title: Kinetics and conformational stability studies of recombinant leucine aminopeptidase.
abstract: Leucine aminopeptidase from Vibrio proteolyticus is a broad specificity N-terminal aminopeptidase that is widely used in pharmaceutical processes where the removal of N-terminal residues in recombinant proteins is required. We previously reported the expression of a heterologous construction of the mature protein fused to a 6-histidine tag that presents a reasonable refolding rate for its use at industrial level. Here, we investigate this recombinant leucine aminopeptidase (rLAP) to explain the gain of activity observed when incubated at 37 °C after its production. Unfolding transitions of rLAP as a function of urea concentration were monitored by circular dichroism (CD) and fluorescence (FL) spectroscopy exhibiting single transitions by both techniques. Free energy change for unfolding measured by CD and FL spectroscopy are 2.8 ± 0.4 and 3.7 ± 0.4 kcal mol(-1), respectively. Thermal stability conformation of rLAP is 2.6 ± 0.1 and 6.1 kcal mol(-1) for CD and Nano-Differential Scanning Calorimetry (Nano-DSC), respectively. Enzyme activity was assessed with L-leucine-p-nitroanilide (L-pNA) as substrate. The catalytic efficiency was 3.87 ± 0.10 min(-1) μM(-1) at 37 °C and pH 8.0. Kinetic and conformation studies show differences between the enzyme native and rLAP; however rLAP is selective and specific to remove N-terminal groups from amino acids.
authors.affiliation: Polymer and Biopolymer Research Group, Cinvestav Querétaro, QRO, Mexico.
authors.forename: Ana V
authors.initials: AV
authors.lastname: Hernández-Moreno
authors.affiliation: Department of Biochemical Engineering, Instituto Tecnológico de Celaya, GTO, Mexico.
authors.forename: Francisco
authors.initials: F
authors.lastname: Villaseñor
authors.affiliation: Research & Development Group, PROBIOMED S.A. de C.V., Edo de México, Mexico.
authors.forename: Emilio
authors.initials: E
authors.lastname: Medina-Rivero
authors.affiliation: Research & Development Group, PROBIOMED S.A. de C.V., Edo de México, Mexico.
authors.forename: Néstor O
authors.initials: NO
authors.lastname: Pérez
authors.affiliation: Research & Development Group, PROBIOMED S.A. de C.V., Edo de México, Mexico.
authors.forename: Luis F
authors.initials: LF
authors.lastname: Flores-Ortiz
authors.affiliation: Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, MOR, Mexico. Electronic address: gsaab@ibt.unam.mx.
authors.forename: Gloria
authors.initials: G
authors.lastname: Saab-Rincón
authors.affiliation: Polymer and Biopolymer Research Group, Cinvestav Querétaro, QRO, Mexico. Electronic address: gluna@qro.cinvestav.mx.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Luna-Bárcenas
mesh_terms: D004789:Enzyme Activation; D004795:Enzyme Stability; D007700:Kinetics; D007931:Leucyl Aminopeptidase / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D058849:Protein Refolding / Q000187:drug effects; D058767:Protein Unfolding / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology; D014733:Vibrio / Q000201:enzymology
chemical_list: D011994:Recombinant Proteins; D014508:Urea; D007931:Leucyl Aminopeptidase
keywords: Conformational stability; Kinetic parameters; Recombinant leucine aminopeptidase