Purification and characterization of the pectin lyase secreted ... - NOPR

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Indian Journal of Biotechnology Vol 5, July 2006, pp 284-291

Purification and characterization of the pectin lyase secreted within the macerating fluid of Rhizopus oryzae (Went & Prinsen Geerligs) grown on orange peel Hossam S Hamdy* Biological Sciences and Geology Department, Faculty of Education, Ain Shams University Roxy, Heliopolis, Cairo 11757, Egypt Received 14 December 2004; revised 16 August 2005; accepted 10 October 2005 Potentiality of Rhizopus oryzae to utilize orange peel, an inexpensive and low-cost substrate, under solid state fermentation (SSF) conditions to produce macerating fluid with high cellulolytic and pectinolytic activities was confirmed in the present work. Addition of NH4NO3 and NH4Cl to the fermentation medium improved the macerating potentiality due to an increase in cellulase and pectinase levels. The pectin lyase (PL) secreted by R. oryzae was also purified to electrophoretic homogeneity, using ammonium sulfate fractionation and 2-step-column chromatography. The PL was purified 22-folds and its specific activity was 2313 U/mg protein. The purified PL expressed its maximum activity at 50°C and pH 7.5, showed good stability in the pH range of 7 to 9.5 and its midpoint of thermal inactivation (Tm) was recorded at 70°C after 45 min of exposure. Presence of Ca2+ enhanced the activity and thermal stability of the purified PL. Ions of Mg, Na and K showed stimulatory effects on the enzyme activity, while ions of Zn, Co, Mn and Hg were inhibitory. The results suggest that possibly SH group in PL structure participated in the activity of the enzyme. Values of Km, Vmax, Kcat and molecular mass of the purified enzyme were 3.87 mg/mL, 297 U/mL, 5.94 mg U-1 min-1 and 31 kDa, respectively. Keywords: cellulase, orange peel, pectin lyase, Rhizopus oryzae, solid state fermentation IPC Code: Int Cl.8 C12N19/88; C12R1/845

Introduction World over, macerating enzymes are rapidly replacing the traditional methods in various applications of biotechnology, such as separating cells from intact walls; production of biologically active substances; extraction of essential oils from medicinal and spicy aromatic raw materials; processing berries, fruits and vegetables; clarifying juices and wine; and also in textile industries1-4. Several enzyme preparations from microbs, viz. Aspergillus alliaceus, A. awamori, Colletotrichum gloeosporioides, C. lindemuthianum, C. magna, Fusarium solani, Pseudomonas fluorescens, 5-8 P. viridiflava and Pythium splendens , were reported to have macerating activity, partially or down to individual cells, against plant tissues. These enzyme preparations typically contain pectinases, cellulases, hemicellulases and other carbohydratases7,9,10. These hydrolytic enzymes, especially pectinases and cellulases, were considered the most active enzymes to which macerating activity could rely7. Among different pectinases, pectin lyase (PL) seems to be the only pectic enzyme capable of breaking down pectin, with a high degree of esterification, into smaller molecules and can work on both methylated and/or non-methylated groups ______________ *E-mail: [email protected]

of galacturonic acid11,12. Therefore, it could be an enzyme of potential interest. Solid state fermentation (SSF) has started to replace the submerged or static fermentation, as an alternative simple technology, due to the excessively increasing cost of enzyme production13. SSF has several advantages over other methods, such as ability to reach high product concentrations, production of less liquid effluents and lesser control of pH3. The SSF has been extensively studied using microorganisms, often fungi, to yield a variety of products including enzymes and food14-15. Moreover, use of inexpensive substrates, such as malt sprouts4, agricultural wastes like orange pulp16 and pectin-containing materials like lemon and orange peels17-18 can further economize the process of fermentation. Orange processing industries generate thousand tons of orange peel per year, which are marketed as animal feed16. The present work was devoted to the production of an extracellular enzymatic complex, having a potent macerating activity, by Rhizopus oryzae (Went & Prinsen Geerligs) in a solid state culture utilizing orange peel. The diversity of enzymes detected under these conditions was also studied and PL was purified and characterized.

HAMDY: PECTIN LYASE FROM R. ORYZAE GROWN ON ORANGE PEEL

Materials and Methods Microorganism and Enzyme Preparation

R. oryzae used in this work was previously isolated from Egyptian soil and identified by Centraalbureau Voor Schimmelcultures, Netherlands. The fungus was maintained on malt extract agar at 4°C by routine culture in the laboratory. Fresh, washed and ground orange peel (20 g) were kept in triplicate sets of 250 mL Erlenmeyer conical flasks with 8 mL distilled water. Each flask was sterilized and inoculated with 1 mL freshly prepared spore suspension (1.95 × 105 spores) of R. oryzae from 7-d-old cultures. Each flask was statically incubated at 30°C for 10 d. The content of each flask was thoroughly mixed with 10 mL cooled distilled water, filtered off through Buchner’s apparatus and served as the crude enzyme preparation. Chemical Analysis of Orange Peel

Orange peel was analyzed in terms of cellulose, hemicellulose and pectin contents as described by Jermyn19. Tissue Maceration Test

Tissue maceration was evaluated and rated as described before7,20. Discs (10×1 mm) of potato tubers and cucumber fruit tissues, used as the substrate, were placed in enzyme preparations buffered at pH 8.0 with 5 mM Tris-HCl buffer and measurements of tissue maceration were made over 100 min incubation period with the enzyme. Discs which were not macerated received a score of 0 and those macerated completely received a score of 5. Enzyme and Protein Assays

Activities of exo-β-(l→4)-glucanase (C1), endo-β(l→4)-glucanase (Cx) and β-glucosidase (C2) were assayed using microcrystalline cellulose, carboxymethylcellulose and cellobiose as substrates, respectively. One mL of culture filtrate was added to 1 mL of 0.2 M acetate buffer (pH 4.8) containing 10 mg of the specific substrate and the total volume was completed to 3 mL. Mixtures were incubated for 30 min at 40°C and assayed by measuring the release of reducing sugars (as glucose) at 575 mμ21. One unit of enzyme was defined as the amount of enzyme that releases 1 μmol min-1 of reducing sugar equivalent under the assay conditions. Polygalacturonase (PG) was determined by measuring the amount of reducing sugar released from sodium polygalacturonate as substrate22. One

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unit of the enzyme was defined as the amount of enzyme that releases 1 μmol min-1 of reducing sugar at 40°C using monogalacturonic acid as standard. PL was assayed by measuring the increase in absorbance of the enzymatic products at 235 mμ23. The reaction mixture containing 1 mL of 0.5% citrus pectin (dissolved in 0.05 M Tris-HCl buffer, pH 8.0) and 0.5 mL of the enzyme solution was incubated at 30°C for 60 min. The reaction was stopped by adding 3.5 mL of 0.5 M HCl. For blank, the acid was added initially to the enzyme solution. One unit of PL was defined as the amount of the enzyme that releases 1 μmol min-1 of 4,5-unsaturated digalacturonic acid24. Pectin methyl esterase (PM) activity was assayed as follows25: 5 mL of 0.5% solution of citrus pectin (prepared in 0.15 M NaCl) was added to 1 mL of 0.01% solution of bromothymol blue (prepared in 0.02 M potassium phosphate buffer, pH 7.5) and 0.5 mL of enzyme solution (adjusted to pH 7.5 with conc. NaOH). The absorbency was determined at 620 mμ. Protein content was assayed using bovine serum albumin as standard26. Enzyme Purification

Protein content of the cell-free filtrate (CFF) was precipitated overnight by 65% ammonium sulfate, collected by centrifugation at 12×103 g for 15 min, desalted by passing through column of Sephadex G25 and then fractionated by 2-steps of column chromatography, where 2 mL solution was cautiously applied to a column (2.5×82 cm) of Sephadex G150 (Pharmacia product) equilibrated with 0.05 M TrisHCl buffer (pH 8.0) for gel filtration and of DEAEcellulose (diethylaminoethyl-cellulose, fast flow, fibrous form—Sigma product) for ion-exchange chromatography. Five mL fraction was eluted with 0.05 M Tris-HCl buffer, pH 8.0 in case of Sephadex column and with a linear gradient of NaCl (0.0 to 0.5 M prepared in the same buffer) in case of DEAEcellulose. Fractions were assayed for protein content and enzyme activity and the most active fractions in terms of their specific activities were pooled, desalted, lyophilized and kept cooled for the subsequent work. Characterization of PA

pH optimization studies were performed by carrying out the reaction at different pH values using different buffers (0.1 M phosphate for pH 6.0-7.0; 0.05 M Tris-HCl for 7.0-9.0 and M sodium bicarbonate-sodium carbonate for 9.5-10) and the activity was measured under the standard assay

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conditions. For pH stability determination, enzyme solution was incubated for variable time periods at fixed pH values ranged from 6 to 10.5 and the residual activity in each treatment was assayed. Similarly, reaction mixture was incubated for 60 min at different temperatures (30 to 65°C) and enzyme activity was measured to determine the optimum temperature for activity. However, the enzyme was incubated for variable durations (0 to 60 min) at fixed temperatures (50 to 85°C) for determination of thermal stability. The molecular mass of the enzyme preparation was estimated by gel filtration27 on Sephadex G75. The elution pattern was calibrated using the following proteins: lysozyme, 14.3; chymotrypsin, 25.1; carbonic anhydrase, 29; ovalbumin, 43; bovin serum albumin, 66; phosphorylase, 94; alcoholic dehydrogenase, 150; and catalase, 232 kDa, and a standard calibration graph was constructed. Results and Discussion After a preliminary investigation, wherein 25 fungal species were tested for their ability to grow on orange peel and/or produce macerating enzymes active against potato and cucumber discs, Rhizopus oryzae was found to be the most potent fungus (data not shown). The enzymes, obtained in appreciable amount, from the macerating fluid of R. oryzae grown on orange peel are given in Table 1. This particular spectrum of enzymes could be related to the relatively high amounts of cellulose and pectin in the fermentation medium. The chemical analysis of orange peel showed that it contained 25.3, 20.5 and 32.8% of cellulose, hemicelluloses and pectin, respectively. In spite, the presence of 20% hemicelluloses present in the orange peel, xylanase activity was only limited to 0.75 U/g orange peel. Ability of R. oryzae strain NBRC 4707 and strain NRRL 395 to produce cellulase and pectinase was previously reported15,18 but it was discussed only in relation to their role in the production of lactic acid and ethanol. An effort was made to improve the yields of cellulase and pectinase through enriching the orange peel medium with some natural additives (0.5%, w/v) or nitrogenous compounds (0.7%, w/v). Table 2 demonstrates that the maximum productivity of cellulolytic and pectinolytic enzymes was recorded in presence of NH4NO3 and NH4Cl, respectively. The similar effect of additives and nitrogenous compounds

on the level of the enzymes detected in the macerating fluid of Aspergillus spp. was reported elsewhere4,7,17. Production of cellulase and pectinase in presence of different concentrations of NH4NO3 and NH4Cl, respectively, was also studied (data not shown). Maximum production of C1 was recorded at 0.7% (w/v) NH4NO3, while maximum production of both Cx and C2 was recorded at 0.6% NH4NO3. Meanwhile, the maximum production of pectinolytic enzymes (PG, PM & PL) was attained at 0.7% (w/v) NH4Cl. Macerating activities of the enzyme preparations produced by R. oryzae in presence of NH4Cl or NH4NO3 was studied. In presence of NH4Cl, macerating activity was at its maximum level, while it was slightly higher or similar to that of control in presence of NH4NO3 (Table 3). As shown in Table 2, there was a sharp increase in the level of pectinolytic enzymes in presence of NH4Cl, while the presence of NH4NO3 resulted in an increase in cellulolytic enzymes and a decrease in pectinolytic enzyme. Thus, the results suggest an important role for the pectinoytic enzymes in the macerating activity. Moreover, the level of PL (17.71 U/g) was higher than that of PG (14.18 U/g) and PME (11.52 U/g; Table 2), which indicates that PL is an enzyme capable of degrading pectin with a high degree of esterification and can work on methylated and/or nonmethylated groups of galacturonic acid11-12. These observations show PL as an enzyme of potential interest. Therefore, the work was extended to purify and characterize PL. Purification of PL

The steps of enzyme purification are summarized in Table 4. Precipitation of the protein content of the CFF was performed by 65% ammonium sulfate. The precipitate was collected by centrifugation that resulted in an active pellet containing 70.79% of the original activity and specific activity of 209.63 U/mg. Further fractionation of the enzyme preparation was done through Sephadex G150. Most of the PL activity was recovered in fractions 15 to 21 representing Table 1—Enzyme profile of the macerating enzyme complex produced by R. oryzae grown on orange peel Enzyme Cellulase (C1) Carboxymethyl cellulose (Cx) Cellobiase (C2) Polygalacturonase (PG) Pectin methyl esterase (PM) Pectin lyase (PL)

Units (U/g orange peel) 2.35 1.48 1.75 4.60 3.27 5.20

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Table 2—Effect of some natural additives and nitrogenous compounds on the level of cell wall degrading enzymes produced by R. oryzae Substance# Malt extract Yeast extract Beef extract Soybean meal Fish meal Peptone Molasses Corn-steep liquor (NH4)2SO4 NH4Cl NH4NO3 NH4H2PO4 (NH4)2HPO4 KNO3 NaNO3 Casein hydrolysate Control

Activity (U/g orange peel) C1

Cx

C2

PG

3.84+0.12* (164.10) 2.87+0.11* (122.65) 2.11 +0.10* (90.17) 2.82 +0.11* (120.51) 1.87 +0.06* (79.91) 3.28 +0.12* (140.17) 1.82 +0.04* (77.77) 3.91 +0.16* (167.09) 3.04 +0.11* (129.91) 3.23 +0.11* (138.03)

2.35+0.10* (159.86) 1.97 +0.05* (134.01) 1.35 +0.06* (91.84) 1.76 +0.09* (119.73) 1.23 +0.06* (83.67) 2.35 +0.07* (159.86) 1.19 +0.04* (80.95) 2.51 +0.11* (170.75) 1.92 +0.04* (130.61) 2.06 +0.06* (140.14)

2.85+0.11* (162.86) 2.33 +0.10* (133.14) 1.66 +0.07* (94.86) 2.11 +0.11* (120.57) 1.44 +0.05* (82.29) 2.28 +0.09* (130.29) 1.39 +0.06* (79.43) 2.95+0.11** (168.57) 2.32 +0.09* (132.57) 2.45 +0.13* (140.0)

*

*

*

5.85 +0.19

(250.0)• 2.57 +0.07* (109.83) 2.34 +0.05* (100.00) 3.16 +0.14* (135.04) 3.99 +0.20* (170.51) 2.62 +0.11* (111.97) 2.34 +0.08*

3.15 +0.08

(214.29)• 1.69 +0.05* (114.97) 1.52 +0.03* (103.40) 2.02 +0.04* (137.41) 2.54 +0.08* (172.79) 1.63 +0.03* (110.88) 1.47 +0.04*

4.03 +0.08

(230.29)• 1.94 +0.06* (110.86) 1.90 +0.08* (108.57) 2.59 +0.08* (148.00) 2.87 +0.11* (164.0) 1.92 +0.06* (109.71) 1.75 +0.07*

PME

PL

12.86+0.65** (278.35) 9.24 +0.48* (200) 2.95 +0.13* (63.85) 7.37 +0.21* (159.52) 4.72 +0.20* (102.16) 5.27 +0.13* (114.07) 5.32 +0.18* (115.15) 7.82 +0.31* (169.26) 7.57 +0.24* (163.85) 14.18+0.31*

9.24+0.30* (282.57) 7.27 +0.26* (222.32) 2.46 +0.10* (75.23) 5.16 +0.23* (157.80) 3.37 +0.16* (103.06) 3.67 +0.11* (112.23) 3.62 +0.11* (110.70) 5.66 +0.08* (173.09) 5.26 +0.16* (160.86) 11.52+0.47*

15.30+0.57* (293.67) 10.94+0.47* (209.98) 2.80 +0.08* (53.74) 8.39 +0.26* (161.04) 5.48 +0.23* (105.18) 6.20 +0.22* (119.00) 5.68 +0.21* (109.02) 8.76 +0.44* (168.14) 8.34 +0.33* (160.08) 17.71+0.69*

(306.93)• 4.07 +0.13* (88.10)

(352.29)• 2.92 +0.06* (89.30)

(339.92)• 4.54 +0.13* (87.14)

12.19+0.09* (263.85) 5.49 +0.09* (118.83) 4.17 +0.06* (90.26) 3.87 +0.07* (83.77) 4.61 +0.14* (99.78) 4.62 +0.17*

7.62 +0.30* (233.03) 3.95 +0.15* (120.80) 2.79 +0.06* (85.32) 3.19 +0.08* (97.55) 3.35 +0.09* (102.45) 3.27 +0.10*

11.36+0.37* (218.04) 5.48 +0.15* (105.18) 4.69 +0.22* (90.02) 4.89 +0.15* (93.86) 5.48 +0.25* (105.18) 5.21 +0.12*

Value in the table represents the mean of 3 different readings expressed as U/g orange peel + standard deviation and value in parenthesis represents % of increase or decrease in enzyme levels as compared to control. • = the highest level of enzyme to which other data were statistically compared (Glantz, 1992)36. Results were considered non-significant, significant or highly significant when P > 0.05, < 0.05 or P < 0.01, respectively and expressed as n = non-significant, ** = significant or * = highly significant, in order. # Natural additives were dissolved before sterilization in the liquid portion of the medium (8 mL) at a final concentration of 0.5%, while the nitrogenous compounds were added as equimolar amounts of nitrogen at 0.7%.

53.22% of the original activity, specific activity of 496.72 U/mg and 4.83-fold purification. These fractions were pooled, lyophilized and subjected to further fractionation onto DEAE-cellulose using linear gradient of NaCl as eluant. A single peak

containing 2313.23 U/mg protein with 22.47-fold purification was obtained in fractions 15-22. This enzyme preparation was salted out twice through Sephadex G25, lyophilized and its purity to electrophoretic homogeneity was confirmed by

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performing SDS-PAGE, where a single major band was obtained. Final concentration of the enzyme was adjusted to 100 U/mL. Effect of pH Value on PL Activity and Stability

Testing the pH-dependence of PL activity revealed that pH 7.5 was optimum for the enzyme activity (Fig. 1). The activity was sharply decreased at the acidic side, while the decline was minor at the alkali side. The same optimum pH was observed for PL from Aureobasidium pullulans23, while pH 6.4 and 5.0 (acidic values) were found optimal for PL from A. niger28 and Curvularia inaequalis29, respectively. As the pH value diverged from the optimum level, the efficiency the enzyme gets affected (Fig. 1). This could be ascribed to decreased saturation of the enzyme with substrate due to decreased affinity

and/or due to the effect of pH on the stability of the enzyme30. This was distinguished experimentally in the present work by testing pH stability, where irreversible destruction was observed at the acidic side and the original activity could not be restored even after readjusting the pH to the original value (Fig. 1). Effect of Temperature on PL Activity and Stability

The experimental data represented in Fig. 2 show that PL from R. oryzae recorded its optimum activity at 50°C. PLs from other sources have been reported to be optimally active in the range of 40-50°C9,23,29, Table 5—Effect of different metal ions and some enzyme inhibitors on the relative activity of the purified PL Metal ions■

Table 3—Tissue maceration of potato and cucumber discs treated with the enzyme preparation produced by R. oryzae grown on orange peel fortified with NH4NO3 or NH4Cl Tissue

Potato

Cucumber

Time (min)

Maceration rate #of the enzyme preparation produced in presence of Orange peel medium*

NH4NO3

NH4Cl

20 40 60 80 100

1 1 2 3 4

1 2 3 3 4

2 4 5 5 5

20 40 60 80 100

0 1 2 3 3

1 2 3 3 3

1 2 3 4 4

* Control Tissue maceration was measured by immersing discs (10×1 mm) of potato tubers or cucumber fruit tissues, as substrates, in 3 mL of enzyme preparation buffered with 5 mM Tris-HCl, pH 8.0 at 30°C (0 = not macerated, 1-4 = progressively macerated, and 5 = completely macerated). All experiments were performed in triplicates. Heat killed enzyme used as control. #

2+

Ba Ca2+ Co2+ Cu2+ Fe3+ Hg2+ K+ Mg2+ Mn+ Na+ Zn+ Enzyme inhibitor` EDTA Iodoacetate Sodium arsenate Sodium arsenite

Relative activity as affected by the concentration of 1 mM

5 mM n

10 mM n

105 + 4.20 119 + 5.39** 86 + 4.69** 99 + 2.62n 101 + 2.01n 21 + 0.32** 113 + 2.14** 114 + 6.46* 99 + 4.08n 110 + 3.16** 88 + 1.45**

99 + 2.62 128 + 5.13** 71 + 1.48** 73 + 2.48** 93 + 3.48* 2 + 0.07** 125 + 3.20** 112 + 2.07** 84 + 1.67** 117 + 5.54** 59 + 2.27**

91 + 4.30* 134 + 3.27** 54 + 1.29** 51 + 1.38** 80 + 3.19** 0 + 0** 130 + 4.46** 102 + 3.25n 73 + 1.53** 130 + 4.15** 47 + 1.66**

80 + 3.00** 65 + 1.03** 73 + 3.00**

71 + 1.53** 48 + 2.00** 69 + 1.23**

68 + 1.57** 16 + 0.86** 44 + 1.00**

63 + 1.63**

42 + 1.75**

30 + 1.02**

The investigated metal ions were added as chloride at the indicated concentration and was incubated with the enzyme for 30 min at 30°C before adding substrate. Activity of the PL in complete absence of such compounds served as control (100% activity). Data represent the mean of 3 readings approximated to the nearest integer number.

Table 4—Summary of the steps followed throughout purification of PL of R. oryzae Step#

Initial volume (mL) Total activity (U)

Total protein (mg)

Specific activity Yield Purification (U/mg) (%) fold(s)

Crude cell filtrate (CFF) 200 3500.00 34.00 102.94 Protein precipitate of NH4(SO4)2 (65%) 200 2477.65 11.82 209.62 1862.7 3.75 496.72 Gel filtration 2* Ion exchange chromatography 2* 1503.6 0.65 2313.23 Total volume used was 200 mL of enzyme preparation obtained from 20 flasks, each containing 20 g orange peel. # Initial enzyme activity in each step is that resulted and given in the previous step. * Concentrated by lyophilization to be applied to the column.

100.0 70.79 53.22 42.96

1 2.04 4.83 22.47

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while 70°C was reported to be optimum for PL from A. niger28. Moreover, mid-point of thermal inactivation (Tm) was determined and it was found to be at 70°C after 45 min of exposure. The enzyme retained its original activity after heating up to 50°C for 1 h (Fig. 3). Above this temperature, a gradual loss in the enzymatic activity was observed and the enzyme completely lost its activity after exposure to 85°C for 45 min. A similar stability was observed for PL from P. splendens8. Fig. 1—Activity and stability of the purified PL produced by R. oryzae as a function of pH value. 0.1 M phosphate (__♦__), 0.05 M Tris-HCl (__□__) and M sodium bicarbonate-sodium carbonate (__x__) buffers were used for the pH values 6-7, 7.5-9 and 9.510.5, respectively. Measurement of the residual activity (__„__) was assessed at pH 7.5 after 60 min of exposure to different pH values. Temperature of the reaction mixture containing 1 mL of 0.5% citrus pectin as substrate was adjusted at 30°C. Each value on the graph represents the mean of 3 different readings and the error bars represent the standard deviation.

Fig. 2—Effect of temperature on the activity of the purified PL produced by R. oryzae. Reaction mixture was held at the indicated temperatures for 60 min and other conditions are those as described in Fig. 1.

Fig. 3—Thermal stability of the purified PL produced by R. oryzae. Residual activity was measured after the enzyme was held at the indicated temperatures for different times of exposure at 50°C and other conditions were similar to those described in Fig. 2.

Effect of Metal Ions and Inhibitors on PL Activity

Effect of some metal ions and enzyme inhibitors on PL activity was investigated (Table 5). Results show that PL activity was pronouncedly increased in presence of Ca2+, Mg2+, Na+ or K+. It is known that alkali-metal cations (Na+ and K+) bind weakly to the enzymes to form ternary complexes between enzyme (E), metal ions (M) and substrate (S) in different ways (M-E-S, E-S-M or E-M-S) that results in enhancing the enzyme activity30-31. Moreover, potassium ions are known to activate many enzymes and also aid in substrate binding31. Ca2+ appears to play a role in maintaining the structural integrity required for catalytic activity of PL as reported in earlier studies6,31-33. The possible role of Ca2+ in maintaining the structural integrity and stability of PL from R. oryzae was studied by investigating thermal inactivation (Tm) of the enzyme in presence or absence of CaCl2 (10 mM) and citrus pectin (0.5%, w/v). Reactions carried out at 70°C, at which Tm of the enzyme was detected (Fig. 3). The thermal stability of PL was significantly enhanced in presence of CaCl2, while it was non-significantly affected or reduced in presence of pectin (Table 6). This suggests that PL from R. oryzae is greatly dependent upon the presence of Ca ions. Further, the activity of PL was moderately inhibited in presence of Zn3+ Co2+ or Mn+ and severely inhibited in presence of Hg2+. However, presence of Ba2+ was almost non-significant. Similar findings were reported for other PLs from P. splendens8 and Aspergillus sp.12. The sever inhibitory effects caused by Hg2+ as well as sodium arsenite and arsenate on the PL activity (Table 5) show that possibly SH group in the structure participates in the enzyme activity. Moreover, the inhibition of PL activity in presence of iodoacetic acid (SH-group specific inhibitor) represents additional evidence. Inhibitory effect of iodoacetic acid on PLs from P. fluorescence and P. viridiflava has also been reported6. Addition of EDTA resulted in considerable

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Table 6—Effect of CaCl2 or citrus pectin on the thermal stability of the purified PL produced by R. oryzae Residual activity (%) after Treatment Control + CaCl2 + Pectine

0 min

15 min

30 min

45 min

60 min

100 + 0 100 + 0n 99+1.7n

81 + 2.43 87.3 + 3.1** 79.19 + 2.8n

65 + 1.62 74.66+2.65* 61.94 + 1.9n

44 + 1.49 52.06 + 1.6* 36.67 + 0.81**

18 + 1.2 28.08+0.95* 15.12 + .3**

Enzyme preparation was incubated at 70°C at the indicated times in mixtures containing CaCl2, citrus pectin or lacking any of them (control). Satistical analysis was performed in comparison to control where n, **, * = non-significant, significant and highly significant, respectively.

splendens and C. lindemuthianum8,9 and 38 kDa for PL from A. niger28. The secreted PL is a glycoprotein, where 2.3% of its composition is carbohydrate as detected by the phenol/sulfuric acid method35. Thus, it can conclude from the present work that there is a possibility to produce macerating enzyme preparation from R. oryzae with a relatively very low cost and economically attractive method. References Fig. 4—Initial velocity of PL (U/mL) against initial pectin concentration (mg/mL) at a constant total enzyme concentration. From the data, 1/s and 1/v were calculated and used to construct the inserted Linweaver-Burk plot.

inhibition of the enzyme activity (Table 5), which is in contradiction with earlier findings where no effect of EDTA was recorded on PL from P. splendens8. Aliquots of EDTA-inhibited enzyme were incubated with Ca2+ (10 mM) for 1 h at 30°C and the residual activity of the enzyme was assayed. Presence of Ca2+ reactivated the PL at 102% of its original activity and this is additional evidence showing the importance of Ca2+ for the PL activity. Other Enzymatic Properties

Fig. 4 shows that at low pectin concentrations, the reaction was first-order but at higher concentrations, it became zero-order with the Vmax of 297 U/mL. From the Linweaver-Burkto plot (inserted within Fig. 4), the apparent Km value of PL for pectin was calculated to be 3.87 mg/mL and the turnover number (Kcat) 5.94 mg U-1min-1. Km values for PLs from Penicillium italicum35; A. niger28 and C. inaequalis29 were earlier recorded to be 4.4 mg/mL, 0.2 mM and 0.35 mM, respectively. Molecular mass of the purified PL was found to be 31+0.5 kDa as estimated by gel filtration, while earlier only 23 kDa was recorded for PL from P.

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