Immobilization Parameters Statistically Optimized for Whole Cells of Pseudomonas putida G7 to Enhance Limonin Biotransformation
Keywords:Response Surface Methodology, Central Composite Design, Na-alginate, Cell load, Bead diameter
This study was aimed for optimizing the immobilization parameters for Pseudomonas putida G7 in Ca-alginate beads, in order to establish a debittering strategy for citrus juices, by biotransforming the bitter principle - Limonin. Response Surface Methodology (RSM) with Central Composite Design (CCD) was employed to model the significant parameters for an enhanced response. An enhanced limonin bioconversion and immobilized bead stability was obtained with alginate concentration (2%), cell load (47.2g/l), and a bead diameter (2.1mm); which had significant effects (p <0.001) on limonin biotransformation. The R2 values of 0.9 showed good agreement between experimental and predicted response. Validation experiments under optimized parameters showed good association between experimental (limonin biotransformation and stability response of 65.8% and 0.97 OD respectively) and predicted responses (limonin biotransformation and stability of 65.1% and 0.094 respectively). Thus, the approach is promising to develop a strategy for debittering citrus juices by biotransforming limonin at a faster rate.
. Birgisson, H., Wheat, J.O., Hreggvidsson, G.O., Kristjánsson, J.K., Mattiasson, B. (2007). Immobilization of a recombinant Escherichia coli producing a thermostable ?-l-rhamnosidase: Creation of a bioreactor for hydrolyses of naringin. Enzyme and Microbial Technology, 40:1181-1187.
. Karel, S.F., Libicki, S.B. and Robertson, C.R. (1985). The immobilization of whole cells: engineering principles. Chemical Engineering Science, 40: 1321–1354.
. Hsiau, L.T., Lee, W.C., & Wang, F.S. (1997). Immobilization of whole-cell penicillin G acylase by entrapping within polymethacrylamide beads. Applied Biochemistry and Biotechnology, 62(2-3): 303-15.
. Babu, P.S. and Panda, T. (1991). Studies on improving techniques for immobilizing and stabilizing penicillin amidase associated with E. Coli cells. Enzyme Microb. Technol., 13: 676–682.
. Bernal, V., Sevilla, Á., Cánovas, M., Iborra, J.L. (2007). Production of L-carnitine by secondary metabolism of bacteria. Microbial Cell Factories, 6: 31–48.
. Hasegawa, S. and Maier, V.P. (1983). Solutions to the limonin bitterness problem of citrus juices [Triterpene derivatives]. Food Technol., 37: 73–77.
. Canovas, M., Garcia-Cases, L. and Iborra, J. (1998). Limonin consumption at acidic pH values and absence of aeration by Rhodococcus fascians cells in batch and immobilized continuous systems. Enz. Microbial. Technol., 22: 111–116.
. Sun, C., Chen, K., Chen, Y. and Chen, Q. (2005). Contents and antioxidant capacity of limonin and nomilin in different tissues of citrus fruit of four cultivars during fruit growth and maturation. Food Chem., 93: 599–605.
. Roy, A. and Saraf, S. (2006). Limonoids: Overview of significant bioactive triterpenes distributed in the plant kingdom. Biol. Pharm. Bull., 29: 191–201.
. Canovas, M., Garcia-Cases, L. and Iborra, J.L. (1996). pH influence on the consumption of limonin species by Rhodococcus fascians cells. Biotechnol. Lett., 18: 423-428.
. Willaert, R.G., De Backer, L. and Baron, G.V. (1996). Mass transfer in immobilised cell systems. In: Willaert, R.G., Baron, G.V., De Backer L. (eds) Immobilised living cell systems: modelling and experimental methods. John Wiley & Sons, Chichester, England, pp 21–45.
. Gervais, T.R., Carta, G. and Gainer, J.L. (2003). Asymmetric synthesis with immobilized yeast in organic solvents: equilibrium conversion and effect of reactant partitioning on whole cell biocatalysis. Biotechnol. Prog., 19: 389–395.
. Li, Y.G., Xing, J.M., Xiong, X.C., Li, W.L., Gao, H.S. and Liu, H.Z. (2008). Improvement of biodesulfurization activity of alginate immobilized cells in biphasic systems. J. Industrial Microbiol. Biotechnol., 35: 145–150.
. Garikipati, S.V.B.J., McIver, A.M., Peeples, T.L. (2009). Whole-cell biocatalysis for 1-naphthol production in liquid–liquid biphasic systems. Appl. Environ. Microbiol., 75: 6545–6552.
. Dwevedi, A. and Kayastha, A.M. (2009). Optimal immobilization of beta-galactosidase from Pea (PsBGAL) onto Sephadex and chitosan beads using response surface methodology and its applications. Biores. Technol., 100: 2667–2675.
. Lee, J.H., Chae, M.S., Choi, G.H., Lee, N.K. and Paik, H.D. (2009). Optimization of medium composition for production of the antioxidant substances by Bacillus polyfermenticus SCD using response surface methodology. Food Sci. Biotechnol., 18: 959–964.
. Potumarthi, R., Subhakar, C., Pavani, A. and Jetty, A. (2008). Evaluation of various parameters of calcium-alginate immobilization method for enhanced alkaline protease production by Bacillus licheniformis NCIM-2042 using statistical methods. Bioresour. Technol., 99: 1776–1786.
. Goksungur, S., Dagbagli, A. and Ucan, A. Güvenç, U. (2005). Optimization of pullulan production from synthetic medium by Aureobasidium pullulans in a stirred tank reactor by response surface methodology. J. Chemical Technol. Biotechnol., 80: 819–827.
. Roig, M.G, Pedraz, M.A., Sanchez, J.M., Huska, J. and Toth, D. (1998). Sorption isotherms and kinetics in the primary biodegradation of anionic surfactants by immobilized bacteria: II. Comamonas terrigena N3H. J. Molecular Catalysis B: Enzymatic, 4: 271–281.
. Tapingkae, W., Parkin, K.L., Tanasupawat, S., Kruenate, J., Benjakul, S. and Visessanguan, W. (2010). Whole cell immobilisation of Natrinema gari BCC 24369 for histamine degradation. Food Chem. 120: 842-849.
. Vaks, B. and Lifshitz, A. (1981). Debittering of orange juice by bacteria which degrade limonin. J. Agric. Food Chem., 29: 1258-1261.
. Ertan, F., Yagar, H. and Balkan, B. (2007). Optimization of a-amylase immobilization in calcium alginate beads. Prep. Biochem. Biotechnol., 37: 195–204.
. Zhang, C.H., Ma, Y.J, Yang, F.X., Liu, W. and Zhang, Y.D. (2009). Optimization of medium composition for butyric acid production by Clostridium thermobutyricum using response surface methodology. Bioresour. Technol., 100: 4284–4288.
. Lu, L., Zhao, M. and Wang, Y. (2007). Immobilization of Laccase by Alginate–Chitosan Microcapsules and its Use in Dye Decolorization. World J. Microbiol. Biotechnol., 23:159–166.
. Urkut, Z., Dagbagli, S. and Goksungur, Y. (2007). Optimization of pullulan production using Ca-alginate-immobilized Aureobasidium pullulans by response surface methodology. J. Chemical Technol. Biotechnol., 82: 837–846.
. Niladevi, K.N. and Prema, P. (2008). Immobilization of laccase from Streptomyces psammoticus and its application in phenol removal using packed bed reactor. World J. Microbiol. Biotechnol., 24: 1215–1222.
. Elibol, M. and Moreira, A.R. (2003). Production of extracellular alkaline protease by immobilization of the marine bacterium Teredinobacter turnirae. Process Biochem., 38: 1445-50.
. Iborra, J.L., Manjón, A. & Cánovas, M. (1997). Immobilization in carrageenans. In Immobilization of Enzymes and Cells. Bickerstaff, G.F. (Ed.), pp. 53–60. Totowa, NJ: Humana Press.