Effect of High-Pressure Processing on Activity and Structure of Alkaline Phosphatase and Lactate Dehydrogenase in Buffer and Milk

Abstract

Changes in the activity and structure of alkaline phosphatase (ALP) and L-lactate dehydrogenase (LDH) were investigated after high pressure processing (HPP). HPP treatments (206-620 MPa for 6 and 12 min) were applied to ALP and LDH prepared in buffer, fat-free milk, and 2% fat milk. Enzyme activities were measured using enzymatic assays, and changes in structure were investigated using far-ultraviolet circular dichroism (CD) spectroscopy and dynamic light scattetering (DLS). Kinetic data indicated that the activity of ALP was not affected after 6 min of pressure treatments (206-620 MPa), regardless of the medium in which the enzyme was prepared. Increasing the processing time to 12 min did significantly reduce the activity of ALP at 620 MPa (P < 0.001). However, even the lowest HPP treatment of 206 MPa induced a reduction in LDH activity, and the course of reduction increased with HPP treatment until complete inactivation at 482, 515, and 620 MPa. CD data demonstrated a partial change in the secondary structure of ALP at 620 MPa, whereas the structure of LDH showed gradual denaturation after exposure at 206 MPa for 6 min, leading to a random coil structure at both 515 and 620 MPa. DLS results indicated aggregation of ALP only at HPP treatment of 206 MPa and not above and enzyme precipitation as well as aggregation at 345, 415, 482, and 515 MPa. The loss of LDH activity with increasing pressure and time treatment was due to the combined effects of denaturation and aggregation.

Isomalt and its diastereomer mixtures as stabilizing excipients with freeze-dried lactate dehydrogenase

“…CD spectroscopy was performed on the unprocessed protein as well as on the freeze-dried protein formulations with excipients before and after storage. The measured CD spectrum of unprocessed LDH with its two ellipticity minima at around 209 and 222 nm agree well with previous CD studies of LDH (Kouassi, et al 2007;Mi, et al 2002;Mi and Wood 2004). Based on the CD spectroscopy measurements, the secondary structure of LDH freeze-dried with sugar excipients was altered to some extent (Figure 2 a, b).…”

“…As an example of the samples having the most changes during storage, the CD spectra of LDH with 1:1 GPM:GPS and sucrose before and after storage are illustrated in Figures 2 e and f. The predominant secondary structure of LDH is α-helix with 39.2% of the residues in this conformation, 22.4% in β-sheet conformation and 38.4% in other secondary structures (Kouassi, et al 2007). Loss in ellipticity observed with LDH both after freeze-drying and after storage with and without the sugars resulted in decrease in the α-helix content and increase in the content of β-sheet and other conformations (Kouassi, et al 2007).…”

High-Pressure Processing Technologies for the Pasteurization and Sterilization of Foods

“…Relatively low pressure (~100-200 MPa) may activate some enzymes while high pressure (400-1,000 MPa) may induce their inactivation. An effective means to accelerate the inactivation of enzymes is by increasing temperature; however, the effect of pressure and temperature has been determined only for a relatively few enzymes and food systems (e.g., Hurtado et al 2002;Verlent et al 2004;Chéret et al 2005;Lakshmanan et al 2005;Rademacher and Hinrichs 2006;Kouassi et al 2007;Iucci et al 2008). The largest contribution of pressure to enzyme inactivation comes from structural rearrangements of proteins under high pressure (Hendrickx et al 1998) such as hydration changes that accompany other intramolecular non-covalent interactions (Mozhaev 1996a,b).…”

Effects of high pressure treatment on glycolytic enzymes of Lactococcus lactis subsp. lactis, Streptococcus thermophilus and Lactobacillus acidophilus

“…Inactivation of LDH by high pressure treatment is attributed to the change in the secondary structure of this enzyme (Kouassi, Anantheswaran, Knabel, & Floros, 2007). The LDH activity of selected starter cultures investigated in this study indicated different responses to pressure treatment.…”

“…acidophilus cells as demonstrated in the current study makes LDH a suitable marker for monitoring the pressureinduced lysis in these cultures at pressures up to 600 MPa. Partial inactivation of rabbit muscle LDH has also been reported by treatment of the enzyme at 206 MPa in buffer and milk with complete inactivation at ≥482 MPa (Kouassi et al, 2007).…”