Caseoperoxidase, mixed β-casein–SDS–hemin–imidazole complex: a nano artificial enzyme

Abstract

A novel peroxidase-like artificial enzyme, named “caseoperoxidase”, was biomimetically designed using a nano artificial amino acid apo-protein hydrophobic pocket. This four-component nano artificial enzyme containing heme-imidazole-β-casein-SDS exhibited high activity growth and kcat performance towards the native horseradish peroxidase (HRP) demonstrated by the steady state kinetics using UV-Vis spectrophotometry. The hydrophobicity and secondary structure of the caseoperoxidase were studied by ANS fluorescence and circular dichroism spectroscopy. Camel β-casein (Cβ-casein), with a flexible structure and exalted hydrophobicity, was selected as an appropriate apo-protein for the heme active site using a homology modeling method. Heme docking into the newly obtained Cβ-casein structure indicated one heme was mainly incorporated with Cβ-casein. The presence of a main electrostatic site for the active site in the Cβ-casein was also confirmed by experimental methods through Wyman binding potential and isothermal titration calorimetry. The existence of Cβ-casein protein in this biocatalyst lowered the suicide inactivation, and indicated that the obtained structure has a good protective role for the heme active-site. Additional further experiments confirmed the retention of caseoperoxidase structure and function as an artificial enzyme.

Hemin-catalyzed oxidative oligomerization of p-aminodiphenylamine (PADPA) in the presence of aqueous sodium dodecylbenzenesulfonate (SDBS) micelles

“…Furthermore, based on previous literature reports on the catalytic activity of hemin, the concentration of hemin used in all measurements was kept constant at [hemin] ¼ 10 mM. [57][58][59] This concentration is also useful for direct spectrophotometric measurements of hemin itself in the region of the Soret band (A 400 (l ¼ 1 cm) z 0.5-1.2), so that information about possible changes in the aggregation state of hemin upon changing the reaction medium (in the absence of PADPA, i.e., without reaction) can be obtained. 17 For reference reactions with HRPC, the PADPA and H 2 O 2 concentrations used were the same as in the case of the reactions with hemin, [PADPA] 0 ¼ [H 2 O 2 ] 0 ¼ 1.0 mM, and the HRPC concentration applied was the one applied in our previous work with AOT vesicles as templates: 5 [HRPC] ¼ 30 nM.…”

Metallo-vesicular catalysis: A mixture of vesicular cysteine/iron mediates oxidative pH switchable catalysis

“…Many attempts have been made to use micelles and vesicles to cover the pocket of an artificial enzyme. Such studies have shown that hydrophobic pockets enhance the catalytic activity of a functional center by stabilizing the intermediate complexes [24,30,44,45]. The kinetic parameters (Table 1) The self-assembly of surfactant molecules constructs various micellar nano-structures [43,46,47].…”

An outlook on suicide enzyme inhibition and drug design

“…To improve catalytic efficiency of HRP in the presence of H 2 O 2, caseoperoxidase (mixed β-casein–SDS–hemin–imidazole complex) has been designed, which is HRP-like enzyme consists of Camel β-casein–SDS. Caseoperoxidas protects HRP’s active sites by one main electrostatic and four minor non-electrostatic (hydrophobic) sites of β-casein [ 110 ]. To determine the protective effect of camel β-casein, heme–imidazole–SDS nanobiocatalyst was designed and compared with caseoproxidase through the suicide parameters [ 111 ].…”