Screening of Endophytic Fungi for Production of Asparaginase Enzyme

Abstract

L-asparaginase (EC 3.5.11. L-asparaginase amidohydrolase) is first enzyme, studied very intensively in human beings with regard to its antitumor potential against tumor of lymphoid precursor, acute lymphoblastic leukemia (ALL). It has profound bearings in medical as well as non-medical fields. The current drugs are suffering from many side effects like immunesuppression,infertility, secondary neoplasm. The immunogenic complications associated with its present microbial sources Escherichia coli, Erwinia carotovora limits its medicinal frontier. So there exists a need of switching to novel natural sources to serve as non immunogenic and better production sources of L-asparaginase. Plants harbor a variety of micro-organisms known as epiphyte, endophytes and pathogenic micro-organisms. Endophytic fungi are reported to be hub of novel bioactive compounds. In the present study, 50 endophytic fungi isolated from medicinally important plants collected from biodiversity hot spots of India are screened for the production of L-asparaginase. The endophytic fungi’s potential to produce this enzyme was appraised by screening them on modified Czapek Dox medium (Patil et al., 2012) and then secondary screening was carried on with L-asparagine as a sole carbon and nitrogen source and phenol red as pH indicator. Four cultures viz. #5AMSTYEL, #1048AMSTITYEL, #6CCSTITD, #17AMSTYEL selected on the basis of primary and secondary screening were subjected for culture filtrate production which was further tested for L-asparaginase production by agar well diffusion assay. #1048AMSTITYEL and #6CCSTITD exhibited appreciable L-asparaginase activity. #5AMSTYEL showed maximum potential for L-asparaginase production. The crude protein of selected four isolates was quantitatively tested for L-asparaginase activity. #5AMSTYEL holds maximum L-asparaginase activity followed by #1048AMSTITYEL. Protein estimation was done by Lowry’s method (Lowry et al., 1951). The selected isolates were then screened for glutaminase contamination. The isolate #5AMSTYEL and #1048AMSTITYEL, isolated from Aegle marmelos was not possessing Glutaminase contamination. The crude sample was separated by SDS- PAGE, stained by silver staining showed single band of approximately 60 kDa. Further studies on L-asparaginase purification,characterization, kinetic parameters and anti-tumor assays would untap L-asparaginase antitumor potential and open up ways for its large scale production.