Response surface methodology based optimized production, purification, and characterization of L-asparaginase from Fusarium foetens.

L-asparaginase is used as one of the prime chemotherapeutic agents to treat acute lymphoblastic leukemia. L-asparaginase obtained from bacteria exhibits hypersensitive reactions including various side effects. The present work aimed to optimize growth parameters for maximum production of L-asparaginase by Fusarium foetens through response surface methodology, its purification, and characterization. The optimization of L-asparaginase production by Fusarium foetens was initially done through a one-factor-at-a-time method. L-asparaginase production was further optimized using a central composite design based response surface methodology. The maximum L-asparaginase activity of 12.83 IU/ml was obtained under the following growth conditions; temperature-27.5 °C, pH-8, inoculum concentration-1.5 × 106 spores/ml, and incubation period-7 days. In comparison with the unoptimized growth conditions (4.58 IU/ml), the optimization led to a 2.65-fold increase in the L-asparaginase activity. The L-asparaginase from Fusarium foetens was purified 15.60-fold, with a yield of 39.89% using DEAE-cellulose column chromatography. After purification, the L-asparaginase activity was determined to be 127.26 IU/ml and the specific activity was found to be 231.38 IU/mg. The molecular mass was estimated to be approximately 37 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme showed optimum activity at pH 5, and a temperature of 40 °C. The enzyme showed 100% specificity towards L-asparagine and no activity towards L-glutamine. Its activity was enhanced by Mn2+, Fe2+, and Mg2, while it was inhibited by ß-mercaptoethanol and EDTA. The Km and Vmax of the purified L-asparaginase were found to be 23.82 mM and 210.3 IU/ml respectively. The results suggest that Fusarium foetens could be a potent candidate for the bioprocessing of L-asparaginase at a large scale.

Insights into diversity and L-asparaginase activity of fungal endophytes associated with medicinal plant Grewia hirsuta.

L-asparaginase is used as one of the prime chemotherapeutic agents to treat acute lymphoblastic leukemia. The present work aimed to study the endophytic fungal diversity of Grewia hirsuta and their ability to produce L-asparaginase. A total of 1575 culturable fungal endophytes belonging to four classes, Agaricomycetes, Dothideomycetes, Eurotiomycetes, and Sordariomycetes, were isolated. The isolates were grouped into twenty-one morphotypes based on their morphological characteristics. Representative species from each group were identified based on their microscopic characteristics and evaluation of the ITS and LSU rDNA sequences. Most of the fungal endophytes were recovered from the leaves compared to other plant parts. Diaporthe sp. was the predominant genus with a colonization frequency of 8.62%. Shannon-Wiener index for diversity ranged from 2.74 to 2.88. All the plant parts showed similar Simpson's index values, indicating a uniform species diversity. Among the sixty-three fungal endophytes screened, thirty-two were identified as L-asparaginase-producing isolates. The enzyme activities of fungal endophytes estimated by the nesslerization method were found to be in the range of 4.65-0.27 IU/mL with Fusarium foetens showing maximum enzyme activity of 4.65 IU/mL. This study for the first time advocates the production of L-asparaginase from Fusarium foetens along with the endophytic fungal community composition of Grewia hirsuta. The results indicate that the fungal endophyte Fusarium foetens isolated in the present study could be a potent source of L-asparaginase.

Endophytic Fungi as a Promising Source of Anticancer L-Asparaginase: A Review.

L-asparaginase is a tetrameric enzyme from the amidohydrolases family, that catalyzes the breakdown of L-asparagine into L-aspartic acid and ammonia. Since its discovery as an anticancer drug, it is used as one of the prime chemotherapeutic agents to treat acute lymphoblastic leukemia. Apart from its use in the biopharmaceutical industry, it is also used to reduce the formation of a carcinogenic substance called acrylamide in fried, baked, and roasted foods. L-asparaginase is derived from many organisms including plants, bacteria, fungi, and actinomycetes. Currently, L-asparaginase preparations from Escherichia coli and Erwinia chrysanthemi are used in the clinical treatment of acute lymphoblastic leukemia. However, they are associated with low yield and immunogenicity problems. At this juncture, endophytic fungi from medicinal plants have gained much attention as they have several advantages over the available bacterial preparations. Many medicinal plants have been screened for L-asparaginase producing endophytic fungi and several studies have reported potent L-asparaginase producing strains. This review provides insights into fungal endophytes from medicinal plants and their significance as probable alternatives for bacterial L-asparaginase.

Molecular profiling and antimicrobial potential of endophytic Gliomastix polychroma CLB32 inhabiting Combretum latifolium Blume.

Fungal endophytes as a source of bioactive metabolites have led to the development of pharmaceutical products finding new applications. In a survey of endophytic fungal biodiversity, an antimicrobial endophytic strain CLB32 was isolated from the leaf of Combretum latifolium Blume (Combretaceae) from the Western Ghats of Southern India. CLB32 was then identified as Gliomastix polychroma (KR704576) by morphological and phylogenetic analysis based on internal transcribed spacer (ITS) nuclear rDNA and intervening 5.8S rRNA gene. CLB32 here constituted the first report on incidence of endophytic fungi from C. latifolium Blume. Ethyl acetate fraction of strain CLB32 was evaluated for antimicrobial activity by disc diffusion assay. Secondary metabolites produced effectively inhibited methicillin-resistant Staphylococcus aureus (18.33 ± 0.33 mm), Pseudomonas aeruginosa (14.66 ± 0.33 mm) and Candida albicans (14.00 ± 0.57 mm). Biosynthesis of these antimicrobial compounds was detected by analytical TLC-bioautography method as depicted by zone of inhibition on intensive the band. These findings suggest that G. polychroma CLB32, as a producer of natural antimicrobial drugs, could help to combat against multidrug-resistant infections and also provide baseline information for industrial applications.