Viscometric Method for Assayingof Total Endodepolymerase Activity of Pectinases

Viscometric Method for Assayingof Total Endodepolymerase Activity of Pectinases

(Parte 1 de 3)

Pectins are important components of many plants [1]. Biodegradation of pectins is catalyzed by a number of enzymes, which differ in their specificities [1 4]. Depolymerization of the pectin backbone formed by residues (usually substituted) of D galacturonic acid is catalyzed by the following pectinases: 1) polymethyl galacturonase and polygalacturonase (EC 3.2.1.15) hydrolyzing randomly the 1,4 α D galactosiduronic linkages in methoxylated or demethoxylated pectins, respectively; 2) pectin lyase (EC 4.2.2.10) depolymerizing pectin by eliminating of the 6 methyl ∆ 4,5 D galactur onate residues; 3) pectate lyase (EC 4.2.2.2) also exhibit ing the elimination mechanism, but towards demethoxy lated pectin (pectate). All the enzymes described are characterized by a random (endodepolymerase) type of action on the polymeric substrate. Additionally, the cleavage of the main polymeric chain as well as fragments of its destruction is catalyzed by the exodepolymerases: exopolygalacturonase (EC 3.2.1.67), exopoly α galac turonosidase (EC 3.2.1.82), and exopolygalacturonate lyase (EC 4.2.2.9). Lastly, biodegradation of pectins involves such enzymes as pectin esterase (EC 3.1.1.1) and pectin acetylesterase cleaving the methoxyl and acetyl bonds in galacturonans containing the correspon ding substituents.

The activity of pectinases of the lyase class can be determined spectrophotometrically by monitoring the formation of the double bond at 232 235nm [3, 5 7]. The activity of hydrolytic pectinases can be determined either by measuring the reducing sugars or using viscometry [3, 6, 8 19]. Other methods of estimation of the pectinase activity, for example turbidimetric ones [3], are not wide ly used.

The most popular procedures for determination of the polygalacturonase activity (dinitrosalicylic and Somogyi–Nelson methods) are based on the analysis for reducing sugars using pectate as the substrate [2, 3, 6, 13]. The use of natural pectins (methoxylated galacturonans with different substitution degree) as substrates is limited, because both methods include heating of a sample in a boiling water bath in an alkaline medium. Under these conditions, methoxylated pectins are unstable and thus give a high background level of optical density while assaying for reducing sugars [1, 3].

Viscometric methods for assaying pectinase activity are free from this disadvantage. These methods allow esti

Biochemistry (Moscow), Vol. 67, No. 6, 2002, p. 676 682. Translated from Biokhimiya, Vol. 67, No. 6, 2002, p. 815 822. Original Russian Text Copyright © 2002 by Gusakov, Markov, Grishutin, Semenova, Kondratyeva, Sinitsyn.

006 2979/02/6706 0676$27.0 ©202 MAIK “Nauka/Interperiodica” * To whom correspondence should be addressed.

Viscometric Method for Assaying of Total Endodepolymerase Activity of Pectinases

A. V. Gusakov*, A. V. Markov, S. G. Grishutin, M. V. Semenova, E. G. Kondratyeva, and A. P. Sinitsyn

School of Chemistry, Lomonosov Moscow State University, Moscow, 119992 Russia; fax: (095) 939 0997; E mail: avgusakov@enzyme.chem.msu.ru

Received April 16, 2001

Abstract—An improved method for assaying of the total endodepolymerase activity of pectinases has been developed. The method is based on the determination of the viscosity of a citrus pectin solution in the presence of the enzyme using an Ostwald viscometer. The depolymerizing activity of different pectinases can be detected including polygalacturonase, poly methylgalacturonase, pectin lyase, and pectate lyase. One unit of the endodepolymerase activity corresponds to the activity resulting in 50% decrease in the relative viscosity of 0.5% citrus pectin solution for 5min at 40°C and the appropriate pH. Depending on the pH optima of the enzymes, two modifications of the method are described: 1) for acid pectinases at pH5.0, and 2) for neutral (mildly alkaline) pectinases at pH8.0. The modifications differed in the control and in the calcu lation of the activity. Six enzyme preparations were used to demonstrate the applicability of the method. The parameter used for the calculation of the enzymatic activity was directly proportional to the enzyme concentration (the dependence was lin ear in the range of at least 10 fold change in the enzyme concentration). The relative error of the method did not exceed 10%.

Key words: pectin, pectinase, polygalacturonase, polymethylgalacturonase, pectin lyase, pectate lyase, endodepolymerase activity, viscometry

BIOCHEMISTRY (Moscow) Vol. 67 No. 62002

ASSAYING OF ENDOPECTINASE ACTIVITY677 mation of the total endopectinase activity of enzyme preparations reflecting the result of depolymerization of pectin by one, two, or more enzymes of hydrolytic or lyase type of action. However, there is controversial infor mation in the literature concerning viscometric methods, conditions of the assay, and the activity calculation. In some works, the time that was necessary to reduce the vis cosity of a pectin solution to a certain extent (for exam ple, 20 or 50%) was used as a measurable parameter for evaluation of the activity [12, 19]. According to other authors, the unit of the activity was defined as the amount of the enzyme that was necessary to achieve a 30 or 50% decrease in the viscosity of a pectin solution after a defi nite time of the reaction (1, 10, 20, 60, or 120min) [8, 1, 15 18]. One of the approaches suggested calculating the exponential coefficient βproportional to pectinase activ ity from the curves of the decrease in the substrate viscos ity [14]. Moreover, pectins from different sources and with different degree of methoxylation were used for vis cometric studies [8, 12 19]. Thus, literature data on the pectinase viscometry are impossible to compare.

The goal of the present study was to develop a visco metric method for assaying of the total endodepoly merase activity of pectinases, to compare different types of pectins in terms of their applicability in the viscometry, and to compare the endopectinase activity of different enzyme preparations.

Enzymes. The following commercial enzyme prepa rations were used: Celloviridine G2X (Belmedprepapaty, Belarus), Bio Prep 3000 L (Novo Nordisk, Denmark), and NCE L 600 (Dyadic International, USA). Culture filtrates of the fungi Aspergillus japonicus, Penicillium ver ruculosum, and P. canescens were obtained from the Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciences (Pushchino). All preparations were in a liquid state except for theP. verruculosum(lyophilized culture liquid).

Substrates. Polygalacturonic acid and citrus and apple pectins were from Sigma (USA); beet pectin was provided by M. V. Gernet (Moscow State University of Food Industry). The content of the methoxyl groups in the citrus pectin was 9%.

Assaying of the polygalacturonase,pectate lyase,and pectin lyase activities. The polygalacturonase activity was assayed using the Somogyi–Nelson method [20, 21]. The procedure was based on the determination of reducing sugars formed during the incubation of 0.5% polygalac turonic acid in the presence of an enzyme preparation (10min at 50°C and pH5.0 or 8.0). The pectate lyase and pectin lyase activities were determined by measuring the initial rate of the accumulation of ∆ 4,5 unsaturated products of degradation of polygalacturonic acid or citrus pectin, respectively [6]. A spectrophotometric cuvette containing 2.9ml of the substrate solution (0.24%) in 0.05M acetate (pH5.0) or Tris HCl (pH8.0) buffer was thermostatted at 40°C; the reaction was started by the addition of 0.1ml of the enzyme solution, and the kinet ics of the accumulation of unsaturated products was recorded at 235nm. In all cases, one unit of the activity was defined as the amount of the enzyme catalyzing for mation of 1 µmol of a product permin of the reaction.

Substrate solution for viscometry. Citrus and apple pectins were dissolved in 0.1M acetate buffer, pH5.0, to yield a 1% stock substrate solution, which was stored for 2 3 days at room temperature. To prepare a working pectin solution (0.5%), the stock solution was diluted twice with 0.1M acetate buffer, pH5.0. The efflux time of the working pectin solution in the viscometer was 115 120sec. In the case of beet pectin, a 1.5% working solu tion was prepared to obtain the same efflux time.

To assay the activity of neutral and mildly alkaline pectinases, the stock and working solutions were prepared in the same way, but using 0.5M Tris HCl buffer, pH8.0. Since pectin is unstable in an alkaline medium, the solu tions were prepared on the day of the experiment.

Viscometric assay of the activity of acid pectinases.

Viscosity of solutions was measured in a thermostatted Ostwald viscometer (capillary diameter of 0.5mm, the efflux time for water was 27sec) using two stopwatches.

To measure the activity of acid pectinases, 5ml of the working solution (0.5% pectin in 0.1M acetate buffer, pH5.0) was placed into the dry and thoroughly washed viscometer thermostatted at 40°C. The solution was pre heated at 40°C for 5min, and then the efflux time T0was determined. The measurements were repeated 2 3 times, so that the difference between the values obtained was no more than 1sec. Then 0.1ml of a diluted enzyme solution was added by a glass pipette, starting the first stopwatch simultaneously. The solution was mixed carefully (so not to splash), blowing an air through the capillary by a rub ber bulb. Then the first measurement of the efflux time of the reaction mixture T1was made using the second stop watch, fixing the time corresponding to the beginning of the first measurement t1by the first stopwatch (the moment when the solution passed the upper mark of the viscometer). After the first measurement was made, the reaction mixture was mixed again and the second meas urement was made to determine T2(the efflux time of the reaction mixture) and t2(the time interval between the moment of the addition of the enzyme and the moment when the solution passed the upper mark of the viscome ter). To determine T3and t3, T4and t4, the third and forth measurements were made in the same way. In the case the efflux time values T3and T4differed from the T0value less than by 30 40sec, all measurements were remade, reduc ing the dilution of the enzyme.

Since the addition of 0.1ml of the enzyme resulted in some dilution of the solution, a control value of the

678GUSAKOV et al.

BIOCHEMISTRY (Moscow) Vol. 67 No. 6 2002 efflux time for the original working pectin solution was determined after the addition of 0.1ml of 0.1M acetate buffer, pH5.0. Usually, 3 4 measurements were made so that the difference between the values did not exceed 2sec. Then the mean value was calculated that was taken as the intrinsic efflux time of the working pectin solution

(T0int). Usually, on the addition of 0.1ml of the buffer, the

∆T0value was 2 3sec (∆T0= T0– T0int). Such a control was performed once a day for each substrate solution used, and the ∆T0value determined was used to calculate the activity of the enzyme investigated.

Calculation of the activity of acid pectinases. For each i th measurement of the efflux time of the pectin solution in the presence of the enzyme (see previous sec tion), a ratio of the relative viscosity of the original pectin solution to the relative viscosity of the same solution con taining the enzyme (FRi) and the reaction time (tri) were calculated using the following equations:

FRi= (T0int– TB)/(Тi– TB) ; tri= ti+ Тi/2 , where T0int= T0– ∆T0and TBis the efflux time for the buffer. All time values are given in seconds.

Then FRiwas plotted against triusing linear regression (various computer programs can be used, for example

Excel or Origin). Typical dependences FRiagainst triare presented in Figs. 1b, 3, and 5. The activity of the enzyme is proportional to the slope of the straight line (tanα). The unit of the activity was taken as the activity resulting in a 50% decrease in the relative viscosity of the pectin solu tion for 5min of the reaction. Such a decrease in the vis cosity corresponded to the change in the FRivalue by 1 for 300sec. Thus, one unit of pectinase activity correspond ed to the slope of 0.03 of the straight line {FRi; tri}. Since we added into the viscometer 0.1ml of the enzyme solu tion, one unit of the pectinase activity was provided by the enzyme solution of 10 U/ml.

Depending on the slope of the straight line {FRi; tri}, the equation for the calculation of the activity is the following:

A (U/ml)= 3000·tanα·R, where Ris the dilution of the enzyme before its addition to the substrate solution in the viscometer.

Viscometric assay of the activity of neutral (mildly alkaline) pectinases at pH8.0. The experimental proce dure for assay of the activity at pH8.0 was similar to that described for pH5.0 (see above). However, since pectin was not quite stable at pH8.0 and enhanced temperature (40°C), it was gradually subjected to spontaneous degra dation by the β elimination mechanism. So, the control and the calculation of the activity were more complex (see “Results” for details).

The method for estimation of the total endopecti nase activity uses similar procedure as the standard visco metric method for assay of the hemicellulase activity [2].

Viscometric assay of acid pectinase activity (pH5.0).

Figure 1a presents dependences of the efflux time of the citrus pectin solution on time of the enzymatic reaction in the presence of different dilutions of the enzyme preparation of A. japonicus. Figure 1b presents the same data as a plot FRiagainst tri. Figure 2 shows the depend ence of the slopes of the straight lines {FRi; tri} on concen tration of the enzyme preparation (1). As seen from the

Fig. 1. a) Changes in the efflux time of a citrus pectin solution in the presence of different dilutions (R= 10, 20, 30, 40, and 100) of A. japonicussample (curves 1 5, respectively); b) lin earization of the data in the coordinates employed for the cal culation of the endopectinase activity: y= 0.0049x+ 1.0526 (1), 0.0028x+ 1.0336 (2), 0.0018x+ 1.034 (3), 0.0013x+ 1.0373 (4), 0.0007x+ 1.0043 (5).

Тi, sec

2 F Ri tri, sec

BIOCHEMISTRY (Moscow) Vol. 67 No. 62002

ASSAYING OF ENDOPECTINASE ACTIVITY679 figures, the data on the change in the viscosity of the pectin solution (Fig. 1a) can be linearized in the suggest ed coordinates reasonably well (Fig. 1b), and the rate of the change in the ratio of the relative viscosities FRi(i.e., tanα) is proportional to the enzyme concentration in a wide range (Fig. 2). In all cases of the linear regression, the correlation coefficient was close to 1 (0.997 0.9), this suggesting the possibility of the use of the described method for assaying of the pectinase activity in practice. In a perfect case, the absolute term of the equation of a linear regression (i.e., the point where the straight line cuts the ordinate) must be 1. In the equations of the lin ear regression obtained using the data presented in Fig. 1b this value was close to 1 (it varied from 1.004 to 1.053), this indicating the adequacy of the method.

Figure 3 demonstrates the changes in the pectin solu tion viscosity in the presence of a number of other enzyme preparations at pH5.0. It should be noted that since the preparations were diluted before the experiment to a dif ferent extent, slopes of the straight lines in Fig. 3 do not reflect the ratio of endopectinase activities of the original preparations. As seen from the figure, the plot FRiversus tr i yielded good linear dependences for all samples. The data for Celloviridine (plot 3) were linearized worse in the given coordinates, and the same character of the dependence was observed for different concentrations (dilutions) of this preparation. For all enzyme preparations, the rate of the change in FRidepended linearly on the enzyme con centration (data for Celloviridine are presented in Fig. 2, data for other enzymes are not shown). The results of the viscometric assay of the endopectinase activity of different preparations are summarized in Table 1.

Comparison between different types of pectins. Three different types of pectin (citrus, apple, and beet) were compared in terms of their applicability in the viscomet ric assay of the pectinase activity using three enzyme preparations: Celloviridine G2X, A. japonicus, and NCE L 600 (at pH5.0). Kinetic curves reflecting the changes in the efflux time of apple and beet pectin solutions in the presence of enzymes were similar to those obtained with citrus pectin (see above), and the data were linearized reasonably well in the suggested coordinates. The activity values of Celloviridine G2X as well as preparations of A. japonicusand NCE L 600 determined using citrus (0.5%), apple (0.5%), and beet (1.5%) pectins were 499, 158, and 30; 478, 141, and 45; and 375, 177, and 6 U/ml, respectively. The data obtained with apple and citrus pectins were in good agreement, while the activity values obtained with beet pectin significantly differed from those obtained using apple and citrus pectins as substrates. Citrus pectin was chosen for subsequent studies as a well characterized and accessible substrate, also being the most convenient one (it provided the most stable efflux time value of the solution).

Viscometric assay of the activity of neutral and mildly alkaline pectinases (pH8.0). As mentioned above, while measuring the pectinase activity in an alkaline medium and enhanced temperature using methoxylated pectins as substrates, special attention should be paid to control experiments, because under such conditions pectins can be subjected to spontaneous degradation by the β elimi nation mechanism [1, 3].

Figure 4 presents the dependence of the efflux time of the citrus pectin solution on time of incubation at 40°C

Fig. 2. Effect of the enzyme concentration on the rate of the change in the ratio of the relative viscosities (tanα) for enzyme preparation from A. japonicus(1, y= 0.00005x+ 0.02, R2= 0.99518) and Celloviridine G2X (2, y= 0.00014x+ 0.00026, R2= 0.98943).

tan α

Fig. 3. Data on changes in the viscosity of the citrus pectin solution in the presence of different enzyme preparations at pH5.0 in the coordinates used for the calculation of the endopectinase activity: 1) NCE L 600 (R= 10, y= 0.001x+ 0.9929); 2) P. canescens(R= 10, y= 0.0015x+ 1.0344); 3) Celloviridine G2X (R= 50, y= 0.003x+ 1.1779); 4) P. verru culosum(20g/liter) (y= 0.0092x+ 1.0855).

F Ri

680GUSAKOV et al.

(Parte 1 de 3)

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