Determining the Optimum Conditions for L-Asparaginase Produced from a Local Isolate of Pseudomonas Aeruginosa

Abstract

L-asparaginase is an enzyme of significant medical importance that is extensively applied in the management of cancers. The enzyme catalyzes the degradation of the amino acid L-asparagine, thereby preventing cancer cells from utilizing it for proliferation. Organisms such as bacteria are crucial sources of the enzyme. Bacteria such as Pseudomonas aeruginosa (P. aeruginosa) are exceptionally adaptable across diverse habitats and can synthesize many biologically essential enzymes. The study aims to isolate and characterize P. aeruginosa from various sources in Baghdad, determine its capacity to synthesize L-asparaginase, and establish favorable conditions for enzyme synthesis. The total number of samples taken was seventy-two (sixty-nine soil samples and three water samples). The samples were cultured on appropriate nutrient medium after dilution. The isolates were characterized morphologically and biochemically, with further confirmation carried out using the VITEK 2 method. Enzyme production by the isolates was initially screened by culturing them on a nutrient medium supplemented with L-asparagine and phenol red indicator. Enzyme activity was estimated using the Nessler test, whereas protein was estimated using the Bradford method. Isolates that exhibited high efficiency were chosen for optimization studies. The results showed great ability in the biosynthesis of L-asparaginase with statistically significant differences. The number of isolates obtained was 72, where 30 showed a positive result for biosynthesis of the enzyme, accounting for 41.7%. According to the first screening test results, 78.1% of the isolates chosen produced the enzyme with different levels according to their ability and the amount of enzyme production index (EPI). The isolates (P6, P25, PW30) proved to be the most efficient ones regarding quality. The enzyme activity varied quantitatively from 4.76 to 76.90 U/mL. The isolate P1 proved to have high activity (76.90 U/mL) and high specific activity (49.30 U/mg), which means that its enzyme production rate is greater than that of other isolates. The best production medium was C1 (lacking asparagine) with the highest enzyme activity and specific activity of 20.47 U/mL and 23.27 U/mg respectively, implying that the deficiency of substrate induces the production of enzyme. In terms of carbon sources, sucrose was the most efficient carbon source with the highest specific activity of 84.32 U/mg. Yeast extract exhibited high efficiency with enzyme activity and specific activity of 75.39 U/mL and 110.87 U/mg respectively, followed by ammonium sulfate, while peptone had low efficiency. The optimum pH was eight with the highest enzyme production of 74.60 U/mL, while the highest specific activity was at 30°C.  The highest enzyme activity and specific activity were 74.60 U/mL and 124.33 U/mg respectively.