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

Md. Mahmuduzzaman Mian^a, Md. Arafat Al Mamun^b, Shakila Nargis Khan^a, Md. Mozammel Hoq^a*

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.

Abstract

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.

References

Abinaya, R., Ramya, P., Sivakami, V., Ponnusami, V., & Sugumaran, K. R. (2017). Alkaline protease production by Bacillus sp. MTCC 511 from cost effective substrate. Journal of Chemical and Pharmaceutical Sciences, 10(1), 488–491.

Adinarayana, K., Bapi Raju, K. V. V. S. ., & Ellaiah, P. (2004). Investigations on alkaline protease production with B. subtilis PE-11 immobilized in calcium alginate gel beads. Process Biochemistry, 39(11), 1331–1339. https://doi.org/10.1016/S0032-9592(03)00263-2

Adinarayana, K., & Ellaiah, P. (2002). Response surface optimization of the critical medium components for the production of alkaline protease by a newly isolated Bacillus sp. Journal of Pharmacy & Pharmaceutical Sciences, 5(3), 272–8. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/12553896

Akcan, N., & Uyar, F. (2011). Production of extracellular alkaline protease from Bacillus subtilis RSKK96 with solid state fermentation. EurAsian Journal of Biosciences, 72(July), 64–72. https://doi.org/10.5053/ejobios.2011.5.0.8

Aksoy, S. Ç., Uzel, A., & Hames Kocabas, E. E. (2012). Extracellular serine proteases produced by Thermoactinomyces strains from hot springs and soils of West Anatolia. Annals of Microbiology, 62(2), 483–492. https://doi.org/10.1007/s13213-011-0280-z

Arslan-Alaton, I., Tureli, G., & Olmez-Hanci, T. (2009). Treatment of azo dye production wastewaters using Photo-Fenton-like advanced oxidation processes: Optimization by response surface methodology. Journal of Photochemistry and Photobiology A: Chemistry, 202(2–3), 142–153. https://doi.org/10.1016/j.jphotochem.2008.11.019

Bayraktar, E. (2001). Response surface optimization of the separation of dl-tryptophan using an emulsion liquid membrane. Process Biochemistry, 37(2), 169–175. https://doi.org/10.1016/S0032-9592(01)00192-3

Bhunia, B., Basak, B., & Dey, A. (2012). A review on production of serine alkaline protease by Bacillus spp . Journal of Biochemical Technology, 3(4), 448–457.

Carvalho, C. M. L., Serralheiro, M. L. M., Cabral, J. M. S., & Aires-Barros, M. R. (1997). Application of factorial design to the study of transesterification reactions using cutinase in AOT-reversed micelles. Enzyme and Microbial Technology, 21(2), 117–123. https://doi.org/10.1016/S0141-0229(96)00245-1

Chang, S.-F., Chang, S.-W., Yen, Y.-H., & Shieh, C.-J. (2007). Optimum immobilization of Candida rugosa lipase on Celite by RSM. Applied Clay Science, 37(1–2), 67–73. https://doi.org/10.1016/j.clay.2006.12.001

Davati, N., & Najafi, M. B. H. (2013). Overproduction Strategies for Microbial Secondary Metabolites?: a Review Production Over- of Microbial. International Journal of Life Science and Pharma Research, 3, 23–37.

Deepak, V., Kalishwaralal, K., Ramkumarpandian, S., Babu, S. V., Senthilkumar, S. R., & Sangiliyandi, G. (2008). Optimization of media composition for Nattokinase production by Bacillus subtilis using response surface methodology. Bioresource Technology, 99(17), 8170–4. https://doi.org/10.1016/j.biortech.2008.03.018

El-Enshasy, H. A., Mohamed, N. A., Farid, M. A., & El-Diwany, A. I. (2008). Improvement of erythromycin production by Saccharopolyspora erythraea in molasses based medium through cultivation medium optimization. Bioresource Technology, 99(10), 4263–8. https://doi.org/10.1016/j.biortech.2007.08.050

Ernst, O., & Zor, T. (2010). Linearization of the bradford protein assay. Journal of Visualized Experiments?: JoVE, (38). https://doi.org/10.3791/1918

Gokhale, D. V., Patil, S. G., & Bastawde, K. B. (1991). Optimization of cellulase production by Aspergillus niger NCIM 1207. Applied Biochemistry and Biotechnology, 30(1), 99–109. https://doi.org/10.1007/BF02922026

Hajji, M., Rebai, A., Gharsallah, N., & Nasri, M. (2008). Optimization of alkaline protease production by Aspergillus clavatus ES1 in Mirabilis jalapa tuber powder using statistical experimental design. Applied Microbiology and Biotechnology, 79(6), 915–23. https://doi.org/10.1007/s00253-008-1508-0

Helal, M., Amer, H., & Abdelwahed, A. (2012). Physiological and Microbiological Studies on Production of Alkaline Protease from Locally Isolated Bacillus Subtilis. Australian Journal of Basic and Applied Sciences, 6(3), 193–203.

Hoq, M. M., Mamun, A. Al, Shishir, M. A., Akand, N. R., Khan, M., & Khan, S. N. (2013). Bioprocess development for eco-friendly microbial products and its impacts on bio-industry establishment in bangladesh. In International Conference on Biotechnology (Vol. 1, pp. 205–217). Dhaka, Bangladesh.: cares.org.bd. Retrieved from http://www.caresbd.org/uploads/book1_14.pdf

Hoq, M. M., Siddiquee, K. A. Z., Kawasaki, H., & Seki, T. (2005). Keratinolytic Activity of Some Newly Isolated Bacillus Species. Journal of Biological Sciences, 5(2), 193–200. https://doi.org/10.3923/jbs.2005.193.200

Huang, K. xue, Badger, M., Haney, K., & Evans, S. L. (2007). Large scale production of Bacillus thuringiensis PS149B1 insecticidal proteins Cry34Ab1 and Cry35Ab1 from Pseudomonas fluorescens. Protein Expression and Purification, 53(2), 325–330. https://doi.org/10.1016/j.pep.2007.01.010

Lakshmi, B., & Hemalatha, K. (2015). Response surface optimization of medium composition for alkaline protease production by Bacillus cereus strain S8. International Journal of Pure and Applied Bioscience, 3(4), 216–223.

Lakshmi, B., & Hemalatha, K. (2016). Production of Alkaline Protease from Bacillus licheniformis through Statistical Optimization of Growth Media by Response Surface Methodology. Fermentation Technology, 5(2), 1–7. https://doi.org/10.4172/2167-7972.1000130

Li, J., Ma, C., Ma, Y., Li, Y., Zhou, W., & Xu, P. (2007). Medium optimization by combination of response surface methodology and desirability function: an application in glutamine production. Applied Microbiology and Biotechnology, 74(3), 563–571. https://doi.org/10.1007/s00253-006-0699-5

Liu, G.-Q., & Wang, X.-L. (2007). Optimization of critical medium components using response surface methodology for biomass and extracellular polysaccharide production by Agaricus blazei. Applied Microbiology and Biotechnology, 74(1), 78–83. https://doi.org/10.1007/s00253-006-0661-6

Mamun, A. Al, Mian, M., Saifuddin, M., Khan, S. N., & Hoq, M. (2017). Optimization of fermenting medium by statistical method for production of alkaline protease by Bacillus licheniformis MZK05M9. Journal of Applied Biology & Biotechnology, 5(6), 24–28. https://doi.org/10.7324/JABB.2017.50604

Md. Mahmuduzzaman Mian. (2014). Optimization of alkaline protease production by Bacillus licheniformis MZK05M9 in batch culture using Response Surface Methodology. Brac University. Brac University. Retrieved from http://hdl.handle.net/10361/4150

Montgomery, D. C. (2006). Design and Analysis of Experiments. Technometrics, 48(1), 158–158. https://doi.org/10.1198/tech.2006.s372

Mourin, M., Shishir, A., Khan, S. N., & Hoq, M. M. (2015). Regulation of major cultural components for designing a cost effective medium to increase δ-endotoxin synthesis by Bacillus thuringiensis. African Journal of Biotechnology, 14(16), 1379–1386. https://doi.org/10.5897/AJB2014.14340

Nahar, M., Asaduzzaman Shishir, M., Waliullah, S., Sanowarul Haque, M., Ilias, M., Manjurul Karim, M., … Mozammel Hoq, M. (2016). Cloning, expression and structure simulation of keratinase from Bacillus licheniformis strain MZK05. Malaysian Journal of Microbiology, 12(2), 182–190. https://doi.org/10.21161/mjm.78515

Nazir, Y., Shuib, S., Kalil, M. S., Song, Y., & Hamid, A. A. (2018). Optimization of Culture Conditions for Enhanced Growth, Lipid and Docosahexaenoic Acid (DHA) Production of Aurantiochytrium SW1 by Response Surface Methodology. Scientific Reports, 8(1), 8909. https://doi.org/10.1038/s41598-018-27309-0

Qureshi, A. S., Bhutto, M. A., Khushk, I., & Dahot, M. U. (2011). Optimization of cultural conditions for protease production by Bacillus subtilis EFRL 01. African Journal of Biotechnology, 10(26), 5173–5181. https://doi.org/10.5897/AJB09.1574

Rahman, A., & Gomes, D. J. (2003). Optimization of medium ingredients for A-mannanase production by Aspergillus sp. isolated from commercial guar gum. Dhaka University Journal of Biological Sciences, 12(2), 153–164.

Rao, K. J., Kim, C.-H., & Rhee, S.-K. (2000). Statistical optimization of medium for the production of recombinant hirudin from Saccharomyces cerevisiae using response surface methodology. Process Biochemistry, 35(7), 639–647. https://doi.org/10.1016/S0032-9592(99)00129-6

Salaheen, S., Mamun, M. A. Al, Khan, S. N., & Hoq, M. M. (2015). Improvement of Bacillus licheniformis MZK05 by mutation for increased production of keratinase. Dhaka University Journal of Biological Sciences, 24(1), 17–23. Retrieved from http://journal.library.du.ac.bd/index.php?journal=dujbs&page=article&op=download&path%5B%5D=1004&path%5B%5D=964

Sarrai, A., Hanini, S., Merzouk, N., Tassalit, D., Szabó, T., Hernádi, K., & Nagy, L. (2016). Using Central Composite Experimental Design to Optimize the Degradation of Tylosin from Aqueous Solution by Photo-Fenton Reaction. Materials, 9(6), 428–438. https://doi.org/10.3390/ma9060428

Saxena, R., & Singh, R. (2010). Statistical optimization of conditions for protease production from Bacillus sp. Acta Biologica Szegediensis, 54(2), 135–141.

Sayyad, S. A., Panda, B. P., Javed, S., & Ali, M. (2007). Optimization of nutrient parameters for lovastatin production by Monascus purpureus MTCC 369 under submerged fermentation using response surface methodology. Applied Microbiology and Biotechnology, 73(5), 1054–8. https://doi.org/10.1007/s00253-006-0577-1

Sen, R. (1997). Response Surface Optimization of the Critical Media Components for the Production of Surfactin. Journal of Chemical Technology & Biotechnology, 68(3), 263–270. https://doi.org/10.1002/(SICI)1097-4660(199703)68:3<263::AID-JCTB631>3.0.CO;2-8

Sharma, K. M., Kumar, R., Panwar, S., & Kumar, A. (2017). Microbial alkaline proteases: Optimization of production parameters and their properties. Journal of Genetic Engineering and Biotechnology, 15(1), 115–126. https://doi.org/https://doi.org/10.1016/j.jgeb.2017.02.001

Song, C., Li, X., Wang, L., & Shi, W. (2016). Fabrication, Characterization and Response Surface Method (RSM) Optimization for Tetracycline Photodegration by Bi3.84W0.16O6.24- graphene oxide (BWO-GO). Scientific Reports, 6(1), 37466. https://doi.org/10.1038/srep37466

Song, Z., Zhao, Z., Qin, X., Huang, J., Shi, H., Wu, B., & Chen, Q. (2007). Highly sensitive choline biosensor based on carbon nanotube-modified Pt electrode combined with sol-gel immobilization. Frontiers of Chemistry in China, 2(2), 146–150. https://doi.org/10.1007/s11458-007-0030-8

Srinivas, M. R. S., Chand, N., & Lonsane, B. K. (1994). Use of Plackett-Burman design for rapid screening of several nitrogen sources, growth/product promoters, minerals and enzyme inducers for the production of alpha-galactosidase by Aspergillus niger MRSS 234 in solid state fermentation system. Bioprocess Engineering. https://doi.org/10.1007/BF00369470

Suganthi, C., Mageswari, A., Karthikeyan, S., Anbalagan, M., Sivakumar, A., & Gothandam, K. M. (2013). Screening and optimization of protease production from a halotolerant Bacillus licheniformis isolated from saltern sediments. Journal of Genetic Engineering and Biotechnology, 11(1), 47–52. https://doi.org/https://doi.org/10.1016/j.jgeb.2013.02.002

Xiao, Z. J., Liu, P. H., Qin, J. Y., & Xu, P. (2007). Statistical optimization of medium components for enhanced acetoin production from molasses and soybean meal hydrolysate. Applied Microbiology and Biotechnology, 74(1), 61–8. https://doi.org/10.1007/s00253-006-0646-5