Stabilization of horseradish peroxidase by covalent conjugation with dextran aldehyde against temperature and pH changes

Abstract

Stabilization of Horseradish Peroxidase (HRP; EC 1.11.1.7) against temperature and pH via the formation of the conjugates obtained by multipoint covalent bonding of dextran aldehyde (DA) to the enzyme were studied. Hence, three different molar weighted dextrans (17.5 kD, 75 kD, 188 kD) were covalently bonded to purified enzyme with different molar ratios (nHRP/nDA 20/1, 10/1, 1/1, 1/5, 1/10, 1/15, 1/20). The thermal stabilities of the obtained conjugates were evaluated with the activities determined at different temperatures (25, 30, 35, 40, 50, 60, 70, 80°C) applying 60 minutes incubation time. Conjugates formed were characterized by gel-permeation chromatography (GPC) and fluorescence techniques. The conjugate synthesized using dextran 75 kDa with nHRP/nDA 1/10 molar ratio showed better thermal stability than other conjugates and purified enzyme at pH 7. This conjugate also has wider activity pH range than purified enzyme. In addition, mentioned conjugate at pH 7 had very long storage lifetime compared to purified enzyme at +4°C and room temperature; which is considered a favorable feature for usage in practice.

Decolorization of Naphthol Blue Black using the Horseradish Peroxidase

“…As the temperature is increased from 30°C to 70°C, at pH 7, a slow decrease in the rate was observed. The reason may be attributed to the fact that the activity of purified HRP enzyme decreases at temperatures above 30°C, as determined in our previous study [31]. As clearly shown in Fig.…”

“…The fractions containing HRP activity were collected and concentrated in ultrafiltration cell with Regenerated Cellulose membrane (Dia 25 mm, Mw 10.000) by washing 2 times with distilled water and 2 times with 0.01 M phosphate buffer, pH 7. RZ value (A 460 /A 280 ) of the enzyme was determined as 2.24 [31]. The analytical determination was carried out in duplicate, and the average was used as a datumpoint.…”

“…HRP activity was assayed by monitoring the oxidation of o-dianisidine at 30°C and 460 nm [31]. One unit (U) of enzyme activity was defined as I μmol of oxidative compound produced in 1 min.…”

Photoinduced Cleavage and Hydrolysis of o‐Nitrobenzyl Linker and Covalent Linker Immobilization in Gelatin Methacryloyl Hydrogels

“…This assumption is supported by different studies trying to enhance the temperature stability of HRP, e.g., by modification of amino acid residues, [73] or conjugation with, e.g., dextran. [74]…”

Enzyme properties and thermal stability of horseradish peroxidase (HRP-DL)

“…The literature reported that by obtaining the stable horseradish peroxidase-dextran aldehyde conjugates can improve the stability of HRP against the inverse environment such as high temperature. Multipoint covalent bonding of dextran to horseradish peroxidase caused the formation of a conjugate whose thermal stability was highest at pH 7 and the molar ratios of HRP and dextrans provided to obtain the conjugate was 1/10 [10] . However, these methods still have limitations in practical applications such as poor resistance activity, and large amount of protective agent.…”