MECHANISM OF ENZYME

INACTIVATION BY
PULSED ELECTRIC FIELD
 A new food processing technology, pulsed electric fields (PEF), is
currently being investigated due to its capability of inactivating
undesirable microorganisms and enzymes with limited increase in food
temperature. As a result, more stable foods with fewer changes in
composition, physicochemical properties and sensory attributes can be
obtained.

 the mechanism of enzyme inactivation is still unclear, it is believed that

PEF processing may affect the native structure of enzymes and therefore
could promote changes in enzymatic activity
• Enzymes are proteins whose catalytic activity relies on the native
configuration of their active site and the conformation of the
surrounding proteins. The amino acid group present in enzyme
proteins creates highly asymmetric spatial distributions of charge
that lead to strongly polar and charged regions in the molecular
structure of proteins
Because of a complex network of non-covalent (electrostatic forces, ion
pairing, Van der Waals forces, hydrogen bonding and interaction
hydrophobic effect) as well as covalent interactions (di sulphide bonds),
the structural stability and catalytic functions of enzymes are maintained
 Changes in enzyme activity may occur due to the effect
on the three-dimensional molecular structure of
enzymes. The behavior of proteins under intense
electric fields is not well known, but it seems that these
fields may cause protein unfolding and denaturation,
breakdown of covalent bonds and oxidation– reduction
reactions, like those between sulphide groups and di
sulphide bonds, because of the charge separation
 Moreover, it is known that electric fields influence the
conformational state of proteins through charge, dipole,
or induced dipole chemical reactions 
 Charged groups and structures are highly susceptible to
various types of electric field perturbations and these
changes cause modifications of its structure and
consequently the loss of activity due to the difficulty of
assembling the substrate with the active site
 Application of PEF affect the forces involved in
maintaining the three-dimensional structure of lipase,
because denaturation of the enzyme was observed.
However, the conformational changes in the molecule
that lead to an activity inhibition were more evident
using a batch PEF system than a continuous PEF
system
 Factors Affecting Pulsed Electric Field
Induced Enzyme Activity

 Although PEF has proven to be an effective technique

for membrane permeabilization and microbial
inactivation, enzymes have been found to be more
resistant to PEF treatments.
 Factors that influence the effect of PEF on
enzyme activity can be as follows:

 • PEF treatment parameters (electric field strength, number of

pulses, pulse duration, electrical energy density, pulse frequency,
shape, width and polarity of pulses, geometry and shape of
treatment chamber, batch or continuous mode treatment, etc.)
 • Physicochemical properties of enzymes (enzyme structure and
composition, covalent and non-covalent interactions, geometry
of active sites, etc.)
 • Initial temperature
 • Media factors (pH, conductivity, viscosity, ionic strength, etc.)
 Effect of PEP Processing on Specific
Enzyme

Alkaline Phosphatase:
 Alkaline phosphatase (ALP) is known as a hydrolase enzyme, which is
responsible
 for cleavage of phosphate groups from many types of biomolecules such as
nucleotides,
 proteins, and alkaloids to give inorganic phosphate and the corresponding
 alcohol, phenol, and sugar. In a study, Jaeger et al. (2009) evaluated the impact of
 PEF processing on reducing ALP activity in raw milk. They reported that the
 inactivation of ALP was reduced from 78% residual activity to 92% after PEF
 treatment. Thermal effects and local temperature peaks associated with PEF
treatment
 were attributed to the enzyme inactivation. However, there are not many
 reports showing the effects of PEF on ALPs derived from various sources to
describe
 the PEF treatment efficacy on ALPs
Lipase:
Lipase is the key enzyme needed for the absorption of dietary triglycerides. Grahl and Markl
(1996) observed around 90% destruction of lipase enzymes using PEF processing of milk at the
following conditions: E, 21.5 kV/cm; frequency, 1–22 Hz; temperature, 45–50 C; and treatment
time, 10–1,000 μs. In a study conducted by Ho et al. (1997), the effect of PEF treatment on lipase
dispersed in distilled water was assayed. In this research, about 90% inactivation of lipase was
observed by applying PEF processing at the following conditions: E, 88 kV/cm; pulse width, 2
μs; treatment duration, 2 s; frequency, 0.5 Hz; number of pulses, 30; and maximum temperature
employed, 20 C. In another study, 62% lipase inactivation was reported in batch mode and 13%
in continuous flow process of PEF-treated simulated milk ultrafiltrate (Bendicho et al. 2002)
under the following conditions: energy density, 27.4–37.3 kV/cm; pulse width, 4 μs; treatment
duration, 314.5 μs; number of pulses, 80–100; and maximum temperature employed, >34 C. The
different inactivation degrees of lipase observed in these studies might be due to different
processing conditions and media used.
Lipoxygenase:
Lipoxygenases (LOX) are a family of nonheme iron-containing enzymes that
catalyze the insertion of dioxygen into polyunsaturated fatty acids. Reports regarding the
effect of PEF treatment on LOX inactivation differ widely, which may be due to the
differences between the properties of the treated materials, processing conditions, or
technical characteristics of the applied PEF equipment. Aguiló-Aguayo et al. (2009)
investigated the effect of PEF treatment on LOX activity in tomato juice at the following
conditions: square-shaped pulses of 35 kV/cm for 1, 000 μs, pulse width from 1 to 7 μs,
frequencies from 50 to 250 Hz, and monopolar or bipolar modes. An increase in
frequency or pulse width resulted in a decrease of residual LOX activity. The lowest
residual LOX activity (81%) was observed in tomato juice treated at 250 Hz for 7 μs in
bipolar mode. The polarity of the treatment had significant impact on LOX activity.
Similarly, Van Loey et al. (2002) reported a minimum activity of soybean LOX in distilled
water when 1, 000 monopolar pulses were used at 1 Hz. In another study, the combination
of preheating to 50 C and a PEF treatment of 100 μsat 40 kV/cm revealed high
inactivation level (84.5%) of soya milk LOX (Riener et al. 2008b).
Thank You