Isolation and Characterization of L-Asparaginase Producing Endophytic Fungi Inhabiting Prunus africana and Periploca linearifolia
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Date
2025-09
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Kenyatta University
Abstract
The clinical use of L-Asparaginase derived from bacterial sources has been hindered by various challenges, including toxicity and repression. This has prompted the exploration of alternative sources, particularly eukaryotic microorganisms like fungi, in an effort to enhance the safety and effectiveness of therapeutic ASNase. In this study, endophytic fungi isolated from medicinal plants, Periploca linearifolia (Apocynacease family) and Prunus africana (Rosaceae family), were investigated for their potential as a source of novel ASNase for therapeutic applications. These isolates were screened for L-Asparaginase production using the plate assay method on modified Czapex dots agar medium. L-Asparaginase activity of the fungal endophytes was determined using the nesslerization method. Identification of the fungal endophytes was performed using morphological characteristics and DNA barcoding with ITS sequencing, followed by BLAST analysis. Additionally, a phylogenetic tree was constructed using MEGA version X software. Twenty-four percent of the fungal endophytes exhibited positive reaction for L-ASNase activity and were identified as Penicillium ubiquetum, Penicillium pancosmium, Phoma sp, Penicillium. crustosum, Fusarium sporotrichioides, Cercospora canescens, Penicillium commune, septoria sp, Fusarium solani, and Colletotrichum sydowii. The fungal endophytes exhibited significant variation in production of L-asparaginase under the inflence of time of incubation and pH. It was observed that the fungal endophytes showed L-asparaginase activity at different day of incubation with Penicillium ubiquetum (2.63±0.47UI/mL), Penicillium pancosmium (1.44±0.1UI/mL), Phoma sp (2.6±0.47UI/mL), Penicillium crustosum (3.80±0.37 UI/mL), Penicillium commune (2.52±0.29 UI/mL), Fusarium sporotrichioides (3.47±0.24 UI/mL), Cercospora canescen (2.24±0.12 UI/mL) showed highest enzyme activity on the 6th day of incubation. Septoria sp and Colletotrichum sydowii exhibited best L-asparaginase activity of 12.6±0.81UI/mL and 4.06±0.23 UI/mL on the 9th day of incubation, respectively. While Fusarium solani showed atmost L-asparaginase activity of 12.4±1.12 UI/mL on the 12th day of incubation. In addition, the ten identified fungal endophytes records the highest activity at pH range 5.0-6.0 with Fusarium solani recording the highest enzyme activity of (6.14±0.01 UI/mL) at pH 6.0. The study revealed that fungal endophytes inhabiting plants with medicinal properties are potential source of L-Asparaginase. Among the fungal isolates, Fusarium solani and Septoria sp. showed the highest ASNase activity under optimized conditions (pH 5-6, incubation 9-12 days), indicating their potential as safer alternative to bacterial L-Asparaginase for anticancer therapy.
Description
A Thesis Submitted in Partial Fulfillment of the Requirements for the Award of the Degree of Master of Science (Biotechnology) in the School of Pure and Applied Sciences of Kenyatta University, September 2025.
Supervisors
1. Dr. George Isanda Omwenga
2. Prof. Eliud NM Njagi