title: Biochemical characterization and immobilization of Erwinia carotovoral-asparaginase in a microplate for high-throughput biosensing of l-asparagine.
abstract: 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 (Km, kcat) 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 10-200μΜ. The biosensor had a detection limit of 10μM for l-asparagine. The method's reproducibility was in the order of ±3-6% and l-asparagine mean recoveries were 101.5%.
authors.affiliation: Laboratory of Enzyme Technology, Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of Athens, 75 Iera Odos Street, GR-11855-Athens, Greece. Electronic address: Lambrou@aua.gr.
authors.forename: Nikolaos E
authors.initials: NE
authors.lastname: Labrou
authors.affiliation: Department of Pharmacognosy, College of Pharmacy, Prince Sattam Bin Abdulaziz University, 11942 Alkharj, Saudi Arabia; Department of Microbiology, College of Science, Al-Azhar University, Nasr City, 11884, Cairo, Egypt.
authors.forename: Magdy Mohamed
authors.initials: MM
authors.lastname: Muharram
mesh_terms: D000595:Amino Acid Sequence; D001215:Asparaginase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001216:Asparagine / Q000032:analysis* / Q000097:blood; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods; D001774:Blood Chemical Analysis / Q000379:methods; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005504:Food Analysis / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016973:Pectobacterium carotovorum / Q000201:enzymology* / Q000235:genetics; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D011198:Solanum tuberosum / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; D001216:Asparagine; D001215:Asparaginase
keywords: Enzyme immobilization; Hydrolase; Kinetic analysis; l-Asparaginase