as hair shaft, hair follicles and glandular structures. The protease showed the stain removing property from blood stained cotton cloth and found to be compatible with six commercially available detergents. The protease could release peptides from natural proteins after digestion of coagulated egg albumin and blood clot.

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

References

  1. Sookkheo B, Sinchaikul S, Phutrakul S, Chen ST (2000) Purification and characterization of the highly thermostable proteases from Bacillus stearothermophilus TLS33. Protein Exp Purif 20:142–151

    Article  CAS  Google Scholar 

  2. Beg QK, Sahai V, Gupta R (2003) Statistical media optimization and alkaline protease production from Bacillus mojavensis in a bioreactor. Process Biochem 39:203–209

    Article  CAS  Google Scholar 

  3. Rao MB, Tanksale AM, Mohini SG, Deshpande VV (1998) Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev 62:597–602

    PubMed  CAS  Google Scholar 

  4. Kumar R, Balaji S, Uma TS, Mandal AB, Sehgal PK (2010) Optimization of influential parameters for extracellular keratinase production by Bacillus subtilis (MTCC9102) in solid state fermentation using horn meal—a biowaste management. Appl Biochem Biotechnol 160:30–39

    Article  PubMed  CAS  Google Scholar 

  5. Cheng-gang CAI, Ji-shuang C, Jiong-jiong QI, Yun Y, Xiao-dong Z (2008) Purification and characterization of keratinase from a new Bacillus subtilis strain. J Zhejiang Univ Sci 9:713–720

    Article  Google Scholar 

  6. Zhu HY, Tian Y, Hou YH, Wang T (2009) Purification and characterization of the cold-active alkaline protease from marine cold-adaptive Penicillium chrysogenum. Mol Biol Rep 36:2169–2174

    Article  PubMed  CAS  Google Scholar 

  7. Cowan DA (1994) Industrial Enzymes. In: Moses V, Cape RE (eds) Biotechnology—the science and the business. Harwood Academic Publishers, Switzerland, pp 326–328

    Google Scholar 

  8. Mizuho N, Tomiko A, Etsuko K, Mam M, Wakako E, Tomoko T, Akio O, Keiko A, Junko F (2008) Application of an Aspergillus saitoi protease preparation to soybean curd to modify its functional and rheological properties. Biosci Biotechnol Biochem 72:587–590

    Article  Google Scholar 

  9. Gupta R, Beg QK, Lorenz P (2002) Bacterial alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotechnol 59:15–17

    Article  PubMed  CAS  Google Scholar 

  10. Han HW, Srinivasan VR (1968) Isolation and characterization of cellulose utilizing bacterium. Appl Microbiol 16:1140–1145

    PubMed  CAS  Google Scholar 

  11. Kunitz M (1947) Crystalline soybean trypsin inhibitor. J Gen Physiol 30:291–310

    Article  PubMed  CAS  Google Scholar 

  12. Bradford MM (1976) A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  13. Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  14. Shafee N, Sayangku NA, Raja NZ B, Mahiran AR, Abu Bakar S (2005) Optimization of environmental and nutritional conditions for the production of alkaline protease by a newly isolated bacterium Bacillus cereus strain 146. J Appl Sci Res 1:21–28

    Google Scholar 

  15. Chu IM, Lee CLi TS (1992) Production and degradation of alkaline protease in batch cultures of Bacillus subtilis ATCC 14416. Enz Microbial Technol 14:755–761

    Article  CAS  Google Scholar 

  16. Nilegaonkar SS, Zambare V, Kanekar PP, Dhakephalkar PK, Sarnai SS (2007) Production and partial characterization of dehairing protease from Bacillus cereus MCM B-326. Biores Technol 98:1238–1245

    Article  CAS  Google Scholar 

  17. El-Safey EM, Abdul-Raouf (2004) Production, purification and characterization of protease enzyme from Bacillus subtilis. International Conferences for Development and the Environment in the Arab World, Assiut University

  18. Chellappan S, Jasmin C, Soorej M, Basheer KK, Elyas SGB, Chandrasekaran M (2006) Production, purification and partial characterization of a novel protease from marine Engyodontium album BTMFS10 under solid state fermentation. Process Biochem 41:956–961

    Article  CAS  Google Scholar 

  19. da Silva CRD, Andria BD, Meire LL (2007) Effect of the culture conditions on the production of an extracellular protease by thermophilic Bacillus sp. and some properties of the enzymatic activity. Braz J Microbiol 38:171–173

    Google Scholar 

  20. Guangrong H, Dai D, Hu W, Jiang J (2008) Optimization of medium composition for thermostable protease production by Bacillus sp. HS08 with a statistical method. Afr J Biotechnol 7:1115–1122

    Google Scholar 

  21. Morita Y, Kenji K, Quamrul H, Toshifumi S, Yuji M, Kenji Y, Eiichi T (1997) Purification and characterization of a cold-active protease from psychrotrophic Serratia marcescens AP3801. J Am Oil Chemists Soc 74:1377–1383

    Article  CAS  Google Scholar 

  22. Doddapaneni KK, Tatineni R, Vellanki RN, Rachcha S, Anabrolu N, Narakuti V, Mangamoori LK (2009) Purification and characterization of a solvent and detergent-stable Bacillus cereus. Microbiol Res 164:383–390

    Article  PubMed  CAS  Google Scholar 

  23. Ghorbel-Frikha B, Sellami-Kamoun A, Fakhfakh N, Haddar A, Manni L, Nasri M (2005) Production and purification of a calcium-dependent protease from Bacillus cereus BG1. J Ind Microbiol Biotechnol 32:186–194

    Article  PubMed  CAS  Google Scholar 

  24. Chaplin MF, Bucke C (1990) Enzyme technology. Cambridge University Press, Cambridge

    Google Scholar 

  25. Rao K, Lakshmi N (2007) Alkaline protease from Bacillus firmus 7728. Afr J Biotechnol 6:2493–2496

    CAS  Google Scholar 

  26. Reddy L, Wee YJ, Ryu HW (2008) Purification and characterization of anorganic solvent and detergent-tolerant novel protease produced by Bacillus sp. RKY3. J Chemical Technol Biotechnol 83:1526–1533

    Article  CAS  Google Scholar 

  27. 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 95:267–271

    Article  PubMed  CAS  Google Scholar 

  28. Pawar R, Zambare V, Barve S, Paratkar G (2009) Application of protease isolated from Bacillus sp. 158 in enzymatic cleansing of contact lenses. Biotechnology 8:276–280

    Article  CAS  Google Scholar 

  29. Kaur S, Vohra RM, Kapoor M, Beg QK, Hoondal GS (2001) Enhanced production and characterization of a highly thermostable alkaline protease from Bacillus sp. P-2. World J Microbiol Biotechnol 17:125–129

    Article  CAS  Google Scholar 

  30. Kamoun AL, Haddar A, Ali NE, Frikha BG, Kanoun S, Nasri M (2008) Stability of thermostable alkaline protease from Bacillus licheniformis RP1 in commercial solid laundry detergent formulations. Microbial Res 163:299–306

    Article  Google Scholar 

  31. Thanikaivelan P, Bharath CK, Saravanabhavan S, Chandrasekaran B, Rao JR, Chandrababu NK, Nair BU (2007) Integrated hair removal and fiber opening process using mixed enzymes. Clean Technol Environ Policy 9:61–68

    Article  CAS  Google Scholar 

  32. Mukhtar H, Haq I (2008) Production of alkaline protease by Bacillus subtilis and its application as a depilating agent in leather processing. Pak J Bot 40:673–1679

    Google Scholar 

  33. Pillai P, Archana G (2008) Hide depilation and feather disintegration studied with keratinolytic serine protease from a novel Bacillus subtilis isolate. Appl Microbiol Biotechnol 78:643–650

    Article  PubMed  CAS  Google Scholar 

  34. Rao CS, Sathish T, Ravichandra P, Prakasham RS (2009) Characterization of thermo- and detergent stable serine protease from isolated Bacillus circulans and evaluation of eco-friendly applications. Process Biochem 44:262–268

    Article  Google Scholar 

  35. Sivasubramanian S, Murali BM, Rajaram A, Puvanakrishnan R (2008) Ecofriendly lime and sulphide free enzymatic dehairing of skins and hides using a bacterial alkaline protease. Chemophere 70:1015–1024

    Article  CAS  Google Scholar 

  36. Macedo AJ, Da Silva W, Gava R, Driemeier D, Henriques JAP, Termignoni C (2005) Novel keratinase from Bacillus subtillus S14 exhibiting remarable dehairing capabilities. Appl Environ Microbiol 71:594–596

    Article  PubMed  CAS  Google Scholar 

  37. Thanikaivelan P, Rao JR, Nair BU, Ramasami T (2004) Progress and recent trends in biotechnological methods for leather processing. Trends Biotechnol 22:181–188

    Article  PubMed  CAS  Google Scholar 

  38. Zambare VP, Nilegaonkar SS, Kanekar PP (2007) Production of an alkaline protease by Bacillus cereus MCM B-326 and its application as a dehairing agent. World J Microbiol Biotechnol 23:1569–1574

    Article  CAS  Google Scholar 

  39. Najafi MF, Dileep D, Deepti D (2005) Potential application of protease isolated from Pseudomonas aeruginosa PD100. Electron J Biotechnol 8:197–203

    Article  CAS  Google Scholar 

  40. Banik RM, Prakash M (2004) Laundry detergent compatibility of the alkaline protease from Bacillus cereus. Res Microbiol 159:135–140

    Article  CAS  Google Scholar 

  41. Brown DA, Cameron BA, Meyer SS (1993) A survey of commercial laundry detergent- How effective they are? Part 1: Powders. J Consumer Studies Home Econom 171:145–152

    Article  Google Scholar 

  42. Shikha AS, Darmwal NS (2007) Improved production of alkaline proteases from a mutant form of alkalophilic Bacillus pantotheneticus using molasses as a substrate. Biores Technol 98:881–885

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore, 54590, Pakistan

    Mahjabeen Saleem & Atiqa Rehman

  2. Sheikh Zayed Fedral Postgraduate Medical Institute, Lahore, 54590, Pakistan

    Riffat Yasmin

  3. Institute of Industrial Biotechnology, Government College University, Lahore, 54590, Pakistan

    Bushra Munir

Authors
  1. Mahjabeen Saleem
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Atiqa Rehman
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Riffat Yasmin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bushra Munir
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mahjabeen Saleem.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 161 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Saleem, M., Rehman, A., Yasmin, R. et al. Biochemical analysis and investigation on the prospective applications of alkaline protease from a Bacillus cereus strain. Mol Biol Rep 39, 6399–6408 (2012). https://doi.org/10.1007/s11033-011-1033-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-011-1033-6

Keywords

Search

Navigation