TY - JOUR
T1 - Kinetics and conformational stability studies of recombinant leucine aminopeptidase
AU - Hernández-Moreno, Ana V.
AU - Villaseñor, Francisco
AU - Medina-Rivero, Emilio
AU - Pérez, Néstor O.
AU - Flores-Ortiz, Luis F.
AU - Saab-Rincón, Gloria
AU - Luna-Bárcenas, Gabriel
PY - 2014/3
Y1 - 2014/3
N2 - 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±0.4kcalmol-1, respectively. Thermal stability conformation of rLAP is 2.6±0.1 and 6.1kcalmol-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±0.10min-1μM-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.
AB - 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±0.4kcalmol-1, respectively. Thermal stability conformation of rLAP is 2.6±0.1 and 6.1kcalmol-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±0.10min-1μM-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.
KW - Conformational stability
KW - Kinetic parameters
KW - Recombinant leucine aminopeptidase
UR - http://www.scopus.com/inward/record.url?scp=84891643227&partnerID=8YFLogxK
U2 - 10.1016/j.ijbiomac.2013.12.023
DO - 10.1016/j.ijbiomac.2013.12.023
M3 - Artículo
SN - 0141-8130
VL - 64
SP - 306
EP - 312
JO - International Journal of Biological Macromolecules
JF - International Journal of Biological Macromolecules
ER -