Kinetics and conformational stability studies of recombinant leucine aminopeptidase

Structural and functional characterization of a recombinant leucine aminopeptidase

“…When tricine buffer at pH 8.0, or citrate buffer at pH 4.0, 5.0 or 6.0 were employed, the enzymatic activity remained below 2.5 U/mg. A subtle increase to 3 U/mg was observed using carbonate buffer at pH 8.5 and 9.0; however, when the pH increased to 10.0 the activity was totally lost after 72 h at 37 • C. An increase to 15 U/mg was observed after 72 h of incubation in phosphate buffer at pH 7.0 and 8.0, which remained similar to our original assessment, performed using a different batch of rLAP uf [22], however, when pH decreased down to 6.0, the specific activity of rLAP augmented to 31 U/mg after 72 h of incubation. The enzymatic activity of rLAP folded in phosphate buffer at pH 6.0, along with the aforementioned results at pH 8.0, suggests that the folding process was reproducible among batches ( Fig.…”

“…The mature 6×-His tag rLAP under study is a non-glycosilated monomeric protein of 304 amino acids, with an apparent averaged mass of 32.8 kDa (by reducing SDS-PAGE) [17]. This enzyme exhibits a characteristic secondary structure, as reported by circular dichroism [22]; furthermore, as in the wild type leucine aminopeptidase (LAP wt ) [19,23], rLAP has only one disulfide bond in its mature form which makes easier to study its folding.…”

“…In a previous study we reported an increase in the enzymatic activity from 1 to 18 U/mg when 3 M rLAP were incubated at 37 • C for 72 h in a buffer of 10 mM phosphate, 10 M ZnCl 2 at pH 8.0 [22]. In this study we conducted exploratory tests by employing different pH values and buffers to assess the folding process of rLAP through its activity toward the optimization of the process.…”

“…In a previous study, we reported an increase in the enzymatic activity of rLAP after an incubation period that was conceived as the best for folding [22]. Herein, we focus on the structural changes of rLAP and the repercussion on its functional attributes headed to the improvement of bioprocesses.…”

The role of conserved Cys residues in Brassica rapa auxin amidohydrolase: Cys139 is crucial for the enzyme activity and Cys320 regulates enzyme stability

“…18 Leucine aminopeptidase revealed a T max almost identical to the herein studied BrILL2 (57 AE 0.5 1C) and a three-fold higher D r H value of 80 AE 0.1 kcal mol À1 (335 AE 0.4 kJ mol À1 ). 19 Thus, the compared enzymes, having similar general functions, and approximately comparable sizes are characterised by similar thermodynamic parameters of denaturation. All enzymes showed lower T max values and consequently certain protein destabilization upon adding a reducing agent.…”