Purification and Characterization of the Pectin Lyase Secreted Within the Macerating Fluid of Rhizopus Oryzae (Went & Prinsen Geerligs) Grown on Orange Peel

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 of galacturonic acid11,12. Therefore, it could be an World over, macerating enzymes are rapidly enzyme of potential interest. replacing the traditional methods in various applications Solid state fermentation (SSF) has started to of biotechnology, such as separating cells from intact replace the submerged or static fermentation, as an walls; production of biologically active substances; alternative simple technology, due to the extraction of essential oils from medicinal and spicy excessively increasing cost of enzyme production13. aromatic raw materials; processing berries, fruits and SSF has several advantages over other methods, vegetables; clarifying juices and wine; and also in textile such as ability to reach high product concentrations, industries1-4. Several enzyme preparations from microbs, production of less liquid effluents and lesser control viz. Aspergillus alliaceus, A. awamori, Colletotrichum of pH3. The SSF has been extensively studied using gloeosporioides, C. lindemuthianum, C. magna, microorganisms, often fungi, to yield a variety of Fusarium solani, Pseudomonas fluorescens, products including enzymes and food14-15. P. viridiflava and Pythium splendens5-8, were reported Moreover, use of inexpensive substrates, such as to have macerating activity, partially or down to malt sprouts4, agricultural wastes like orange pulp16 individual cells, against plant tissues. These enzyme and pectin-containing materials like lemon and preparations typically contain pectinases, cellulases, orange peels17-18 can further economize the process hemicellulases and other carbohydratases7,9,10. These of fermentation. hydrolytic enzymes, especially pectinases and cellulases, Orange processing industries generate thousand were considered the most active enzymes to which tons of orange peel per year, which are marketed as macerating activity could rely7. Among different animal feed16. The present work was devoted to the pectinases, pectin lyase (PL) seems to be the only pectic production of an extracellular enzymatic complex, enzyme capable of breaking down pectin, with a high having a potent macerating activity, by Rhizopus degree of esterification, into smaller molecules and can oryzae (Went & Prinsen Geerligs) in a solid state work on both methylated and/or non-methylated groups culture utilizing orange peel. The diversity of ______enzymes detected under these conditions was also *E-mail: [email protected] studied and PL was purified and characterized. HAMDY: PECTIN LYASE FROM R. ORYZAE GROWN ON ORANGE PEEL 285

Materials and Methods unit of the enzyme was defined as the amount of Microorganism and Enzyme Preparation enzyme that releases 1 μmol min-1 of reducing sugar R. oryzae used in this work was previously isolated at 40°C using monogalacturonic acid as standard. from Egyptian soil and identified by Centraalbureau PL was assayed by measuring the increase in Voor Schimmelcultures, Netherlands. The fungus was absorbance of the enzymatic products at 235 mμ23. maintained on malt extract agar at 4°C by routine The reaction mixture containing 1 mL of 0.5% citrus culture in the laboratory. pectin (dissolved in 0.05 M Tris-HCl buffer, pH 8.0) Fresh, washed and ground orange peel (20 g) were and 0.5 mL of the enzyme solution was incubated at kept in triplicate sets of 250 mL Erlenmeyer conical 30°C for 60 min. The reaction was stopped by adding flasks with 8 mL distilled water. Each flask was 3.5 mL of 0.5 M HCl. For blank, the acid was added sterilized and inoculated with 1 mL freshly prepared initially to the enzyme solution. One unit of PL was 5 spore suspension (1.95 × 10 spores) of R. oryzae defined as the amount of the enzyme that releases 1 from 7-d-old cultures. Each flask was statically μmol min-1 of 4,5-unsaturated digalacturonic acid24. incubated at 30°C for 10 d. The content of each flask Pectin methyl esterase (PM) activity was assayed was thoroughly mixed with 10 mL cooled distilled as follows25: 5 mL of 0.5% solution of citrus pectin water, filtered off through Buchner’s apparatus and (prepared in 0.15 M NaCl) was added to 1 mL of served as the crude enzyme preparation. 0.01% solution of bromothymol blue (prepared in 0.02 M potassium phosphate buffer, pH 7.5) and 0.5 Chemical Analysis of Orange Peel mL of enzyme solution (adjusted to pH 7.5 with conc. Orange peel was analyzed in terms of cellulose, NaOH). The absorbency was determined at 620 mμ. hemicellulose and pectin contents as described by 19 Protein content was assayed using bovine serum Jermyn . albumin as standard26.

Tissue Maceration Test Enzyme Purification Tissue maceration was evaluated and rated as 7,20 Protein content of the cell-free filtrate (CFF) was described before . Discs (10×1 mm) of potato tubers precipitated overnight by 65% ammonium sulfate, and cucumber fruit tissues, used as the substrate, were collected by centrifugation at 12×103 g for 15 min, placed in enzyme preparations buffered at pH 8.0 with desalted by passing through column of Sephadex G- 5 mM Tris-HCl buffer and measurements of tissue 25 and then fractionated by 2-steps of column maceration were made over 100 min incubation chromatography, where 2 mL solution was cautiously period with the enzyme. Discs which were not applied to a column (2.5×82 cm) of Sephadex G macerated received a score of 0 and those macerated 150 (Pharmacia product) equilibrated with 0.05 M Tris- completely received a score of 5. HCl buffer (pH 8.0) for gel filtration and of DEAE-

Enzyme and Protein Assays cellulose (diethylaminoethyl-cellulose, fast flow, fibrous form—Sigma product) for ion-exchange Activities of exo-β-(l→4)-glucanase (C1), endo-β- chromatography. Five mL fraction was eluted with (l→4)-glucanase (Cx) and β-glucosidase (C2) were assayed using microcrystalline cellulose, 0.05 M Tris-HCl buffer, pH 8.0 in case of Sephadex carboxymethylcellulose and cellobiose as substrates, column and with a linear gradient of NaCl (0.0 to 0.5 respectively. One mL of culture filtrate was added to M prepared in the same buffer) in case of DEAE- 1 mL of 0.2 M acetate buffer (pH 4.8) containing cellulose. Fractions were assayed for protein content 10 mg of the specific substrate and the total volume and enzyme activity and the most active fractions in was completed to 3 mL. Mixtures were incubated for terms of their specific activities were pooled, desalted, 30 min at 40°C and assayed by measuring the release lyophilized and kept cooled for the subsequent work. 21 of reducing sugars (as glucose) at 575 mμ . One unit Characterization of PA of enzyme was defined as the amount of enzyme that pH optimization studies were performed by releases 1 μmol min-1 of reducing sugar equivalent carrying out the reaction at different pH values using under the assay conditions. different buffers (0.1 M phosphate for pH 6.0-7.0; Polygalacturonase (PG) was determined by 0.05 M Tris-HCl for 7.0-9.0 and M sodium measuring the amount of reducing sugar released bicarbonate-sodium carbonate for 9.5-10) and the from sodium polygalacturonate as substrate22. One activity was measured under the standard assay 286 INDIAN J BIOTECHNOL, JULY 2006

conditions. For pH stability determination, enzyme on the level of the enzymes detected in the macerating solution was incubated for variable time periods at fluid of Aspergillus spp. was reported elsewhere4,7,17. fixed pH values ranged from 6 to 10.5 and the Production of cellulase and pectinase in presence residual activity in each treatment was assayed. of different concentrations of NH4NO3 and NH4Cl, Similarly, reaction mixture was incubated for 60 respectively, was also studied (data not shown). min at different temperatures (30 to 65°C) and Maximum production of C1 was recorded at 0.7% enzyme activity was measured to determine the (w/v) NH4NO3, while maximum production of both optimum temperature for activity. However, the Cx and C2 was recorded at 0.6% NH4NO3. Meanwhile, enzyme was incubated for variable durations (0 to 60 the maximum production of pectinolytic enzymes min) at fixed temperatures (50 to 85°C) for (PG, PM & PL) was attained at 0.7% (w/v) NH4Cl. determination of thermal stability. Macerating activities of the enzyme preparations produced by R. oryzae in presence of NH Cl or The molecular mass of the enzyme preparation was 4 NH NO was studied. In presence of NH Cl, estimated by gel filtration27 on Sephadex G . The 4 3 4 75 macerating activity was at its maximum level, while it elution pattern was calibrated using the following was slightly higher or similar to that of control in proteins: lysozyme, 14.3; chymotrypsin, 25.1; presence of NH NO (Table 3). As shown in Table 2, carbonic anhydrase, 29; ovalbumin, 43; bovin serum 4 3 there was a sharp increase in the level of pectinolytic albumin, 66; phosphorylase, 94; alcoholic enzymes in presence of NH Cl, while the presence of dehydrogenase, 150; and catalase, 232 kDa, and a 4 NH NO resulted in an increase in cellulolytic standard calibration graph was constructed. 4 3 enzymes and a decrease in pectinolytic enzyme. Thus,

the results suggest an important role for the Results and Discussion pectinoytic enzymes in the macerating activity. After a preliminary investigation, wherein 25 Moreover, the level of PL (17.71 U/g) was higher fungal species were tested for their ability to grow on than that of PG (14.18 U/g) and PME (11.52 U/g; orange peel and/or produce macerating enzymes Table 2), which indicates that PL is an enzyme active against potato and cucumber discs, Rhizopus capable of degrading pectin with a high degree of oryzae was found to be the most potent fungus (data esterification and can work on methylated and/or non- not shown). The enzymes, obtained in appreciable methylated groups of galacturonic acid11-12. These amount, from the macerating fluid of R. oryzae grown observations show PL as an enzyme of potential on orange peel are given in Table 1. This particular interest. Therefore, the work was extended to purify spectrum of enzymes could be related to the relatively and characterize PL. high amounts of cellulose and pectin in the fermentation medium. The chemical analysis of Purification of PL orange peel showed that it contained 25.3, 20.5 and The steps of enzyme purification are summarized 32.8% of cellulose, hemicelluloses and pectin, in Table 4. Precipitation of the protein content of the respectively. In spite, the presence of 20% CFF was performed by 65% ammonium sulfate. The hemicelluloses present in the orange peel, xylanase precipitate was collected by centrifugation that activity was only limited to 0.75 U/g orange peel. resulted in an active pellet containing 70.79% of the Ability of R. oryzae strain NBRC 4707 and strain original activity and specific activity of 209.63 U/mg. NRRL 395 to produce cellulase and pectinase was Further fractionation of the enzyme preparation was 15,18 previously reported but it was discussed only in done through Sephadex G150. Most of the PL activity relation to their role in the production of lactic acid was recovered in fractions 15 to 21 representing and ethanol. An effort was made to improve the yields of Table 1—Enzyme profile of the macerating enzyme complex produced by R. oryzae grown on orange peel cellulase and pectinase through enriching the orange peel medium with some natural additives (0.5%, w/v) Enzyme Units (U/g orange peel) or nitrogenous compounds (0.7%, w/v). Table 2 Cellulase (C1) 2.35 demonstrates that the maximum productivity of Carboxymethyl cellulose (Cx) 1.48 cellulolytic and pectinolytic enzymes was recorded in Cellobiase (C2) 1.75 Polygalacturonase (PG) 4.60 presence of NH4NO3 and NH4Cl, respectively. The Pectin methyl esterase (PM) 3.27 similar effect of additives and nitrogenous compounds Pectin lyase (PL) 5.20 HAMDY: PECTIN LYASE FROM R. ORYZAE GROWN ON ORANGE PEEL 287

Table 2—Effect of some natural additives and nitrogenous compounds on the level of cell wall degrading enzymes produced by R. oryzae Activity (U/g orange peel) Substance# C1 Cx C2 PG PME PL Malt extract 3.84+0.12* 2.35+0.10* 2.85+0.11* 12.86+0.65** 9.24+0.30* 15.30+0.57* (164.10) (159.86) (162.86) (278.35) (282.57) (293.67) Yeast extract 2.87+0.11* 1.97 +0.05* 2.33 +0.10* 9.24 +0.48* 7.27 +0.26* 10.94+0.47* (122.65) (134.01) (133.14) (200) (222.32) (209.98) Beef extract 2.11 +0.10* 1.35 +0.06* 1.66 +0.07* 2.95 +0.13* 2.46 +0.10* 2.80 +0.08* (90.17) (91.84) (94.86) (63.85) (75.23) (53.74) Soybean meal 2.82 +0.11* 1.76 +0.09* 2.11 +0.11* 7.37 +0.21* 5.16 +0.23* 8.39 +0.26* (120.51) (119.73) (120.57) (159.52) (157.80) (161.04) Fish meal 1.87 +0.06* 1.23 +0.06* 1.44 +0.05* 4.72 +0.20* 3.37 +0.16* 5.48 +0.23* (79.91) (83.67) (82.29) (102.16) (103.06) (105.18) Peptone 3.28 +0.12* 2.35 +0.07* 2.28 +0.09* 5.27 +0.13* 3.67 +0.11* 6.20 +0.22* (140.17) (159.86) (130.29) (114.07) (112.23) (119.00) Molasses 1.82 +0.04* 1.19 +0.04* 1.39 +0.06* 5.32 +0.18* 3.62 +0.11* 5.68 +0.21* (77.77) (80.95) (79.43) (115.15) (110.70) (109.02) Corn-steep liquor 3.91 +0.16* 2.51 +0.11* 2.95+0.11** 7.82 +0.31* 5.66 +0.08* 8.76 +0.44* (167.09) (170.75) (168.57) (169.26) (173.09) (168.14) * * * * * * (NH4)2SO4 3.04 +0.11 1.92 +0.04 2.32 +0.09 7.57 +0.24 5.26 +0.16 8.34 +0.33 (129.91) (130.61) (132.57) (163.85) (160.86) (160.08) * * * * * * NH4Cl 3.23 +0.11 2.06 +0.06 2.45 +0.13 14.18+0.31 11.52+0.47 17.71+0.69 (138.03) (140.14) (140.0) (306.93)• (352.29)• (339.92)• * * * * * * NH4NO3 5.85 +0.19 3.15 +0.08 4.03 +0.08 4.07 +0.13 2.92 +0.06 4.54 +0.13 (250.0)• (214.29)• (230.29)• (88.10) (89.30) (87.14) * * * * * * NH4H2PO4 2.57 +0.07 1.69 +0.05 1.94 +0.06 12.19+0.09 7.62 +0.30 11.36+0.37 (109.83) (114.97) (110.86) (263.85) (233.03) (218.04) * * * * * * (NH4)2HPO4 2.34 +0.05 1.52 +0.03 1.90 +0.08 5.49 +0.09 3.95 +0.15 5.48 +0.15 (100.00) (103.40) (108.57) (118.83) (120.80) (105.18) * * * * * * KNO3 3.16 +0.14 2.02 +0.04 2.59 +0.08 4.17 +0.06 2.79 +0.06 4.69 +0.22 (135.04) (137.41) (148.00) (90.26) (85.32) (90.02) * * * * * * NaNO3 3.99 +0.20 2.54 +0.08 2.87 +0.11 3.87 +0.07 3.19 +0.08 4.89 +0.15 (170.51) (172.79) (164.0) (83.77) (97.55) (93.86) Casein hydrolysate 2.62 +0.11* 1.63 +0.03* 1.92 +0.06* 4.61 +0.14* 3.35 +0.09* 5.48 +0.25* (111.97) (110.88) (109.71) (99.78) (102.45) (105.18) Control 2.34 +0.08* 1.47 +0.04* 1.75 +0.07* 4.62 +0.17* 3.27 +0.10* 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 containing 2313.23 U/mg protein with 22.47-fold 496.72 U/mg and 4.83-fold purification. These purification was obtained in fractions 15-22. This fractions were pooled, lyophilized and subjected to enzyme preparation was salted out twice through further fractionation onto DEAE-cellulose using Sephadex G25, lyophilized and its purity to linear gradient of NaCl as eluant. A single peak electrophoretic homogeneity was confirmed by 288 INDIAN J BIOTECHNOL, JULY 2006

performing SDS-PAGE, where a single major band and/or due to the effect of pH on the stability of the was obtained. Final concentration of the enzyme was enzyme30. This was distinguished experimentally in adjusted to 100 U/mL. the present work by testing pH stability, where irreversible destruction was observed at the acidic Effect of pH Value on PL Activity and Stability side and the original activity could not be restored Testing the pH-dependence of PL activity revealed even after readjusting the pH to the original value that pH 7.5 was optimum for the enzyme activity (Fig. (Fig. 1). 1). The activity was sharply decreased at the acidic side, while the decline was minor at the alkali side. Effect of Temperature on PL Activity and Stability The same optimum pH was observed for PL from The experimental data represented in Fig. 2 show Aureobasidium pullulans23, while pH 6.4 and 5.0 that PL from R. oryzae recorded its optimum activity (acidic values) were found optimal for PL from at 50°C. PLs from other sources have been reported to 28 29 A. niger and Curvularia inaequalis , respectively. be optimally active in the range of 40-50°C9,23,29, As the pH value diverged from the optimum level, the efficiency the enzyme gets affected (Fig. 1). This Table 5—Effect of different metal ions and some enzyme could be ascribed to decreased saturation of the inhibitors on the relative activity of the purified PL enzyme with substrate due to decreased affinity Relative activity as affected by the concentration of Metal ions■ Table 3—Tissue maceration of potato and cucumber discs treated 1 mM 5 mM 10 mM with the enzyme preparation produced by R. oryzae grown on Ba2+ 105 + 4.20n 99 + 2.62n 91 + 4.30* 2+ ** ** ** orange peel fortified with NH4NO3 or NH4Cl Ca 119 + 5.39 128 + 5.13 134 + 3.27 2+ ** ** ** # Co 86 + 4.69 71 + 1.48 54 + 1.29 Tissue Time Maceration rate of the enzyme 2+ n ** ** (min) preparation produced in presence of Cu 99 + 2.62 73 + 2.48 51 + 1.38 Fe3+ 101 + 2.01n 93 + 3.48* 80 + 3.19** Orange peel Hg2+ 21 + 0.32** 2 + 0.07** 0 + 0** NH NO NH Cl + ** ** ** medium* 4 3 4 K 113 + 2.14 125 + 3.20 130 + 4.46 Mg2+ 114 + 6.46* 112 + 2.07** 102 + 3.25n 20 1 1 2 Mn+ 99 + 4.08n 84 + 1.67** 73 + 1.53** 40 1 2 4 Na+ 110 + 3.16** 117 + 5.54** 130 + 4.15** Potato 60 2 3 5 Zn+ 88 + 1.45** 59 + 2.27** 47 + 1.66** 80 3 3 5 Enzyme

100 4 4 5 inhibitor` EDTA 80 + 3.00** 71 + 1.53** 68 + 1.57** 20 0 1 1 Iodoacetate 65 + 1.03** 48 + 2.00** 16 + 0.86** Cucumber 40 1 2 2 Sodium 73 + 3.00** 69 + 1.23** 44 + 1.00** 60 2 3 3 arsenate 80 3 3 4 Sodium 63 + 1.63** 42 + 1.75** 30 + 1.02** 100 3 3 4 arsenite * Control #Tissue maceration was measured by immersing discs (10×1 mm) The investigated metal ions were added as chloride at the of potato tubers or cucumber fruit tissues, as substrates, in 3 mL indicated concentration and was incubated with the enzyme for 30 of enzyme preparation buffered with 5 mM Tris-HCl, pH 8.0 at min at 30°C before adding substrate. Activity of the PL in 30°C (0 = not macerated, 1-4 = progressively macerated, and 5 = complete absence of such compounds served as control (100% completely macerated). All experiments were performed in activity). Data represent the mean of 3 readings approximated to triplicates. Heat killed enzyme used as control. the nearest integer number.

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

Step# Initial volume (mL) Total activity Total protein Specific activity Yield Purification (U) (mg) (U/mg) (%) fold(s) Crude cell filtrate (CFF) 200 3500.00 34.00 102.94 100.0 1 Protein precipitate of NH4(SO4)2 (65%) 200 2477.65 11.82 209.62 70.79 2.04 Gel filtration 2* 1862.7 3.75 496.72 53.22 4.83 Ion exchange chromatography 2* 1503.6 0.65 2313.23 42.96 22.47 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. HAMDY: PECTIN LYASE FROM R. ORYZAE GROWN ON ORANGE PEEL 289

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.

Effect of Metal Ions and Inhibitors on PL Activity Fig. 1—Activity and stability of the purified PL produced by R. Effect of some metal ions and enzyme inhibitors on oryzae as a function of pH value. 0.1 M phosphate (__♦__), 0.05 M Tris-HCl (__□__) and M sodium bicarbonate-sodium carbonate PL activity was investigated (Table 5). Results show (__x__) buffers were used for the pH values 6-7, 7.5-9 and 9.5- that PL activity was pronouncedly increased in 10.5, respectively. Measurement of the residual activity (__„__) presence of Ca2+, Mg2+, Na+ or K+. It is known that was assessed at pH 7.5 after 60 min of exposure to different pH alkali-metal cations (Na+ and K+) bind weakly to the values. Temperature of the reaction mixture containing 1 mL of enzymes to form ternary complexes between enzyme 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 (E), metal ions (M) and substrate (S) in different ways error bars represent the standard deviation. (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

Fig. 2—Effect of temperature on the activity of the purified PL stability of PL was significantly enhanced in presence produced by R. oryzae. Reaction mixture was held at the indicated of CaCl2, while it was non-significantly affected or temperatures for 60 min and other conditions are those as reduced in presence of pectin (Table 6). This suggests described in Fig. 1. 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 Fig. 3—Thermal stability of the purified PL produced by R. oryzae. Residual activity was measured after the enzyme was held at the evidence. Inhibitory effect of iodoacetic acid on PLs indicated temperatures for different times of exposure at 50°C and from P. fluorescence and P. viridiflava has also been 6 other conditions were similar to those described in Fig. 2. reported . Addition of EDTA resulted in considerable 290 INDIAN J BIOTECHNOL, JULY 2006

Table 6—Effect of CaCl2 or citrus pectin on the thermal stability of the purified PL produced by R. oryzae Residual activity (%) after Treatment 0 min 15 min 30 min 45 min 60 min Control 100 + 0 81 + 2.43 65 + 1.62 44 + 1.49 18 + 1.2 n ** * * * + CaCl2 100 + 0 87.3 + 3.1 74.66+2.65 52.06 + 1.6 28.08+0.95 + Pectine 99+1.7n 79.19 + 2.8n 61.94 + 1.9n 36.67 + 0.81** 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 1 Rombouts F M & Pilnik W, Enzymes in fruit and vegetable juice technology, Process Biochem, 13 (1978) 9.

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