Characterization of an alkaline protease with high quality bating potential in leather processing from Bacillus licheniformis MZK05M9 mutant

  • Authors

    • Md. Arafat Al Mamun Centre for Advanced Research in Sciences (CARS), University of Dhaka, Dhaka-1000, Bangladesh.
    • Md. Murad Khan Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh
    • Md. Nahinur Rahmam Akand Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh
    • Shakila Nargis Khan Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh
    • Md. Mozammel Hoq Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh
    2015-03-24
    https://doi.org/10.14419/ijbr.v3i1.4250
  • Alkaline Protease, Bacillus licheniformis MZK05M9 (BlM9), Bating, Characterization, Eco-Friendly.
  • An alkaline protease from Bacillus licheniformis MZK05M9 (BlM9), a mutant strain developed in our laboratory, has been partially purified and characterized for its robustness and eco-friendly application potential in processing of hides and skins for leather manufacturing and detergent industries. The enzyme was purified 2.70 fold with specific activity of 1624U/mg in comparison to crude enzyme extract by using ammonium sulfate precipitation, dialysis and Sephadex G-75 column chromatography. The molecular mass of the enzyme was 27.2 kDa as judged by SDS–PAGE. The purified protease had a pH optimum of 8.5 and temperature optimum of 55°C. According to the inhibition profiles obtained with the various protease inhibitors, it was confirmed that the partially purified protease belongs to the serine protease type. The activity of partially purified enzyme was enhanced by calcium, magnesium, barium, potassium and manganese ions and strongly inhibited by mercury ion. In addition, the protease showed remarkable stability in the presence of 1% SDS; 1, 3 and 5% Triton X-100 and H2O2, which comprise the common bleach-based detergent formulation. The enzyme was found equally efficient to a commercial enzyme Oropon K (one of the commercial enzymes imported into Bangladesh for bating purpose) in bating of animal hide as proved by different comparative qualitative tests such as tensile strength, percent of elongation, stitch tears strength, water vapor permeability, grain crack strength and tongue tear  strength tests. In addition, the stability profile (pH, temperature and surfactants) and blood stain removal data also revealed its suitability for application in detergent industry.

  • References

    1. [1] Adinarayana K & Ellaiah P (2004), Investigations on alkaline protease production with Bacillus subtilis PE-11 immobilized in calcium alginate gel beads. Process Biochemistry 39, 1331-1339. http://dx.doi.org/10.1016/S0032-9592(03)00263-2.

      [2] Anwar a & Saleemuddin M (2000), Alkaline protease from Spilosomaoblique: potential applications in bioformulations. Biotechnology and Applied Biochemistry 31, 85-89. http://dx.doi.org/10.1042/BA19990078.

      [3] Aretz W, Koller KP & Riess G (1989), Proteolytic enzymes from recombinant Streptomyces lividans TK24. FEMS Microbiology Letter 65, 31-36. http://dx.doi.org/10.1111/j.1574-6968.1989.tb03592.x.

      [4] Arulmani M, Arumugan P, Kalaichelvan PT, Aparanjini K, Vasanthi K & Arivuchelvi M (2007), Purification and partial characterization of serine protease from thermostable alkalophilic Bacillus laterosporus-AK1. World Journal of Microbiology and Biotechnology 23, 475-481. http://dx.doi.org/10.1007/s11274-006-9249-7.

      [5] Banik RM & Prakash M (2004), Laundry detergent compatibility of the alkaline protease from Bacillus cereus. Microbiological Research 159,135-140. http://dx.doi.org/10.1016/j.micres.2004.01.002.

      [6] Beg QK & Gupta R (2003), Purification and characterization of an oxidation-stable, thiol-dependant serine alkaline protease from Bacillus mojavensis. Enzyme and Microbial Technology 32, 294-304. http://dx.doi.org/10.1016/S0141-0229(02)00293-4.

      [7] Bradford MM (1976), A rapid and sensitive method for the quantitation of microgram quantities of protein-dye binding. Applied Biochemistry 72, 80-85.

      [8] Dhandapani R & Vijayaragavan R (1994), Production of a thermophilic, extracellular alkaline protease by Bacillus stearothermophilus AP-4. World Journal of Microbiology and Biotechnology 10, 33-35. http://dx.doi.org/10.1007/BF00357559.

      [9] Dutta SS (1990), An introduction to the principles of physical testing of leather, 1st edn. Indian Leather Technologists’ Association, Mercantile Buildings, Lalbazar Street, Calcutta.

      [10] Gupta A, Joseph B, Mani A & Thomas G (2008), Biosynthesis and properties of an extracellular thermostable serine alkaline protease from Virgibacillus pantothenticus. World Journal of Microbiology and Biotechnology 24, 237-243. http://dx.doi.org/10.1007/s11274-007-9462-z.

      [11] Haki GD & Rakshit SK (2003), Developments in industrially important thermostable enzymes: a review. Bioresource Technology 89, 17-34. http://dx.doi.org/10.1016/S0960-8524(03)00033-6.

      [12] Kaur S, Vohra RM, Kapoor M, beg Qk & Hoondal GS (2001), Enhanced production and characterization of highly thermostable alkaline protease from Bacillus sp. P-2. World Journal of Microbiology and Biotechnology 17, 125-129. http://dx.doi.org/10.1023/A:1016637528648.

      [13] Kreger AS & Lockwood D (1981), Detection of extracellular toxins produced by Vibrio vulnificus. Infection and Immunity 33, 583-590.

      [14] Kumar CG (2002), Purification and characterization of a thermostable alkaline protease from alkalophilic Bacillus pumilus. Letters in Applied Microbiology 34, 13-17. http://dx.doi.org/10.1046/j.1472-765x.2002.01044.x.

      [15] Kumar CG & Takagi H (1999), Microbial alkaline proteases: from a bioindustrial viewpoint. Biotechnology Advances 17, 561-594. http://dx.doi.org/10.1016/S0734-9750(99)00027-0.

      [16] Kumar PKp, Mathivanan V, Karunakaran M, Renganathan S & Sreenivasan RS (2008), Studies on the effects of pH and incubation period on protease production by Bacillus spp. Using groundnut cake and wheat bran. Indian Journal of Science Technology 1, 1-4.

      [17] Laemmli UK (1970), Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. http://dx.doi.org/10.1038/227680a0.

      [18] Margesin R, Palma N, Knausedar F & Schinner F (1992), Purification and characterization of an alkaline protease produced by a psychrotrophic Bacillus sp. Journal of Biotechnology 24, 203-206. http://dx.doi.org/10.1016/0168-1656(92)90124-R.

      [19] Najafi MF, Deobagkar D & Deobagkar D (2005), Potential application of protease isolated from Psedomonas aeruginosa PD100. Electronic Journal of Biotechnology 8, 197-203. http://dx.doi.org/10.2225/vol8-issue2-fulltext-5.

      [20] Paliwal N, Singh SP & Garg SK (1994), Cation induced thermal stability of an alkaline protease from a Bacillus sp. Bioresource Technology 50, 209-211. http://dx.doi.org/10.1016/0960-8524(94)90091-4.

      [21] Rao MB, Tanksale AM, Ghatge MS, Deshpande VV (1998), Molecular and biotechnological aspects of microbial proteases. Microbiology and Molecular Biology Reviews 62, 597-635.

      [22] Shimogaki H, Takeuchi K, Nishino T, ohdera m, kudo t, ohba k, iwama m &trie m (1991), Purification and properties of a novel surface-active agent and alkaline resistant protease from Bacillus sp. Y. Agricultural and Biological Chemistry 55, 2251-2258. http://dx.doi.org/10.1271/bbb1961.55.2251.

      [23] Singh J, Batra N & Sobti CR (2001), Serine alkaline protease from a newly isolated Bacillus sp. SSR1. Process Biochemistry 36, 781-785. http://dx.doi.org/10.1016/S0032-9592(00)00275-2.

      [24] Tsujibo H, Miyamoto K, Hasegawa T & Inamori Y (1990), Purification and characterization of two types of alkaline serine proteases produced by an alkalophilic Actinomycete. Journal of Applied Bacteriology 69, 520-529. http://dx.doi.org/10.1111/j.1365-2672.1990.tb01544.x.

      [25] Uddin MN, Ilias M, Rahman A & Hoq MM (2006), Compatibility and Stability of alkaline protease from Bacillus licheniformisMZK-05 with commercial detergent. Bangladesh Journal of Microbiology 23, 19-23.

      [26] Zambare VP, Nilegaonkar SS & Kanekar PP (2013), Protease production and enzymatic soaking of salt-preserved buffalo hides for leather processing. IIOAB Letters 3, 1-7. http://dx.doi.org/10.5195/iioablett.2013.19.

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    Mamun, M. A. A., Khan, M. M., Akand, M. N. R., Khan, S. N., & Hoq, M. M. (2015). Characterization of an alkaline protease with high quality bating potential in leather processing from Bacillus licheniformis MZK05M9 mutant. International Journal of Biological Research, 3(1), 36-41. https://doi.org/10.14419/ijbr.v3i1.4250