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Research Journal of Agriculture and Biological Sciences, 4(5): 575-584, 2008 © 2008, INSInet Publication

Antagonistic Activities of Potato Associated Bacteria via Their Production of Hydrolytic Enzymes with Special Reference to Pectinases 1

Doaa A.R. Mahmoud, 2Abeer A. Mahmoud and 3A.M. Gomaa

1

Department of chemistry of Natural and Microbial Products, National Research centre, Cairo, Egypt. 2 Department of Btany, Plant Physiology Section, Faculty of Agriculture, Cairo University, Egypt. 3 Dempartment of Agricultural Microbiology, National Research Centre, Cairo, Egypt. Abstract: Fifty two potato-associated bacteria were tested for their ability as biological control agents against the plant pathogens Phoma betae and Rhizoctonia solani. Forty six of tested bacterial strains have high antagonistic activities towards the two pathogens. The antagonistic mechanisms were examined in vitro via their production of hydrolytic enzymes. The study was focused on pectinolytic enzymes and their production, characterization and applications. Among the fifty two microbial isolates, Paenibacillus polymyxa showed the highest pectinolytic activities and the exo-polygalactouronase was the major in the culture filtrate. The maintenance of nutritional requirements such as inoculume size, incubation time and nitrogen sources were found to be highly significant factors on production of endo- and exo-polygalacturonase. The partially purified exo-polygalacturonase was active over a wide pH range (3.0 - 0.9). The optimal temperature of the enzyme indicated maximal activities at 60-70 ° C which makes it a good candidate for application in industrial processes. In addition it increased the amount of reducing sugar content of fruit pulps from 20 mg sugar per ml apple juice to 44mg and from 15mg sugar per banana juice to 62mg at 65 ° C after 2hr .The dual roles of antagonistic activity of Paenibacillus polymyxa against different plant pathogens make it appealing alternatives to hazardous fumigants and fungicides. Key words: Antagonistic activities, potato associated bacteria, hydrolytic enzymes, pectinases they include polygalacturonase (PGase), pectin esterase, pectin lyase and pectate lyase on the basis of their mode of action [4] and are widely distributed in higher plants and microorganisms. These enzymes are essential in the decay of dead plant material by nonpathogenic microorganisms and thus assist in recycling carbon compounds in the biosphere [3] . Pectinases have widespread applications in food and textile industries, wastewater treatment, degumming of natural fibers[13,5,16]. Also they are widely used in extraction of oils from vegetable and citrus peels[6] and in bleaching of kraft pulp [2]. The present investigation deals with antagonistic activity of some potato-associated bacteria and their production for hydrolytic enzymes. The study was focused on production and characterization of pectinolytic enzymes and their applications.

INTRODUCTION Biological control offers an environmentally friendly approach to the management of plant disease [21 ] . The microbiologists and plant pathologists try to gain a better knowledge of biocontrol agents, to understand their mechanisms of control and to explore new biotechnological approaches [ 1 9 ] . A screening approach was developed to assess the potential of plant-associated bacteria to control plant pathogens[8] . The study of plant-associated bacteria and their antagonistic potential is important not only for understanding their ecological role and the interaction with plants and plant pathogens but also for any biotechnological application. Plant-associated bacteria can be used directly for biological control of soil borne plant pathogens or indirectly for the productions of active substances (e.g., antibiotics, hydrolytic enzymes, osmoprotective substances [ 9 ] ). Th e a n t a gonistic mechanisms towards fungal pathogens in vitro include their glucanolytic, chitinolytic, cellulolytic, proteolytic and pectinolytic activity [8]. P ectinolytic enzymes are heterogonous groups of related enzymes that hydrolyze the pectic substances;

MATERIALS AND METHODS Microorganisms: Fifty two microbial strains were kindly provided by Jana lottmann, Microbiology Department, University of Rostock, Germany. These

Corresponding Author: Doaa A.R. Mahmoud, Chemistry of Natural and Microbial Products Dept., National Re s e a rc h centre, Cairo, Egypt. Email: [email protected]

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Silley[22] . One pectin-lyase activity unit was defined as the quantity of enzyme needed to increase the absorbance by one unit per minute per milliliter of enzyme extract.

organisms were isolated from the rhizosphere of potato plants (Solanum tuberosum cv. Cilena) in RostockGermany. Screening for Hydrolytic Enzymes: Potato associated bacteria were tested for their ability to produce the hydrolytic enzymes: chitinases, cellulases, proteases, and pectinases in a plate assay method using chitin, carboxymethyl cellulose, skim milk, pectin as a sole carbon sources in a minimal agar media respectively according to Berg et al.[9] with one modification in pectin assay method is that, at the end of incubation period, iodine-potassium iodide solution (1.0g iodine, 5.0g potassium iodide and 330 ml H2O) was added to detect clearance zones[24] .

Determination of Growth: The growth of tested organism was determined by separating the cells from the culture medium using a centrifuge at 6000 rpm, 10 min. The precipitated cells were dried at 60 ° C to complete dryness. Applications: Two applications have been tested for the activity of polygalacturonase of Paenibacillus polymyxa i.e., its ability to increase sugars and its ability as antifungal agent. Fresh fruits (apple and banana) were obtained from the local market and the pulp was used. Pulp of apple and banana were treated with polygalacturonase (3U/ml enzyme) at 35°C. The treated fruit pulps were incubated for 2 h in an incubator shaker at 150 rpm according to the method of Kaur et al.[16] , dead enzyme used as control. The amounts of reducing sugars in the fruit juice were determined by using dinitrosalicylic acid reagent. In attempt to examine the antifungal activity of polygalacturonase, paper-disk agar diffusion method was used. Different concentrations of polygalacturonase were loaded on filter paper disks and applied to the plant pathogens (Phoma betae and Rhizoctonia solani).

Production of Pectinases Enzymes: Strains presenting large clearing zones in iodine test were used for enzyme production on liquid medium containing citrus pectin as a sole carbon source. Its composition was as follows: 1% citrus pectin with 67% methoxylation, 0.14 % (NH 4 ) 2 SO 4 , 0 . 20 % K 2 HP O 4 , 0.02% MgSO4 .7H2 O and pH was adjusted to be 6.5 before autoclaving. Shaking techniques were used and 250 ml Erlenmeyer flask filled with 50ml of the medium. Each flask was inoculated with 0.5 ml of bacterial suspension of 24hr old slant. At the end of incubation period at 30 ° C, the biomass was separated by centrifugation at 6000 rpm for 10 min and pectinolytic activities determined.

RESULTS AND DISCUSSION Screening for Biological Control Agents: Bacterial isolates were screened for their activity towards plant pathogens Phoma betae and Rhizoctonia solani by a dual – culture in vitro assay on Waksman agar medium containing 5g peptone, 10g glucose, 3g meat extract, 5g NaCl, 20g agar, pH 6.8. Zones of inhibition were measured after 5 days according to the method of Berg[7] . The results in Table (1) indicated that forty six bacteria have high antagonistic activities towards the two tested pathogens and four strains of Pseudomonas fluorescens, one strain of Pseudomonas marginalis and one strain of Pseudomonas putida showed no inhibition activity toward the pathogens. The inhibition zones of most of the potato-associated bacteria were 10-15 mm, encourged the use of plant-associated bacteria as biological agents.

Assays for Pectinolytic Activities in Culture Filtrate: The activities of the hydrolytic enzymes including exopolygalacturonase, endo-polygalacturonase and pectin lyase were estimated by the following methods: Estimation of Exo-polygalacturonase Activity: The exo-polygalacturonase was assayed by determining the liberated reducing end products by dinitrosalicylic acid reagent [18] from the reaction mixture containing 0.2 ml enzyme (clear culture filtrate) and 0.8 ml (1.0%) of citrus pectin in 0.2M acetate buffer (pH 4.5) for 20 min at 45° C. Estimation of Endo-polygalacturonase Activity: Endpolygalacturonase activity of clear culture filtrate was measured at 40° C viscometrically by measuring the relative change in viscosity of 1.0% pectin in Ostwald viscometer[1] . One unit of endo-polygalacturonase activity was defined as the amount of enzyme which reduced the viscosity of 10 ml of pectin solution by 50% in 10 min at pH 4.5.

Screening for Hydrolytic Enzymes: Production of cell-wall degrading enzymes and secondary metabolites are common mechanisms bacteria used to inhibit fungal growth. The present study examined the antagonistic bacterial isolate and their potential to produce hydrolytic enzymes. The results in Table (2) indicated that the majority of isolates were able to degrade chitin

Estimation of Pectin-lyase: Pectin- lyase activity was determined spectrophometrically as described by

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Res. J. Agric. & Biol. Sci., 4(5): 575-584, 2008 Antagonistic activity of potato-associated bacteria. Antagonistic Antagonistic No. Strain Potato-associated against: Strain Potato-associated against: number Bacteria ---------------number Bacteria -----------------P. betae P. betae R. solani R. solani 1 L10-1-8 Paenibacillus polymyxa +++ ++ 27 L31-6-9 Pseudomonas fluorescens ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2 L11-1-3 Pseudomonas putida +++ + 28 L18-4-2 Pseudomonas putida ++ ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------3 L3-2-1 Pseudomonas fluorescens + 29 L20-5-3 Pantoea agglomerans ++ +++ ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------4 L7-2-3 Pseudomonas putida +++ ++ 30 L1-6-11 Pseudomonas fluorescens + ++ ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------5 L7-2-9 Pseudomonas fluorescens ++ + 31 L19-6-1 Serratia grimesii ++ + ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------6 L9-2-5 Pseudomonas chlororaphis +++ ++ 32 L24-6-12 Pantoea agglomerans ++ ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------7 L5-3-11 Burkholderia cepacia ++ +++ 33 L27-6-10 Pseudomonas putida ++ + ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------8 L16-3-3 Serratia grimesii +++ 34 L32-6-8 Pseudomonas putida +++ + ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------9 L30-3-7 Pseudomonas marginalis +++ 35 LC20-6-11 Pseudomonas putida +++ ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------10 L2-4-4 Pseudomonas fluorescens + +++ 36 L13-6-12 Pseudomonas putida ++ + ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------11 L9-5-1 Pseudomonas fluorescens ++ 37 LC21-3-3 Erwinia chrysanthemi + ++ ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------12 L9-5-9 Pseudomonas fluorescens ++ ++ 38 4Kc13 Pseudomonas putida + ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------13 L1-6-11 Pseudomonas fluorescens + + 39 6Kp10 Pseudomonas chlororaphis + ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------14 L12-6-1 Pseudomonas fluorescens + 40 3Rc3 Serratia fonticola ++ ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------15 L31-6-11 Pseudomonas fluorescens ++ ++ 41 4Rr41 Pseudomonas syringae ++ +++ -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------16 LC1-3-4b Burkholderia cepacia + + 42 9Er15 Pseudomonas fluorescens ++ +++ ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------17 LC31-3-6b Burkholderia cepacia + ++ 43 1Pe4-13 Bacillus subtilis ++ ++ ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------18 L16-3-11 Burkholderia cepacia +++ +++ 44 2R1-7 Pseudomonas fluorescens ++ + ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------19 L12-3-12b Burkholderia cepacia +++ 45 2Re2-6 Pseudomonas fluorescens ++ ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------20 L1-3-5b Serratia proteamaculans ++ 46 3R1-19 Pseudomonas putida +++ ++ ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------21 QC11-6-10 Pseudomonas putida +++ +++ 47 3Re2-7 Pseudomonas reactans +++ + ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------22 L10-6-1 Pseudomonas fluorescens 48 3Re4-18 Serratia plymuthica ++ + ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------23 L10-6-5 Pseudomonas putida + 49 4R3-101 Pseudomonas fluorescens + ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------24 L10-6-6 Pseudomonas putida 50 7R15-501 Pseudomonas fluorescens ++ ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------25 L18-6-1 Pseudomonas fluorescens 51 3R12-302 Pseudomonas fluorescens ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------26 L22-6-1 Pseudomonas marginalis 52 3R12-102 Pseudomonas fluorescens + Result indicates the width of the zone of inhibition as follows: +, 0-5 mm, ++, 5-10mm, +++ 10-15mm. This measurement according to Berg et al. [7 ]. Table 1:

in plate assays and most of them have antagonistic effect toward either Phoma betae or Rhizoctonia solani. Chitinases appear to be potential candidates for management of fungal disease, however not all chitinases show antifungal action against pathogens of the host from which the chitinases have been isolated [27] . cellulolytic activity was observed for four isolates, twelve isolates were proteolytic and seven

were pectinolytic . Screening for Pectinolytic Activity: Among the fifty two microbial isolates, only ten strains were able to grow on medium containing citrus pectin as the only carbon source, and they were: Paenibacillus polymyxa, Pseudomonas fluorescens, P. marginalis, P. chlororaphis, P. putida, P. syringae, 577

Res. J. Agric. & Biol. Sci., 4(5): 575-584, 2008 Table 2: Hydrolytic activity of potato-associated bacteria. No. Chitinases Pectinases Proteases Cellulases No. Chitinases Pectinases Proteases Cellulases activity activity activity activity activity activity activity activity 1 + + 27 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2 + 28 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------3 + 29 + + -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------4 + 30 + -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------5 + 31 + + -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------6 + 32 + + + -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------7 + 33 + -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------8 + + + 34 + -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------9 + 35 + -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------10 36 + -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------11 37 + + -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------12 + 38 + -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------13 + 39 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------14 + 40 + + -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------15 + 41 + + -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------16 + 42 + -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------17 + 43 + + + -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------18 44 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------19 45 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------20 + 46 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------21 + + 47 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------22 + + 48 + -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------23 + 49 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------24 50 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------25 + 51 + -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------26 + + 52 + + The results indicated that (+) mean have hydrolytic active and (–) mean no hydrolytic activity. Few reports applied pec tino lytic enzymes as antifungal agent, so it was of interest to attempt the examination of pectinolytic activities of the potent isolate in this field.

Pantoea agglomerans, Erwinia chrysanthemi, Serratia fonticola and Bacillus subtilis. These strains were tested for pectin hydrolysis by plate assay, at pH 6.5. The strains were considered to be producers of pectinolytic enzymes when gave clear zone around colonies. Only three of these strains (Table 3)

presented clear halos around colonies of at least 1.5 cm, therefore classified as very good producers[10] . Production of Polygalacturonase on Liquid Medium: Three microbial strains demonstrated high pectinolytic activity when assayed by plate method and were

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Res. J. Agric. & Biol. Sci., 4(5): 575-584, 2008 Table 3: Microbial isolates producing pectinolytic enzymes detected by clear halos around colonies. Code number Strains Diameter of clear zones around colonies (cm) 1Pe4-13 Bacillus subtilis 1.49 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------LC21-3-3 Erwinia chrysanthemi 1.54 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------L10-1-8 Paenibacillus polymyxa 1.63 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------L24-612 Pantoea agglomerans 0.59 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------6Kp10 Pseudomonas chlororaphis 0.00 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------L18-6-1 Pseudomonas fluorescens 0.00 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------L22-6-1 Pseudomonas marginalis 0.00 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------4Kc13 Pseudomonas putida 0.83 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------4Rr41 Pseudomonas syringae 0.33 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------3Rc3 Serratia fonticola 0.29 Table 4: Pectinolytic activities of the selected strains on liquid medium. Microbial isolate Exopolygala-turonase (U/ml) Endopolygala- turonase (U/ml) pectin lyase (U/ml) Bacillus subtilis 1.12 0.94 1.01 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Erwinia chrysanthemi 0.36 0.56 0.99 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Paenibacillus polymyxa 4.70 1.36 0.29

Fig. 1: Effect of inoculume size of Paenibacillus polymyxa on production of pectinolytic enzymes. P G = polygalacturonase. c u lt iv a t e d f o r p r o d u c t i o n o f e n d o - , e x o Polygalacturonase and pectin lyase in liquid medium. The data obtained are presented in Table (4). Among all the tested strains, Paenibacillus polymyxa showed the highest exo- and endopolygalactronase activities. It also showed a low pectin

lyase. However, these results indicated that the polygalactronase activities are the major one in the culture filtrate of Paenibacillus polymyxa .Soriano et al.[25] reported that Paenibacillus sp BP-23 secreted a system for pectin degradation which comprised of pectate lyase. The data recorded in Table (4) also 579


indicated that culture filtrates of Erwinia chrysanthemi had high pectin lyase activity. However culture filtrates of Bacillus subtilis had low pectin lyase and low endogalacturonase. The different sets of enzymes for the degradation of pectin shown by the studied strains reflect the diversity of enzymes and strategies for pectin depolymerization among microorganisms. According to the previous results, Paenibacillus polymyxa was selected as the best organism to carry out further experiments. Inoculum level for optimal production of pectinolytic enzymes by Paenibacillus polymyxa (Fig. 1) showed that the increase of the inoculums from 0.1ml to 1ml per 50ml medium enhanced productivity yielding. At low concentration, the numbers of Paenibacillus polymyxa cells were not well enough to utilize essential amount of substrate to produce enzyme. The influence of the incubation time on the produc t iv it y of Paenibacillus poly my x a polygalacturonase activity was investigated. It was noticed that a good growth and production of polygalacturonase (endo- and exo-) enzymes increased by increasing time (Fig. 2). The activity increased and reached its highest value after 72hr. and longer incubation time showed a gradual decrease of polygalacturonase activity.

Properties of Partially Purified Polygalacturonase: Partially purified polygalacturonase was prepared by precipitation with acetone at 60% concentration according to the method of Kaur et al. [16] . An improved knowledge of the properties of microbial pectinases is important in commercialization of industrial production and to apply these enzymes in various potential fields[12] . Since any biotechnological process is likely to be based on the use of crude or partially purified enzymes, it is important to determine the optimal temperature for activity and thermostability under these conditions[17] . Hence, this study was focused on polygalacturonase performance in different pH and temperature degrees. The exo-polygalacturonase of Paenibacillus polymyxa was active over a wide pH range (3.0 - 9.0) with optimum at 7 (Fig. 4). A similar pH range for the activity of the enzyme was reported in Bacillus subtilis[2] Sporotrichum thermopile [16] , Streptomyces sp. QG11-3 and Verticillum abo-atrum [14]. The optimum pH of polygalacturonase of Bacillus licheniformis[23] and Fusarium oxysporum [20] was 11. Assay for determination of the optimal temperature of exo-polygalacturonase activity of Paenibacillus polymyxa indicated maximal activities at 60-70 ° C; this high temperature makes it a good candidate for a p p lic a t io n in industrial proce sse s. O p t im u m polygalacturonase activity of Thermoascus aurantiacus was 65° C [17] and optimum pectin lyase activity of Paenibacillus sp. BP-23 was 65 ° C [25] . To determine the effect of temperature on the stability of exo-polygalacturonase, enzyme solutions (3U/ml) were incubated for 60 min at temperatures between 30 ° C and 90 ° C and the residual activity was assayed after cooling. Results in Fig. (6) indicated that no changes in pectinolytic activities were observed when the incubation temperature was 65 ° C and lower. While at 70 ° C for 60 min, 93 % of original polygalacturonase a c t ivity was retained. After incubation at 80 ° C for 1h 45% of polygalacturonase activity was lost while no activity detected after 1h of incubation at 90 ° C. These results to some extant were similar to thermostable polygalacturonase of S p o r o t r i c h u m t h e r m o p h i l e a n d t h e r m o a sc u s aurantiacus[16,17] and similar to the alkaliphilic pectinase of Bacillus subtilis[2] . P ectinases play a major role in fruit juice extraction and increasing the libration of reducing sugars[11] . The current study showed that treatment of fruit pulps (apple and banana) with Paenibacillus polymyxa polygalacturonase was found to be economically feasible, that polygalacturonase increased the amount of reducing sugar content from 20 mg sugar per ml apple juice to 44 mg and from 15 mg sugar per ml banana juice to 62 mg at 65 ° C after 2hr.

Effect of Pectin Concentration on Production of (Exo- and Endo-) Polygalacturonase: Different pectin concentration (0.5-5 %; w/v) were used in the culture medium. The results in Fig. (3) showed that a concentration of 1% pectin led to maximal production of exo-polygalacturonase. There was a consistent increase in exo-polygalacturonase of Sporotichum thermophile production with increase in pectin concentration up to 5% [16]. However the maximum production of endo-polygalacturonase enzyme activity was obtained in cultures containing 3% pectin. Effect of Nitrogen Source on Production of Exopolygalacturonase: Nitrogen constituent has a profound effect on production of pectinolytic activity in culture medium. The data in Table (5) shows the effect of different nitrogen (peptone, yeast extract, urea, NH4Cl and (NH4)2 SO 4. Among all the nitrogen sources tested, yeast extract gave maximum production of pectinolytic activity (for both exo- and endopolygalacturonase). The optimum concentration of yeast extract (not published data) was found to be 2.5 g/l. In contrast polygalacturonase of thermophilic mould Sporotichum thermopile increased with increase in the level of yeast extract up to 0.5% and thereafter it decline [16].

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Fig. 2: Effect of incubation time on production of pectinolytic enzymes

Fig. 3: Effect of pectin concentration on production of (exo- and endo-) polygalacturonase.

Fig. 4: Effect of pH on exo-polygalacturonase of Paenibacillus polymyxa.

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Fig. 5: Optimum temperature of polygalacturonase.

Fig. 6: Thermal stability of exo-polygalacturonase of Paenibacillus polymyxa Martins et al.[17] reported that enzymatic clarification of juice may be carried out at 15 ° C for 12hr or at 54° C for 1-2hr, intermediated temperatures are not used because yeast growth is at maximum in this range. Although the higher temperature is more adequate owing to the high viscosity of the juice and the fact that it eliminates the pasteurization step, the limiting factor is the temperature stability of the enzyme mixture and polygalacturonase of Paenibacillus polymyxa proved to be highly thermo stable at 65° C.

The dual roles of antagonistic activity against plant pathogens make Paenibacillus polymyxa appealing alternatives to hazardous fumigants and fungicides. Unfortunately the direct application of different concentrations of partially purified polygalacturonase of Paenibacillus polymyxa to the plant pathogens (Phoma betae and Rhizoctonia solani) was not effective, this is probably because the mechanism of inhibition is due to the pectinolytic and proteolytic activities together as the result indicated before that Paenibacil l u s p o l y m yxa characterized by the production of both enzymes. In unpublished data, the inhibition zone in pectin agar plate was larger than that in skim milk agar plate indicating higher production a of pectinolytic enzyme than proteolytic enzyme. In contrast, immobilized alkaline pectinase prepared by Ibrahim et al. [ 1 5 ] was active against gram-negative and gram-positive bacteria, filamentous and non-filamentous fungi.

Biotechnological Application: The application of Paenibacillus polymyxa it self to the plant pathogens in vitro dual culture test as shown in Fig. (7) indicated large inhibition zone and hence Paenibacillus polymyxa was effective as biological control of fungal pathogens, Pythium ultimum, Rhizoctonia solani, Aphanomyces cochlioides Phoma betae.

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Res. J. Agric. & Biol. Sci., 4(5): 575-584, 2008 Table 5: Effect of nitrogen source on production of exo-polygalacturonase. Nitrogen source Exopolyga-laturonase (U/ml) Endopolygal-aturonase (U/ml) Peptone 4.60 1.42 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Yeast extract 8.56 1.47 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Urea 0.90 0.00 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------NH4Cl 2.20 0.89 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------(NH4)2 SO4(control) 6.77 1.39

Fig. 7: Antagonistic activity of P aenibacillus polymyxa against plant pathogens, A (Phoma betae), B (Rhizoctonia solani), C (Pythium ultimum), D (Aphanomyces cochlioides). Sunish et al.[26] isolated new strain of Pseudomonas aeruginosa exhibits broad-spectrum antifungal activity towards plant pathogen and characterized by production of phosphatase. Antifungal activity of fluorescent Pseudomonas lacked activity of cellulases and pectinase indicated the production of other antifungal metabolite [28] . The antifungal activity may be due to the production of other metabolites (degraded by acetone used in precipitation of enzyme).

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