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

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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.
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