Bioprocess development for L-asparaginase production by Streptomyces rochei, purification and in-vitro efficacy against various human carcinoma cell lines.

In the near future, the demand for L-asparaginase is expected to rise several times due to an increase in its clinical and industrial applications in various industrial sectors, such as food processing. Streptomyces sp. strain NEAE-K is potent L-asparaginase producer, isolated and identified as new subsp. Streptomyces rochei subsp. chromatogenes NEAE-K and the sequence data has been deposited under accession number KJ200343 at the GenBank database. Sixteen different independent factors were examined for their effects on L-asparaginase production by Streptomyces rochei subsp. chromatogenes NEAE-K under solid state fermentation conditions using Plackett-Burman design. pH, dextrose and yeast extract were the most significant factors affecting L-asparaginase production. Thus, using central composite design, the optimum levels of these variables were determined. L-asparaginase purification was carried out by ammonium sulfate followed by DEAE-Sepharose CL-6B ion exchange column with a final purification fold of 16.18. The monomeric molecular weight of the purified L-asparaginase was 64 kD as determined by SDS-PAGE method. The in vitro effects of L-asparaginase were evaluated on five human tumor cell lines and found to have a strong anti-proliferative effects. The results showed that the strongest cytotoxic effect of L-asparaginase was exerted on the HeLa and HepG-2 cell lines (IC50 = 2.16 ± 0.2 and 2.54 ± 0.3 U/mL; respectively). In addition, the selectivity index of L-asparaginase against HeLa and HepG-2 cell lines was 3.94 and 3.35; respectively.


Results and Discussion
In this study, the medium ingredients and environmental conditions that play a critical role in maximizing the production of L-asparaginase have been optimized. The critical factor for solid state fermentation (SSF) is the choice of the appropriate solid substrate for fermentation. Process economization of solid state fermentation aiming at increasing L-asparaginase production by using cheaper materials to reduce the production costs. The soybean substrate was the perfect for L-asparaginase production and this could be explained by the fact that there are adequate quantities of carbohydrates, proteins, lipids and minerals 26 . El-Naggar et al. 26 have reported that soybean was used as substrate for production of L-asparaginase under SSF conditions by Streptomyces brollosae NEAE-115.
Several strains of actinomycetes were isolated from soil samples collected from Egypt and Saudi Arabia and evaluated for their L-asparaginase activities using the plate assay method. The presence of L-asparaginase activity was detected by the formation of pink areas around the colonies (Fig. 1). Among the tested strains, Streptomyces sp. strain NEAE-K is promising and was therefore selected for further studies. L-glutaminase activity was determined for the selected strain and the results demonstrated that the produced enzyme is glutaminase free L-asparaginase. In our previous study 26 , a comparative study was performed to assess the suitability of soybean and wheat bran for L-asparaginase production as carbon sources whether individually or mixed. It was clear from the results that the two substrates support L-asparaginase production (wheat bran; 16.49 U/gds and soybean meal; 21.84 U/gds). However, maximum L-asparaginase production under SSF was achieved with mixed soybean and wheat bran (1:1; w/w) (44.821 U/gds) (Fig. 2). Isaac and Abu-Tahon 27 reported that of the seven natural substrates, wheat bran was the most potential substrate used by Fusarium solani AUMC 8615 to produce maximum L-asparaginase activity of 187.9 U/mL.
Morphological and cultural features of the strain neAe-K. The morphological features of the strain NEAE-K are compatible with the genus Streptomyces 28 . Strain NEAE-K grew well on all tested ISP media 2-7. The organism is aerobic and mesophilic. Color of the aerial mycelium was gray on all media tested (Fig. 3, Table 1). Colony reverse side has brown pigments on oatmeal agar, yeast-malt agar, inorganic salts-starch agar and glycerol-asparagine agar media. Faint brown pigment released in oatmeal agar, glycerol-asparagine agar and inorganic salts-starch agar media, but these pigments were not produced in yeast-malt agar, peptone-yeast extract iron agar or tyrosine agar medium. These pigments are pH indicator that changes from faint brown by adding HCl (0.05 M) to colorless. The morphology of the strain NEAE-K was seen on inorganic salt-starch agar; there are  A, B) production of the enzyme indicated by color change in the medium from yellow to pink zone surrounding the colony after two and five days; respectively (C) Control plates were prepared as medium without dye. www.nature.com/scientificreports www.nature.com/scientificreports/ NEAE-K belongs to Streptomyces genus, on the basis of the physiological, cultural, morphological and chemotaxonomic characteristics ( Table 2). phylogenetic analysis. The sequence of the 16 S rRNA gene for the NEAE-K strain has been estimated to be 1532 bp. The sequencing product has been deposited under the accession number KJ200343 in the database of the GenBank. The GenBank database search by BLAST 29 revealed its similarity to the Streptomyces genus. A phylogenetic tree (Fig. 5) was constructed with MEGA software version 4.0 30 using neighbor-joining algorithm method 31 . Phylogenetic tree shows that the NEAE-K strain joins a clad with Streptomyces rochei strain NBRC 12908 (NR_041091.1), Streptomyces plicatus strain NBRC 13071 (AB184291.1) and Streptomyces olivaceus strain NBRC 3119 (AB184730.1).
A comparison between NEAE-K strain and its closest phylogenetic neighbors (Streptomyces rochei, Streptomyces olivaceus and Streptomyces plicatus) showed significant variations (Table 2) in that it produced faint brown diffusible pigment and has maximum NaCl tolerance of 7% (w/v). Strain NEAE-K varied also in its pattern of utilization of some carbon sources from its closest phylogenetic neighbors. It has been clear that the NEAE-K strain is a new sub-species of Streptomyces rochei and the proposed name for this strain is Streptomyces rochei subsp. chromatogenes NEAE-K.
The relationship between L-asparaginase production and the independent factors is determined by the statistical analysis of the Plackett-Burman design results ( Table 4). The interpretation of the data was based on the signs of the coefficients and main effects (positive or negative effects on L-asparaginase production). On the basis of the calculated main effects of the factors (Table 4), ten of the sixteen factors affect the production of L-asparaginase positively; they are yeast extract, fructose, soybean meal + wheat bran, temperature, incubation time, MgSO 4 .7H 2 O, K 2 HPO 4 , KNO 3 , FeSO 4 . 7H 2 O and CaCl 2 ; while the other six factors (pH, dextrose, moisture content, inoculum size, L-asparagine and NaCl) exerted negative effects on the production of L-asparaginase.
When the determination coefficient (R 2 ) value is closer to 1, the design used is better and stronger for predicting the response 32 . The value of R 2 in this study is 0.9974, indicating the fitness of the model. In addition, the value of the adjusted R 2 of 0.9795 is also very high, implying that the model has a great significance. A higher predicted R 2 (0.9014) implying that this model is adequate for predicting the value of L-asparaginase production in the range of factors used.
In order to assess the significance of the variables, both P and F-values were calculated (Table 4). In the current experiment, variables evidencing P-values of less than or equal to 0.05 (confidence levels of more than or equal to 95%) were considered to have significant impacts on L-asparaginase production. The most significant factor was dextrose, with a confidence level of 99.4, P-value of 0.0060 and F-value of 164.01, followed by yeast extract (confidence level 99.02, F-value 101.05 and P-value 0.0098), pH (confidence level 98.88, F-value 87.54 and Pvalue 0.0112). The F-value of the model of 55.69 (Table 4)   where the L-asparaginase production is Y and the independent factors are: A (incubation time), C (inoculum size), D (pH), E (temperature), G (dextrose), H (fructose), J (L-asparagine), K (K 2 HPO 4 ), M (yeast extract), O (NaCl) and Q (CaCl 2 ). The significance order of the factors influencing the production of L-asparaginase illustrated in the Pareto chart (Fig. 6). Among the variables screened; G (dextrose), M (yeast extract), and D (pH) have been defined as the most significant factors for the production of L-asparaginase and chosen for central composite design optimization.
To evaluate the precision of Plackett-Burman design, the medium of the following composition was used in the confirmatory experiment (g/L): L-asparagine 7, yeast extract 4, Wheat bran + soybean meal 15, fructose 4, dextrose 2, FeSO 4 . 7H 2 O 0.02, CaCl 2 0.01, NaCl 0.1, MgSO 4 .7H 2 O 0.5, K 2 HPO 4 2, KNO 3 1. The moisture content was 50% and pH 6.5. Two mL of the inoculum was used for medium inoculation and the inoculated flasks were incubated at a temperature of 37°C for 7 days. The obtained maximum production of L-asparaginase was 145.4 U/ gds, which was increased 3.24 times compared with production of L-asparaginase achieved prior to the application of Plackett-Burman design (44.821 U/gds). Singh and Srivastava 33 reported that the optimization of the solid state fermentation media and parameters resulted in a 14% increase in the L-asparaginase activity.
Optimization of L-asparaginase production by central composite design (CCD). Central composite design of response surface methodology (RSM) is an effective experimental technique designed for optimizing the production conditions. The main advantage of RSM is that it requires little experimental runs for multiple parameters to provide sufficient information for statistically acceptable results. It is faster, reduces the study's time and effort and avoids misinterpretation that occurs with the conventional approach. The regression analysis of the RSM helps in identifying the significant factors, exploring their relationships and predict the response 34 .
The optimal levels of the three variables; pH (X 1 ), dextrose (X 2 ) and yeast extract (X 3 ) and their mutual effects on L-asparaginase production were estimated by CCD. Table 5, includes the design matrix for 20 experimental runs, the central point was repeated six times (2, 5, 7, 8, 14, and 20). Table 5 also presents different combinations of the three independent factors and L-asparaginase production (predicted and observed values).
Based on the levels of the three independent variables, the results revealed considerable variability in the L-asparaginase production. The maximum L-asparaginase production was obtained in the central run no. 2 with a value of 177.64 U/gds where pH was 7, yeast extract was 3 g/L and dextrose was 2.5 g/L. While the minimum L-asparaginase production (88.36 U/gds) has been obtained in the central run no. 12, where pH was 8, yeast extract was 4 g/L and dextrose was 2 g/L.
Statistical analysis of the data. Table 6 presents the statistical analysis of the data, coefficients, P-value and F-value. The model determination coefficient (R 2 ) of 0.9898 means that the model could explain 98.98 percent of the variance in the production of L-asparaginase. Data were interpreted based on the significance (P < 0.05). Statistical analysis of the data shows that the model is very significant, as demonstrated by the Fisher's F-value (107.65) and a very low P-value (0.0001 < 0.05). It is evident from the significance values ( Table 6) that the linear coefficients of pH (X 1 ), dextrose (X 2 ) and quadratic effects of pH (X 1 ), dextrose (X 2 ) and yeast extract (X 3 ) are significant. The P-values indicate that the linear coefficient of yeast extract (X 3 ) is not significant (P-value

Residuals Actual Predicted
106.14 102.71 3.43 Variable level Days % %; v/v pH°C g,1:1 g/L g/L g/L g/L g/L g/L g/L g/L g/L g/L   www.nature.com/scientificreports www.nature.com/scientificreports/ = 0.1947). On the other hand, the different interactions between the three variables studied (X 1 , X 2 ; X 1 X 3 and X 2 X 3 ) are not significant (P-values of 0.1225, 1552, 0.0763; respectively), indicating that they have not significantly contributed to the enhancement of L-asparaginase production. Table 7 shows the fit summary results, the quadratic model is a highly significant and adequate model fitting L-asparaginase production with a very low probability value (P-value < 0.0001) and non-significant lack of fit   Table 5. Central composite design for L-asparaginase production as influenced by pH (X 1 ), dextrose (X 2 ) and yeast extract (X 3 ).
To evaluate the correlation between different factors and to calculate the highest production of L-asparaginase corresponding to the optimal values of pH, dextrose and yeast extract, the equation of the second-order polynomial model (Eq. 2) has been suggested. Depending on the optimum levels of the independent variables (X 1 , X 2 and X 3 ), the maximum L-asparaginase production is predictable: where Y is the value of predicted production of L-asparaginase, X 1 is the coded value of pH, dextrose (X 2 ) and yeast extract (X 3 ).
Model accuracy checking. Some statistics were carried out for confirmation of the design's accuracy. Figure 7A shows, the Box-Cox graph, the green line represents the best lambda value (Lambda = −0.07) and the blue line represents the current transformation (Lambda = 1). While the red lines represent the lowest and the highest values of confidence intervals between −1.71 and 1.51. As Lambda's best value lies between the minimum and maximum confidence intervals, no transformations of the data were recommended. The blue line lies between the two red lines; it implies that the model is well fitting to the experimental data obtained. Figure 7B Source of variance   www.nature.com/scientificreports www.nature.com/scientificreports/ displays NPP (the normal probability plot) of the residuals. The residuals have been drawn versus the normal scores which are the cumulative probability. The resulting points are shown in the NPP of the residues close the diagonal line in such a way that they are normally distributed, indicating a good model fit. Figure 7C shows a plot of the predicted L-asparaginase production against the residuals. The residuals scattered uniformly and randomly above and below the center line of zero with no obvious pattern, indicating that the residuals have a constant variance, confirming the model's adequacy. Figure 7D shows a plot of predicted versus the actual L-asparaginase production, showing the points close to the fitted line, supporting a significant correlation between the predicted L-asparaginase production of the model and its actual results. three-dimensional surface plots. The three-dimensional surface plots were designed to identify the optimal concentrations and the interaction between the factors for maximal L-asparaginase production. L-asparaginase production was plotted on the Z-axis against two factors, while the third factor was set at its zero level. (Fig. 8A-C). Figure 8A illustrates the production of L-asparaginase as a response of pH (X 1 ), dextrose (X 2 ) by keeping yeast extract (X 3 ) at zero level. It has been shown that the highest and lowest levels of dextrose were accompanied by low L-asparaginase production. At the moderate dextrose level, maximum L-asparaginase production was obtained. L-asparaginase production was maximized at the mid-pH levels. By solving the Eq. (2), the highest production of 172.15 U/gds L-asparaginase could be gained by using 3 g/L yeast extract at the optimal predicted concentration of dextrose (2.6 g/L) and pH (6.7).
The production of L-asparaginase by different microorganisms strongly depends on the medium pH. The diffusion of different components throughout the cell membrane and regulation of the metabolic processes in the cells are fundamentally affected by the pH 35 . Dharmaraj 36 reported that the highest Streptomyces noursei MTCC 10469 production of L-asparaginase was achieved at pH 8. While, the maximum Serratia marcescens (NCIM 2919) L-asparaginase production was obtained at pH 7.4 37 . Varalakshmi and Raju 38 recorded Aspergillus terreus MTCC 1782 highest production of L-asparaginase (191.3 U/gds) was achieved at pH 8 under SSF conditions. Carbon sources are used in the formulations of microbial fermentation media to enhance the growth of the desired microorganism and subsequently resulted in a higher synthesis of primary metabolites (such as the enzymes) during fermentation process 39 . Carbon source acts as a major nutrient for cell proliferation and for www.nature.com/scientificreports www.nature.com/scientificreports/ production of L-asparaginase. Glucose, sucrose and fructose are found to be promising carbon sources for production of L-asparaginase 40 . The influence of carbon source on microbial growth and production of metabolites depends on several factors, including the carbon concentration 41 .
The role of glucose (D-glucose is also known as dextrose) in L-asparaginase synthesis is controversial. In general, glucose at higher concentrations was seen as a catabolic repressor in the bacterial L-asparaginase synthesis by Erwinia aeroideae and Escherichia coli 42,43 . This may be due to the suppression of catabolites of lactate transport components that stimulated synthesis of L-asparaginase 44 . The maximum production of L-asparaginase by Escherichia coli was achieved by using 0.25% glucose supplementation 45 . In contrast, the highest synthesis of L-asparaginase by Bacillus circulans was achieved when the strain was grown in the presence of 1-1.5 g of glucose supplementation in the fermentation medium 18 . Serratia marcescens 46 , Bacillus sp. WF67 47 and Pectobacterium carotovorum MTCC 1428 48 produce L-asparaginase maximally when glucose was used as a source of carbon. Glucose and starch were stated to be good sources of carbon for the highest production of L-asparaginase by Streptomyces ginsengisoli 49 . Dextrose (glucose) has been identified as the best carbon source at a concentration of 3 g/L having a positive influence on the production of Streptomyces olivaceus NEAE-119 glutaminase free L-asparaginase 50 .
Literature on fungal L-asparaginase production reported that Fusaium oxysporum had the highest L-asparaginase production with 0.3 percent glucose as a carbon source 51 . Maximum L-asparaginase production by Bipolaris sp. BR438 was obtained using the modified Czapek-Dox medium supplemented with 0.4% glucose 52 . Aspergillus terrus MTCC 1782, grown in the modified Czapek-Dox medium complemented by 0.6% soybean flour and 0.2% glucose, achieved the highest L-asparaginase synthesis 53 . Figure 8B illustrates L-asparaginase production as a response of pH (X 1 ), yeast extract (X 3 ) by keeping dextrose (X 2 ) at zero level. At the moderate levels of both pH and yeast extract, maximum L-asparaginase production has been obtained and a gradual decline in the production of L-asparaginase has been observed at higher levels of both pH and yeast extract. By analyzing Fig. 8B and predicting Eq. (2), the L-asparaginase production of 171.94 U/gds could be achieved with 2.5 g/L dextrose at the optimum predicted levels of pH (6.7) and 2.95 g/L yeast extract.
L-asparaginase production increases with supplementation of nitrogen sources, indicating that it is regulated by nitrogen; therefore it requires supplementary nitrogen sources in addition to the carbon source to enhance L-asparaginase production. Many microorganisms consume both organic and inorganic sources of nitrogen to generate components of the cell wall, proteins, nucleic acids and amino acids, industrial enzymes. Di-ammonium hydrogen phosphate (0.16%) was identified as a good source of nitrogen for L-asparaginase production by Enterobacter aerogenes 54 . As reported by Pallem et al. 51 , malt extract was the preferred source of nitrogen for maximum L-asparaginase production by Fusarium oxysporum. On the other hand, yeast extract was found to be a good source of nitrogen for the production of L-asparaginase by Amycolatopsis CMU-H002 (1.5%) 55 , Streptomyces albidoflavus (2%) 13 , Erwinia aroideae 56 and Erwinia carotovora EC-113 57 . Liu and Zajic 56 reported that Erwinia aroideae production of L-asparaginase was dependent on the concentration of yeast extract and the optimal concentration of 1.5 percent gave the maximum L-asparaginase. In addition, Verma et al 3 . indicated that the yeast extract was essential for cell growth as well as L-asparaginase production, which was inhibited at high concentrations of yeast extract 42,53 . Figure 8C illustrates L-asparaginase production as a response of dextrose (X 2 ), yeast extract (X 3 ) by keeping pH (X 1 ) at zero level. It has been shown that the larger production of L-asparaginase has been achieved at the middle level of dextrose (X 2 ), yeast extract (X 3 ) and the higher levels resulted in a decline in L-asparaginase production. By analyzing Fig. 8C and predicting Eq. (2), the maximal L-asparaginase production of 170.18 U/ gds could be achieved with pH (7) and the optimal predicted levels of yeast extract concentration (2.97 g/L) and 2.55 g/L dextrose.
Verification of the experimental model. The validation of the experimental results of Streptomyces rochei subsp. chromatogenes NEAE-K L-asparaginase production has been evaluated under optimum conditions from the CCD and compared to the predicted data. The highest predicted L-asparaginase production (172.15 U/ gds) could be gained by using 3 g/L yeast extract, 2.6 g/L dextrose and pH 6.7. The experimental L-asparaginase activity (175.96 U/gds) was very close to that predicted by the regression model (172.15 U/gds), which confirmed the model's validity.
El-Naggar et al. 26 used a central composite design based optimization to investigate and analyze the combined effect of three variables that were significant for Streptomyces brollosae NEAE-115 L-asparaginase production. The highest production of L-asparaginase was 149.65 U/gds at the optimal levels of K 2 HPO 4 (2.5 g/L), L-asparagine (12 g/L) and soybean + wheat bran (16 g/L). The optimized levels of process variables for maximum experimental L-asparaginase production (3.74 U) by Cladosporium sp. were found to be incubation temperature (30 °C), pH (5.8) and moisture content (58%) using wheat bran as substrate 58 . Ghosh et al. 37 used traditional one variable at a time method and Box-Behnken design to evaluate the effect of different nutritional ingredients and cultural conditions on Serratia marcescens L-asparaginase production. The highest production of L-asparaginase (5.86 U/ gds) using Box-Behnken design based optimization was obtained by using the optimal levels of the most significant variables: initial substrate moisture content 50%, pH 7.4, temperature 35.5°Cand 7.5 g of coconut oil cake. On the other hand, Kumar et al. 59 found that the maximum production of L-asparaginase by Serratia marcescens was 83.16 U/g under the optimum conditions of time (48 h), temperature (28°C), pH (7.5) and moisture content (60%) when the fermentation medium supplemented with 0.6% L-asparagine and 1.5% sucrose. Furthermore, Hosamani and Kaliwal 60 found that, maximum production of Fusarium equiseti L-asparaginase was 8.51 U which obtained under the following conditions: yeast extract (0.5%, w/v), glucose (0.5%, w/v), inoculum volume (20%, v/w), particle size (3 mm), initial moisture content (70%, v/w), ammonium sulphate (0.5%, w/v) and incubation period (48 h). Singh and Srivastava 33 found that the maximum production of Bacillus aryabhattai strain ITBHU02 L-asparaginase was 16.1 U/gds and obtained on wheat bran supplemented with 0.25% (w/w) L-asparagine and 1% (w/w) yeast extract. The moisture content was 100% (v/w) and pH 7.5. The inoculated medium had been incubated for 36 hours at a temperature of 30 °C.
L-asparaginase purification. For purification of L-asparaginase, crude filtrate of culture with overall activity 24410.1 U, protein content 3304 mg and specific activity of 7.39 U/mg proteins was used. The ammonium sulfate precipitation partially purified L-asparaginase and the dialyzed enzyme had 226.8 mg protein content, 32.37 U/mg protein specific activity and enzyme recovery of 30.07% with 4.38 purification fold ( Table 8). The purified enzyme was obtained with the packed column of DEAE Sepharose CL-6B, resulting in 80 fractions with a single peak (Fig. 9). The purified L-asparaginase collected from the packed column of DEAE Sepharose CL-6B has 4.8 mg protein content with overall activity 573. 41 Table 8. Purification steps summary.  www.nature.com/scientificreports www.nature.com/scientificreports/ purification folds. Sahu et al. 61 reported the purification of L-asparaginase produced by an actinomycete strain LA-29 with 1.9% protein recovery and 13.57 U/mg protein specific activity and 18 purification fold.

SDS-PAGE and determination of monomeric molecular weight of the protein. Protein separa-
tion by SDS-PAGE method was used to determine the monomeric molecular weight of L-asparaginase 62 . Only one protein band has been resolved with a molecular mass of 64 kDa (Fig. 10). L-asparaginase molecular weight was shown to be different depending on the source of the enzyme. Purified L-asparaginase from Streptomyces gulbargensis 63 , Streptomyces fradiae NEAE-82 64 , Streptomyces PDK2 65 , Streptomyces noursei 36 and Streptomyces albidoflavus 13 exhibited a molecular weight of 85, 53, 140, 102 and 112 kDa; respectively. cytotoxic activity of L-asparaginase against human cancer cell lines. The purified enzyme has been assessed on five human cancer cell lines for its in vitro anticancer effect via the standard MTT assay. The cancer cell lines, namely; epitheliod carcinoma (HeLa), Hep2 (epidermoid larynx carcinoma), MCF-7 (breast carcinoma), Caco2 (Colorectal adenocarcinoma), and hepatocellular carcinoma (HepG-2). Human lung fibroblast, WI-38 (normal cell line) was also used. The results showed that L-asparaginase displayed varying degrees of inhibitory activity against the tested human cell lines of the tumors and normal cell line (Fig. 11).
Sudarkodi and Sundar 67 reported that L-asparaginase showed a good anticancer activity against HeLa and HepG-2 cell lines. The purified L-asparaginase showed a dose-depended cytotoxic effect on HeLa cells 68 . On the other hand, D'Souza et al. 69 reported that L-asparaginase showed a significant cytotoxic activity against MCF-7, HeLa, HepG-2 and 3T3L1 cell lines. However, Moharib 70 reported that L-asparaginase has higher cytotoxic activity against HepG-2 and HCT-116 than HeLa and MCF7 carcinoma cell lines. The anticancer activity of the purified L-asparaginase showed significant toxic activity toward HepG-2 cells, MCF-7 and HCT-116 cells 71 . Variations in the cytotoxic effects of L-asparaginase on different cell lines may be depend on the genetic nature of the microbial strain used for L-asparaginase production. The L-asparaginases obtained from various plant and microbial sources have different physiochemical and kinetic characteristics 72 .

Materials and Methods
Microorganism used in this study and cultural conditions. Streptomyces sp. strain NEAE-K used in this study was isolated from a soil sample obtained from Taif in Saudi Arabia. Isolation of Streptomyces sp. strain NEAE-K has taken place on the agar plates of starch nitrate medium. The plates have been incubated at 30°C for seven days. The isolate was preserved in 20% (v/v) glycerol as a spore suspension for subsequent studies. evaluation of L-asparaginase production by agar plates assay method. The assessment of L-asparaginase production was performed using the agar plates of asparagine dextrose salts consisting of (%): Dextrose 0.2 g; K 2 HPO 4 0.1 g; L-asparagine 1.0 g; MgSO 4 0.05 g; Agar 2 g per 100 mL water; pH was adjusted to 6.8-7. Phenol red as an indicator of pH (0.009 per cent final concentration in ethanol) was added to the medium 73 and sterilized. The plates were inoculated, inverted and incubated at 30 °C for 5-7 days. The production of L-asparaginase resulted in a rise in pH of culture medium causing changes in color of the pH indicator from yellow to pink. Isolate surrounded by pink zone was considered as L-asparaginase producing strain and were chosen for further studies. Plates of control were prepared as a dye-free medium. inoculum preparation. The spore inoculum was made from a seven-day old culture which grew on a slope of starch-nitrate agar and the spores were suspended by a sterile loop in 10 mL sterile medium of production supplemented with 0.01% (v/v) of Tween 80 74 . comparative assessment of substrates for L-asparaginase production and solid state fermentation. Local wheat bran and soy bean grains have been obtained from Mansoura local market, Egypt and were used as substrate for production of L-asparaginase. The grains were ground to produce around 1-3 mm of particle size. The efficacy of agro-industrial residues as a carbon source (wheat bran and soybean meal; separately and in combination) for the production of L-asparaginase was assessed. Solid state fermentation was performed in 250 mL Erlenmeyer flasks sets containing 10 g of each and mixed wheat bran and soybean meal were added in 1:1 proportions, to which freshly prepared asparagine dextrose salts broth medium was supplemented. The contents were thoroughly mixed and sterilized. The sterilized medium components were inoculated with 2 mL of inoculum to get the desired moisture content. The flasks contents were thoroughly mixed with a sterile wooden spoon and incubated in a stationary position at 30 °C for 5-7 days. crude enzyme preparation. After incubation, the crude enzyme was extracted from the fermented substratum using a 90 mL sodium phosphate buffer (pH 7, 0.1 M). The fermented substratum and sodium phosphate buffer were homogenized with a constant stirring by agitation in a rotary shaker at 150 rpm for 1 h. The crude enzyme was centrifuged in a cooling centrifuge (at 4 °C) for 15 min at 5000 × g. The resulting supernatant was used for L-asparaginase activity assay.  www.nature.com/scientificreports www.nature.com/scientificreports/ Assay of L-asparaginase activity. L-asparaginase activity was calculated from estimates of the quantities of ammonia released from L-asparagine hydrolysis by direct nesslerization using Wriston and Yellin 75 method. One blank was prepared for each sample tested to exclude the quantity of ammonia generated throughout the microbial fermentations. One unit (U) of L-asparaginase is described as the quantity of the enzyme releasing 1 μmole of ammonia as a result of L-asparagine hydrolysis at 37 °C per minute using pH 8.6. The enzyme activity was expressed in terms of units per gram dry fermented substrate (U/gds).
Assay of L-glutaminase. Imada et al. 76 technique was used to estimate the L-glutaminase activity of both culture filtrate and purified Streptomyces rochei L-asparaginase using L-glutamine as substrate. The reaction mixture containing 0.5 mL of the enzyme preparation and 1.5 mL of 0.04 M L-glutamine (prepared in 0.05 M Tris-HCl buffer, pH 8.6). The reaction mixture tubes were incubated in a water bath shaker for 30 minutes at 37 °C. The reaction was stopped by adding 0.5 mL of 1.5 M Trichloroacetic acid (TCA). The blank was prepared by adding the enzyme after the addition of TCA, one blank has been conducted for each sample tested. The precipitated proteins were separated by centrifugation at 10,000 × g for 10 minutes. The amount of released ammonia was determined colorimetrically by direct nesslerization by adding 1 mL of Nessler's reagent to tubes containing 0.5 mL of clear supernatant diluted with 7 mL of distilled water and incubated at room temperature for 20 min. The formation of the yellow color reflects the presence of ammonia and was measured against a blank at 450 nm using Optizen Pop-UV/Vis spectrophotometer. The amount of ammonia released was determined by using the standard curve of ammonium chloride.
Spore chain morphology and cultural characteristics. The spore chain morphology of Streptomyces sp. strain NEAE-K was examined on ISP medium 4 cultured at 30 °C for 7-14 days. The dehydrated gold covered samples had been investigated for various magnifications with Jeol JSM-6360 LA Scanning Electron Microscope. The color of the aerial spores, pigmentation of the substrates mycelia and the production of diffusible pigments have been detected at ISP media 2-7 as illustrated by Shirling and Gottlieb 77 .
Whole cell sugar analysis. Sugars were identified by High-Performance Liquid Chromatography (HPLC).
The whole dried cells (50 mg) were hydrolyzed for 10 h at 100 °C in 1 mL of 2 mol/L trifluoroacetic acid in a sealed Pyrex tube. The residual acid was removed in a water-bath at 60 °C under a stream of N 2 after the addition of methanol and taking the contents to dryness three successive times. The residue was dissolved by adding 1 mL of distilled water. Monosaccharides analysis in the extract of the whole-cell hydrolyzate was done with High-Performance Liquid Chromatography (HPLC, Agilent 1100 with Refractive Index Detector) using 4.6 × 250 mm Hypersil ASP-2 column with a mobile phase of 80: 20 acetonitrile-water and a flow rate of 0.4 mL min −1 . The optical unit temperature and column were set at 40°C and 35°C; respectively. The injection volume of the standard and sample hydrolyzates was 10 μL.
The monosaccharides in the sample hydrolyzate was identified by comparing the obtained retention times with that of the standards for nine monosaccharides (fructose, xylose, mannose, arabinose, glucose, galactose and rhamnose) which injected in a concentration of 10 mg mL −1 under the same HPLC conditions. physiological characteristics. The physiological features of Streptomyces sp. strain NEAE-K have been carried out according to the guidelines of Shirling and Gottlieb 77 . The strain's ability to inhibit the growth of two yeasts "(Candida albicans, Sacchromyces cerevisiae), five strains of fungi (Fusarium oxysporum, Aspergillus niger, Bipolaris oryzae, Alternaria solani, Rhizoctonia solani) and five strains of bacteria (Klebsiella, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Escherichia coli)" was determined. The chitosanase 78 , asparaginase 73 and uricase 79 activities were also assessed.
16S rRNA analysis. The PCR reaction was conducted and the reaction mixture was purified and sequenced according to El-Naggar et al. 80 methods. The neighbor-joining phylogenetic tree was generated using version 2.1 of MEGA4 software 30 . Selection of the significant variables using Plackett-Burman design. In the optimization of bioprocesses, Plackett-Burman design 81 is commonly used to screen and assess the significant variables influencing the response. It reduces the experiments to a minimum by screening n variables in n + 1 runs. It is often used to study more than five independent variables. The design of Plackett-Burman can rapidly identify the key physicochemical variables that have large effects on the response of multiple independent variables in a very small number of experiments with regard to their main effects 82 . Plackett-Burman design disadvantage is that it does not describe the mutual interactions among the independent variables.