Production of a Novel Marine Pseudomonas aeruginosa Recombinant L-Asparaginase: Insight on the Structure and Biochemical Characterization

The present study focused on the cloning, expression, and characterization of L-asparaginase of marine Pseudomonas aeruginosa HR03 isolated from fish intestine. Thus, a gene fragment containing the L-asparaginase sequence of Pseudomonas aeruginosa HR03 isolated from the fish intestine was cloned in the pET21a vector and then expressed in Escherichia coli BL21 (DE3) cells. Thereafter, the recombinant L-asparaginase (HR03Asnase) was purified by nickel affinity chromatography, and the enzymatic properties of HR03Asnase, including the effects of pH and temperature on HR03Asnase activity and its kinetic parameters, were determined. The recombinant enzyme HR03Asnase showed the highest similarity to type I L-asparaginase from Pseudomonas aeruginosa . The three-dimensional (3D) modeling results indicate that HR03Asnase exists as a homotetramer. Its molecular weight was 35 kDa, and the maximum activity of the purified enzyme was observed at pH8 and at 40 °C. The k m and V max of the enzyme obtained with l -asparagine as substrate were 10.904 mM and 3.44 × 10 −2 mM/min, respectively. The maximum activity of HR03Asnase was reduced by 50% at 90 °C after 10-min incubation; however, the enzyme maintained more than 20% of its activity after 30-min incubation. This enzyme also maintained almost 50% of its activity at pH 12 after 40-min incubation. The evaluation of pH and temperature stability of HR03Asnase showed that the enzyme has a wide range of activity, which is a suitable characteristic for its application in different industries. Overall, the results of the present study indicate that marine sources are promising biological reservoirs for enzymes to be used for biotechnological purposes, and marine thermostable HR03Asnase is likely a potential candidate for its future usage in the pharmaceutical and food industries.