EMAN RESEARCH PUBLISHING | Journal | <p>Efficient Medium for Protease Production by <em>Bacillus licheniformis</em> MZK05M9 Optimized through Response Surface Methodology </p>
Microbial and anti-microbial compound biology

Efficient Medium for Protease Production by Bacillus licheniformis MZK05M9 Optimized through Response Surface Methodology 

Md. Mahmuduzzaman Miana, Md. Arafat Al Mamunb, Shakila Nargis Khana, Md. Mozammel Hoqa*

+ Author Affiliations

Microbial Bioactives 1(1) 022-028 https://doi.org/10.25163/microbbioacts.11003A0425300718

Submitted: 04 March 2018  Revised: 25 July 2018  Published: 30 July 2018 

The efficiency of the enzyme in solubilizing the whole feathers was also assessed which indicated that the enzyme produced with cheap substrates could be utilized as a cost effective and eco-friendly agent in poultry feed formulation, leather processing etc.


Background. Due to certain limitations, the bioprocess development for protease production needs more convenient and realistic statistical approach instead of conventional optimization technique. For an economic bioprocess with enhanced protease yield, Response Surface Methodology (RSM) based on Central Composite Design (CCD) was employed and evaluated in this study. Materials and methods. The fermentation was performed with a mutant strain, Bacillus licheniformis MZK05M9 (BlM9) using molasses, urea and CaCl2.2H2O as carbon, nitrogen and trace element sources respectively in shake flask. The conditions for fermentation were maintained with temperature, pH and agitation at 37 °C, 7.5 and 150 rpm respectively. The required number of trials were determined by investigating each variable (Molasses, Urea and CaCl2) at five levels: -α, -1, 0, +1 and +α through CCD with protease yield as the response function and the interaction effects as well as optimal parameters were obtained by using Minitab software. The significance of the independent variables and their interactions were tested by means of analysis of variance (ANOVA) with a 95% confidence level and 3-D surface plots were developed through RSM. Results. Upon 20 trials, the optimum values of the tested variables for maximum alkaline protease production as predicted through CCD and RSM were as 0.63%, 0.16%, and 0.11% (w/v) for Molasses, Urea and  CaCl2.2H2O respectively. The protease activity in Conventionally Optimized (CO) medium was 410 U/ ml and it was predicted as 463.1 U/ ml for statistically optimized medium. Upon experiments with the optimized medium, the protease activity was estimated as 560 U/ ml which was 36.6% (i.e. 1.36 fold) higher than that of CO medium. Conclusion. The efficiency of the enzyme in solubilizing the whole feathers was also assessed which indicated that the enzyme produced with cheap substrates could be utilized as a cost effective and eco-friendly agent in poultry feed formulation, leather processing etc.

Key words: Bacillus licheniformis MZK05M9, Central Composite Design (CCD), Response Surface Methodology (RSM), Protease, Economic bioprocess.

Abbreviations: RSM, Response Surface Methodology; CCD, Central Composite Design; ANOVA, Analysis of Variance; TCA, Trichloroacetic acid; BSA, Bovine Serum Albumin; rpm, Rotation per minute; BlM9, Bacillus licheniformis MZK05M9.

Significance: Economic production of protease by statistical approach.


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