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Production and Characterization of Keratinolytic Protease of Bacillus Licheniformis Mzk-03 Grown on Feather Mill

Debasish Paul
Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh

Alamgir Rahman
Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh

Mohammad Ilias
Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh

M Mozammel Hoq
Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh

Abstract/ Executive Summary:

Keratinolytic protease is an inducible specific proteolytic enzyme, which is produced by Bacillus licheniformis MZK-03 in presence of keratin (feather mill) as sole carbon and nitrogen source in growth medium. Maximum level of keratinolytic protease was produced after 42 h at 37°C over a wide range of initial pH (5.0 to 12.0) under continuous agitation (200 rpm). Keratinolytic protease worked best at 37°C and at pH 8.5. The enzyme was quite stable over a wide range of pH (7.0 to 11.0) but activity dropped drastically beyond this level (enzyme activity dropped to 8.8% and 19.3% at pH 6.5 and 12.0, respectively). Half-life of keratinolytic protease at 70° and 60°C were found to be 3 and 7 min respectively. The enzyme showed highest stability at 40°C (>90% after 3 h). The half-life at 4°C was 34 days. The presence of metal ions (5 mM) like Mg²⁺, Mn²⁺, Ca²⁺ and K⁺ had no remarkable effect on the keratinolytic protease activity but the activity decreased in presence of Hg²⁺ and Cu²⁺. The enzyme might belong to serine protease group as it was inhibited by serine protease inhibitor phenyl methyl sulphonyl fluoride (PMSF). The enzyme was as compatible as other commercially available enzymes used in leather industry with tannery chemicals. It was completely incompatible with Na₂S and CaO for their high alkalinity (pH >13.0), which was also observed for other commercial enzymes except the commercial enzyme supplemented with ammonium sulphate.

Keywords: Bacillus licheniformis MZK-03, Keratinolytic protease, Keratin, Production, Characterization

Location: Department of Microbiology, University of Dhaka

Start Date:

End Date:

Program Area: Knowledge Management

Commodity: Fermenters

Objectives:

To partially characterize the keratinolytic protease produced by Bacillus licheniformis MZK-03.
To optimize the physical conditions of keratinolytic protease production

Methodology:

Bacillus licheniformis strain MZK-03 was previously isolated from feather-decomposed soil and identified by 16S rRNA typing5. Stock culture of the organism was maintained at -70°C in nutrient broth containing 10% glycerol. A single colony from a freshly subcultured nutrient agar plate was transferred into 250-ml conical flask containing 50 ml of nutrient broth. The flask was incubated for 6-10 h at 37°C and 200 rpm in an orbital shaker until it reached to an absorbance of 0.5- 0.8 at 600 nm.The seed culture (5 ml) was transferred to 95 ml of feather mill medium in a 500-ml Erlenmeyer flask. Feather mill medium contained 0.075% NaCl, 0.21% K₂HPO₄, 0.105% KH₂PO₄, 0.015% MgCl₂.6H₂O, 0.009% CaCl₂, 0.15% molasses and 0.75% feather mill (initial pH 7.5). The inoculated flasks were placed in a thermostated orbital shaker for 48 h, at 37°C and 150 rpm. Samples were withdrawn at regular intervals and centrifuged at 5,000 rpm for 20 min. The cell free supernatant was preserved at 4°C and used for enzyme assay and protein estimation (in duplicate). Soluble protein in the culture supernatant was estimated according to the Bradford method using bovine serum albumin (BSA) as standard. Keratinolytic protease activity was measured by modified azocasein (Sigma, USA) digest method. The enzyme (400 ml) was incubated with 200 ml of 1% azocasein in 0.05 mM Tris-HCl buffer (pH 8.5) at 37°C for 30 min in a shaking water bath. The reaction was terminated by adding 1.4 ml of 10% trichloroacetic acid (TCA) and the mixture was kept at 4°C for 15 min. After centrifugation at 10,000 rpm for 10 min, 1 ml of the supernatant was decanted to a fresh tube and mixed with 1 ml of 0.5 M NaOH. The absorbance of the reaction mixture was read at 440 nm against a control prepared in the same way, except that the TCA was added before addition of enzyme. One unit of enzyme activity was defined as an increase of 0.01 absorbance units per min under the given conditions.

Source of Information: Bangladesh J Microbiol, Volume 24, Number 1, June 2007, pp 57-61

Article Link (Online Journal):

Funding Source:

1.

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The remarkable high production ability of keratinolytic protease by B. licheniformis MZK-03 validated the potential of using this strain in industries for large scale production of the enzyme. The broad pH stability (6.0-11.0) of the produced enzyme provides some advantages of using in many industrial processes. Its stability under ambient condition for few weeks allows it to be used and stored in crude form under limited facilities. The use of (NH4)₂SO₄ as stabilizing agent provided a viable mechanism of increasing the storage stability of the enzyme which could make the enzyme more compatible with the commercially available chemicals used in tannery industry under high alkaline condition.

Knowledge Management: Journal