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Title: L-Asparaginase producing endophytic fungi isolated from medicinal plants
Authors: Bedi, Sonali
Supervisor: Vasundhara, M
Keywords: Acute lymphoblastic leukemia;L-asparaginase, L-Asparagine, Modified Czapex dox medium, Terminalia arjuna
Issue Date: 19-Aug-2019
Abstract: Endophytic bioactive compounds from medicinal plants can be integrated in novel drug discoveries due to their wide variety of biological activities such as antibiotic, anticancer, antioxidant and anti-inflammatory agents (Jawahar et al. 2014). Acute Lymphoblastic Leukemia (ALL) is a cancer of the bone marrow in which early lymphoid precursors multiply and supplant the typical hematopoietic cells of the marrow. Larger part of these cases are in kids and youthful grown-ups. Asparaginases have been the foundation of ALL treatments for most recent 4 decades (Seiter et al. 2018). Medicinal plants are home of numerous proficient bioactive metabolites producing endophytic fungi that have incredible medicinal and therapeutic worth. L-Asparaginase is one of the enzymes produced by endophytic fungi. The objectives of this study were to screen endophytic fungal isolates for L-Asparaginase activity, optimization of parameters of fermentation for the production of L-Asparaginase. Identification of endophytic fungus producing maximum amount of L-Asparaginase was also done. In this study, pre-isolated fungal cultures of Tinospora cordifolia (Giloy), (GR, GR3, GS1, GS3, GS4, GL2), Terminalia arjuna (AL2, AL4, AJ4, AF2, AL3), and Taxus baccata (T6) were taken and analysed for L-Asparaginase activity. Out of 12 endophytic fungal cultures, 10 fungal cultures gave positive result in qualitative assay after 3 days of incubation at 26±2°C. These positive cultures were then subjected to quantitative analysis. Out of the ten endophytic isolates tested for quantitative analysis, AL4 showed maximum L-Asparaginase activity of 16.407 U/mg/mL. Afterwards, optimization of fermentation parameters for maximum production of L-Asparaginase from AL4 was done. The specific enzyme activity was determined on 2nd, 3rd, 5th, 7th and 8th day of incubation. Maximum enzyme activity of 137.80 U/mg/mL was recorded at pH 5 on the 5th day. Optimum temperature was found to be 30°C with the specific enzyme activity of 31.58 U/mg/mL. Identification of AL4 was done by using microscopic, macroscopic and molecular method. AL4 grown on PDA plate showed that the fungus was white, cottony and reached 8 cm diameter of the plate in ten days. Long branched hyphae were observed when seen under microscope. Flattened oval shaped spores were also observed. PCR amplification of ITS regions were done using ITS1 as forward primer and ITS4 as reverse primer. By running electrophoresis PCR product was evaluated. The ITS amplified sequence of 569 bp obtained was run through blast n. Homologous sequences were obtained in FASTA format that were then aligned using Multalign. Evolutionary relationships between homologous sequences were found by neighbor-joining method with bootstrapping of 1000. MEGA 10 software was used to construct phylogenetic tree. It confirmed the clustering of AL4 with Fusarium proliferatum. Endophytic fungi thus can be a novel source for the production of L-Asparaginase enzyme and can play a very important role in food, pharmaceuticals and other industries.
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