Glucose oxidase stabilization against thermal inactivation using high hydrostatic pressure and hydrophobic modification

Abstract

High hydrostatic pressure (HHP) stabilized glucose oxidase (GOx) against thermal inactivation. The apparent first-order kinetics of inactivation of GOx were investigated at 0.1-300 MPa and 58.8-80.0°C. At 240 MPa and 74.5°C, GOx inactivated at a rate 50 times slower than at atmospheric pressure at the same temperature. The apparent activation energy of inactivation at 300 MPa was 281.0 ± 17.4 kJ mol or 1.3-fold smaller than for the inactivation at atmospheric pressure (378.1 ± 25.6 kJ mol ). The stabilizing effect of HHP was greatest at 74.5°C, where the activation volume of 57.0 ± 12.0 cm mol was highest compared to all other studied temperatures. Positive apparent activation volumes for all the treatment temperatures confirmed that HHP favors GOx stabilization. A second approach to increase GOx stability involved crosslinking with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and either aniline or benzoate. The modified enzyme remained fully active with only slight increases in K (1.3-1.9-fold increases for aniline and benzoate modification, respectively). The thermal stability of GOx increased by 8°C with aniline modification, while it decreased by 0.9°C upon modification with benzoate. Biotechnol. Bioeng. 2017;114: 516-525. © 2016 Wiley Periodicals, Inc.

Stabilization of galactose oxidase by high hydrostatic pressure: Insights on the role of cavities size

“…The apparent E ai at 0.1 MPa (167 kJ mol −1 ) was 3.2 times smaller than the apparent E ai at 150 MPa (532 kJ mol −1 ). This contrasts the results of XOx (Halalipour et al, 2017a) and GOx (Halalipour et al, 2017b) which reported a decrease in E ai with increased pressure. However, the labile fraction of AOx from Pichia pastoris followed a similar trend as GaOx (Buchholz-Afari et al, 2019).…”

“…The effect of pressure on k inact of GaOx at each temperature is represented in Figure 2 (Halalipour et al, 2017a), GOx (Halalipour et al, 2017b), AOx (Buchholz-Afari et al, 2019 and a pectinase cocktail (Tomlin et al, 2013), which were 9.5-28.9 cm 3 mol −1 , 22.8-57.0 cm 3 mol −1 , and 13.7-33.7 cm 3 mol −1 , respectively. A greater ΔV ‡ corresponds to a greater stabilizing effect of pressure is; therefore, pressure has a stronger stabilization effect on GaOx than on other reported enzymes.…”

“…Most structural studies of the effects of HHP in enzymes in solution have been done on small proteins (< ~30 kDa) in part because they are amenable to a variety of structural analyses including nuclear magnetic resonance (NMR) (Pastore & Temussi, 2023). We have studied the effects of HHP on the thermal stability of glucose oxidase (GOx) a 160 kDa dimer (Yang et al, 2020), alcohol oxidase (AOx) a 675 kDa octamer (Buchholz-Afari et al, 2019), and xanthine oxidase (XOx), a 283 kDa dimer (Halalipour et al, 2017a(Halalipour et al, , 2017b) by studying the decrease in the rate of inactivation. The optimal pressure for stability and the extent of stabilization were different for each oxidase.…”

ABE-Stat, a Fully Open-Source and Versatile Wireless Potentiostat Project Including Electrochemical Impedance Spectroscopy

“…Rapid advances in the understanding of molecular interactions, [1][2][3][4] development of novel materials, [5][6][7][8][9][10] and stabilization of biologically active compounds [11][12][13] is increasing the scope for practical electroanalytical tools in the field. 14,15 These advances coupled with simple electrochemical techniques are especially compelling for diagnostic applications related to food, 4,6,7,[16][17][18][19][20][21] agriculture, 16,[22][23][24][25][26][27][28] and the environment 22,[28][29][30][31] that are highly sensitive to cost.…”

The Core–Shell Structure, Not Sugar, Drives the Thermal Stabilization of Single-Enzyme Nanoparticles

“…It should be noted that small changes in unfolding temperature can correlate to very large differences in stability at a given temperature. Although the thermal unfolding of all the GOx samples is irreversible, the DSC scans can still be fitted with a model, indicating independent two-state transitions (native to unfolded). , Figure shows the buffer-subtracted DSC scans of unmodified GOx, SENs, and cross-linked SENs after normalization using the GOx concentration. The enthalpy and temperature of the denaturation are summarized in Table .…”