“Endophytes,” the microbes residing within the plant tissues, are important sources of secondary metabolites.
Natural products derived from the medicinal plants are used across the globe as pharmaceutical drugs, cosmetics, fertilizers, insecticides, and pesticides. The overexploitation of medicinal plants is a threat to their existence with several taxa becoming extinct. Alternate sources of important metabolites have focused on the ability of microbes associated within the living tissues of plants “the endophytes” since two decades after the discovery of taxol-producing endophytic fungus
Enzymes produced by microorganisms are employed in the treatment of cancerous cells. L-asparaginase is one such enzyme routinely employed in chemotherapy particularly for cancerous tumors of white blood cells. The enzyme deprives the cancer cells of an essential amino acid asparagine by catalyzing its breakdown ultimately leading to starvation and death. L-asparaginase is specific in its action and does not pose threat to the survival of normal cells. Bacterial asparaginases are currently in use under different trade names such as Elspar from
Bark, twig, leaf, and fruit samples (
Surface sterilization was performed by sequential immersion of samples in 70% ethanol for one min, 3.5% sodium hypochlorite (NaOCl) for three min, and rinsed three times in sterile distilled water to remove traces of sterilants [
The relative frequency of colonization (% CF) of endophytes was calculated as the number of isolates of taxon from each segment divided by the total number of segments plated × 100 [
The plate assay method of Gulati et al. [
The positive isolates were cultured in MCD broth medium incubated at 30°C in orbital shaker (GeNei, Bangalore) set at 120 rpm for five days. L-asparaginase was estimated by Nesslerization as described by Imada et al
A total of 727 isolates of endophytic fungi belonging to 20 taxa were obtained from the plating of 1000 tissue segments. The relative per cent isolation was highest for isolates of leaf samples (43%) and least for isolates of bark samples (11%). The per cent colonization frequency of endophytes differed for the plant parts used and is represented in Table
Colonization frequency of endophytic fungi from the plant parts of
Endophytic fungi | Bark* | Twig* | Leaf* | Fruit* | Seed* | Total | DE (%) |
---|---|---|---|---|---|---|---|
|
— | — | — | 4.5 | — | 9 | 14.07 |
|
— | — | — | — | 18.5 | 37 | 28.90 |
|
— | — | — | — | 5.0 | 10 | 7.81 |
|
19.5 | — | — | — | — | 39 | 61.90 |
|
— | 15.0 | — | — | — | 30 | 15.54 |
|
1.5 | 4.5 | — | — | — | 12 | 9.42 |
|
1.5 | — | — | — | — | 3 | 4.76 |
|
— | — | — | — | 39.5 | 79 | 61.71 |
|
1.0 | — | — | — | — | 2 | 3.17 |
|
— | 3.5 | — | — | — | 7 | 3.62 |
|
— | 5.0 | — | — | — | 10 | 5.18 |
|
— | — | — | 4.5 | — | 9 | 33.33 |
|
— | — | — | — | 1.0 | 2 | 1.56 |
|
— | 6.5 | — | — | — | 13 | 6.73 |
|
— | — | 44.5 | — | — | 89 | 33.20 |
|
— | — | 23.5 | — | — | 47 | 17.53 |
|
— | — | 14.0 | — | — | 28 | 10.44 |
|
— | 46.0 | — | — | — | 92 | 47.66 |
|
— | — | 52.0 | — | — | 104 | 38.80 |
|
— | 2.5 | — | — | — | 5 | 2.59 |
Number of isolates | 68 | 133 | 268 | 80 | 78 | 627 | — |
Note: *200 segments of each sample (
†Reference number assigned in the laboratory.
Colony morphology of dominant endophytes isolated from
Rate of isolation of endophytic fungi from the plant parts of
Preliminary screening for the enzyme activity by plate assay revealed the enzyme producing ability of nine endophytes. A pink zone was observed around the colonies suggesting that endophytic fungi were able to utilize the substrate asparagine by secreting the enzyme asparaginase which catalyzes the breakdown of the substrate (Figure
L-asparaginase activity detected by plate assay. (a) Colour change in the medium (yellow to pink) around colony indicates production of enzyme, (b) nonproducer isolate (C—control, T—test).
The reaction between the ammonia in the reaction mixture and Nessler’s reagent was indicated by the formation of an orange colored solution. The enzyme activities were found to occur in the range of 0.006–1.136 unit/mL (Table
Estimation of L-asparaginase production by the endophytic fungi.
Endophytic fungi | Absorbance at 450 nm | Enzyme in unit/mL |
---|---|---|
|
0.755 | 1.006 |
|
0.713 | 0.950 |
|
0.201 | 0.268 |
|
0.852 | 1.136 |
|
0.171 | 0.228 |
|
0.589 | 0.784 |
|
0.478 | 0.637 |
|
0.048 | 0.064 |
Note: ttwig isolate; bbark isolate; ffruit isolate; lleaf isolate.
Research on endophytes in the past few decades has amounted to our understanding of their nature, interaction with host, and roles played by them in deterring insects, plant pathogens, and other environmental stress. Plant defense via endophytes is attributed to the production of secondary metabolites. The metabolites derived from endophytes have attracted researchers since their roles in medicine have been addressed. Tropical trees have received less attention with regard to endophytic studies in comparison to temperate trees probably due to inaccessible locations. However, in recent years several attempts have been made to bioprospect endophytes of tropical trees and have yielded fruitful outcomes. Thus, tropical regions known for the diversity of plant species also have the prospect of housing microbes with great diversity [
In the current investigation
The per cent colonization of endophytes was high in the leaf tissues in comparison to other plant parts. Similar observations were made by Kharwar et al. [
So far, 12 species of the family Apocynaceae,
Endophytes of
L-asparaginase is produced by plants, animals, and microorganisms. The microbes are better sources of L-asparaginase because of the ease with which they can be cultured, extracted, and purified, also facilitating the industrial scale production.
The authors declare that no conflict of interests is involved in the publication of this paper.
This work was supported by the University Grants Commission (UGC) major research project under Grant no. F-40-307/2011. The authors thank Dr. K. K. Sampath Kumara, Faculty, Government College, Davanagere, for his help in plant identification. The authors acknowledge the assistance provided by the Chairman, Department of Studies in Biotechnology and facilities at Department of Studies in Botany Manasagangotri, University of Mysore. This work is a part of the dissertation work in Master of Philosophy Programme (M. Phil), by the first author at the University of Mysore.