Full Text: 1 Introduction Medicinal plants have been utilized and recognizes all through mankind's history. Natural products of medicinal plants are usually used in preparation of cosmetics, pharmaceutical drugs, insecticides, pesticides, and fertilizers (Manasa & Nalini, 2014). Since two decades following the discovering of taxol-producing fungal endophyte Taxomyces andreanae, researchers have concentrated on the endophytes; microbes living within the healthy tissues of plants; as alternatives in the production of the valued metabolites (Stierle et al., 1993). Endophytes are microorganisms including fungi, bacteria and actinomycetes which reside in the internal tissue of plants for all or parts of its life cycle. Endophytes have capability of colonizing in the internal tissues of healthy plant’s leaves, stems, bark, root, twigs, flower, seeds and fruit without causing any obvious harmful effect or pathogenic infection to the host plants. Endophytes considered valuable because of their ability to synthesize various important bioactive compounds. These bioactive compounds were basically involved in defence mechanisms of plants against plant pathogens (Fouda et al., 2015; Chow & Ting, 2015). L-asparaginase enzyme (L-asparagine amidohydrolase, E.C. 3.5.1.1.) is well accepted as an antitumour agent used in combination therapy with other drugs in the therapy of some lymphomas and leukemias (Jha et al., 2012;Arjun et al., 2015; El-Naggar et al., 2015). It has been used for more than 30 years in the treatment of acute lymphoblastic leukemia (ALL) (Ghasemi et al., 2017). It acts as catalyzing agent for the conversion of amino acid L-asparagine to ammonia and L-aspartatic acid (Arif & Hussain, 2014; Kavya & Madhu, 2019). This hydrolysis process generally occurred in two steps through an inter-mediate product: beta-acyl-enzyme (Figure 1). In first step, a strong base activates the enzyme nucleophilic residue and the amide carbon atom of L-asparagine (substrate) is attacked, and a product beta-acyl-enzyme intermediate is generating. In the second reaction step the ester carbon is attacked by a nucleophile which is activated by a water molecule (Cachumba et al., 2016). The principle behind the use of L-asparaginase in the tumours therapy depends upon the fact that tumour cells lack the Asparagine synthetase activity which restricts their ability to synthesis the normally non-essential amino acid L-asparagine. The leukemic cells metabolism depends on the circulating L-asparagine. L-asparaginase action does not affect the normal cells functions which are able to synthesis its own requirements of L-asparagine but it reduces the concentration of the free exogenous circulating L-asparagine and consequently induces the fatal starvation state in the susceptible leukaemic cells which then be destroyed (Ahmed et al., 2015; Alrumman et al., 2019).  L-asparaginase is wide spread enzyme found in many plants, bacteria, and in the serum of certain rodents, but not in man (Emmanuel et al., 2015). Commonly, L-asparaginase produced from microbial sources, because they are easily cultured and L-asparaginase extraction and purification from them is also favourable, and facilitates the production at industrial scale (Ahmad et al., 2012).  Major part of L-asparaginase activity has been predominantly reported in bacterial species like Escherichia coli (Mercado & Arenas, 1999) Erwina carotovora (Maladkar et al., 1993) and Serratia marcescens (Sukumaran et al., 1979), actinomycetes like Streptomyces venezuelae and  S. karnatakensis (Mostafa & Salama, 1979), yeast includes Saccharomyces cerevisiae (Oliveira et al., 1999)  Candida utilis (Kil et al.,1995) while from fungi it has been  reported from Aspergillus tamari, A. terreus, and A. niger (Mishra, 2006), and Fusarium sp (Gaddad et al., 2016). L-Asparaginase from bacterial sources is available now in market which used in the tumours therapy is sold under diverse trade names like Erwinase from Erwinia chrysanthemi and Elspar from Escherichia coli (Manasa & Nalini, 2014). But it is clear that prokaryotic L-asparaginase has currently reported to cause a lot of side effects upon use in tumour therapy which causes allergic reactions (Patil et al., 2012; Pola et al., 2018; Alrumman et al., 2019).  On the otherhands, eukaryotic L-asparaginase derived from yeast and filamentous fungi may induce relatively less toxicity and weak immune response (Asthana & Azmi 2003; Sarquis et al., 2004). Recently, the use of fungal endophytes as novel new promising L-asparaginase sources is interesting and considered relatively new and there are only few studies which have been reported on it (Chow & Ting, 2015). The present study focus on the isolation and screening of novel Glutaminase free L-asparaginase producing fungal endophytes from wild medicinal plants associated with antitumour activity. 2 Materials and Methods 2.1 Medicinal plant hosts species collection Four medicinal plants viz., Curcuma longa, Murraya koenigii, Catharanthus roseus, and Withania somnifera, traditionally known to be associated with antitumour activity were selected for this study. The plants were collected from different housing gardens of Nanded city, Maharashtra, India during the month of July, 2016. Different healthy plants parts viz., flowers, leaves, stem, rhizomes and roots were collected and placed in polyethylene bags and then stored at 4ºC. The processing of the plant samples was carried out under laboratory condition within 48h of its collection. 2.2 Isolation and identification of fungal endophytes The plant parts were first washed with running tap water for 20 minutes. Using a sterile scalpel, the tissues of leaf and flowers were cut into segments with the dimensions of 2cm × 2cm whereas the tissues of stem, rhizomes and roots were cut into a length of 2cm each (Chow & Ting, 2015). Surface sterilization of plants parts was carried out by immersion of the samples for 20 min in 1.5% sodium hypochlorite and for 30 s in 70% ethanol, and finally rinsed for three times in sterile deionised water to remove traces of the sterilants. The process was repeated triple times on each plant tissues batches (Jan et al., 2013). The sterilized plants segments were injured and placed on Rose Bengal agar medium which was supplemented with 0.05 g L^-1 of chloramphenicol in order to selection only fungal isolates. Sterilized but non-injured plants segments were used as control and placed on Rose Bengal agar plates where the absence of mycelial growth indicated the plants segments surface effective sterilization whereas mycelial growth presence in the plates seeded with injured tissues was considered as endophytes (Theantana et al., 2007). All the plates were incubated at 30°C/14 days. Emerged colonies from injured plants segments were subsequently sub-cultured to Potato Dextrose Agar (PDA) plates to get pure cultures and then maintained in slants at 4° C for later investigations. Fungal endophytes with Glutaminase free L-asparaginase producing activity were identified according to their morphological and cultural characteristics. 2.3 Plate Screening of Glutaminase free L-Asparaginase Production In general, it is observed that production of Glutaminase and L-asparaginase is accompanied by an increase acidity of the culture filtrates (Arif & Hussain, 2014). The plate screening assay was based on Gulati et al. (1997) method in which  pH indicator phenol red (prepared in ethanol) is incorporated in medium containing Glutamine and L-asparagine (sole nitrogen source) for screening the production of glutaminase and L-asparaginase respectively. This method was performed for screening of Glutaminase and L-asparaginase qualitatively. The phenol red is yellow at acidic pH and turns pink at alkaline pH; hence a pink zone is created around microbial producer colonies and considered as a positive result. Fungal endophytes isolates were screened for L-asparaginase production using Modified Czapek Dox medium (McDox) which is composed of: (g/L): 10 L-asparagine, 2 glucose, 1.52 KH2PO4, 0.52% MgSO4.7H2O, 0.52 KCl, 0.05 ZnSO4.7H2O, 0.03 CuNO3. 3H2O, 0.03 FeSO4.7H2O, 20  agar and supplementing the medium with 0.05  phenol red dye and the pH was adjusted to 6 (Chow & Ting, 2015). The medium was sterilized at 1.5 atmospheric pressured for 20 minutes. Inoculated plates were incubated at 28ºC for 5days. Plates containing L-asparagine were examined for pink zones surrounding producers isolates which indicates the positive asparaginase activity, and the isolates were considered as L-asparaginase producing strains (Figure 2). Isolates exhibiting L-asparaginase activity were selected for Glutaminase production screening to select only Glutaminase free L-asparaginase producer strains following same procedure except for L-asparagine which was replaced by Glutamine. Later on, production of L-asparaginase form the Glutamianse free L-asparaginase producing fungal endophytes was estimated quantitatively using Nesslerization method. 2.4 Nesslerization Method for L- Asparaginase Estimation The glutamianse free L-asparaginase  producing fungal endophytes isolates were cultured  in McDox broth medium and incubated at 28ºC in shaker incubator set at 120 rpm for 5days. The spore suspension containing ~ 1x10?spores ml^-1 was prepared using distilled water containing 0.1 % of Tween-80 and was used as inoculums. Estimation of L-asparaginase was performed quantitatively by Nesslerization method reported by Imada et al. (1973). In a tube, a prepared mixture was containing  0.5mL of 0.05M L-asparagine, 0.5mL of enzyme,  0.5mL of 0.5M Tris-HCl buffer (pH 8.2), and 0.5mL distilled water and this mixture was kept 30min at 37ºC. After incubation time, 0.5mL of 1.5M trichloroacetic acid (TCA) was added to the mixture in order to stop the reaction. 0.1mL from the reaction mixture was taken into a tube contain 0.2mLof Nessler’s reagent in 3.7mL distilled water and incubated for 20 min. At 450nm, the optical density was read using UV-Visible spectrophotometer (Kumar et al., 2016). To prepare blank tubes, enzyme was added after the addition of TCA. To get the enzyme activity, standard curve of ammonium chloride gradual concentration was prepared and one international unit (IU) of L-asparaginase was considered as the amount of enzyme which liberates one ????mol min^-1 ml^-1 of ammonia at 37ºC.  2.5 Protein Estimation To calculate the protein specific activity, protein content was quantified following the method described by Bradford (1976) in which the bovine serum albumin was used as a standard. 2.6 Statistical Analysis The experiments were performed in triplicate and the all results are expressed in terms of mean ± SD. Minitab 18 statistical software was used to perform the statistical analysis. 3 Results 3.1 Isolation of fungal endophytes Total seventy eight (78) fungal endophytes were isolated from different plant parts of the four medicinal plants viz., Curcuma longa, Murraya koenigii, Catharanthus roseus and Withania somnifera used. Among the used medicinal plants, high isolating percentage of 60% was reported from Curcuma longa this was followed by Murraya koenigii (23%), Catharanthus roseus (14%) and Withania somnifera (3%). Hence, Curcuma longa different plant parts may be considered as a good source for fungal endophytes. 3.2 Plate Screening of Glutaminase  free L-Asparaginase Production Twenty nine (29) isolates only among the seventy eight (78) fungal endophytes isolates were able to produce L-asparaginase. Five (5) isolates from these 29 isolates were showing production ability of L-asparaginase without Glutaminase activity. Five fungal strains which identified according to morphological characteristics probably were Fusarium solani (from Curcuma longa rhizomes), F. oxysporum (from Murraya koenigii stem ), Aspergilus sp (Murraya koenigii leaves), Penicillium sp (Curcuma longa rhizome), Alternaria sp. (Curcuma longa rhizome). These five isolates were selected for further procedures. 3.3 Nesslerization Method for L- Asparaginase Estimation The enzyme activities were found to occur in the range of 619.102–149.057 IU ml^-1 and a specific activity ranging from 8.807-3.035 IU mg^-1. F. solani from Curcuma longa rhizomes exhibited high asparaginase activities of 619.102 IU ml^-1 with a highest specific activity of 8.807 IU mg^-1 as shown in (Table. 1). The experiments were performed in triplicate and the all results are expressed in terms of mean ± SD. 4 Discussion and Conclusion Fungal endophytes isolated from medicinal plants considered as promising sources of L-asparaginase production with novel characteristics at high therapeutic index. In this study some medicinal plants with antitumour properties has been selected and used for isolation of fungal endophytes which have been screened thoroughly for production of the Glutaminase free L-asparaginase therapeutic enzyme. Results in the present study revealed that fungal endophyte Fusarium solani isolated from Curcuma longa rhizomes showed L-asparaginase activity of 619.102 IU ml^-1 which is higher than which previously reported enzyme activities. Similarity, El Refai et al. (2018) and El-Hadi et al. (2017) reported F. solani as a potent source of L-asparaginase       enzyme with an enzyme activity of 121 U ml^-1  and 254 U ml^-1 respectively. Gaddad et al. (2016) reported that Fusarium sp. (SMGR-F1) isolated from the papaya leaves produced 111.07±1.53 IU ml^-1 L-asparaginase activity. Sanjotha & Manawadi (2017) reported production of the enzyme by Aspergillus sp with an activity of 155U ml^-1. Fungal strain C-7 reported by Doriya & Kumar (2016), was shown L-asparaginase activity of 33.59 U mL^-1. Yadav & Sarkar (2014) have reported that the activity of L-asparaginase from F. oxysporum was 182 IU ml^-1.  Hence findings in the present study reported that Fusarium solani fungal endophyte may be considered as a potential source for production of Glutaminase free L-asparaginase at industrial scale. L-asparaginase enzyme is clinically acceptable in the treatment of tumours especially in the treatment of childhood blood cancers and considered to have exceptional therapeutic properties (Albertsen et al., 2019). Findings of the present study clearly showing that fungal endophytes isolated from medicinal plants which having anti-tumour activities may considered as a better potent alternative source for the L-asparaginase enzyme production which may has a clinically novel properties. These organisms must be taken into consideration through any subsequent future research on Glutaminase free L-asparaginase and may be used in the large scale production. The therapeutic Glutaminase free L- asparaginase enzyme from these organisms may show a higher therapeutic index with fewer side effects according to its novel properties comparing with the enzymes available nowadays in the markets which are from bacterial sources. Acknowledgments The authors would sincerely thank the Director of the School of Life Sciences S. R. T. M. University, Nanded, India for his support and encouragement. This work is a part of the dissertation work in Doctor of Philosophy degree (PhD), by the second author at SRTM University, India. Conflicts of interest  We have no conflict of interest to declare.