An Improved Diffusion Assay for Quantifying the Polygalacturonase Content of Erwinia Culture Filtrates

Taylor, R. J., and Secor, G. A. 1988. An improved diffusion assay for quantifying the polygalacturonase content of Erwinia culture filtrates. Phytopathology 78:1101-1103. The agar diffusion procedure for quantifying pectolytic enzyme activity punched in the agarose with a # 1 cork borer. After incubation at 37 C for 17 was modified to optimize assay sensitivity and simplify its implementation. hr, the gel was developed with 10 ml of 0.05% ruthenium red for 30 min, and The revised assay was run in 100 X 50 mm petri plates containing 20 ml of the diameter of the clear zone of activity was measured microscopically. 1% agarose (Type II), ammonium oxalate (0.5%), and sodium azide (0.2%) Polygalacturonase equivalents as low as 2.3 X 10-4 units were detected. The in phosphate buffer (0.2 M, pH 5.3) with polygalacturonic acid (0.0 1%) as modified assay required less sample and reduced the problem of gel the substrate. Samples (35 yl) were pipetted into 4.1 mm diameter wells dehydration associated with the standard assay. Additional keywords: cup plate assay, pectinase. The agar "cup plate" diffusion assay of Dingle et al (7) can be resulting clear areas (rings) of activity were measured at 7X used to quantify the activity of a variety of enzymes, including magnification with a standard dissecting microscope containing a amylase, arabanase, cellulase, lipase, pectinesterase, polycalibrated ocular micrometer. Two diameter measurements (at galacturonase, protease, and xylanase. Although it was developed right angles) were taken for each well from duplicate plates and over 30 years ago, this assay is still used to determine enzyme averaged. activity. Since 1980, Dingle's original paper has been cited Preparation of the polygalacturonase standard curve. A approximately 50 times as a source for polygalacturonase assays. standard curve (ring diameter vs. concentration of standard) was The procedure has been utilized to determine the pectolytic activity prepared with polygalacturonase, poly [1, 4-a-D-galacturonide] of fungal (2,9,10,12,15,16,19,20) as well as bacterial (5,11,13) glycanohydrolase: EC 3.2.1.15 (Sigma Chemical Co.) at the culture filtrates. This system has also proven useful in evaluating following concentrations: 5.0, 0.5, 0.05, 0.005, and 0.0005 mg/ml host tissue for the presence of components that inhibit pectinases in dH 20. According to Sigma Chemical Co. analysis, the (1,3,4,12). The major criticism of the agar diffusion technique is its lyophylized polygalacturonase had a specific activity of 455 units relative inaccuracy when compared with more sophisticated quanti(U)/gm. (One unit of polygalacturonase liberates 1.0 umole of tative procedures (8). galacturonic acid per min from polygalacturonic acid at pH 4.0 We have made several refinements in the standard assay and 25 C.) Pectolytic activity was expressed as U/ml. procedure (18) to simplify its implementation and optimize its Preparation of samples. Control samples were prepared at high sensitivity. The assay is easy to run and has the advantage of being and low enzyme levels by arbitrarily mixing an Erwinia culture able to be performed with minimal equipment. The modified filtrate with commercial polygalacturonase. There was no specific procedure provides consistent results when crude Erwinia culture relationship between the high and low controls. The culture filtrates are assayed. filtrates were prepared by growing Erwinia carotovora subsp. carotovora (strain 71) in Chatterjee's (6) minimum salts medium MATERIALS AND METHODS (MinS) with citrus pectin (Sigma Chemical Co.) directly substituted for glucose. Bacteria were rinsed from a 24-hr nutrient Assay procedure. The assay medium contained ammonium agar slant culture and diluted to 7 X 108 colony-forming units in oxalate (0.5%), sodium azide (0.2%), Type II agarose (1.0%) sterile dH 20. One ml of the suspension was added to 250 ml of (Sigma Chemical Company, St. Louis, MO) in 0.2 M phosphate MinS in a 1-L Erlenmeyer flask. After culture at 24 C for 72 hr, the buffer (adjusted to pH 5.3), with the sodium salt of polybacteria were removed by sequential filtration through membrane galacturonicacid (0.01%) (United States Biochemical Corporation, filters VM-1 (5.0 Atm), BA-6 (0.45 ym), and GA-8 (0.2 pm) Cleveland, OH) as the substrate. The medium was heated to (Gelman Sciences, Inc., Ann Arbor, MI) and the remaining filtrate dissolve the polygalacturonic acid and agarose, then transferred to mixed with polygalacturonase. The high and low control samples 100 X 15 mm petri plates (20 ml per plate). A #1 cork borer was were segregated into 1-ml aliquots and frozen at-20 C. An aliquot used to punch five holes, 4.1 mm in diameter and 2.5 cm apart, in of each sample was thawed and included as an internal control with the solidified medium. The holes were arranged in three rows in a each enzyme assay. I X 3 X I matrix, and the wells were filled (35 /.l) with standard, Interassay variation was estimated by assaying the high and low control, or unknown (filtrate) solutions. The assay was incubated samples over several assays at approximately 2-wk intervals. Variaat 37 C for 17 hr. tion within the assay was examined by assaying the low control The gel was developed after incubation by flooding the assay several times within a single assay. plate with 10 ml of 0.05% ruthenium red (Sigma Chemical Co.) for 30 min at 25 C. Excess dye was removed by washing the plate RESULTS several times with deionized water (dH 20). The diameters of the Typical standard curves obtained by using the original assay procedure and the modified cup plate assay are given in Figure 1. ©1988 The American Phytopathological Society The modified assay was linear from 2.28 X 10-4 to at least 45.5 Vol. 78, No. 8,1988 1101 U/ml. The equation for the regression line of the standard curve diameter (Fig. 2). compares favorably with the average regression line based on 10 Storage conditions may affect enzyme stability, so we also assays (y = 4.18x X 24.92). Standard errors of the mean slope and monitored pectolytic activity in samples that were frozen and mean Y intercept of the 10 standard curves were 0.08 and 0.13, thawed numerous times and samples that were stored for an respectively. The standard curve from the original assay was also extended period of time at refrigerator temperature (10 C). An linear to at least 45.5 U/ml, but the lower limit of resolution was Erwinia filtrate taken through eight freeze thaw cycles lost 77% of only 2.28 X 10-3 U/ml. Although ring development did occur below its pectolytic activity. Assayable enzyme activity of two other this concentration, the margin of the ring was too close to the edge filtrates was reduced by 82% and 71% after refrigeration for 110 of the well and not distinct enough to be measured accurately. In days. addition, the slope of the standard curve from the original assay was not as great as that of the revised assay. DISCUSSION Mean polygalacturonase concentrations obtained from the standard samples are listed in Table 1. When low control was tested The sensitivity of the diffusion cup plate assay is related to 12 times within a single assay, the mean polygalacturonase value factors such as the substrate used, concentration of the substrate, was 4.50 X 10-3 U/ml. This control was also tested in seven separate pH and nature of the gel, temperature, and length of the incubation assays with a mean approximately twice that of the intraassay period. Changing the substrate from citrus pectin to polymean. A mean concentration of 5.18 U/ml was obtained when the galacturonic acid, reducing the substrate concentration, using high control was analyzed in nine separate assays, but the agarose as the gel, and developing the plates with ruthenium red interassay standard deviation, variance, and standard error of the stain all contributed to optimizing the sensitivity of the modified mean were not as great as those obtained for the control with the assay. The revised diffusion assay is actually more sensitive than lower pectolytic activity, the original assay because both the slope of the standard curve and Decreasing the substrate (polygalacturonic acid) concentration the lower limit of resolution have been increased. caused an upward shift in the standard curve, but the slope The modified assay is apparently linear to 2.28 X 10U/mi remained virtually unaffected (Table 2). Even at a low polygalacturonase equivalents; however, sensitivity ultimately concentration of enzyme standard (2.28 X 10-' U/ml), there was a depends upon accurately measuring the diameter of the rings good correlation between substrate concentration and ring marking the zone of enzyme activity. Reducing the substrate concentration promotes greater ring development, as indicated by an increase in the Y intercept of the regression line (Table 2). The increase is consistent throughout the range of enzyme concentrations used in the standard curve. Accurate measurement of ring diameter is particularly important at the lowest portion of the 25 a TABLE 2. Effect of substrate (polygalacturonic acid) concentration on the E standard curve parameters E 20Polygalacturonic acid ~ 20 mg/ml Slope Y intercept r value 1.0 4.43 19.44 0.9877 E 0.5 4.48 20.96 0.9942 20.25 4.49 22.39 0.9969 15 0.1 4.42 24.31 0.9995