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. 2010 Mar 16;50(3):309–317. doi: 10.1007/s12088-010-0024-y

Partial purification and properties of a laundry detergent compatible alkaline protease from a newly isolated Bacillus species Y

M Mala 1,✉, S Srividya 2
PMCID: PMC3450070  PMID: 23100846

Abstract

Alkaline protease production by a newly isolated Bacillus species from laundry soil was studied for detergent biocompatibility. From its morphological and nucleotide sequence (about 1.5 kb) of its 16S rDNA it was identified as Bacillus species with similarity to Bacillus species Y (Gen Bank entry: ABO 55095), and close homology with Bacillus cohnii YN-2000 (Gen Bank entry: ABO23412). Partial purification of the enzyme by ammonium sulfate (50–70% saturation) yielded 8-fold purity. Casein zymography and Sodium dodecylsulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) of the partially purified enzyme revealed two isozymes of molecular sizes approximately 66 kDa and 18 kDa, respectively. The enzyme was most active at pH 12 and 50°C. At pH 12 the enzyme was stable for 5 h and retained 60% activity. The enzyme retained 44% activity at 50°C up to 2 h. The protease showed good hydrolysis specificity with different substrates tested. The presence of Mn2+, Co2+ and ethylenediaminetetracetic acid (EDTA) showed profound increase in protease activity. The protease of Bacillus species Y showed excellent stability and compatibility with three locally available detergents (Kite, Tide and Aerial) up to 3 h retaining almost 70–80% activity and 10–20% activity at room temperature (30°C) and 50°C, respectively, indicating the potential role of this enzyme for detergent application.

Keywords: Alkaline protease, Bacillus species Y

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References

  • 1.Layman P.L. Industrial enzymes: battling to remain specialties. Chem Eng News. 1986;64:11–14. [Google Scholar]
  • 2.Gupta R., Beg Q.K., Loranz P. Bacterial alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotechnol. 2002;59(1):15–32. doi: 10.1007/s00253-002-0975-y. [DOI] [PubMed] [Google Scholar]
  • 3.Prakasham R.S., Rao C.S., Rao R.S., Sarma P.N. Alkaline protease production by an isolated Bacillus circulans under solid state fermentation using agro-industrial waste: Process Parameters Optimization. Biotechnol Prog. 2005;21:1380–1388. doi: 10.1021/bp050095e. [DOI] [PubMed] [Google Scholar]
  • 4.Adinarayana K., Ellaiah P., Prasad D.S. Purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus subtilis PE-11. AAPS Pharm Sci Tech. 2003;4(4):1–9. doi: 10.1208/pt040456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Holt J.G., Krieg N.R., Sneath P.H.A., Staley J.T., Williams S.T., editors. Bergey’s Manual of Determinative Bacteriology. 9th edition. Maryland: Williams & Wilkins; 1993. pp. p559–564. [Google Scholar]
  • 6.Sumantha A., Sandhya C., Szakacs G., Carlos R., Soccol, Ashok P. Production and partial purification of a neutral metalloprotease by fungal mixed substrate fermentation. Food Technol Biotechnol. 2005;43(4):313–319. [Google Scholar]
  • 7.Kumar C.G., Tiwari M.P., Jany K.D. Novel alkaline serine proteases from alkalophilic Bacillus species: purification and characterization. Proc Biochem. 1999;34:441–449. doi: 10.1016/S0032-9592(98)00110-1. [DOI] [Google Scholar]
  • 8.Bradford M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Chem. 1976;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  • 9.Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  • 10.Oliver G.W., Stetler-Stevenson W.G., Kleiner D.E. In: Proteolytic Enzymes:Tools and Targets. Sterchi E., Stoker W., editors. Berlin: Springer-Verlag; 1999. pp. 63–76. [Google Scholar]
  • 11.Leber T.M., Balkwill F.R. Zymography: A single step staining method for quantitation of proteolytic activity on substrate gels. Anal Biochem. 1997;249:24–28. doi: 10.1006/abio.1997.2170. [DOI] [PubMed] [Google Scholar]
  • 12.Kumar C.G., Takagi H. Microbial alkaline proteases: from a bioindustrial viewpoint. Biotechnol Adv. 1999;17:561–594. doi: 10.1016/S0734-9750(99)00027-0. [DOI] [PubMed] [Google Scholar]
  • 13.Kumar C.G., Malik R.K., Tiwari M.P. Novel enzyme based detergents: an Indian perspective. Curr Sci. 1998;75:1312–1318. [Google Scholar]
  • 14.Towatana N.H., Painupong A., Suntinanaler P. Purification and characterization of an extracellular protease from alkaliphilic and thermophilic Bacillus species PS719. J Biosci Bioeng. 1999;87:581–587. doi: 10.1016/S1389-1723(99)80118-2. [DOI] [PubMed] [Google Scholar]
  • 15.Kato T., Yamagata Y., Arai T., Ichishima E. Purification of a new extracellular 90-kDa serine proteinase with isoelectric point of 3.9 from Bacillus subtilis (natto) and elucidation of its distinct mode of action. Biosci Biotech Biochem. 1992;56:1166–1168. doi: 10.1271/bbb.56.1166. [DOI] [PubMed] [Google Scholar]
  • 16.Gouda K.M. Optimization and purification of alkaline proteases produced by marine Bacillus species MIG newly isolated from Eastern Harbour of Alexandria. Polish J Microbiol. 2006;55(2):119–126. [PubMed] [Google Scholar]
  • 17.Paliwal N., Singh S.P., Garg S.K. Cation-induced thermal stability of an alkaline protease from a Bacillus species. Bioresource Technol. 1994;50:209–211. doi: 10.1016/0960-8524(94)90091-4. [DOI] [Google Scholar]
  • 18.Ghorbel B., Kamoun A.S., Nasri M. Stability studies of protease from Bacillus cereus BGI. Enzyme Microbiol Technol. 2003;32:513–518. doi: 10.1016/S0141-0229(03)00004-8. [DOI] [Google Scholar]
  • 19.Beg Q.K., Gupta R. Purification and characterization of an oxidation-stable, thiol-dependent serine alkaline protease from Bacillus mojavensis. Enzyme Microbiol Technol. 2003;32:294–304. doi: 10.1016/S0141-0229(02)00293-4. [DOI] [Google Scholar]
  • 20.Ammar MS, Bayoumi RA, El-Kasaby AMH and Soliman AM (2003) Purification and properties of thermostable protease by B. brevis geltinoamylolyticus using fish wastes (Fi.W.) and poultry wastes (Po.W) under solid state fermentation (S.S.F.) conditions 5th Int. Sic. Conf. Al-Azhar Univ. Fac. Sci. March 25–27, 2003, Cairo, Egypt, pp 54
  • 21.Freeman S.A., Peek K., Prescott M., Daniel R. Characterization of a chelator-resistant proteinase from Thermus strain Rt4A2. Biochem J. 1993;295:463–469. doi: 10.1042/bj2950463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Rahman R.N.Z.A., Razak C.N., Ampon K., Basri M., Yunus W.M.Z.W., Salleh A.B. Purification and characterization of a heat stable alkaline protease from Bacillus Stearothermophilus F1. Appl Microbiol Biotechnol. 1994;40:822–827. doi: 10.1007/BF00173982. [DOI] [Google Scholar]
  • 23.Venugopal M., Saramma A.V. An alkaline protease from Bacillus circulans BM15, newly isolated from a mangrove station: characterization and application in laundry detergent formulations. Indian J Microbiol. 2007;47:298–303. doi: 10.1007/s12088-007-0055-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Singh J., Batra N., Sobti R.C. Serine alkaline protease from newly isolated Bacillus species SSR1. Proc Biochem. 2001;36:781–785. doi: 10.1016/S0032-9592(00)00275-2. [DOI] [Google Scholar]
  • 25.Bhosale S.H., Rao M.B., Deshpande V.V., Srinivasan M.C. Thermostability of high-activity alkaline protease from Conidiobolus coronatus (NCL 86.8.20) Enzyme Microbiol Technol. 1995;17:136–139. doi: 10.1016/0141-0229(94)00045-S. [DOI] [Google Scholar]
  • 26.Ingale S.S., Rele M.V., Srinivasan M.C. Alkaline protease production by Basidiolobus (NCL 97.1.1): Effect of ‘darmform’ morphogenesis and cultural conditions on enzyme production and preliminary enzyme characterization. World J Microbiol Biotechnol. 2002;8:403–408. doi: 10.1023/A:1015596108518. [DOI] [Google Scholar]
  • 27.Banik R.M., Prakash M. Laundry detergent compatibility of the alkaline protease from Bacillus cereus. Microbiol Res. 2004;159:135–140. doi: 10.1016/j.micres.2004.01.002. [DOI] [PubMed] [Google Scholar]

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