Exopolygalacturonase

Erwinia chrysanthemi synthesizes and secretes a large number of pectinases. The major pectinases include a pectin methylesterase PemA and five isoenzymes of endo-pectate lyases PelA, PelB, PelC, PelD and PelE. In addition, secondary pectinases were identified a pectin methylesterase PemB, two endo-pectate lyases PelL and PelZ, an exo-pectate lyase PelX and an exopolygalacturonase, PehX. The regulation of pectinase synthesis is very complex and dependent on many environmental conditions. It is induced by pectin catabolic products and affected by growth phase, catabolite repression, osmolarity, iron or oxygen starvation... 

Endopolygalacturonases PGI and PGII isolated from a recombinant Aspergillus, niger and exopolygalacturonase (PGX) isolated from A. tubingensis are described elsewhere in this volume (see Benen et al. and Kester et al., respectively). For NMR spectroscopy the enzymes were lyophilized three times from DjO. 

Four forms of exopolygalacturonase were found in carrot juice and one additive form more in carrot roots pulp. They differ in pH optima (3.6 3.8 4.7 ... 

Although the first study of exopolygalacturonases from carrots [11] indicated the presence of multiple forms of this enzyme based on the three present pH optima, the latter studies supported the idea of one form of exopolygalacturonase [2-4]. The present study deals with the whole spectrum of multiple forms of exopolygalacturonase, with forms described sooner and with forms found only now. 

Fig. 1. Separation of two exopolygalacturonase groups (Fraction A, Fraction B) on CM-Sephadex C-50. Column size, 20x250 mm. Stepwise elution with 0.05 M acetate buffer, pH 3.8 (starting at arrow marked a), 0.10 M acetate buffer, pH 4.8 (at arrow marked b), 0.15 M acetate, pH 5.6 (at arrow marked c) and the latter buffer plus 1.0 M NaCl (at arrow marked d). Fraction size 6 ml per half hr. Exopolygalacturonase activity determined with sodium pectate, pH 5.0 (o—O) 2nd expressed as A,, .

The course of separation of the lyophilisate from pulp by this method was very similar to separation of the juice lyophilisate but led to only one activity peak. The pH optima determination (Fig. 2) and gel chromatography on Superose 12 (Fig. 3 a,b,c) showed these fractions to be partially purified, complex mixture of more exopolygalacturonase forms with different molecular masses and pH optima. 

Fig. 4. Molecular mass distribution of Fraction A purified on Concanavalin A -cellulose on Superose 12 column. Buffer - 0.05 M phosphate, pH 7.0, 0.15 M NaCl, fraction size 0.5 ml/min. Exopolygalacturonase activity determined with penta(D-galactosiduronic) acid pH 5.0 and pH 3.8 (0—0)-...

The inicial reaction rates of exopolygalacturonase forms distinguished on the base of their pH optima on substrates with various degree of polymerization [(GA)j - di(D-galactosiduronic) acid, (GA)5 - penta(D-galactosiduronic) acid. 

Other difference between these two exopolygalacturonases was their action pattern expressed by various initial rates on substrates with various DP (Tab. 1), where exopolygalacturonase with pH optimum 5.0 preferred substrates with... 

The courve of pH optima determination indicated a presence of acidic exopolygalacturonase form as it was in Fraction A but with slight shift to pH 3.6 (Fig. 2). It was impossible to commute this enzyme form with the acidic exopolygalacturonase from Fraction A because of its molecular mass about 30000 and action pattern identical with form with pH optimum 5.4. Further characterization of this form was not made because of its low content in lyophilizate. 

The third described enzyme form with pH optimum about 4.7 [11, 4], we found in Fraction C - the fraction from carrot roots pulp (Fig. 2). We supposed that this form of exopolygalacturonase is relatively strongly bound on carrot cell walls and so it can be released only by higher salt concentrations. The approximative molecular mass determination on Superose 12 (Fig. 3c) showed the molecular mass about 50 000 for this form and the second, with more acidic pH optimum, form present in the fraction. The further characterization of these enzymes showed the exopolygalacturonase with pH optimum 4.7 to be identical with enzyme described sooner by Pressey and Avants [4] and exopolygalacturonase with pH optimum 3.8 to be identical with the enzyme from Fraction A. In conclusion, the exopolygalacturonase form with pH optimum 3.8 can be considered to be the main enzyme form present in carrot roots. 

The affinity chromatography on ConA - cellulose indicated the presence of small N-glycosylation of all forms of exopolygalacturonases present in carrot roots (unpublished results). This method was usefull for purification of these enzymes from other protein inpurities but was completely uneffective by separation of individual forms (Fig. 4). 

In contrast with previous result [4] all exopolygalacturonase forms were inhibited by their product, D-galactopyranuronic acid [19] however the extent (Tab. 1) and the type of inhibition was various (competitive for enzyme with pH optimum 3.8 and mixed for the others). 

This work should be considered as an introduction to plant exopolygalacturonase multiple forms structure studies. 

The molecular masses of polygalacturonase and exopolygalacturonase were approximately determined by gel chromatography on Superose 12 using FPLC device (Pharmacia, Sweden) and the Calibration proteins II kit (Boehringer-Mannheim, Germany). 

The activities of pectic enzymes present in cultivation medium (98 mg of protein extracted from 2.5 1 of pectin medium) were poor, not leading to the clarification of cultivation medium indicating the cleavage of pectate chains, with values 0.024 pmol/min.mg for polygalacturonase, 0.004 pmol/min.mg for exopolygalacturonase, 0.034 pmol/min.mg for pectinesterase and 0.005 pmol/min.mg for pectin lyase. The production of individual pectic enzymes was dependent on the C-source used in the cultivation medium (Tab. 1). 

The pH optima determination showed at least three pH regions with increased polygalacturonase activity (pH optima 4.0, 4.6 and pH 5.6) the pH 4.0 being the pH optimum of exopolygalacturonase (Fig. 4). 

The approximate determination of molecular masses of polygalacturonases present in the medium (Fig. 5) showed two activity peaks corresponding the values of about 40 kDa for polygalacturonase and 50 kDa for exopolygalacturonase. 

Pectinesterase activity expressed as a unit corresponding to the microequivalent of ester bonds of pectin molecule, which were hydrolyzed during 1 min. at 45 °C and pH 5.0 under the conditions, which were optimum for these enzymes. Endopolygalacturonase and exopolygalacturonase activities were determined using a technique determined by Ufshitz [8]. Activity of pectintranseliminase was determined by procedure (lOJ. 

Liao CH, Revear L, Hotchkiss A and Savary B. 1999. Genetic and biochemical characterization of an exopolygalacturonase and a pectate lyase from Yersinia enterocolitica. Can J Microbiol 45 396-403. 

Hydrolysis pectate pectin endopolygalacturonase endopolymethylgalacturonase exopolygalacturonase exopolymethylgalacturonase... 

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