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A contribution to the characterization of protopectinase SE, an endopolygalacturonase with pectin-releasing activity from Geotrichum klebahnii

 

Una contribución a la caracterización de protopectinasa-SE, una endo-polygalacturonasa de Geotrichum klebahnii con actividad solubilizadora de pectina

 

S.F. Cavalitto*, R.A. Hours and C.F. Mignone

 

Research and Development Center for Industrial Fermentation (CINDEFI; UNLP, CONICET La Plata), School of Science, La Plata National University, 47 y 115 (B1900ASH), La Plata, Argentina. *Corresponding author. E-mail: cavali@biotec.org.ar.

 

Received May 15, 2013.
Accepted December 31, 2013.

 

Abstract

Hydrolysis of polygalacturonic acid (1.8 g.L⁻¹ ) with 1.25 mg.L⁻¹ of a commercial preparation (Pectinase SE from Shikibo Ltd., Japan) containing protopectinase SE (PPase-SE) activity for 80 min released a reducing power equivalent to ≈ 400 mg.L⁻¹ of galacturonic acid monohydrate, yielding oligogalacturonates with an average polymerization degree of around 4.

Thermostability of PPase-SE was positively affected by enzyme concentration. Enzyme activity of a solution containing 12.5 mg.L⁻¹ of Pectinase SE in 20 mM sodium acetate buffer, pH 5.0, quickly dropped to less than 20 % after 80 s of vortexing. Enzyme pre-incubation (37°C, 30 min) with Ca²⁺, Mg²⁺, Co²⁺, Cu²⁺, Fe²⁺, Zn²⁺ and Mn²⁺ (0.1, 1.0 and 10.0 mM) did not affect the activity except in the case of Cu²⁺ (10 mM). Ca²⁺ (2.5 mM) and Mg²⁺ (2.5 mM) in the reaction mixture caused inhibition and activation, respectively. Ca²⁺ inhibition was partially reverted by Mg²⁺. The enzyme is not inhibited by products. K_m and V_max value for PGA was determined to be 0.198 ± 0.013 g.L⁻¹ and 0.0509 ± 0.0032 µmol.mL⁻¹.min⁻¹ respectively. This K_m value is one of the lowest reported for microbial PGases from different origins.

Keywords: endo-polygalacturonase, kinetic characterization, metal inhibition.

 

Resumen

La hidrólisis de ácido poligalacturónico (1.8 g.L⁻¹ en buffer acetato de sodio pH 5) con una solución 1.25 mg.L⁻¹ de un preparado comercial (Pectinase SE, Shikibo Ltd., Japan) de protopectinasa SE (PPasa-SE) por 80 minutos produce un poder reductor equivalente a ≈ 400 mg.L⁻¹ de ácido galacturónico monohidrato, lo que representa un grado de polimerización de aproximadamente 4. La estabilidad térmica de la enzima resulta ser proporcional a la concentración de la misma. Una solución conteniendo 12.5 mg.L⁻¹ de pectinasas SE en 20 mM de buffer acetato de sodio pH 5 pierde el 80 % de la actividad al ser agitada en vortex por un período de 80 s a temperatura ambiente. La preincubación de la enzima (37°C, 30 min) con Ca²⁺, Mg²⁺, Co²⁺ Cu²⁺, Fe²⁺, Zn²⁺ and Mn²⁺ (0.1, 1.0 and 10.0 mM) no afecta su actividad a excepción del Cu²⁺ (10 mM). El Ca²⁺ (2.5 mM) and Mg²⁺ (2.5 mM) en la mezcla de reacción causan inhibición y activación de la enzima respectivamente. La inhibición causada por el Ca²⁺ es parcialmente revertida por el Mg²⁺. La enzima no es inhibida por producto. Los valores de K_m y V_max para PGA fueron determinados, resultando en 0.198 ± 0.013 g.L⁻¹ y 0.0509 ± 0.0032 µmol.mL⁻¹.min⁻¹ respectivamente. Este valor de K_m es uno de los más bajos reportados para PGasas microbianas de diferentes orígenes.

Palabras clave: endo-poligalacturonasa, caracterización cinética, inhibición metálica.

 

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References

Blanco, P., Sierio, C., Diaz, A., and Villa, T.G. (1994). Production and partial characterization of an endopolygalacturonase from Saccharomyces cerevisiae. Canadian Journal of Microbiology 40, 974-977.         [ Links ]

Burns, J.K. (1991). The polygalacturonases and lyases. In: The Chemistry and Technology of Pectin, (Walter, R. H. eds.), Pp. 165-188. Academic Press, Inc., San Diego, CA.         [ Links ]

Cavalitto, S.F., Hours, R.A., and Mignone, C.F. (1997). Quantification of pectin-releasing activity of protopectinase-SE from Geotrichum klebahnii. Biotechnology Techniques 11, 331-334.         [ Links ]

Cavalitto, S.F., Hours, R.A., and Mignone, C.F. (1999). Quantification of protopectinase SE, an endopolygalacturonase with pectinreleasing activity from Geotrichum klebahnii. Biotechnology Techniques 13, 385-390.         [ Links ]

Cavalitto, S.F., Hours, R.A., and Mignone, C.F. (2000). Growth and protopectinase production of Geotrichum klebahnii in batch and continuous cultures with syntetic media. Journal of Industrial Microbiology & Biotechnology 25, 260-265.         [ Links ]

Cavalitto, S.F. and Mignone, C.F. (2007). Application of factorial and Doehlert designs for optimization of protopectinase production by a Geotrichum klebahnii strain. Process Biochemistry 42, 175-179.         [ Links ]

Colombie, S., Gaunand, A., Rinaudo, M., and Lindet, B. (2000). Irreversible lysozyme inactivation and aggregation induced by stirring: kinetic study and aggregates characterization. Biotechnology Letters 22, 277-283.         [ Links ]

Ferreyra, O., Cavalitto, S.F., Hours, R.A., and Ertola, R.J. (2002). Influence of trace elements on enzyme production: protopectinase expression by a Geotrichum klebahnii strain. Enzyme and Microbial Technology 31, 498-504.         [ Links ]

Fry, S.C. (1988). Wall polymers: Chemical characterisation. In: The Growing Plant Cell Wall: Chemical and Metabolic Analysis, (102-187. Longman Scientific & Technical, New York.         [ Links ]

Kubi, S.A. (1991). Some complex kinetic mechanisms and treatment of enzyme kinetic data. In: A study of enzymes, (341-376. CRC Press, Boca Raton, Florida.         [ Links ]

Nakamura, T., Hours, R.A., and Sakai, T. (1995). Enzymatic maceration of vegetables with protopectinases. Journal of Food Science 60, 468-472.         [ Links ]

Rojas, N.L., Cavalitto, S.F., Mignone, C.F., and Hours, R.A. (2008). Role of PPase-SE in Geotrichum klebahnii, a yeast-like fungus able to solubilize pectin. Electronic Journal of Biotechnology 11, 1-8.         [ Links ]

Rombouts, F.M. and Pilnik W. (1980). Pectic enzymes. In: Microbial enzymes and Bioconversions, Economis Microbiology, (Rose, A. H. eds.), Pp. Academic Press, London.         [ Links ]

Sakai, T. (1992). Degradation of pectins. In: Microbial degradation of naturals products., (Winkelmann, G. eds.), Pp. 57-81. Weinheim, Germany.         [ Links ]

Sakai, T. and Okushima, M. (1978). Protopectin solubilizing enzyme from Trichosporon penicillatum. Agricultural and Biological Chemistry 42, 2427-2429.         [ Links ]

Sakai, T. and Okushima, M. (1982). Purification and crystallization of protopectin-solubilizing enzime from Trichosporon penicillatum. Agricultural and Biological Chemistry 46, 667-676.         [ Links ]

Sakai, T., Sakamoto, T., Hallaert, J., and Vandamme, E.J. (1993). Pectin, pectinase, protopectinase: production, properties, and applications. Advances in Applied Microbiology 39, 213-294.         [ Links ]

Thomas, C.R. and Geer, D. (2011). Effects of shear on protein solution. Biotechnology Letters 33, 443-456.         [ Links ]

Wang, M.C. and Keen, N.T. (1970). Purification and characterization of endopolygalacturonase from Verticillium albo-atrum. Archives of Biochemistry and Biophysics 141, 749-757.         [ Links ]

Whitaker, J.R. (1990). Microbial pectolytic enzymes. In: Microbial Enzymes and Biotechnology, (Fogarty, W. and Kelly, C. T. eds.), Pp. 133-176. Elsevier Science Publisher, New York.         [ Links ]

Zapata Zapata, A.D., Gaviria Montoya, A.A., Cavalitto, S.F., Hours, R.A., and Rojano, B.A. (2012). Enzymatic maceration of albedo layer from sour orange (Citrus aurantium L.) with protopectinase-SE and measurement of antioxidant activity of the obtained products. LWT -Food Science and Technology 45, 289-294.         [ Links ]