Optimizing the Production of Glutaminase-Free L-asparaginase by Halotolerant Penicillium sp. Isolated from Halophyte Cogongrass Rhizosphere

L-asparaginase is an enzyme included in the treatment of acute lymphoblastic leukemia (ALL). Its therapeutic mechanism is the hydrolyzes of L-asparagine, which is an essential amino acid for neoplastic cells while it can be synthesized by normal cells. The problem of L-asparaginase from microbial origin represented in its association with glutaminase activity. The search for new eukaryotic L-asparaginase-producers among microorganisms with low glutaminase affinity is important. This study investigated the biotechnological potential of filamentous fungi isolated from the cogongrass ( Imperata cylindrical ) rhizosphere soil. Fifteen


Introduction
The most prevalent cause of cancer-

Quantitative assay of L-asparaginase and L-glutaminase
Using Nessler's reagent, the L-

Statistical analysis
The measured data were subjected to the analysis of variance (ANOVA) using a one-way, completely randomized design in triplicates, and the mean values with standard deviation were calculated. Using the CoStat statistical software, the critical difference at a 5% level of probability was compared with the significant differences between treatments using Duncan's test.

The isolated Fungi
Halophilic

Primary screening analysis using phenol red indicator
The plate assay is an efficient and dependable technique that allows for the immediate observation of L-asparaginase production. The L-asparaginase is free of glutaminase-producing fungi initially screened from marine water and soil samples. The results obtained showed pink color. One fungal isolate out of the fifteen fungal isolates can produce the lLasparaginase free of glutaminase after 7 days. The isolate that showed growth in asparagine media was chosen to complete the study.

Semi-quantitative assay
The development of the pink zone around the isolates' wells was a sign that Lasparaginase free of glutaminase was being produced. Appearing of the colored zones around the colonies indicating the pH elevation caused by ammonia build up in the media because of the production of Lasparaginase free of glutaminase. The value of the zone index obtained using phenol red was 13.57. The fungal isolate showed no growth in glutamine media (Fig. 2).

L-asparaginase quantitative assay
This experiment was carried out to investigate the potentiality of the different isolated species to produce L-asparaginase free of glutaminase in their culture filtrate.
The productivity of the L-asparaginase

The incubation period's impact
The findings demonstrated that after 4 days of incubation, the fungal Lasparaginase activity peaked at 11.30 U/ml. In contrast, dry weight (0.24 g/20ml) was recorded at the end of 6 days. The results also showed that Lasparaginase activity and dry weight were decreased gradually with a further extension of the incubation periods to 7 days, Fig. (4).

Effect of PH values
The pH value of the culture medium is essential in improving L-asparaginase production efficacy. The influence of five initial medium pH values (5, 6, 7, 8, and 9) on L-asparaginase production was studied. The fungal results showed that pH 7.0 was optimal for L-asparaginase activity (11.28 U/ml). Above and below pH 7 the activity of L-asparaginase was substantially lower and the optimal dry weight (0.35 g/20ml) was at pH 6.0. The experimental fungus can develop and generate intracellular L-asparaginase at a variety of pH levels (Fig. 5).

Effect of NaCl concentrations
The outcomes depicted in Fig. 6 showed that L-asparaginase activity and dry weight were obtained at different concentrations of sodium chloride, where the maximum enzyme activity was 16.43 U/ ml at 75 gL -1 of NaCl and the dry weight was 0.06 g/20ml at 25 gL -1 of NaCl. Further increasing or decreasing in sodium chloride concentration led to a reduction in dry weight and Lasparaginase activity (Fig. 6).

Impact of various carbon sources
Production of L-asparaginase is dependent on the presence of both carbon and nitrogen sources in the medium. Both influence how enzyme synthesis is regulated. According to the findings in Fig. (7), sucrose was the most efficient carbon source for the activity of the fungus L-asparaginase. (35.78U/ml), and the maximum fungal dry weight was produced using glucose as a carbon source. While L-asparaginase activity was present in the other carbon sources as well, they did not produce as much as sucrose did (Fig. 7).   The minimum fungal dry weight was sodium nitrate (0.24 g/ 20 ml) (Fig. 9).

Effect of incubation temperature
The effect of temperatures ranging from 25-45 o C was studied on lasparaginase productivity and fungal development. The results showed that the L-asparaginase production increased as the incubation temperature was raised, with the maximum enzyme productivity occurring at 35°C (26.28 U/ml). The L-asparaginase activity was significantly lower both above and below this temperature. Maximum dry weight was obtained at 35°C and 30°C; there is no significant value between the two degrees. Around these degrees, the fungal dry weight decreased (Fig. 11).