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Optimization of Medium Ingredients for Keratinolytic Protease Production by Bacillus Licheniformis Mzk-03 Using Statistical Experimental Designs

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

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

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

Abstract/ Executive Summary:

A culture medium was optimized for the production of keratinolytic protease by a newly isolated strain of Bacillus licheniformis MZK-03 in shake-flask culture. Based on the results of preliminary experiments, feather mill, molasses and trace elements were found to be major variables in keratinolytic protease production. The concentrations of these ingredients were optimized by using two statistical approaches, namely Box-Wilson method and central composite design. The optimized culture medium, finally determined by using the statistical approaches, composed of 0.95% feather mill, 0.12% molasses and 1.44% trace elements. The keratinolytic protease production was increased by approximately 2-fold when the strain was grown in the optimized medium (95.2 U/ml) compared to the un-optimized medium (56.05 U/ml).

Keywords: Keratinolytic protease, Optimization, Bacillus licheniformis MZK-03, Statistical designs

Location: Department of Microbiology, University of Dhaka

Start Date:

End Date:

Program Area: Knowledge Management

Non-commodity: Performance

Objectives:

To assess some medium ingredient using statistical techniques for production of keratinase by Bacillus licheniformis MZK-03 as a matter of optimization.

Methodology:

The microorganism used was Bacillus licheniformis MZK-03. This organism was previously isolated from feather-decomposed soil collected from a local poultry farm in Dhaka, Bangladesh and identified using 16S rRNA typing by the International Centre for Biotechnology, Osaka University, Japan. Stock culture of the organism was maintained at -70°C in nutrient broth containing 10% glycerol. A single colony from the agar plate was aseptically transferred to 50 ml nutrient broth in 250-ml Erlenmeyer flask. The flask was incubated for 7-8 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. To induce enzyme production 5 ml of the seed culture was transferred to 95 ml of feather degrading enzyme-producing broth in a 500-ml Erlenmeyer flask. The feather degrading enzyme-producing broth contained 0.75 g/l NaCl, 0.09 g/l CaCl₂, 1.50 g/l MgCl₂.6H₂O, 1.05 g/l KH₂PO₄ and 2.10 g/l K₂HPO₄ supplemented with 1% feather mill corresponding to 10 g/l (initial pH 7.5). The inoculated flasks were incubated at 37°C at 150 rpm in an orbital shaker unless otherwise stated. Crude enzyme was collected after 42 h of incubation by centrifuging at 5,000 rpm for 10 min. The supernatant was preserved at 4°C and assayed for enzymes and protein (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 determined by azocasein (Sigma, USA) hydrolysis method with some modifications.One unit of enzyme activity was defined as an increase of 0.01 absorbance unit per min at 440 nm under the assay conditions. Experimental design - Based on the results obtained in preliminary experiments, feather mill, molasses and trace element solution were found to be major variables in the keratinolytic protease production. Hence, these variables were selected to find the optimized conditions for higher keratinolytic protease production using two well known statistical methods namely, Box-Wilson Method and central composite design. According to the Box-Wilson Experimental method if the proportions of three ingredients x₁, x₂ and x₃ are to be optimized and y is the yield, then y can be related to the three ingredients as follows: y = b_o + b₁x₁ + b₂x₂ + b₃x₃ + b₁₂x₁x₂ + b₁₃x₁x₂ + b₁₃x₁x₃ + b₂₃x₂x₃ + b₁₁x_{1 2} + b₂₂x_{1 2} + b₃₃x_{1 2}……. b represents the regression coefficients. If yield is taken to be the surface of a mountain, the method explores a small part of the yield surface, in which the linear function equation, y = b_o+ b₁x₁ + b₂x₂ + b₃x₃, is calculated. Computer software Statistical edition 99 (StatSoft Inc) was used to map contours of the whole yield surface. In the first experiment of the Box-Wilson Method, which begins at some distance from the yield summit, the regression coefficient b was determined or the direction of the steepest slope to the summit was established. The goal was to optimize a medium containing feather mill, molasses and trace elements. In the first step a factorial experiment was designed in which two levels of feather mill, molasses and trace elements are used. Both levels of each were run in combination with all the others. The levels chosen varied the same amount above and below a chosen mean. The results from this experiment indicated how much the concentrations of each ingredient should be increased or decreased proportionally in the next experiment. In the second experiment, by changing the levels of the medium ingredients in accordance with the obtained regression coefficient values the ‘ascent’ was actually made. Finally, the medium was optimized by using a central composite design, which was basically a two factor experimental design where each factor was studied at three levels. After completion of the experiment, the optimum concentration of each nutrient were calculated using defined statistical equations.

Source of Information: Bangladesh J Microbiol, Volume 24, Number 1, June 2007, pp 52-56

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Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Bacillus licheniformis MZK-03 was found to be a potent producer of keratinolytic enzyme. Optimization of the medium by two sophisticated statistical approaches certainly increased the efficiency of enzyme production.

Knowledge Management: Journal