Elucidation of the L-asparaginase-synthesising fungal endophyte
Screening for L-asparaginase production, i.e., both qualitative and quantitative was done using the isolated fungi. The formation of a pink zone is a clear indication of a positive response in the plate assay due to the presence of phenol red. Among all the isolates, MJS2 (Figure 1a, b) exhibited the highest zone diameter in the plate assay method (Figure 1c). In qualitative estimation, the asparaginase action of MJS2 was 13.97 U mL^-1, which was identified as Fusarium oxysporum based on the ITS-based molecular method (Figure 1d). Endophytic F. oxysporum isolated from the medicinal plant Murraya koenigii is known to exhibit L-asparaginase action of 0.013 µM mL^-1 min^-1.⁴² Meanwhile, other Fusarium species such as F. solani, and F. proliferatum with asparaginase enzyme activity of 3.58 IU mL^-1 and 16.75 IU mL^-1 have also been reported from Withania somnifera and Cymbopogon citratus, respectively.^43,44 Ashok et al. suggested that Trichosporon asahii IBBLA₁, isolated from arctic soil, showed the L-asparaginase activity of 20.57 U mL^-1 at an optimum condition of 30 °C and pH 7.⁴⁵ Chakraborty and Shivakumar reported about the L-glutaminase and urease-free L-asparaginase producer, Ganoderma australe GPC191 which, showed enzyme action of 1.57 U mL^-1.⁴⁶ The study of de Andrade et al. reported about the glutaminase and urease free L-asparaginase enzyme activity of many microbes as Aspergillus, Diaporthe, Xylaria, Epicoccum, and Trametes, obtained from Antarctic mosses.⁴⁷ It is seen that our presently studied isolate produces L-asparaginase higher than the contemporary ones. An isolate of Fusarium, i.e., F. equiseti, has produced an enzyme of 40.78 U/mL, which is somewhat near to our presently reported one (32.47 U/mL), but to the best of our knowledge, F. oxysporum has not been reported for such an activity earlier.

Fusarium oxysporum has been reported as an endophytic fungus by several researchers from various types of plants⁴⁸ and is known to synthesise a variety of bioactive metabolites.^49,50 Though they are potent plant pathogens but not all the strains are virulent, and F. oxysporum, isolated from Catharanthus roseus, is known to produce anticancer drugs vincristine and vinblastine.⁵¹ Different anticancer compounds i.e., oxysporidinone analogue, 3-hydroxyl-2-piperidinone derivative, -4,62 -anhydrooxysporidinone, (+)-fusarinolic acid, gibepyrone D, beauvercin, cerevisterol, fusaruside, and (2S,22 R,3R,32 E,4E,8E)-1-O-Dglucopyranosyl-2-N-(22-hydroxy-32-octadecenoyl)-3-hydroxy-9-methyl-4,8-sphingadienine, obtained from F. oxysporum, an isolate of Cinnamomum kanehirae, exhibited a cytotoxicity against PC-3, PANC-1, and A549 cell lines with an IC₅₀ dosage of 10.4 ± 1.6-49.5 ± 3.8 µM.⁵² Kour et al. isolated podophyllotoxin (a precursor of anticancer medicine-etoposide, teniposide and etopophose phosphate) from F. oxysporum, an endophyte of Juniperus recurva.⁵³ Not only in the pharmaceutical sector, but also F. oxysporum is effective in controlling weeds in wheat crop fields and stimulating wheat growth.⁵⁴ So, in the present investigation, the F. oxysporum MJS2-derived L-asparaginase will not be a safety issue.

Precipitation of crude L-asparaginase using ammonium sulphate
The initial stage in the L-asparaginase purification was precipitating the crude enzyme with ammonium sulphate.⁵⁵ It was noted that 80% of ammonium sulphate showed the highest recovery of the L-asparaginase enzyme. The highest enzymatic activity was 56.69 U mg^-1 after 80% fractional precipitation following the gel filtration technique. There are several techniques related to the precipitation and purification of the enzyme L-asparaginase, of which ammonium sulphate precipitation is the most popular one and adopted by the majority of the researchers.^56-58 Methanol-based precipitation is performed for L-asparaginase from Cladosporium species, which has a lower yield than the former.¹²

Characterization of MJS2-produced L-asparaginase
Evaluation of the molecular weight (MW)
A pre-stained protein ladder (HIMedia Laboratories MW 11-245) was used to compare the MW of the purified L-asp. Figure 2 shows the SDS-PAGE of the purified L-asparaginase, showing one protein band, and the approximate molecular weight was 35 KDa. The results are comparable to those of purified Lactobacillus casei subsp. casei ATCC 393, which had a molecular weight of 35 kDa.⁵⁹ The study of Yap et al. examined the extracellular L-asparaginase-producing activity of an endophytic fungus Colletotrichum gloeosporioides that consists of monomeric units of 25 kDa.⁶⁰ An endophytic fungus Lasidiplodia theobromae, which can produce asparaginase with a molecular weight of 70 kDa, has been shown to have antileukemic activity.⁶¹ Jenila et al. identified L-asparaginase enzyme-producing endophytic species of Fusarium sp. with a molecular weight of 66 kDa.⁶² Arredondo-Nunez et al. reported that the MW of type-II L-asp from halotolerant Bacillus subtilis was 38 kDa in the SDS-PAGE study.⁶³ Darwesh et al. reported that the recombinant L-asp isolated from Burkholderia pseudomallei had a MW of 33.6 kDa in SDS-PAGE.⁶⁴ Alongside, researchers screened Trichoderma species obtained from soil and agricultural waste for their ability to produce antileukemic L-asparaginase, which revealed that the purified enzyme had molecular weights of 69.3 kDa and 72 kDa, respectively.⁶⁵ All these studies unveiled the different ranges in molecular weight of L-asp from distinct microorganisms. Actually, L-asparaginase enzyme is a dimer of dimers, highlighting the anticancer activity related to four active sites found at the interface of two subunits.⁶⁶ The molecular weight of L-asparaginase was found to differ based on the enzyme’s source.

Influence of different culture compounds on L-asparaginase action
Enzymatic change of L-asparagine monohydrate to the corresponding aspartate and ammonia are among the most interesting bio-transformations. Therefore, L-asparaginase production needs to be optimized using a quick and effective methodology. Reports state that enzyme production could be increased severalfold by a statistics-based experimental design. Culture conditions and concentration parameters during fermentation at the shake flask level have shown a gradual increase in enzyme activity. The statistical experimental design allows examining numerous variables at the same time, making it faster and more cost-effective.

This present study showed that endophytic isolate MJS2 exhibits the highest enzyme production of 32.47 ± U mL^-1 at a fermentation time of 120 h, 37 °C of fermentation temperature, and an initial medium pH of 7. The effect of various sugar and protein sources on L-asparaginase synthesis was evaluated (Table 1). The growth of the studied endophytic fungi MJS2 was assessed by the estimation of biomass and is represented in Table 1. The biomass development (i.e., growth of the fungus) is mostly associated with L-asparaginase activity of the isolate, and a higher production of L-asparaginase is also accompanied by increased biomass. 120 hours of fermentation time has a biomass output of 1.35 ± 0.04 g/L and enzymatic activity of 18.05 ± 0.03 U/mL and at 37 °C of incubation temperature 1.39 ± 0.01 g/L of biomass is recorded with a L-asparaginase action of 19.39 ± 0.04 U/mL. This same trend has been followed for other parameters also, i.e., at glucose concentration of 2 g/L (1.41 ± 0.09 g/L and 21.87 ± 0.16 U/mL), ammonium sulphate of 6 g/L (1.49 ± 0.06 g/L and 22.87 ± 0.03 U/mL). The increase in enzyme production is simultaneous with the biomass generation in all the cases.

Table (1):
Optimization of fermentation conditions regarding L-asparaginase production from MJS2

This observation is related to the result of Moubasher et al., who reported that the most suitable temperature is 37 °C for the purified asparaginase enzyme of Lasidiplodia theobromae.⁶¹ Furthermore, recent studies by Luhana and Bariya stated the optimum growth situations for the L-asparaginase action by Trichoderma viridae were 37 °C and a medium pH of 8.0. They also showed the influence of different metal ion concentrations, such as zinc, copper, iron, and manganese, and how they affected enzyme production under control conditions.⁶⁵ Glucose and ammonium sulphate at values of 5 gL^-1 and 7 gL^-1 maximally influenced the production more than the other tested sources, e.g., maltose, sucrose, fructose, mannitol, xylose, starch, ammonium nitrate, urea, and ammonium chloride. Medium substrate concentration (L-asparagine monohydrate) at a value of 12.5 gL^-1 yielded a prominent synthesis (25.54 ± 0.06 UmL^-1) of L-asparaginase. Jenila and Gnanadoss also reported glucose and ammonium sulphate as the best carbon and nitrogen sources at a concentration of 3 gL^-1 and 20 gL^-1, respectively, for the highest production of L-asparaginase by the endophytic Fusarium sp.⁶⁷ Ruma et al. observed the maximum enzyme activity of 3.58 IU by F. solani with fermentation temperature 35 °C at medium pH 5.0 with 0.002% of magnesium ions, and 1% of sucrose as the best carbon source.⁶⁸ A lot of studies on L-asparaginase are reported through solid state fermentation only, but here we have adopted submerged fermentation as it is more reliable and stable. The OVAT/OFAT (One Factor At A Time) technique has been adopted by several workers related to the optimum production of L-asparaginase.^43,69 120 or even 144 hours of incubation time is found to be effective for L-asparaginase production from Fusarium sp. (F. solani, F. proliferatum, and F. oxysporum) by several authors.^20,43,70,71

Values of these most influential factors obtained from the OVAT table 1 were therefore selected as the zero-coded values to determine optimum model values using the Response Surface Methodology technique. The impact of various fermentation factors on the enzymatic action is represented in Figure 3 (a-i). The optimum enzyme production at glucose may be the reason that the isolate MJS2 prefers the simplest sugar (monosaccharide) instead of complex sugars (i.e., disaccharides). In the present study, in most cases, we have seen that L-asparaginase production drops after reaching the peak of any contributing parameters, i.e., incubation time, pH, carbon and nitrogen source, substrate content, etc. This may be driven due to several factors, i.e., limited nutrient availability, accumulation of toxic/inhibitory end products (e.g., ammonia), turbidity of liquid media, interactions of additive nutrients, and change in the pH of culture medium.^42,72 A lower pH (<7, i.e., acidic) is not suitable for the enzyme production as it affects the fungal metabolic processes, cell membrane permeability, initiate irreversible modifications in the active site of the enzyme by damaging particular amino acids at the active site, and triggers the hydrolysis of the peptide bond, which initiates irreversible damage to the synthesized enzyme.⁷³

RSM-based optimization of L-asparaginase synthesis
A central composite design was adopted to obtain the statistically valid model related to L-asparaginase synthesis using the obtained OVAT data. The polynomial model evaluated the relation of independent variables and enzyme activity. The best model to fit the data was the quadratic model that statistically compared the actual and predicted values at p < 0.0001. Both actual and predicted L-asparaginase activity (U mL^-1) were determined using the three most influential variables i.e., glucose, ammonium sulphate, and substrate concentration (L-asparagine) in 20 experimental runs are shown in Table 2. As described in Table 3, quadratic model was recommended to be the best-fit model using the sum of square analysis by comparing the ratio of mean square regression and square residual. This model is best fit in quadratic analysis as the predicted R² (0.95) and adjusted R² (0.99) values are high. Adequate Precision for this model has a signal-to-noise ratio value of 104.25 indicating an adequate signal, i.e. this model can be used to navigate the design space.

Table (2):
Tabulated representation of expected and observed values associated with the synthesis of L-asparaginase from MJS2

Table (3):
Representation of ANOVA outcomes related to L-asparaginase production

Table 3 shows the nine variance interactions of three variables that are A, B, C, AB, AC, BC, A², B² and C². Each variance was checked through Analysis of Variance (ANOVA). The high model F-value of 1127.12, suggests its significance. The ANOVA table 3 shows the values of A (value of glucose), B (value of substrate concentration), C (value of ammonium sulphate), A² (quadratic terms of glucose), B² (quadratic terms of substrate concentration), and C² (quadratic terms of ammonium sulphate concentration).

The perturbation plot (Supplementary Figure 1) contrasts the influence of three different factors at a specific point within the design space. A perturbation plot at the center point (glucose content of 4.76 gL^-1, ammonium sulphate content of 5.25 gL^-1, and substrate concentration of 12.03 gL^-1) was obtained to show the interaction of three variables as OVAT on L-asparaginase production. The perturbation plot indicates that B represents L-asparagine concentration, has the most influential impact on L-asparaginase synthesis (steepest slope), and A (glucose) has a lesser effect. Figure 4(a-f) represents the three-dimensional (3D) plots displaying the amalgamated effect of two separate factors for L-asparaginase production, where the third factor remained as a zero coded value.

Based on the above model, the optimum glucose concentration was 4.76 gL^-1, ammonium sulphate 5.25 gL^-1, and substrate concentration 12.03 gL^-1 at 37 °C for a fermentation time of 5 days, resulting in an assumed L-asparaginase activity of 32.47 U mL^-1. The expected yields were confirmed under optimized conditions in three independent experiments. Thus, overall, 2.32-fold L-asparaginase activity was observed after adopting the RSM optimization method. Endophytic Fusarium sp. LCJ273 showed an increase in L-asparaginase enzyme production up to 2-fold when 3 gL^-1 D-glucose, 20 gL^-1 ammonium sulphate, and 2.5 gL^-1 wheat bran were used as culture conditions.⁶⁵ Farag et al. demonstrated the optimised culture conditions for fungal isolate Aspergillus terreus that increased 1.33-fold as compared to unoptimized conditions.⁷⁴ Here, we have adopted the RSM technique, but some researchers, while working on medicinal plant endophytes (fungi, bacteria, and actinobacteria), adopt Plackett-Burman experiments, Taguchi model, and Central Composite Designs too.^{61,67,69,75,76} The contemporary reports highlight that the requirement of basic media compositions is higher than our present investigational outcome. Such a lesser requirement of carbon, nitrogen, metal ions, and substrate concentration by endophytic fungi is evident, that it is economically feasible and can be brought forward further for industrial exploitation.

Biochemical characterization of MJS2 produced L-asparaginase
Influence of various substrate concentrations
The impact of asparagine monohydrate concentration (10-100 mM) on semi-purified L-asparaginase enzyme was recorded. A change in L-asparagine concentration (from 10 to 50 mM) causes a gradual elevation in the enzymatic action, but after a certain point, the activity remains constant or decreases slightly. The optimum substrate concentration was 50 mM, with the highest activity of 56.69 U mL^-1 min^-1. The impact of different conditions on the enzymatic action is represented in Figure 5 a-d. An earlier study by Chow and Ting estimated the optimum substrate concentration for L-asparaginase production in the endophyte Penicillium simplicissimum PBL13, which was 30 mM. Another isolate, Fusarium oxysporum MKS1 exhibited maximum response at 10 mM of L-asparagine concentration.⁷⁷ Kathivaran et al. also reported the positive impact of L-asparagine on enzyme synthesis over other inducer molecules, i.e., L-arginine, L-glutamic acid, L-tyrosine, and L-tryptophan. The maximum L-asparaginase production of 18.3 U mL^-1 was achieved at 10 g L^-1 of substrate concentration.⁷⁸ MJS2 exhibits optimum action at a low substrate requirement, which is indicative of the fact that it has a low Km (Michaelis-Menten Constant) value. This highlights that a low substrate concentration is needed to attain the maximum enzymatic action, and it can work in a patient’s body even at lower doses, and also, this chemotherapeutic agent will not get resistant quickly against the cancer cells.⁸

Effect of Temperature
The activity and stability of the semi-purified enzyme were studied at seven different temperatures with a 5 °C difference from 20 °C to 50 °C. It was found that 35 °C has the most positive impact on enzymatic activity, and then three other slightly variable temperatures, 36 °C, 37 °C, and 39 °C, also have potent L-asparaginase activity. Figure 3 shows the effect of different incubation temperatures on L-asparaginase production. The maximum L-asparaginase activity was obtained at 37 °C. Darwesh et al. suggested that maximum L-asparaginase action was seen at 30 °C and 37 °C temperatures.⁶⁴ Type-II L-asparaginase obtained from a rhizospheric microbe Stenotrophomonas maltophilia, also showed maximum activity at 37 °C of medium temperature.⁷⁹ A maximum/optimum activity at 37 °C provides an opportunity to be used at in vivo systems, i.e., for initial trials in mice/rat models and finally for eradication of ALL in the human body.⁶¹ As the majority of the reported L-asparaginase functions at the same temperature (Lasidiplodia theobromine, Aspergillus flavus HK03, A. niger, A. lentulus, Mucor sp., and Rhizopus sp.), and our investigated enzyme follows the same pattern, our result is valid as per contemporary outcomes and bears biotechnological interest.^61,65,80,81

Effect of H⁺ ion concentration
To evaluate the best pH, seven varied pH ranges (5-11) were reacted with the semi-purified enzyme. Figure 4 shows the impact of the various pH values on L-asp action. The highest L-asparaginase action was acquired at pH 7.⁸² Enzyme from marine Aspergillus flavus was stable at a pH of 7.0 to 8.0.⁸³ The enzyme studied in the present investigation has a wide range of pH adjustability, which makes it suitable for industrial use. The outcomes are similar to the outcomes of Dev et al., who elucidated the enzyme action at pH 7.0 for the bacterial isolate Cupriavidus oxalaticus.⁸⁴ L-asp from Streptomyces fradie acts at an optimum pH of 8.5.⁸⁵ Alrumman et al. suggested that the best pH for L-asp activity isolated from Bacillus licheniformis was 6.5.⁸⁶ Darwesh et al. suggested that the enzyme from Burkholderia pseudomallei works optimum pH 8.⁶⁴ Rhizomucor miehei, Penicillium sp., Mucor hiemalis, Corynebacterium glutamicum, Bacillus megaterium, Bacillus pumilus synthesised enzymes are known to function at their optimum at pH 7 and it is the mostly physiological pH of human body, so it is a higher chance of working fine when injected into a patient’s body.^87-91

Effect of metal ions, chelating agents, inhibitors, and other additives
Among the tested metal and chelating agents, MgSO_4, KCl, and NaCl caused the increase of the L-asparaginase activity by 25%, 36.48%, and 48.03%, respectively. These characteristics are crucial to be exploited for the food industry.⁹² A strain of Aspergillus oryzae was screened for the maximum L-asparaginase producing activity, which revealed the greater effect of KCl and MgSO₄ at 0.052% concentration.⁹³ The presence of CaCO₃, MnSO₄, SnCl₂, CaCL₂, BaCl₂, EDTA, and urea had weak or no effect on L-asparaginase activity. Vala et al. and Monica et al. also showed the decrease in activity in presence of Mn²⁺ and EDTA in the case of Aspergillus niger and Mucor hiemalis, respectively.^94,95 The steep decrease in L-asparaginase action in the presence of PbNO₃, HgCl₂, and AgNO₃ was 25.53%, 29.58%, and 24.83%, respectively. Dias et al. highlighted the impact of different salts and other activators on the asparaginase enzyme-producing ability of Aspergillus spp. MnCl₂ highly affected the L-asparaginase production at 1 mM concentration.⁹⁶ Ei-Naggar et al. showed that Cd⁺, Pb⁺, and Hg⁺ by 145.15%, 143.04%, and 121.53%, respectively, have a negative impact on purified L-asp action synthesised by rhizospheric isolate S. bollosae NEAE-115.⁸⁵ Monovalent metal, i.e., K⁺ and divalent ones like Mg²⁺ and Ca²⁺ function as activators, whereas Hg²⁺ and EDTA are enzymatic inhibitors.⁹⁷ Actually, some of these metal ions function as co-factors (as they act as electron donor or acceptor) for the enzyme and modify its stability, functionality, and turnover number.⁸⁰