A Peptidase Enzyme from Bacillus cereus with Antimicrobial Properties: Optimizing the Immobilization in Chitosan Beads Using Box-Behnken Design

Catalina Kotlar^{1, 2}, Sara Inés Roura^{1, 2} and Alejandra Graciela Ponce^{1, 2,}

¹Grupo de Investigación en Ingeniería en Alimentos, Departamento de Ingeniería Química y en Alimentos, Facultad de Ingeniería, Universidad Nacional de Mar del Plata, Juan B. Justo 4302, 7600 Mar del Plata, Argentina

²Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina

Cite this paper:
Catalina Kotlar, Sara Inés Roura and Alejandra Graciela Ponce. A Peptidase Enzyme from Bacillus cereus with Antimicrobial Properties: Optimizing the Immobilization in Chitosan Beads Using Box-Behnken Design. Journal of Polymer and Biopolymer Physics Chemistry. 2016; 4(1):28-39. doi: 10.12691/jpbpc-4-1-4

Abstract

Enzymes are exploited as catalysts in many industrial, biomedical, and analytical processes. There has been considerable interest in the development of carrier systems for enzyme immobilization because immobilized enzymes have enhanced stability compared to soluble enzymes, and can easily be separated from the reaction. In the current study, microbial peptidases liberated by B. cereus were immobilized in cross-linked chitosan beads and characterized using azocasein as a substrate. The Box-Behnken design was applied to determine the optimal conditions to maximize proteolytic activity. An empirical second-order model was determined by multiple regression analysis of the experimental data to describe the relationship between tested variables and the response. The determination coefficients (R²) were above 90%. Under optimal conditions (2.2 mm bead diameter, 1.06 enzyme/ bead ratio, 5.82% v/v glutaraldehyde and 18°C) the proteolytic activity was 0.938 U/ml. The retained immobilized enzyme can be reused up to five times. The storage stability of immobilized peptidases at 4°C was up to 10 days, while at 32°C the enzyme lost its activity within three days. Finally, novel antimicrobial properties of the immobilized peptidases were found. These results could have important benefit for the food industry.

Keywords:
Bacillus cereus Box-Behnken experimental design immobilization enzymes proteolytic enzymes immobilization process optimization Response surface methodology

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References:

[1]	Wellington, S.A., Edilson, H.F., James, A.S., Gonçalves, L.R. (2005). Appl. Biochem. Biotech. 41, 201-207.
[2]	Kalisz, H.M., Ed. (1988). Advances in Biochemical Engineering/ Biotechnology, Springer-Verlag, Berlin Heidelberg, pp: 1-65.
[3]	Potumarthi, R., Subhaker, C., Pavani, A. (2008). Bioresour Technol. 99, 1776-1786.
[4]	Dutta, P.K., Dutta. J., Tripathi. V.S. (2004). J. Sci. Ind. Res. 63, 20-31.
[5]	Ward, O.P. (1983). Proteinases. In Microbia1 Enzymes and Biotechnology, Applied Science Publishers, London, 251-317.
[6]	de Azeredo, H.M.C. (2009). Food Research International 42, 1240-1253.
[7]	Sotitopoulou, G., Chaimotakis, N. A. (2005). Biomat. 26, 6771-6779.
[8]	Chatterjee, S., Lee, D.S., Lee, M.W., Woo, S.H. (2009). J. Hazard Mater. 166, 508-513.
[9]	Gupta, R., Chaudhury, N.K. (2007). Biosens. Bioelectron 22, 2387-2399
[10]	Myres, R.H., Montgomery, D.C. (2008). Response surface methodology: process and product optimization using designed experiments, Hoboken, pp. 290-296.
[11]	Kotlar, C.E., Ponce, A.G., Sansevero, R., Roura, S.I. (2009). Internet J. Microbiol. 8 (1), 1937-8289.
[12]	Mundra, P., Desai, K., Lele, S.S. (2007). Bioresour Technol. 98, 2892-2896.
[13]	Pérez Borla, O., Davidovich, L., Roura, S.I. (2010). LWT - Food Sci. and Technol. 3(2), 298-301.
[14]	Khuri, A., Cornell, J.A., Eds. (1987). Response Surface Design and Analysis of Experiment, AQSA Quality Press, New York, pp. 177-186.
[15]	Masson, R.L., Gunst, R.F., Hess, J.L., Eds. (1989). Statistical Design and Analysis of Experiments, New York, pp.518-526.
[16]	Kotlar, C.E., Ponce, A.G., Roura, S.I. (2013). LWT - Food Sci. and Technol. 50, 378-385.
[17]	Kotlar, C.E., Agüero, M.V., Roura, S.I. (2010). Ind. Biotechnol. 6(6), 364-374.
[18]	Box, G.E.P., Hunter, J.S. (1957). Ann. Stat. 28, 195-241.
[19]	Huiping, L., Guoqun, Z., Shanting, N., Yiguo, L. (2007). Comp. Mater. Sci. 38, 561-570.
[20]	Nahit, A. (2005). Enzyme Micro. Tech. 37, 441-447.
[21]	Chiou, M.S., Li, H.Y. (2002). J. Hazard Mater. B93, 233-248.
[22]	Blanco, R.M., Guisán, J.M. (1988). Enzyme Microb. Tech. 10, 227-232.
[23]	Blanco, R.M., Calvete, J.J., Guisán, J.M. (1989). Enzyme Microb. Tech. 11, 353-359.
[24]	Bryjac, J. (2003). Biochem. Eng. J. 16, 347-355.
[25]	Vidinha, P., Augusto, V., Almeida, M. (2006). J. Biotechnol. 121, 23-33.
[26]	Wang, Q., Zhang, J.,Wang, A. (2009). Carbohyd. Polym. 78, 731-737.
[27]	Mansfield, S.H., Mooney, C., John, N., Saddler, J.N. (1999). Biotechnol. Prog. 15, 804-816.
[28]	Durango, A.M., Soares, N.F.F., Andrade, N.J. (2006). Food Cont. 17, 336-341.
[29]	Anwar, A., Qader, S.A., Raiz, A., Iqbal, S., Azhar, A. (2009). J. Appl. Behav. Sci. 7, 1281-1286.