Optimization of Protease Production by Bacillus mojavensis A21 on Chickpea and Faba Bean

Samiha Mhamdi¹, Anissa Haddar¹, Ibtissem Hamza Mnif¹, Fakher Frikha², Moncef Nasri¹, Alya Sellami Kamoun^1,2*

¹Laboratoire de Génie Enzymatique et de Microbiologie, Université de Sfax, Ecole Nationale d’Ingénieurs de Sfax, Sfax, Tunisia.
²Université de Sfax, Faculté des Sciences de Sfax, Sfax, Tunisia.

DOI: 10.4236/abb.2014.514120                

Abstract

Response surface methodology (RSM) was employed to optimize the medium composition and culture conditions for the production of alkaline proteases by Bacillus mojavensis A21 on uncommon substrates: chickpea (CF) and faba bean (FF) flours. A significant positive influence of temperature, CF, FF, incubation time and inoculums size on the protease production was evaluated by Plackett Burman Design. Among these, CF was the most influential factor. The enhancement of protease to 9127 U/ml was achieved with the optimization procedure on the medium composed of (g/l): CF, 40; FF 30, NaCl 2.0; KH2PO4 1; K2HPO4 1; CaCl2, 0.1; MgSO4 0.1. The cultures were conducted for 72 hours with an IS of 2%, at 30°C, an agitation speed of 150 rpm and an initial pH of 8.0. More interestingly, the optimization was accomplished using two cheap and local fermentation substrates, CF and FF, which could result in a significant reduction in the cost of medium constituents. The maximum alkaline protease production was 9127 U/ml after 72 h of incubation and showed 5-fold increase in protease production over the initial level.

Keywords

Bacillus mojavensis, Protease, Chickpea, Faba Bean, Plackett Burman Design

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Banerjee, U.C., Sani, R.K., Azmi, W. and Soni, R. (1999) Thermostable Alkaline Protease from Bacillus brevis and Its Characterization as a Laundry Detergent Additive. Process Biochemistry, 35, 213-219. http://dx.doi.org/10.1016/S0032-9592(99)00053-9
[2]	Gupta, R., Beg, Q.K. and Lorenz P. (2002) Bacterial Alkaline Proteases: Molecular Approaches and Industrial Applications. Applied Microbiology and Biotechnology, 59, 15-32. http://dx.doi.org/10.1007/s00253-002-0975-y
[3]	Johnvesly, B. and Naik, G.R. (2001) Studies on Production of Thermostable Alkaline Protease from Thermophilic and Alkaliphilic Bacillus sp. JB-99 in a Chemically Defined Medium. Process Biochemistry, 37, 139-144. http://dx.doi.org/10.1016/S0032-9592(01)00191-1
[4]	Kirk, O., Borchert, T.V. and Fuglsang, C.C. (2002) Industrial Enzyme Applications. Current Opinion in Biotechnology, 13, 345-351. http://dx.doi.org/10.1016/S0958-1669(02)00328-2
[5]	Wang, Q., Hou, Y., Xu, Z., Miao, J. and Li, G.O. (2007) Optimization of Cold-Active Protease Production by the Psychrophilic Bacterium Colwellia sp. NJ341 with Response Surface Methodology. Bioresource Technology, 99, 1926-1931. http://dx.doi.org/10.1016/j.biortech.2007.03.028
[6]	Chu, I.M., Lee, C. and Li, T.S. (1992) Production and Degradation of Alkaline Protease in Batch Cultures of Bacillus subtilis ATCC 14416. Enzyme and Microbial Technology, 4, 55-61.
[7]	Sandhya, C., Sumantha, A., zakacs, G.and Pandey, A.(2005) Comparative Evaluation of Neutral Protease Production by Aspergillus oryzae in Submerged and Solid-State Fermentation. Process Biochemistry, 40, 2689-2694. http://dx.doi.org/10.1016/j.procbio.2004.12.001
[8]	Prakasham, R.S., Rao, Ch.S. and Sarma, P.N. (2006) Green Gram Husk an Inexpensive Substrate for Alkaline Protease Production by Bacillus sp. in Solid-State Fermentation. Bioresource Technology, 97, 1449-1454. http://dx.doi.org/10.1016/j.biortech.2005.07.015
[9]	Haaland, P.D. (1989) Statistical Problem Solving. In: Haaland, P.D., Ed., Experimental Design in Biotechnology, Marcel Dekker, New York, 1-18.
[10]	Reddy, L.V.A., Wee, Y.J., Yun, J.S. and Ryu, H.W. (2008) Optimization of Alkaline Protease Production by Batch Culture of Bacillus sp. RKY3 through Plackett-Burman and Response Surface Methodological Approaches. Bioresource Technology, 99, 2242-2249. http://dx.doi.org/10.1016/j.biortech.2007.05.006
[11]	Bhunia, B. and Dey, A. (2012) Statistical Approach for Optimization of Physicochemical Requirements on Alkaline Protease Production from Bacillus licheniformis NCIM 2042. Enzyme Research, 2012, Article ID: 905804. http://dx.doi.org/10.1155/2012/905804
[12]	Kumar, R.S., Ananthan, G. and Prabhu, A.S. (2014) Optimization of Medium Composition for Alkaline Protease Production by Marinobacter sp. GACAS9 Using Response Surface Methodology—A Statistical Approach. Biocatalysis and Agricultural Biotechnology, 3, 191-197.
[13]	Haddar, A., Bougatef, A., Agrebi, R., Sellami-Kamoun, A. and Nasri, M. (2009) A Novel Surfactant-Stable Alkaline Serine-Protease from a Newly Isolated Bacillus mojavensis A21. Purification and Characterization. Process Biochemistry, 44, 29-35. http://dx.doi.org/10.1016/j.procbio.2008.09.003
[14]	Haddar, A., Agrebi, R., Bougatef, A., Hmidet, N., Sellami-Kamoun, A. and Nasri, M. (2009) Two Detergent Stable Alkaline Serine-Proteases from Bacillus mojavensis A21: Purification, Characterization and Potential Application as a Laundry Detergent Additive. Bioresource Technology, 100, 3366-3373. http://dx.doi.org/10.1016/j.biortech.2009.01.061
[15]	Hmidet, N., Balti, R., Nasri,R., Sila, A.,Bougatef, A. and Nasri, M. (2011) Improvement of Functional Propreties and Antioxidant Activities of Cuttlefish (Sepia officinalis) Muscle Proteins Hydrolyzed by Bacillus mojavensis A21 Proteases. Food Research International, 44, 2703-2711. http://dx.doi.org/10.1016/j.foodres.2011.05.023
[16]	Younes, I., Ghorbel-Bellaaj, O., Nasri, R., Chaabouni, M., Rinaudo, M.and Nasri, M. (2012) Chitin and Chitosan Preparation from Shrimp Shells Using Optimized Enzymatic Deproteinization. Process Biochemistry, 47, 2032-2039. http://dx.doi.org/10.1016/j.procbio.2012.07.017
[17]	Nasri, R., Chataigné, G., Bougatef, A., Karra Chaabouni, M., Dhulser, P., Nasri, M. and Nedjar-Arroume, N. (2013) Novel Angiotensin I-Converting Enzyme Inhibitory Peptides from Enzymatic Hydrolysates of Goby (Zosterisessor ophiocephalus) Muscle Proteins. Journal of Proteomics, 91, 444-452. http://dx.doi.org/10.1016/j.jprot.2013.07.029
[18]	Haddar, A., Fakhfakh-Zouari, N., Hmidet, N., Frikha, F., Nasri, N. and Sellami Kamoun, A. (2010) Low-Cost Fermentation Medium for Alkaline Protease Production by Bacillus mojavensis A21 Using Hulled Grain of Wheat and Sardinella Peptone. Journal of Bioscience Bioengineering, 110, 288-294. http://dx.doi.org/10.1016/j.jbiosc.2010.03.015
[19]	Souissi, N., Bougatef, A., Triki-Ellouz, Y. and Nasri, M. (2007) Biochemical and Functional Properties of Sardinella (Sardinella aurita) By-Product Hydrolysates. Food Technology and Biotechnology, 45, 187-194.
[20]	Triki-Ellouz, Y., Bayoudh, A., Kammoun, S., Gharsallah, N. and Nasri, M. (2001) Production of Protease by Bacillus subtilis Grown on Sardinelle Heads and Viscera Flour. Bioresource Technology, 80, 49-51. http://dx.doi.org/10.1016/S0960-8524(01)00057-8
[21]	Fakhfakh, N., Ktari, N., Haddar, A., Hamza Mnif, I., Dahmen, I. and Nasri, M. (2011) Total Solubilisation of the Chicken Feathers by Fermentation with a Keratinolytic Bacterium, Bacillus pumilus A1, and the Production of Protein Hydrolysate with High Antioxidative Activity. Process Biochemistry, 46, 1731-1737. http://dx.doi.org/10.1016/j.procbio.2011.05.023
[22]	Miller, J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
[23]	Kembhavi, A.A., Kulkarni, A. and Pant, A.A. (1932) Salt-Tolerant and Thermostable Alkaline Protease from Bacillus subtilis NCIM No 64. Applied Biochemistry and Biotechnology, 38, 83-92. http://dx.doi.org/10.1007/BF02916414
[24]	Plackett, R.L. and Burman, J.P. (1946) The Design of Optimum Multifactorial Experiments. Biometrika, 33, 305-325. http://dx.doi.org/10.1093/biomet/33.4.305
[25]	Box, G.E.P., Hunter, W.G. and Hunter, J.S. (1978) Statistics for Experiments: An Introduction to Design Data Analysis and Model Building. John Wiley, New York.
[26]	Khuri, A.I. and Cornell, J.A. (1987) Response Surfaces: Design and Analysis. Marcel Decker Inc., New York.
[27]	Li, X., Xu, T., Ma, X., Guo, K., Kai, L., Zhao, Y., Jia, X. and Ma, Y. (2008) Optimization of Culture Conditions for Production of Cis-Epoxysuccinic Acid Hydrolase Using Response Surface Methodology. Bioresource Technology, 99, 5391-5396. http://dx.doi.org/10.1016/j.biortech.2007.11.017
[28]	Ewers, E. (1965) Determination for Starch by Extraction and Dispersion with Hydrochloric Acid. International Organisation for Standardization (ISO/TC 93/WGL).
[29]	Joo, H.S., Kumar, C.G., Park, G.C., Kim, K.T., Paik, S.R. and Chang, C.S. (2002) Optimization of the Production of an Extracellular Alkaline Protease from Bacillus horikoshii. Process Biochemistry, 38, 155-159. http://dx.doi.org/10.1016/S0032-9592(02)00061-4
[30]	Sellami-Kamoun, A., Ghorbel-Frikha, B., Haddar, A. and Nasri, M. (2011) Enhanced Bacillus cereus BG1 Protease Production by the Use of Sardinelle (Sardinella aurita) Powder. Annals of Microbiology, 61, 273-280. http://dx.doi.org/10.1007/s13213-010-0134-0
[31]	Hanlon, G.W., Hodges, N.A. and Russel, A.D. (1982) The Influence of Glucose, Ammonium and Magnesium Availability on the Production of Protease and Bacitracin by Bacillus licheniformis. Journal of Genetic and Microbiology, 128, 845-851.
[32]	Puri, S., Khali, O. and Gupta, R. (2002) Optimization of Alkaline Protease Production from Bacillus sp. by Response Surface Methodology. Current Microbiology, 44, 286-290. http://dx.doi.org/10.1007/s00284-001-0006-8
[33]	Tari, C., Genckal, H. and Tokatli, F. (2006) Optimization of a Growth Medium Using a Statistical Approach for the Production of an Alkaline Protease from a Newly Isolate Bacillus sp. L21. Process Biochemistry, 41, 659-665.
[34]	Oskouie, S.F.G., Tabandeh, F., Yakhchali, B. and Eftekhar, F. (2008) Response Surface Optimization of Medium Composition for Alkaline Protease Production by Bacillus clausii. Biochemical Engineering Journal, 39, 37-42. http://dx.doi.org/10.1016/j.bej.2007.08.016
[35]	Agrebi, R., Haddar, A, Hajji, M., Frikha, F., Manni, L., Jellouli, K. and Nasri, M. (2009) Fibrinolytic Enzymes from a Newly Isolated Marine Bacterium Bacillus subtilis A26: Characterization and Statistical Media Optimization. Canadian Journal of Microbiology, 55, 1049-1061. http://dx.doi.org/10.1139/W09-05