الفهرس 
Biochemical and medical significance of L-asparagineOnly 14 pages are availabe for public view
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Abstract
L-Asparaginase is a hydrolytic enzyme that catalyzes the conversation of L-asparagine to L-aspartic acid and release ammonia. L-asparaginase is a therapeutic enzyme used in the treatment of certain human cancers, especially acute lymphoblastic leukemia, as a chemotherapeutic agent. Other than as an anticancer agent, it has many applications, including in the treatment of autoimmune disorders, infectious diseases, antioxidant property and antimicrobial activity.
The objective of the present study was to formulate the production medium and to determine the growth proper conditions for the chosen microorganism producing highly active L-asparaginase enzyme. The general properties of the crude and the partially purified enzyme preparation were determined to define the proper conditions for the enzyme action. Under the specified conditions, the opportunity of the two forms of the enzyme for antimicrobial and antioxidant activities was duly pinpointed as well as the cytotoxic effect of the partially purified L-asparaginase was also determined.
Eight recommended microbial isolates three bacteria (Bacillus subtilis, Echerichia coli and Pseudomonas aeuroginosa) and five fungi (Asperigellus niger, Asperigellus terreus, Asperigellus oryzae, Penicillium sp and Penicillium janthinellum Biourge) were screened for L-asparaginase enzyme productivity. Among all the eight organisms and based on the enzyme production ability, P. janthinellum Biourge was the most potent producer and was selected for the succeeding studies.
The effects of the culture media composition and other fermentation conditions for optimization of L-asparaginase production by the selected organism were duly studied. The maximum L-asparaginase activity of (17.85 U/reaction) was obtained after 3 days incubation on rotary shaker (100 rpm) with the medium containing glucose (2 g/L), L-asparagine (10 g/L) as C & N sources respectively, at 30oC and pH 6.2.The partial purification of the crude P. janthinellum Biourge L-asparaginase was carried out and the superior enzymatic partially purified fraction was 60-80% ethanol.
In addition, the important properties of the crude and the partially purified P. janthinellum Biourge L-asparaginase were duly pinpointed as follows: optimum enzyme protein and substrate concentrations were 1.0, 0.4 mg/mL and 1.0, 0.8 % (w/v), respectively, optimum reaction pH for both enzyme forms was 10.7 and optimum reaction temperatures were 45°C and 37°C, respectively.
The partially purified L-asparaginase was stable for 60 min at 37°C and retained more than 95% of its original activity, also the enzyme had a good stability at the elevated temperatures, where it retained about 87.69 and 84.11 % of its original activity after heating for 60 minutes at 45 & 50ºC, respectively. Also, the partially purified enzyme was more stable at the alkaline pH than at the acidic one as it retained 86 and 85.85% of its original activity at pH 8.6 and 9.5 even after incubation for 120 min at 37°C, respectively. The partially purified L-asparaginase Michaelis constant (Km) and maximum velocity constant (Vmax) were 3 mM (0.046 %, w/v) and 105.5 U mg-1 protein, respectively, applying the Woolf plot.
Any of Mn2+ or Fe2+ was excellent enzyme activator and led to the maximum activation of the partially purified enzyme at 100 mM with relative activity of 986 & 569 %, respectively. On the other hand, any of NaN3 or urea inhibited L-asparaginase enzyme activity to 89 and 87 % at 100 mM concentration, while, Hg2+ led to the complete enzyme activity inhibition at the same concentration.
Under the all specified conditions the crude or the partially purified enzyme exhibited aconsiderable antioxidant activity at varying concentrations. In addition, the partially purified enzyme form exhibited an excellent cytotoxic effect against Hep-G2 (Human Hepatocellular Carcinoma Cell Line).
scavenging activity, anticancer activity.