Kinetics and conformational stability studies of recombinant leucine aminopeptidase

详细信息    查看全文

• 作者：Ana V. Hern谩ndez-Moreno ; Francisco Villase帽or ; Emilio Medina-Rivero ; N茅stor O. P茅rez ; Luis F. Flores-Ortiz ; Gloria Saab-Rinc贸n ; Gabriel Luna-B谩rcenas
• 关键词：Recombinant leucine aminopeptidase ; Kinetic parameters ; Conformational stability
• 刊名：International Journal of Biological Macromolecules
• 出版年：March, 2014
• 年：2014
• 卷：64
• 期：Complete
• 页码：306-312
• 全文大小：1025 K

文摘

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 掳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 卤 0.4 and 3.7 卤 0.4 kcal mol<sup>鈭?sup>, respectively. Thermal stability conformation of rLAP is 2.6 卤 0.1 and 6.1 kcal mol<sup>鈭?sup> 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 卤 0.10 min<sup>鈭?sup> 渭M<sup>鈭?sup> at 37 掳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.