بهینه سازی تولید، خالص‏سازی و شناسایی پلی گالاکتوروناز از قارچ عامل بیماری ساق سیاه لوبیا

Introduction

The plant cells are bound by a rigid structure called cell wall that is made from cellulose microfibrils and matrix polysaccharides with relatively equal amount that are embedded in a gel of pectin with 40-60% of wall content (1). Although pectins have certain advantages in food industry specifically as gelling agents, stabilizers and thickeners (2), in beverage industry it has been considered as nuisance trapping some of juice, making the juice look murky and further leading to inefficiencies within the filtering systems. Pectinases next to other wall hydrolytic enzymes, i.e., cellulases, glucanases and xylanases, improve the clarification and liquefaction of fruits, improving the quality and yield of beverages and concentrates in fruit juice factories, eventually reducing the waste product (3).

Amongst many forms of pectinases, polygalacturonases (PG) hydrolyze the α-1,4-D-galacturonan backbone and appear in two forms of endo- (E.C. 3.2.1.15), and exo- (E.C. 3.2.1.67) hydrolases (4); with greater contribution of endopolygalacturonases in pectin hydrolysis through random breakage of glycosidic linkages between galacturonic acid residues. Plants, bacteria and fungi are the known sources of organisms producing PGs for fruit ripening and softening (5), abscission (6), growth (7) and microbial infection (8, 9). Additionally, some industrial applications have been considered for polygalacturonases when they have been used alone or in combination of other lytic enzymes (10). Producing oligogalacturonides as functional food components (11), extraction of vegetable oils (12), recovering more must/wine in oenology and improving the wine quality, i.e., color and flavor (13), biobleaching of kraft pulp to improve the paper quality (14, 15), producing high quality linen fibers (16), and bioethanol production (17) next to the fruit and vegetable maceration in fruit juice industries (18- 22) are some of these applications.

Since 1930s commercial enzymes have been widely used within every industry transforming fruit juice. However, looking for enzymes with greater potentiality from other microorganisms seems to be inevitable (23- 25). In addition to find new enzymatic sources, one needs to optimize the condition towards obtaining more functional enzymes with fewer resources. This can be achieved through factorial experiments in which many factors with a range of levels are considered simultaneously (26). However as the number of factors grows, the number of experiments will increase. In contrast, Taguchi (27) developed a rather cost-effective approach to reduce the number of experiments via implementing orthogonal arrays that appreciates the signal-to-noise (S/N) ratio (28).

In the current study, fungal phytopathogens (9) were screened for primarily polygalcturonase activity and further the fungus with functional PG at lower pH and higher thermal stability was selected (data not shown). Amongst fungal strains, Macrophomina phaseolina was chosen and the production of PG was analyzed to determine the best time-course, temperature, carbon, nitrogen, and phosphorous sources. Following the initial determination of the suitable condition, Taguchi’s statistical method was applied to determine the best levels of each factor. Finally, for the first time Macrophomina phaseolina PG was purified and analyzed for its biochemical characteristics.

Materials and Methods

Chemicals and fungal strains

Polygalcturonic acid, α-D-galacturonic acid, and 3,5-dinitrosalisylic acid (DNS) were purchased from Sigma (St. Louis, MO, USA). DEAE-Sepharose was obtained from Pharmacia (Stockholm, Sweden). All other chemicals were obtained from Merck (Darmstadt, Germany). Macrophomina phaseolina, Bipolaris sp., Alternaria raphini, Alternaria brassicicola, Fusarium graminearum, Sclerotinia sclerotiorum, Rhizoctonia solani, Botrytis cinerea, and Phytophtora sp. were kindly provided by Dr. Sarpele at Phytopathology Research Institute, Tehran, Iran. Primary colorimetric screening of the fungal strains using rhetinum red (Sigma Chemical Co, St Louis, USA) was carried out according to Torres et al. (29) (data not shown).

Culture media

The fungal strain was cultured on potato dextrose agar plates and transferred into production medium. The production medium (1 l) was contained 5 g polygalcturonic acid, 3 g ammonium sulfate, 10 g potassium dihydrogen phosphate, 2 g magnesium sulfate, 0.7 mg sodium borate, 0.5 mg ammonium molybdate, 10 mg iron sulfate, 0.3 mg copper sulfate, 0.11 mg manganese sulfate, 17.6 mg zinc sulfate and 100 mg amoxyciline at pH 7.2. Cultures were incubated for 24-144 h at 30 ^°C and passed through a filter paper were in 24 h intervals. The filtrate was centrifuged at 10,000 ×g for 20 min at 4^°C and the resulting supernatants were collected and used for subsequent polygalcturonase assay according to Chaplin and Kennedy (30). In each step the protein concentration was measured using a commercial assay kit (Bio-Rad Protein Assay kit) with bovine serum albumin as standard, following the procedure described by Bradford (31).

Polygalacturonase activity assay

In this colorimetric assay the increase of reducing end of galacturonic acid residues during the depolymerization of substrate, 1% (w/v) polygalacturonic acid in 20 mM sodium acetate buffer (pH = 6.0), was determined in the presence of 1% 3,5-dinitrosalicylic acid (DNS) (32).

The substrate (200 μl, 1% polygalacturonic acid) was added to the supernatant (200 μl) and incubated at 35 ^°C for 15 min. DNS reagent was added to the reaction mixture and boiled at 100 ^°C for 5 min. The absorbance was read at 530 nm using a UV spectrophotometer (Beckman DU530, USA). One unit (U) of polygalacturonase activity was defined as the amount of enzyme required to produce 1 micromole/min of reducing sugar once galcturonic acid is being used as standard.

Determination of factors required for optimal PG production

The optimization of medium constituents to improve production of PG by the fungus was carried out. Sugars (1% w/v) including monosaccharides (glucose, fructose, galactose, mannose, arabinose), disaccharaides (lactose, sucrose, maltose), and polysaccharides (starch, pectin, chitin) were used as carbon source in the medium. The sugar molecules were filter-sterilized and added to the production medium lacking polygalcturonic acid. The cultures were aerated at 160 rpm at 30 ^°C for 72 h. Similar conditions for nitrogen source were applied using 1% (w/v) of each pepton, (NH₄)₂SO₄, NH₄NO₃, NH₄Cl, CaNO₃.4H₂O, C₄H₁₁NO₃, (NH₄)₂HPO₄, trypton, and yeast extract following the removal of ammonium sulfate from the production medium. KH₂PO₄, K₂HPO₄, Na₂HPO₄, NaH₂PO₄ (1% w/v) were used separately as phosphate source. The best sources of the carbon, nitrogen, and phosphate were determined and used in combination with other factors in the following experiment.

Statistical optimization of growth condition

The PG production was measured in L27 in Taguchi’s orthogonal arrays considering combinatorial effects of seven factors with three levels (Table 1). On the basis of L27, 27 experiments were carried out in triplicate (Table 2) and the enzymatic assay was conducted as stated above. The results were analyzed using MiniTab software (version 14, USA). The mean values of data were analyzed with Qualiteck-4^TM (Nuteck Inc., MI, USA). It is worth stating that in the full-factorial experimental design, a total of 343 (7³) experiments needed to be carried out to provide the same conclusions as the Taguchi’s array method with 27 experiments. In order to determine the statistically significant factors influencing the PG production, analysis of variance was performed.

Table 1- Factors and their levels used to evaluate polygalacturonase production by M. phaseolina

Trace elements were 0.7 mg sodium borate, 0.5 mgammonium molibdate, 10 mgiron sulfate, 0.3 mgcopper sulfate, 0.11 mgmanganese sulfate and 17.6 mgzinc sulfate per liter.

Isolation and purification of M. phaseolina polygalacturonase via anion exchange chromatography

M. phaseolina  secretome, grown in the availability of polygalacturonic acid, was precipitated by 85% (w/v) ammonium sulfate. The precipitate was dialyzed against 20 mM sodium acetate buffer (pH = 7) at 4 ^°C for 24 h and fractionated by DEAE-Sepharose column. The column was equilibrated with 20 mM Tris buffer (pH = 8.5) and following loading the dialyzed solution, 2 mL fractions were eluted from the column by 20 mM Tris buffer (pH = 8.5) in a gradient of 0 to 1 M NaCl over a 16.5 h period. The fractions were checked at 280 and 530 nm for the presence of protein and polygalacturonase activity (as stated above), respectively. The fractions with the enzyme activity were further purified with the same column, but with a different gradient of NaCl (0-0.5 M). Samples of fractions with enzyme activity were visualized on SDS-PAGE following silver nitrate staining.

Table 2- Variable factors (7), their relative levels (3) and assignment of experimental factors of the L27 Taguchi’s array and relative enzyme activities

Temp: temperature, PGA: poly galacturonic acid, AS: ammonium sulfate, DPHP: dipotassium hydrogen phosphate, TE: trace element, MS: Manganese sulfate

pH profile

Purified polygalcturonase was used in assay reactions in the presence of 50 mM mixed buffer (glycine, sodium phosphate and sodium acetate) prepared in a range of pH from 3 to 12. To determine the pH profile, the enzyme solution incubated for 90 min at any particular pH was added to the substrate, prepared at certain pH (3- 12), and the mixture was incubated at 35 °C for 15 min. The enzyme activity was assayed colorimetrically at 530 nm.

In order to determine the pH stability, the enzyme fraction was incubated in 5 mM mixed buffer for 90 min and substrate was added to the reaction mixture. The enzyme stability was assessed in the same buffering condition, but in a time point manner for 180 min in 30 min intervals. At each time point the substrate, prepared in three pHs (3, 7, 12), was added and enzyme assay was conducted.

Temperature profile

To establish the temperature profile, 200 μl of each of substrate (1% w/v) and enzyme were incubated individually at the particular temperature (10 to 90 ^°C) for 5 min and combined with each other at the same temperature and incubated for 15 min. Subsequently, DNS reagent (400 μl) was added, the mixture was boiled at 100 ^°C for 5 min. The mixture was centrifuged at 6000×g for 15 min and the absorbance was measured at 530 nm.

Enzyme kinetics

The polygalacturonase enzyme kinetics was assayed in the presence of various concentrations of polygalcturonic acid as the substrate and plotted using Michaelis-Menton after measuring the catalytic parameters by Lineweaver-Burk curve. The assay reaction was repeated trice.

The effects of cations, chelators and chemicals on enzyme activity

A range of cations (NaCl, NiSO₄, MnCl₂, KCl, BaCl₂, MgSO₄, LiCl, FeSO₄, CaCl₂, CuSO₄, CoCl₂, ZnCl₂) in 1, 5, 10, and 15 mM, and 1%  of detergents (Urea, Tween 80, Tween 20, Triton X100), chemicals (glycerol, citric acid, ascorbic acid, Tris-HCl, naphtaline acetic acid, iodoacetamid, iodoacetic acid, PMSF) and EDTA as chelator were prepared in 20 mM acetate buffer (pH = 4) and enzyme activity were recorded and compared with control with no extra additives.

Results

Preliminary optimization of PG production

M. phaseolina was cultured in production medium for six days and in 24 h intervals samples were collected and assayed for PG activity. The results demonstrated that at 72 h the highest production of the enzyme was achieved (Fig. 1). Polygalcturonase production started to decline after 72 h. This decline may be explained either by diminishing the nutrients within the culture medium or through PG turnover via the hydrolytic activity of proteases possibly after 72 h (30). Analysis of carbon sources (Fig. 2a) revealed that in the availability of glucose and chitin, PG had the lowest activity that is an indication of lower production. Since glucose is the preferred choice of carbon by relatively all microorganisms, the production decline can easily be explained. However in the presence of chitin, the only reason that may explain the reduced PG production is its complex structure that may have trapped the enzyme, inhibiting it from properly performing its hydrolytic activity. In the presence of other sugars, polygalacturonase production was high. Although the highest production was noted when the polygalacturonic acid was included in the reaction, the production was indifferent from the other mono-, di-, and polysaccharides, presenting an unspecific response (Fig. 2a).

Amongst nitrogen sources that used in this experiment, the highest polygalacturonase production was seen in the presence of ammonium sulfate and peptone (Fig. 2b). Ammonium sulfate was chosen as the nitrogen source and used for Taguchi analysis, which also caused a stabilizing effect on the enzymes (33). From the different phosphate sources that used, dipotassium hydrogen phosphate was demonstrated a significant effect on PG production (Fig. 2c).

Fig. 1- Determination of the best incubation period to achieve the highest PG production. The highest production was noticed after 72 h. The experiment was performed in three replicates

Fig. 2- Determination of the best carbon (a), nitrogen (b) and phosphate (c) sources for PG production. Each experiment was carried out in three replicates

Taguchi’s analysis of variance

In order to provide the best condition for M. phaseolina to produce more polygalacturonase, seven factors including temperature, pH, polygalcturonic acid as substrate, trace elements, ammonium sulfate, dipotassium hydrogen phosphate and magnesium sulfate in three levels were analyzed using Taguchi’s L27 orthogonal array method (Tables 1 and 2). The PG activity within each experiment was measured as stated above (Table 2). Analysis of variance was showed that temperature (P value <0.01), pH (P< 0.01), ammonium sulfate (P < 0.05) and trace elements (P < 0.05) had significant effects on polygalacturonase production (Table 3). Response values of means were compared to determine the best level of each factor. The data was demonstrated that the highest production rate for polygalacturonase can be achieved at pH = 6.0, 35 ^°C, in the presence of 2 g.l^-1 ammonium sulfate and either 0.1% or 0.2% (v/v) trace elements. For small scale product optimization, the enzyme activity that is an indication of enzyme production improved by almost 100% (from 0.537 enzyme unit to 1.116; Table 2).

Isolation of polygalcaturonase and partial characterization

Anion exchange chromatography in two steps was used to isolate the ammonium sulfate precipitated secretome of M. phaseolina polygalacturonase. Following the second chromatography, two peaks (fractions 47 and 56) with polygalacturonase activity were evident (Fig. 3a).  The purified fractions were separated on SDS-PAGE side by side to the ammonium sulfate precipitated secretome. The presence of two purified polygalcturonases was apparent with molecular weights of 120 and 60 kDa (Fig. 3b). The enzyme with relative molecular mass of 60 kDa (fraction 56) was used for further analysis for its higher purity.

Table 3- Analysis of variance of seven factors to determine their significant effect on polygalacturonase production by M. phaseolina.

SOV: sources of variance, df: degrees of freedom, SS: sum of squares, MS: mean of squares, F: Fischer’s Test. *: significant level at 0.05%, **: significant level at 0.01%, ns: non-significant.

The purified polygalacturonase of M. phaseolina grown in the availability of polygalacturonic acid was analyzed to determine the pH optima. The optimum pH was appeared to be 3 (Fig. 4a), while the polygalcturonase activity was appeared to be stable in a relatively wide range of pH (2-8, data not shown). Enzyme was stable at pH 4 and 7 for 180 min without a significant decline in its activity. The enzyme was assayed for polygalacturonase activity at different temperatures and the highest activity was achieved at 30 ^°C (Fig. 4b).

Based on substrate concentrations, Michaelis-Menton curve (Fig. 5) was plotted and the enzyme catalytic parameters were calculated with Lineweaver-Burk standard line (Fig. 5, right panel: y = 0.425x + 4.55, R² = 0.904). Accordingly V_max and k_m were 0.219 U/mL and 0.093 mg/ml, respectively.

Fig. 3- Anion exchange chromatography to isolate secreted polygalacturonases from M. phaseolina. a) DEAE-Sepharose was used to separate secreted proteins using NaCl gradient (0-0.5 M) in a period of 80 min, fractions 47 and 56 demonstrated polygalacturonase activity (a). SDS-PAGE separation of fractions 47 (lane 2), 56 (lane 3), and ammonium sulfate precipitated secretome prior to column chromatography (lane 4) (b).

Fig. 4. Temperature and pH profile of purified polygalacturonase with relative molecular mass of 60 kDa. The PG was active at pH range of 2-6 and the highest activity was observed at pH = 3 (a). PG was active at temperatures ranging from 20 to 50 ^°C with the highest activity at 30 ^°C (b). Each experiment performed in three replicates.

Fig. 5. The curve represents the Michaelis-Menton equation in different substrate concentrations. The linear graph represents the Lineweaver-burk equation. The calculated V_max and K_m for PG with MW = ~ 72 kDa are 0.219, and 0.093, respectively. The data were collected from three replicates (R² = 0.904).