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.

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.

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