Engineering substrate specificity of E. carotovora l-asparaginase for the development of biosensor

l-Asparaginase (E.C.3.5.1.1, l-ASNase) is an enzyme extensively used as an anti-neoplastic agent in the chemotherapy of acute lymphoblastic leukemia (ALL). In the present study, we report the use of in vitro directed evolution for the creation of a new l-ASNase variant lacking glutaminase activity. A library of enzyme variants was constructed by staggered extension process (StEp) using the genes that code for the l-ASNases from Erwinia chrysanthemy (ErL-ASNase) and Erwinia carotovora (Ecal-ASNase) and screened using activity assays. A variant of the E. carotovora enzyme lacking detectable glutaminase activity was identified. Sequence analysis showed that this variant contained a single point mutation (Leu71Ile). Steady-state kinetic measurements and the analysis of the pH dependence of Vmax and Vmax/Km of l-Asn hydrolysis showed that the mutation causes significant alterations in binding and catalytic properties. Analysis of the molecular model of the mutant enzyme showed that Ile71 may perturb the conformation of important amino acid residues in the linker region which directly affects the catalytic function. The Leu71Ile mutant enzyme was used to assemble a cuvette-based biosensor specific for l-Asn. The enzyme was immobilized by crosslinking with glutaraldehyde on the side of a transparent plastic cuvette. The sensing scheme was based on the colorimetric measurement of ammonia formation using the Nesslerʹs reagent. Calibration curve was obtained for l-Asn, with useful concentration range of 0–100 μM for l-Asn. The methodʹs reproducibility was in the order of ±2–5% and l-Asn recoveries were 102.1%.