Year : 2010 | Volume
: 2 | Issue : 1 | Page : 38--43
Isolation and purification of fungal pathogen (Macrophomina phaseolina) induced chitinase from moth beans (Phaseolus aconitifolius)
Neelima Garg1, Himanshu Gupta2,
1 Banasthali University, Rajasthan - 304022, India
2 Jamia Hamdard, Hamdard University, New Delhi-110 062, India
Jamia Hamdard, Hamdard University, New Delhi-110 062
Objective : Chitinase (EC 184.108.40.206) is one of the major pathogenesis-related proteins, which is a polypeptide that accumulates extracellularly in infected plant tissue. An attempt was made to isolate and purify the chitanase enzyme using moth beans as an enzyme source. Materials and Method : The enzyme was isolated and purified from moth beans against the fungal pathogen Macrophomina phaseolina strain 2165. The isolation and purification was done in both in vitro and in vivo conditions. Purification of chitinase was carried out to obtain three fractions, viz. 50°C heated, ammonium sulfate precipitated and sephadex G-25 column-eluted fractions. The molecular mass of Chitinase was directly estimated by sodium dodecyl sulfate-polyacryamide gel electroresis (SDS-PAGE). Result : The yield is sufficient for initial characterization studies of the enzyme. The molecular study of the enzyme shows the possibility of generating the defense mechanism in plants in which it cannot occur. Chitinase was purified by gel filtration chromatography with 20.75-fold and 32.78-fold purification in the in vitro and in vivo conditions, respectively. The enzyme shows a maximum activity after 90 min with 0.1 ml of colloidal chitin as a substrate and 0.4 ml of crude chitinase extract. The optimum pH of 5.0 and an optimum temperature of 40°C was found for maximal activity. The molecular weight of purified chitinase was estimated to be 30 kDa by SDS-PAGE. Conclusion : The chitinase isolated in both in vitro and in vivo conditions is stable andactive.
|How to cite this article:|
Garg N, Gupta H. Isolation and purification of fungal pathogen (Macrophomina phaseolina) induced chitinase from moth beans (Phaseolus aconitifolius).J Pharm Bioall Sci 2010;2:38-43
|How to cite this URL:|
Garg N, Gupta H. Isolation and purification of fungal pathogen (Macrophomina phaseolina) induced chitinase from moth beans (Phaseolus aconitifolius). J Pharm Bioall Sci [serial online] 2010 [cited 2020 Apr 8 ];2:38-43
Available from: http://www.jpbsonline.org/text.asp?2010/2/1/38/62708
Chitinases are ubiquitous enzymes of bacteria, fungi, animals and plants. Chitinase (EC 220.127.116.11) is one of the major pathogenesis-related proteins, is a polypeptide that accumulates extracellularly in infected plant tissue and exhibits high resistance to proteolytic degradation.  Chitinases have been implicated in the defense reactions of plants against potential pathogens. , In many plants chitinases rapidly accumulate following pathogen attack, after elicitor treatment and in response to the plant stress hormone ethylene. Chitinase catalyzes the hydrolysis of chitin, a linear homopolymer of b-1,4 linked N-acetyl-d-glucosamine (NAG) residues. Chitin is not found in plant cells, although it is a component of many fungal cell walls and arthropod exoskeletons. Isolation of genes of chitinases from Trichoderma spp. has been reported and these genes have been transferred to plants in order to increase the resistance against phytopathogens.  Microbial chitinases are widely distributed and produced in bacteria such as Serratia, Chromobacterium, Klebsiella, Pseudomonas, Vibrio, Arthrobacter, Beneckea, Aeromonas and Streptomyces. They are also found in the fungi Trichoderma, Penicillium, Lecanicillium, Neurospora, Mucor, Beauveria, Lycoperdon, Aspergillus, Myrothecium, Conidiobolus, Metharhizium, Stachybotrys and Agaricus. Microorganisms produce chitinases in higher amounts than that produced by animals and plants, generally as inducible extracellular enzymes that are of two types: endochitinases and exochitinases. Chitinase has been produced and purified from Trichothecium roseum, Streptomyces thermoviolaceus, Arabidopsis thaliana, Bacillussp. BG-11, planktonic bacteria, Stenotrophomonas maltophiliaC3.,,,,, The opportunistic fungal pathogen Aspergillus fumigatus contains at least 11 conserved active-site domains for chitinases of glycosyl hydrolase family. These enzymes may be divided into 'fungal/bacteria' chitinases, similar to chitinases from plants. 'Fungal/plant' chitinases of Aspergillussp. and Coccidioides immitis displayed molecular mass from 83 to 97 kDa, whereas fungal/bacterial chitinases have been found to have molecular masses of approximately 46-48 kDa.
Moth bean (Phaseolus aconitifolius) is an important kharif pulse as well as a fodder crop in arid and semiarid regions, particularly in north western states. It belongs to the family leguminaceae and subfamily papillionaceae. Moth bean is drought resistant, hard and is able to survive under dry conditions. It can be used as green manure, vegetable and snack. The plant conserves moisture, protects soil erosion and fixes atmospheric nitrogen through symbiosis with nodule bacteria. The main contribution of moth bean as a food is based on its protein content, which ranges from 20 to 30%. This plant is infected by fungi like Fusarium sp., Macrophomina phaseolina, Pythium sp. etc.
M. phaseolina is a common root parasite found in warmer lands. It is omnivorous and is a widely infecting pathogen. Mycelium spreads through soil causing seedling blight; stem and root rot of herbaceous plant and also decay of woody plant.
Although enormous work has been done on the defense response of plants, the present study is confined to extraction/isolation of chitinase from RMO 40 variety of moth beans (RMO 40 is the genotype of moth beans) through in vitro and in vivo procedures by induction of fungal pathogen M. phaseolina and evaluation of different parameters like time, enzyme concentration and substrate concentration. There has been report on the isolation of chitinase by induction of M. phaseolina in RMO 40 variety of moth beans.
Hence, the objective of the present study is to isolate and purify chitinase from P. aconitifolius in both in vitro (detached from the soil) and in vivo (intact plant in soil) conditions, against the fungal pathogen M. phaseolina strain 2165. The optimum activity of chitinase on substrate concentration, using colloidal chitin as a substrate, its time kinetics, molecular weight etc. were also determined In this work we also illustrate the effect of fungal infection on the protein content of moth bean plant.
Materials and Methods
P. aconitifolius was procured from the Agriculture research station Durgapura, Jaipur. Culture strain of M. phaseolina (MTCC 2165) was procured from IMTECH Chandigarh, India. Sodium citrate, sodium phosphate, potassium phosphate, sodium borate, ammonium hydroxide, di-methyl amino benzaldehyde (DMAB) were procured from Sigma, USA. All other chemicals were of analytical grade and were purchased from local vendors.
P. aconitifolius was grown after surface sterilization with 0.1% HgCl 2 for 5 min in pots in a green house at 28±2°C and 60% relative humidity (RH). The culture strain of M. phaseolina (MTCC 2165) was maintained on potato dextrose agar (PDA) slants at 4 o C and incubated at 37 o C for 10 days for the proper growth and sporulation. For chitinase production in the plant, inoculum was prepared by preparing the spore suspension under aseptic condition in sterilized distilled water. Proper distribution of spores was carried out in the incubator shaker (28±2 o C) at 120 rpm for 2 h. The spores were counted with hemocytometer (Fein optica Jena, Germany) and the concentration was maintained at 10 5 spores per milliliter.
In vitro and in vivo studies
In vitro studies were carried out in laboratory conditions (28±2 o C, RH 60%). Leaves of 15-day-old moth bean plant were separated from pots and taken in petri plates (1 g each time). Then the surface epithelium of leaves was injured mildly with an abrasive to facilitate the entry of fungal pathogen (M. phaseolina). Fungal spore suspension was inoculated over the surface injured leaves using the thin layer chromatography (TLC) sprayer. Enzyme extraction of both control and inoculated leaves was done 0, 4, 24, 48, 72 and 168 h after inoculation. All conditions of in vitro experiments were kept the same for in vivo experiments except the fact that the in vivo experiments were done in intact plants in pot in the green house at 28±2 o C at 60% RH.
Assay of chitinase activity
In both in vitro and in vivo conditions, 1 g weighed portion of the control and inoculated leaf tissues of 15-day-old plants of RMO-40 variety of P. aconitifolius was taken after 0, 4, 24, 48, 72 and 168 h. The tissue was homogenized in a prechilled mortar pestle in 4 ml of 0.1 M sodium citrate buffer, pH 5.0. The homogenate was filtered through premoistened (in 0.1 M sodium citrate buffer) four-layered cheesecloth and the filtrate was centrifuged at 10,000 rpm for 20 min. The supernatant was collected and the pellet was redissolved in 2 ml sodium citrate buffer pH 5.0 and recentrifuged at 10,000 rpm for 20 min. This constitutes the crude enzyme extract.
Determination of chitinase activity
Chitinase activity was measured by the release of NAG using colloidal chitin as a substrate according to the previous reported method.  Briefly, to 0.4 ml of crude enzyme solution, 10 mM sodium phosphate buffer pH 5.0 was added. The reaction was carried out at 37C in a shaking water bath by adding 0.1 ml colloidal chitin as a substrate. After 1.5 h, the reaction was stopped and the reaction medium was centrifuged at 1000 rpm for 3 min. After centrifugation, 0.3 ml of supernatant was transferred to a glass tube containing 30μl of 1 M potassium phosphate buffer pH 7.1 and incubated with 89.5 μl of snail gut enzyme for 1 h to change the pH to 6.8. The pH was increased to 8.9 by the addition of 70 μl of 1 M sodium borate buffer pH 9.8. Mixture was incubated in a boiling water bath for exactly 3 min and then rapidly transferred to ice bath. After cooling, 2 ml of DMAB reagent was added and the mixture was incubated for 20 min at 38±2°C. Release of NAG from the substrate was determined by measuring the absorbance at 585 nm against the enzyme as blank. One unit of chitinase is defined as the amount of enzyme that released 1 μmol of NAG per minute under the specific conditions. The amount of monomer released was extrapolated from the standard graph of NAG.
Purification of chitinases
Purification of chitinases was carried out to obtain three fractions, viz. 50 o C heated, ammonium sulfate precipitated and sephadex G-25 column-eluted fractions.
In the first step of purification, the enzyme extract was heated up to 50°C with vigorous stirring for 20 min. After heating, the extract was cooled first at room temperature, and then cooled below 5°C in an ice bath. After cooling, it was centrifuged at 10,000 rpm for 10 min at 4°C to remove denatured protein. In the second step of purification, the enzyme was precipitated up to 60% by adding ammonium sulfate powder with vigorous stirring at 0°C for 1 h and then centrifuged at 10,000 rpm for 20 min. The supernatant was discarded and the pellet obtained was dissolved in 10 mM sodium acetate buffer pH 5.0. In the third step of purification, this protein was loaded on a sephadex G-25 column (1.0 Χ 40.0 cm) previously equilibrated with 10 mM sodium acetate buffer pH 5.0. Elution was done using the 0.05 M ammonium hydroxide solution and the fractions were collected at a flow rate of 4 ml/h. Fractions (1.0 ml) were collected and assayed for chitinolytic activity using colloidal chitin as the substrate. Fractions showing maximum chitinolytic activity toward colloidal chitin were checked for protein purity by SDS-PAGE on a 12.0% polyacrylamide gel by the method of Laemmli.  The gel was stained by Coomassie blue.
Effect of temperature on activity and stability of chitinase
Effect of temperature on the activity of chitinase was studied by incubating the reaction mixture at various temperatures ranging from 20 to 60°C and assaying the enzyme by the standard method. The effect of temperature on stability of chitinase was determined by exposure of the enzyme solution to 10 mM sodium phosphate buffer pH 5 at a different temperature. Residual enzyme activity was then measured under assay conditions using colloidal chitin as the substrate.
Effect of time on chitinase activity
Influence of incubation time on chitinase activity was studied by varying the incubation time from 60 to 150 min. The residual activity was determined using the standard assay method.
Effect of substrate on chitinase activity
To analyze the effect of colloidal chitin concentration on chitinase activity, studies have been carried out by varying the concentrations of colloidal chitin from 0.025 to 0.15 ml. The residual enzyme activity was then measured under standard assay conditions.
Effect of crude enzyme on chitinase activity
To determine the production of chitinase at maximum concentration, an enzyme assay was carried out by varying the concentration of crude enzyme from 0.1 to 0.5 ml and assaying the enzyme using colloidal chitin as substrate and the amount of NAG released was measured by the method of Reissig et al. 
Protein content determination
The protein content was determined in the crude and partially purified fractions according to the previously reported procedure,  by plotting the standard curve using bovine serum albumin (BSA) as the sample.
Separation of proteins by SDS-PAGE
To determine the purity and molecular weight of chitinase, SDS-PAGE  was carried out in 12% resolving gel (1.5 M Tris-HCl pH 8.8, D.W, 10% SDS, 10% ammonium per sulphate (APS), tetramethylethylenediamine (TEMED); 30 : 0.8 acrylamide : bisacrylamide) and 5% stacking gel (1 M Tris-HCl, pH 6.8, D.W, 10% SDS, 10% APS, TEMED; 30 : 0.8 acrylamide : bisacrylamide).
Results and Discussion
Plant chitinases have different degrees of antifungal activity against different fungi. Although rapid accumulation of high levels of chitinases (together with numerous other pathogenesis-related proteins) occur in resistant tissues expressing a hypersensitive reaction.
In vitro and in vivo chitinase activity
In vitro and in vivo chitinase activity of RMO 40 variety of moth bean plants at different time intervals, i.e. 0, 4, 24, 48, 72 and 168 h in both control and inoculated plants is given in [Table 1]. The data indicate that the chitinase activity is higher in the inoculated plants when compared with the control. In in vitro condition, the maximum activity of chitinase production was observed in 24 h after inoculation of moth bean plants, whereas for in vivo conditions maximum chitinase activity was observed in 168 h. This may be attributed to the gradual loss of biosynthetic machinery due to detaching of plants in in vitro conditions. One may also anticipate that, on the one hand, NAG is possibly used by fungus as a nutritional source, but on the other hand, greater amounts of accumulated NAG may hamper further production of the enzyme. For in vivo conditions, the delayed increase in chitinase activity may be due to slow absorption of the fungal spores after inoculation. Some researchers reported maximum chitinase activity in Bacillus pabuli K I after 120 h incubation, whereas in Aeromonas it was observed only after 50 h incubation time. ,
Purification of chitinase enzyme
Crude chitinase enzyme extracted from both in vitro and in vivo experiments was purified in three steps. In the first step, crude chitinase enzyme was heat treated at 50 o C for 20 min. In the second step, 60% salt saturation of heat-treated sample with (NH 4 ) 2 SO 4 was carried out. In the third step, the inoculated as well as control fractions were passed through sephadex G-25 column for further purification. After each purification step, the purified fractions were assayed for chitinase enzyme activity and protein content of both control as well as inoculated plants in both in vitro and in vivo conditions. The data are given in [Table 2]. A gradual decrease in the enzyme activity as well as the protein content after each purification step has been observed.
In in vitro condition, the purification of the crude enzyme is 20.75-fold when compared with 1.04-fold in control (in vitro). Similarly, a high purification of 32.78-fold was obtained in in vivo conditions when compared with 0.32-fold for the control. In in vitro experiments the excised plant shows an early increase in chitinase activity with a maximum at 24 h after which it decreases gradually, as compares to 168 h in in vivo conditions.
Optimized crude enzyme activity
The graph of enzyme kinetics was plotted using different aliquots of the chitinase enzyme [Figure 1]. The maximum chitinase activity was obtained using 0.4 ml of the crude chitinase extract.
Effect of temperature and time on chitinase activity
The effect of temperature on chitinase activity is shown in [Figure 2], the optimum temperature for chitinase activity was found at 40°C. A drop in activity above 40 o C temperature was possibly due to heat inactivation of the enzyme. Similar temperature values were described for the purified chitinases from mycoparasitic fungi except for Aphanocladium album. 
The chitinase activity determined using colloidal chitin at different time intervals ranging from 60 to 150 min is shown in [Figure 3]. It shows that maximum chitinase activity was at 90 min, which gradually decreases with time.
Effect of colloidal chitin on chitinase activity
To analyze the effect of colloidal chitin on chitinase activity, we varied the volume of colloidal chitin from 0.025 to 0.15 ml. The effect of substrate on chitinase activity [Figure 4] shows that maximum chitinase activity was obtained using 0.1 ml of colliodal chitin, after which it falls slightly. This indicates that chitin activity correlated with substrate concentration and therefore maximum activity was recorded at 0.1 ml of colloidal chitin volume in the medium. A drop in chitinolytic activity at 0.15 ml colloidal chitin volume may indicate a hindering reaction of colloidal chitin serving as substrate, or accumulation of intermediates that results from chitin decomposition into medium which make up a synthetic inhibitor of chitinase itself.
Determination of protein content
Protein content of RMO 40 variety of moth bean plants at different time intervals is given in [Table 1]. The data indicate that the protein content is higher in the inoculated plants as compared to the control. The maximum protein content was observed in 24 h in in vitro conditions, whereas in in vivo condition it was observed in 168 h.
Separation by SDS-PAGE
SDS-PAGE profile gives bands of extracted enzyme at 30 kDa [Figure 5]. Similar results were observed for purified chitinase from chick pea induced Pseudomonas aeruginosa and Fusarium oxysporum where the molecular weights of purified chitinases were 31 and 62 kDa, respectively.  The reported range of chitinase is 26-43 kDa,  which proves that the isolated and purified enzyme is chitinase. According to the current system of chitinase nomenclature, the one with 32 kDa is an exochitinase and the one with 48 kDa is primarily an exochitinase, with only slight endochitinase activity.  Our results are in line with findings provided by the ammonium hydroxide fraction. It also confirms that this system of purification is reproducible, as the pattern of SDS-PAGE was nearly the same when the experiment was repeated.
Chitinase from RMO 40 variety of moth beans (P. aconitifolius) against the fungal pathogen M. phaseolina strain 2165 can be easily synthesized and efficiently purified. The chitinase isolated in both in vitro and in vivo conditions is stable and active. The yield is sufficient for initial characterization studies of the enzyme. With respect to the present results and comparison with previous reports on other chitinase producers, this plant seems to have the capability for production of chitinase. It would be interesting to study the molecular level of the chitinase enzyme, which can make generating the defense mechanism in plants in which it cannot generate.
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