L-Asparaginases (L-ASNase, E.C. 3.5.1.1) catalyze the conversion of L-asparagine to L-aspartic acid and ammonia. In the present work, a new form of L-ASNase from a strain of Erwinia carotovora (EcaL-ASNase) was cloned, expressed in Escherichia coli as a soluble protein and characterized. The enzyme was purified to homogeneity by a single-step procedure comprising ion-exchange chromatography. The properties of the recombinant enzyme were investigated employing kinetic analysis and molecular modelling and the kinetic parameters (K-m, k(cat)) were determined for a number of substrates. The enzyme was used to assemble a microplate-based biosensor that was used for the development of a simple assay for the determination of L-asparagine in biological samples. In this sensor, the enzyme was immobilized by crosslinking with glutaraldehyde and deposited into the well of a microplate in 96-well format. The sensing scheme was based on the colorimetric measurement of ammonia formation using the Nessler's reagent. This format is ideal for micro-volume applications and allows the use of the proposed biosensor in high-throughput applications for monitoring L-asparagine levels in serum and foods samples. Calibration curve was obtained for L-asparagine, with useful concentration range 10-200 mu M. The biosensor had a detection limit of 10 mu M for L-asparagine. The method's reproducibility was in the order of +/- 3-6% and L-asparagine mean recoveries were 101.5%. (C) 2016 Elsevier Inc. All rights reserved.