Horseradish peroxidase (HRP) is an important heme-containing glyco-enzyme that has been used in many biotechnological fields. Valuable proteins like HRP can be obtained in sufficient amounts using Escherichia coli as an expression system. However, frequently, the expression of recombinant enzyme results in inclusion bodies, and the refolding yield is generally low for proteins such as plant peroxidases. In this study, a recombinant HRP was cloned and expressed in the form of inclusion bodies. Initially, the influence of few additives on HRP refolding was assessed by the one factor at a time method. Subsequently, factors with significant effects including glycerol, GSSG/DTT, and the enzyme concentration were selected for further optimization by means of the central composite design of response surface methodology (RSM). Under the obtained optimal condition, refolding increased about twofold. The refolding process was then monitored by the intrinsic fluorescence intensity under optimal conditions (0.35 mM GSSG, 0.044 mM DTT, 7 % glycerol, 1.7 M urea, and 2 mM CaCl2 in 20 mM Tris, pH 8.5) and the reconstitution of heme to the refolded peroxidase was detected by the Soret absorbance. Additionally, samples under unfolding and refolding conditions were analyzed by Zetasizer to determine size distribution in different media.

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In the present study, trypsin from Plodia interpunctella (Hübner) is characterized to discover sequence, biochemical and structural features. This enzyme is purified by ion exchange chromatography using fast protein liquid chromatography on proteins from fifth‐instar larvae. The enzyme is optimally active at 50 °C and pH 11.0. The kinetic parameters (Km and Vmax) of the enzyme are 5.3 ± 0.6 µm and 31 ± 1.3 nmol min−1 mg−1, respectively (using Nα‐benzoyl‐l‐arginine ρ‐nitroanilide hydrochloride as substrate). The enzyme is inhibited by the addition of Cu2+ and Mn2+, whereas it is activated by Li+ at high concentrations. Moreover, the enzyme is almost completely inhibited in the presence of Nα‐tosyl‐l‐lysine chloromethyl ketone hydrochloride and phenylmethanesulphonyl fluoride. To understand some characteristics of P. interpunctella trypsin, including active site structure and alkaline pH profile, a reliable structural model of P. interpunctella trypsin is built based on the Fusarium oxisporum (Schlecht) trypsin cystal structure (Protein Data Bank code: 1GDU). The secondary structure content of the purified trypsin from near‐ultraviolet circular dichroism data shows considerable similarities with that of P. interpunctella trypsin predicted structure. Analysis of pKa values of active site residues, a type of amino acid residue in the active site cleft and the surface charges of the model and Tribolium castaneum (Herbst) trypsin structure as an insect species from different orders reveals some differences between them. These differences might effect on the microenvironment of the active site cleft and consequently shift its pH profile. The application of multiple theoretical and experimental techniques is well adapted to predict the enzyme structure with high accuracy and this could help in the design of a powerful inhibitor for trypsin with ideal properties.