Effect of high-pressure processing on activity and structure of alkaline phosphatase and lactate dehydrogenase in buffer and milk

Article abstract of DOI:10.1021/jf071518q

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.

Full text of DOI:10.1021/jf071518q

9520 J. Agric. Food Chem. 2007, 55, 9520–9529
Effect of High-Pressure Processing on Activity and
Structure of Alkaline Phosphatase and Lactate
Dehydrogenase in Buffer and Milk
GILLES K. KOUASSI,*,† RAMASWAMY C. ANANTHESWARAN,‡
STEPHEN J. KNABEL,‡ AND JOHN D. FLOROS*,‡
Department of Chemistry, Western Illinois University, 324B University Circle, Macomb, Illinois
61455, and Department of Food Science, 206 Food Science Building, The Pennsylvania State
University, University Park, Pennsylvania 16802
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.
KEYWORDS: Alkaline phosphatase; L-lactate dehydrogenase; high-pressure processing; circular dichro-
ism; dynamic light scattering; structure; inactivation; denaturation; aggregation
INTRODUCTION
There is a growing interest in the use of high pressure for
inactivation of indigenous milk enzymes because these enzymes
serve as process indicators to assess the impact of HPP on
milk (3, 5). Alkaline phosphatase (ALP, E.C.3.1.3.1) is a dimer
with a molecular weight of about 140 kDA (6). The effect of
HPP treatments on ALP have been extensively studied, because
its inactivation temperature is slightly above that of most
microorganisms likely to be found in milk (3, 7, 8). Inactivation
of this enzyme might also indicate the absence of pathogens in
pressure-treated milk (9). The effects of HPP on enzymes have
been attributed to the fact that HPP affects hydrogen bonds and
ruptures the three-dimensional configuration of proteins (9, 10).
To provide a clear understanding of the effect of HPP on enzyme
inactivation, it is important to explore a possible relationship
between enzyme activity and the structure of the enzyme after
HPP.
Milk is an ideal medium for the proliferation of microorgan-
isms that cause human disease or spoilage. Hence, the quality
and safety of milk products depend on the proper inactivation
of pathogenic and spoilage microorganisms present in milk.
Pathogen inactivation in milk is achieved by pasteurization,
which traditionally has involved thermal treatments. Although
highly effective for microbial destruction, thermal treatments
of milk have often been associated with reduction in aroma and
loss of nutritional and sensory qualities of food products (1–3).
Therefore, nonthermal technologies such as high-pressure
processing (HPP) have received considerable attention recently
because of the possibility of destroying microorganisms without
significantly affecting the flavor or nutritional components of
foods (2–4).
The effects of HPP on the stability of alkaline phosphatase
have often been investigated using the combined effect of
pressure and temperature (4, 9). Studies reporting exclusively
the effect of HPP on ALP are very limited. Musa and
* Corresponding author. Phone: (814) 865-6842 (J.D.F.); (309) 298-
1727 (G.K.K.). Fax: (814) 863-6132 (J.D.F.); (309) 298-2180 (G.K.K.).
† Western Illinois University.
‡ The Pennsylvania State University.
10.1021/jf071518q CCC: $37.00
2007 American Chemical Society
Published on Web 10/19/2007

High-Pressure Processing and Enzyme Structure and Activity
J. Agric. Food Chem., Vol. 55, No. 23, 2007 9521
the bags were immediately frozen in liquid nitrogen to stop inactivation
and avoid reactivation of the enzyme and were then stored at
-20 °C.
Ramaswany (3) investigated the effects of HPP on the activity
of ALP. They found that the enzyme was inactivated at 200,
250, 493, and 400 MPa when pressure treatments lasted 1039,
751, 250, 493, and 301 min, respectively. In their study, the
time required to inactivate ALP was considered too long and
led to over-processing of the milk (4). Other milk enzymes
including γ-glutamyltransferase (GGT) and phosphohexosei-
somerase have been inactivated at 600 MPa at room temperature
(9). Likewise, lactoperoxidase was found to be pressure-resistant,
because activity was only slightly affected after pressure
treatments up to 600 MPa. The pressure resistance of this
enzyme was greater than that of ALP and was attributed to the
monomeric structure of lactoperoxidase (11).
Enzymatic Assays. ALP assays were conducted as follows: a
working substrate solution containing 150 mM p-nitrophenyl phosphate
(p-NPP) and 0.5 mM magnesium chloride from Sigma Aldrich (St.
Louis, MO) was prepared in 100 mL of 1.0 M diethanolanine buffer
(pH 9.8). The pressure-treated enzyme in buffer, fat-free milk, or 2%
fat milk was dissolved in 2 mL of 1.0 M diethanolanine buffer (pH
9.8) and 0.15 mL of enzyme solution was added to 3.35 mL of substrate
solution. The reaction mixture contained 210 units of ALP and 15 mmol
of substrate. One unit is defined as the amount of enzyme that oxidizes
one micromole of p-NPP in 1 min. The reacting solution was stored at
room temperature (25 °C), and 100 µL aliquots of the mixture were
taken at time intervals of 30 min. Sample mixtures were voxtexed and
acetonitrile (60 µL) HPLC grade from Sigma Aldrich (Pittsburg, PA,
USA) were immediately added to stop any enzymatic reaction. After
dilution (8–12 times depending on sample concentration), the samples
were placed into a model Du 530 Beckman spectrophotometer
(Beckman Coulter, Fullerton, CA) with a cuvette path of 1 cm and the
absorbance was measured at 405 nm to determine the concentration of
p-nitrophenol (p-NP) formed by oxidation of p-nitrophenyl phosphate.
The blank solution used for measurements was similar to the reaction
mixture except that it contained no enzyme. Absorbances of five
different concentrations of p-NP from Sigma Aldrich (St. Louis, MO)
were measured spectrophotometrically and used to construct a standard
curve that was used to determine the concentration of p-NP in each
sample. Each data point was the average of three measurements. The
oxidation of p-nitrophenyl to p-nitrophenol with formation of inorganic
phosphate (pi) in the presence of alkaline phosphatase proceeds
according to the following equation:
L-Lactate dehydrogenase (LDH, E.C.1.1.1. 27) is a tetramer
with a molecular weight of approximately 140 kDa (12). It is
one of the most abundant enzymes in milk (1). LDH was
designated along with ALP and γ-glutamyltransferase as a
potential marker for heat inactivation of pathogens in buffalo
milk (13). Thus, it was considered as a possible indicator of
adequate pasteurization by HPP (15). Inactivation of LDH by
HPP has been widely studied (15–17). However, these studies
were conducted at pressures not exceeding 200 MPa. The
thermal resistance exhibited by these enzymes implies that they
might also exhibit resistance to HPP. It is therefore necessary
to study them in a wide range of pressures to gain insights into
their activity and structure following HPP. The use of structural
information to investigate protein function is becoming com-
monplace (18). Relating information on an enzyme’s folding,
dynamics, and structure to enzyme kinetic data following HPP
treatments will help to define more precisely the appropriate
conditions for HPP pasteurization of milk, milk products, and
food in general.
p-nitrophenlyphosphate + H2O ALP8 p-nitrophenol + pi (1)
In the presence of L-lactate dehydrogenase, pyruvate was transformed
to L-lactate, whereas ꢀ-NADH was oxidized to ꢀ-NAD+. The enzymatic
reaction can be described as:
Therefore, the objectives of this research were to study the
residual enzyme activity of ALP and LDH following a wide
range of HPP treatments and investigate subsequent changes
in secondary structure using circular dichroism spectroscopy
(CD) and quaternary structure using dynamic light scattering
(DLS). Possible relationships between enzyme activity and
changes in enzyme structure are discussed.
pyruvate + ꢀ-NADH LDH8 L-lactate + ꢀ-NAD+
(2)
The change in the concentration of ꢀ-NADH was measured
spectrophotometrically at 340 nm. A working substrate solution
containing 0.11 mM ꢀ-nicotinamide reduced form (ꢀ-NADH), 620 mM
sodium pyruvate solution and 10 g/L BSA obtained from Sigma (St.
Louis, MO, USA) were prepared in 100 mM PBS pH 7.4. Fifteen
milliliters of pressure-treated LDH in buffer, fat-free milk, or 2% fat
milk was dissolved in 100 mL of 100 mM buffer, 200 µL of which
was added to 0.3 mL of substrate solution. In this assay, ꢀ-NADH
was the measurable substrate. One unit of LDH was defined as the
amount of LDH required to oxidize 1 µmol of ꢀ-NADH in one minute.
The reacting solution was stored at room temperature (25 °C) and
vortexed at time intervals of 10 min; 100 µL of the mixture was sampled
and assayed. Acetonitrile (60 µl) was added immediately after sampling
to stop the enzymatic reaction. After dilution, the sample was placed
into a model Du 530 Beckman spectrophotometer, and the absorbance
was measured at 340 nm. To construct a standard curve, we prepared
five different concentrations of ꢀ-NADH and measured their absor-
bances spectrophotometrically at 340 nm. Each data point of the
standard curve was the average of three measurements. The standard
curve was used to calculate the concentration of remaining ꢀ-NADH
in the samples. The activity of ALP and LDH were determined by
measuring reaction rate constants of p-NP formation and substrate
(ꢀ-NADH) disappearance, respectively, using a first-order kinetic model
as described by Ludikhuyze (6) and Pandey and Ramaswany (19).
Circular Dichroism Analysis. Circular dichroisnm (CD) spectra
of pressure-treated ALP and LDH were recorded with a Jasco 810
spectrophotopolarimeter (Wayne Kottkamp, Easton, MD) equipped with
a Peltier thermostatic system under constant nitrogen flux at 25 °C using
a 0.2 cm quartz cuvette. Scannings were performed at wavelengths
MATERIALS AND METHODS
Preparation of Enzyme Solutions in Buffers and Milk. Alkaline
phosphatase type VI-1 from bovine intestinal mucosa and L-lactate
dehydrogenase from rabbit muscle were obtained from Sigma Aldrich,
St. Louis, MO, and used to prepare 1.28 µg/mL stock solutions in 1.0
M diethanolamine buffer at pH 9.8 and 2.6 mg/mL solutions in 100
mM sodium phosphate buffer at pH 7.4, respectively. Buffers were
purchased from Sigma Aldrich. St, Louis, MO. Enzymes were prepared
in buffer and milk with 0 and 2% fat. Pasteurized bovine milk
containing 2% fat and pasteurized fat-free milk were obtained from
The Pennsylvania State University Berkey Creamery. Milks with 0 and
2% fat were tested to determine if the level of fat affected the rate of
enzyme inactivation by HPP. About 438 and 1300 µg of ALP and LDH,
respectively, were transferred from the stock solutions into plastic
stomacher bags (VWR, West Chester, PA) containing 0.5 mL of buffer,
0% fat milk, or 2% fat milk. The bags were heat sealed using a MP-12
Impulse heat sealer (Midwest Pacific, St. Louis, MO) and placed into
4× plastic stomacher bags (VWR, West Chester, PA) containing
water.
High-Pressure Treatments. Enzymes in buffer, fat-free milk, and
2% fat milk were divided into two sets. One set was pressure-treated
for 6 min and the other for 12 min, at 206, 345, 415, 482, 515, and
620 MPa, at room temperature (∼20–24 °C) using a 2 L pilot-scale
high-pressure processing (HPP) unit (Avure Technologies, Kent, WA)
and water as the pressure-transmitting fluid. After pressure was released,

9522 J. Agric. Food Chem., Vol. 55, No. 23, 2007
Kouassi et al.
between 190 and 250 nm, at a rate of 20 nm per/min. The enzyme
concentration was 0.1 mg/mL and spectra were measured in 100 mM
PBS, pH 7.4, instead of diethanolamine buffer to avoid interference
with the strong near-UV signal from the amine bond in diethanolamine.
The composition of the secondary structure of pressure-treated enzymes
was determined using the K2d algorithm (20, 21).
Statistical Analysis. Origin software (Origin 7.5 software, Origin
Laboratory) was used to assess the means and standard deviations.
Minitab Software (Minitab, State College, PA) was used for analysis
of variance and differences between treatment means. Tukey’s test was
used to analyze differences between treatments pairs, with a 95%
confidence limit.
Circular dichroism measures the differential adsorption of left- and
right-handed circular polarized light. It is a form of spectroscopy used
to determine the optical isomerism and secondary structure of molecules.
At a given wavelength
RESULTS AND DISCUSSION
Enzyme Activity. Figure 1 shows first-order plots of ALP
activity determined after 6 and 12 min HPP in buffer, fat-free
milk, and 2% fat milk. The rate constant k of formation of p-NP
was 6.3 × 10-3 min-1 for non-pressure-treated (untreated) ALP
in buffer. When the enzyme was subjected to various pressures
from 206 to 620 MPa, reaction rate constants were practically
the same (P > 0.1). In fat-free and 2% fat milk, k values for
ꢀ-NADH oxidation by untreated ALP were 6.4 × 10-3 and
6.3 × 10-3 min-1, respectively. Similar to ALP in buffer, k
did not change significantly when submitted to higher HPP (P
> 0.1). Furthermore, at each pressure treatment, k values for
ALP in buffer, fat-free milk, and 2% fat milk were not
significantly different (p > 0.05), indicating that changing the
suspending medium did not affect the activity of ALP. When
∆A ) AL - AR
(3)
where ∆A is the difference between absorbance of left circularly
polarized (LCP) and right circularly polarized (RCP) light. This equation
is also expressed by applying Beer’s law, as
∆A ) (εL - εR)Cl
(4)
where εL and εR are the molar extinction coefficients for RCP and LCP
light, C is the molar concentration, and l is the path length in centimeters
(cm). The molar circular dichroism that corresponds to the circular
dichroism of the substance is expressed as
∆ε ) εL – εR
(5)
ALP was treated for 12 min, k was 6.3 × 10-3, 6.3 × 10-3
,
∆A and the molar ellipticity, [θ], are readily interconverted using
the equation
and 6.8 × 10-3 min-1 in buffer, fat-free milk, and 2% fat milk,
respectively. Increasing pressure treatments did not make any
significant difference in the rate constants (P > 0.09) except at
620 MPa, where k values for ALP in buffer, fat-free milk, and
2% fat milk were decreased to 3.2 × 10-3, 2.6 × 10-3, and
4.2 × 10-3 min-1, respectively (p < 0.005). This suggested
that ALP was resistant to pressures below 515 MPa and a
combination of time (12 min) and pressures as high as 620 MPa
was necessary to induce change in activity. However, ALP did
not entirely lose its activity at 515 and 620 MPa (Figure 1).
Our results agree with those of Mussa and Ramaswamy (3),
who reported no inactivation of ALP at 200–482 MPa, and with
Rademacher et al. (10), who reported that 99% inactivation of
ALP was achieved when the enzyme was subjected to HPP at
600 MPa.
[ ]
θ ) 32982∆ε
(6)
Dynamic Light Scattering. DLS measurements were performed
using a Viscotek 802 DLS (Houston, TX) equipped with a class 3B
60mW laser diode with a wavelength of 830 nm and model 802 DLS
Omnisize 2.0 software. One milliliter of 0.2 mg/mL pressure-treated
ALP and LDH in Tris-HCl, pH 8.5, and PBS, pH 7.4, buffers,
respectively, was centrifuged for 5 min at 15 000 g using a table-top
microcentrifuge (Beckman, Coulter, Fullerton, CA), and filtered prior
to DLS measurements. Tris-HCl buffer was chosen for DLS measure-
ments instead of diethanolanine buffer to avoid interference between
the amine groups in diethanolanine and the enzyme that could affect
measurement of protein content. A volume of 100 µL of supernatant
was transferred to a clean Eppendorf polypropylene tube (VWR,
International, Chester, PA) and filtered through a Whatman syringe
filter of pore size 1 µm (VWR International, Chester, PA). Fifteen
microliters of the filtrate was transferred into a 1 cm path quartz cuvette
and placed into the DLS cell for measurement. Ten measurements were
made for each sample and the data were analyzed using the model 802
DLS Omnisize 2.0 software. DLS measures the autocorrelation function
g1(τ), which is related to the size distribution of particles in solution
(18).
The activity of LDH in buffer, fat-free milk, and 2% fat milk
expressed as the changes in the reaction rate constant was
measured using a first-order kinetic model after 6 and 12 min
of HPP. Non-pressure-treated (untreated) LDH was used as the
control. The slope of the plots of the logarithm of the
concentration of ꢀ-NADH versus time was taken as reaction
rate constant k (Figure 2). Rate constants of LDH treated in
buffer (PBS), fat-free milk, and 2% milk are shown in Table
1. The k values of untreated LDH in PBS, fat-free milk, and
∞
g
(1)(τ) ) m2P(q, Γ)G(Γ)e-ΓτdΓ,
(7)
∫
2% milk were 1.9 × 10-2, 1.8 × 10-2, and 2 × 10-2 min-1
,
0
respectively. When LDH was subjected to HPP of 206 MPa, k
dropped to 1.0 × 10-2 min-1 in all three processing media and
continued decreasing as HPP increased. Upon HPP of 415 MPa,
k values were 9 × 10-3, 8.6 × 10-3, and 9 × 10-3 min-1, in
PBS, fat-free milk, and 2% fat milk, respectively (p < 0.005).
Increasing HPP up to 620 MPa did not induce further reduction
of k, which remained between 4.7 × 10-3 and 5.2 × 10-3
min-1. After 12 min of HPP treatement, k values of LDH were
1.8 × 10-2, 1.9 × 10-2, and 1.9 × 10-2 min-1, in PBS, fat-
free milk, and 2% fat milk, respectively, and decreased as
pressure increased in a manner similar to the decrease observed
for 6 min (Figure 2). In the 415–620 MPa range, k did not
change significantly in all media (p > 0.05,) suggesting that
complete enzyme inactivation had occurred in this pressure
range. In contrast, a significant decrease in the rate constant of
oxidation of ꢀ-NADH by LDH was observed at 206 MPa and
at higher-pressure treatments. These results revealed that in the
where G(Γ) is the normalized number distribution function for the decay
constants Γ, mΓ the particle scattering factor, Γ ) q2DT, the decay constant,
q ) (4πn/(λ sin(θ/2)), θ the angle between the incident and scattering
beam, n the refractive index of the medium, and DT the translational
diffusion coefficient related to the hydrodynamic radius Rh by
kBT
DT )
(8)
6πηRh
where kB is the Boltzmann’s constant, T the temperature, and η the
viscosity of the sample.
The concentration of enzyme in the filtrate was measured using the
Biorad Protein Assay Reagent concentrate, using bovine serum albumin
(BSA) as a protein standard. Aliquots of the filtrates (20 µL) were taken
and their absorbances were measured at 505 nm using a model Du
530 Beckman Spectrophotometer. The concentration of protein in the
filtrate was calculated using a standard curve made of known
concentrations of BSA.

High-Pressure Processing and Enzyme Structure and Activity
J. Agric. Food Chem., Vol. 55, No. 23, 2007 9523
Figure 1. Activity of ALP in buffer, fat-free milk, and 2% fat milk, measured after 6 and 12 min at 206, 345, 415, 482, 515, and 620 MPa using a
first-order kinetic model.
pressure range of 206–415 MPa, the activity of LDH was
gradually affected and total inactivation occurred at 482 MPa
and above. When LDH was treated for 12 min, the activity
change at 206 and 345 MPa was similar to treatment at 206–482
MPa for 6 min treatment. The rate constants of inactivation of
LDH in buffer, fat-free milk, and 2% milk were not significantly
different, regardless of the suspending media used (P > 0.05).
Our results did not agreed with those of Seyderhelm et al. (22),
who claimed that bovine milk has a protective effect on enzyme
inactivation by HPP. Furthermore, the difference in fat composi-
tion (2%) in the milk did not have any significant effect on
ALP or LDH activity (p > 0.05).
Circular Dichroism. Changes in the secondary structure of
ALP and LDH after HPP were studied using the far-ultraviolet
CD spectrum in the 190–250 nm range. In general, protein
activation is observed as a shift downward of the CD
spectrum (23–26), whereas inactivation shifts the CD spectrum
upward (28–30). A denatured protein is thus observed as an
upward shift in ellipticity. Figure 3a–d show CD spectra of
ALP and LDH treated at various pressures.
respectively, and a positive ellipticity in the 190–210 nm region.
After 6 min of HPP, similar CD spectra were obtained at 206,
345, 415, 482, 515, and 620 MPa, suggesting that no major
change in ellipticity occurred as a result of HPP. When ALP
was treated for 12 min, CD spectra were similar to those
obtained after 6 min of HPP except at 620 MPa, where an
increase in ellipticity of 4.9 deg cm2 was observed (Figure 3b).
This change in ellipticity indicates a modification in the
composition of the secondary structure of ALP (26). However,
the overall CD spectra of ALP did not suggest a complete
denaturation of the enzyme. Figure 3c shows the CD spectra
of untreated LDH (1) as well as LDH treated during 6 min at
206, 345, 415, 482, 515, and 620 MPa (2–6). The spectrum of
untreated LDH has characteristics of R-helix/ꢀ-sheet/unordered
structure, with a negative ellipticity in the 205–240 nm region
and showed two peaks at 208 and 224 nm and one major peak
at 195 nm. The ellipticity of the spectrum increased with HPP,
indicating a loss of the native structure of the enzyme. When
LDH was subjected to 515 and 620 MPa, the shift upward of
the ellipticity was almost total, synonymous to loss of the
secondary structure of the enzyme. Increasing the treatment time
to 12 min (Figure 3d) produced similar effects. This indicated
that the secondary structure of LDH was gradually lost as LDH
was subjected to higher-pressure treatments.
The CD spectrum of untreated enzyme ALP (Figure 3a 1)
is typical of a blend of R-helix, ꢀ-sheets, and unordered structure
as described by Brahms et al. (30), having a negative ellipticity
in the 205–240 nm region with two peaks at 210 and 222 nm,

9524 J. Agric. Food Chem., Vol. 55, No. 23, 2007
Kouassi et al.
Figure 2. Activity of LDH in buffer, fat-free milk, and 2% fat milk, measured after 6 and 12 min at 206, 345, 415, 482, 515, and 620 MPa using a
first-order kinetic model.
Table 1. Reaction Rate Constants of ALP and LDH after 6 and 12 min HPP in Buffer, Fa-Free Milk, and 2% Fat Milk (rate constants expressed in min-1
were calculated using the enzyme assay data for ALP and LDH)
alkaline phosphatase
L-lactate dehydrogenase
6 min
12 min
6 min
12 min
pressure
(MPa)
fat-free
milk
2% fat
fat-free
milk
2% fat
milk
fat-free
milk
2% fat
fat-free
milk
2% fat
buffer
milk
buffer
buffer
milk
buffer
0
6.3 × 10-3 6.4 × 10-3 6.3 × 10-3 6.3 × 10-3 6.3 × 10-3 6.8 × 10-3 1.9 × 10-2 1.8 × 10-2 2.0 × 10-2 1.8 × 10-2 1.9 × 10-2 1.9 × 10-2
6.3 × 10-3 6.0 × 10-3 6.2 × 10-3 6.4 × 10-3 6.2 × 10-3 6.6 × 10-3 1.0 × 10-2 1.0 × 10-2 1.0 × 10-2 1.1 × 10-2 1.0 × 10-2 9.5 × 10-3
6.8 × 10-3 6.1 × 10-3 5.6 × 10-3 6.2 × 10-3 6.2 × 10-3 6.4 × 10-3 9.0 × 10-3 8.0 × 10-3 9.0 × 10-3 8.2 × 10-3 8.5 × 10-3 9.0 × 10-3
5.7 × 10-3 6.0 × 10-3 5.4 × 10-3 5.9 × 10-3 6.5 × 10-3 5.7 × 10-3 9.0 × 10-3 7.9 × 10-3 9.0 × 10-2 5.8 × 10-3 6.5 × 10-3 5.6 × 10-2
5.3 × 10-3 5.7 × 10-3 5.6 × 10-3 6.7 × 10-3 6.0 × 10-3 6.0 × 10-3 5.2 × 10-3 5.0 × 10-3 6.7 × 10-2 4.0 × 10-3 4.3 × 10-3 4.0 × 10-2
5.3 × 10-3 5.0 × 10-3 5.3 × 10-3 5.8 × 10-3 5.5 × 10-3 5.5 × 10-3 5.0 × 10-3 5.0 × 10-3 4.7 × 10-3 4.0 × 10-3 4.0 × 10-3 4.0 × 10-3
5.5 × 10-3 4.8 × 10-3 5.3 × 10-3 3.2 × 10-3 2.6 × 10-3 4.2 × 10-3 5.0 × 10-3 5.0 × 10-3 4.1 × 10-3 5.1 × 10-3 4.4 × 10-3 5.0 × 10-3
206
345
415
482
515
620
In summary, CD results revealed that the secondary
effect of HPP on myofibrilar protein structure. The composi-
tion of the secondary structure of ALP and LDH obtained
using the K2.d program provided an estimation of the
percentage of R-helix, ꢀ-sheet, and others, including turn and
coil, and are shown in Table 2. The structure of untreated
ALP was composed of 24.7% R-helix, 14.2% ꢀ-sheet, and
61% others (turn and coil). Yan et al. (25) reported a similar
secondary structure composition for this enzyme. This
composition changed very little as pressure treatment in-
creased. In the 0–620 MPa range, R-helix composition
decreased only from 24.7 to 22.3%, whereas the ꢀ-sheet
structure of ALP was only affected after 6 min at 620 MPa,
but not at pressures below this threshold. When the same
treatments were applied for 12 min, HPP effects were also
only significant at 620 MPa. In contrast, the secondary
structure of LDH was affected by 206 MPa and gradually
lost as pressure was increased. The loss of the secondary
structure observed in this study following pressure treatments
is in agreement with the finding of Kinsho et al. (28) in their
investigation of the effects of HPP treatment on carboxypep-
tidase and with Chapleau et al. (30) in their study on the

High-Pressure Processing and Enzyme Structure and Activity
J. Agric. Food Chem., Vol. 55, No. 23, 2007 9525
Figure 3. CD spectroscopy of ALP and LDH. (a, b) CD spectra of ALP after 6 and 12 min HPP, respectively. (c, d) CD spectra of LDH after 6 and 12
min HPP, respectively. Numbers 1–7 denote spectra of untreated enzyme and treated enzyme at 206, 345, 415, 482, 515, and 620 MPa, respectively.
Table 2. Secondary Structure of ALP and LDH Measured from Circular Dichroism Spectra; Data are Expressed as Percentages of Total Composition
Structurea
allcaline phosphatase
L-lactic dehydrogenase
6 min
12 min
6 min
12 min
pressure
(MPa)
R -helix
ꢀ-sheet
other
R-helix
ꢀ-sheet
other
R-helix
ꢀ-sheet
other
R-helix
ꢀ-sheet
other
0
24.7 a
23.5 a
23.2 a
23.1 a
23.1 a
22.8 a
22.3 a
14.2 a
14.7 a
14.6 a
14.7 a
14.5 a
14.8 a
15.1 a
61.1 a
61.7 a
62.2 a
62.3 a
62.3 a
62.9 a
62.6 a
24.6 a
23.3 a
23.1 a
23.1 a
22.9 a
18.6 b
11.6 c
14.2 a
14.5 a
14.6 a
14.7 a
14.9 a
16.2 a
21.4 b
61.2 a
62.2 a
62.7 a
62.3 a
62.2 a
65.1 a
67.0 a
39.2 a
28.5 b
26.3 b
25.0 b
24.5 b
09.6 c
04.8 d
22.4 a
25.2 a
27.1 a
28.4 a
28.6 a
13.3 b
07.7 c
38.4 a
46.3 b
46.1 b
46.3 b
46.9 b
77.1 b
94.4 b
37.6 a
25.3 b
21.2 b
19.4 b
19.0 b
03.0 c
01.6 a
24.4 a
29.5 b
32.0 b
35.2 b
35.4 b
0.76 c
0.34 a
37.5 a
45.1 a
46.8 a
46.2 a
45.0 a
96.2 c
98.1 d
206
345
415
482
515
620
a Identical letters next to values within a column indicate that they are not significantly different.
fraction and others increased only from 14.2 to 15.1% and
62.6 to 71.1%, respectively.
ꢀ-sheet, and others, respectively. Such a composition was
consistent with the secondary structure of LDH reported by
Brahms et al. (30). As HPP treatment increased, the R-helix
content decreased from 22.4 to 4.8%, whereas the ꢀ-sheet
fraction first increased from 38.4 to 46.3 % at 345 MPa, and
the contents of others increased from 46.3 to 77. 1%. After
12 min of HPP in the 0 to 206 MPa range, a more severe
reduction occurred from 37.7 to 1.6% in the R-helix
composition; meanwhile, the ꢀ-helix fraction increased from
24.4% to a maximum of 35.4% at 482 MPa, followed by a
These results indicate that the secondary structure of ALP
was preserved after 6 min of HPP, which suggests that at
these processing conditions no change in the secondary
structure of ALP was observed except in the 515–620 MPa
pressure range where the R-helix concentration dropped from
18.6 to 11.6%, leading to a subsequent increase in ꢀ-sheet
and unordered fraction. The secondary structure of the
untreated LDH contained 39.2, 22.4, and 38.4% of R-helix,

9526 J. Agric. Food Chem., Vol. 55, No. 23, 2007
Kouassi et al.
Table 3. Pearson Correlation Coefficients (r) of Reaction Rate Constants
with Components of the Secondary Structure of ALP and LDH at State
Pressures for 6 mina
other structures from 37.5 to 98.1%. LDH lost most of its
R-helix and ꢀ-sheet components to the detriment of other
unordered structures. The increased in ellipticity observed
in this study as a consequence of HPP is in accordance with
Sun et al. (4) and Brahms et al. (30) and may be ascribed to
the occurrence of changes in water molecules surrounding
proteins in the proteins hydration shell that gives the enzymes
a molten-globule-like structure. The moderate change ob-
served in the secondary structure of ALP in contrast to the
severe changes in the LDH structure were reflected in the
CD spectra, as well as enzyme activity, and suggested a
possible link between pressure inactivation of the LDH due
to loss of the secondary structure in this enzyme. The Pearson
correlation coefficient (r) between the reaction rate constant
ALP
LDH
r
p
r
p
R-helix
ꢀ-sheet
others
0.772
-0.674
-0.804
0.042
0.097
0.022
0.828
0.304
-0.628
0.021
0.507
0.131
a p is the probability of getting a value for the test statistic that is as extreme
as or more than the observed value.
drastic reduction to 0.76 and 0.34% at 515 and 620 MPa,
respectively. This led to an increase in the composition of
Figure 4. Mass distribution histogram of ALP obtained from DLS measurements after 6 min held at 206, 345, 415, 482, 515, and 620 MPa.

High-Pressure Processing and Enzyme Structure and Activity
J. Agric. Food Chem., Vol. 55, No. 23, 2007 9527
Table 4. Characteristics of the Quaternary Structure Observed from DLS
Analysis of Untreated and Treated ALP and LDH at 206, 345, 415, 482,
515, and 620 MPa for 6 min
MW 17429.9 was characteristic of an aggregate of ALP.
However, this aggregate represented only 0.55% of the
protein in solution, indicating that 99.5% of ALP in filtrates
remained in its initial state. At 345–620 MPa, the distribution
of the enzyme was monomodal and no aggregation was
detected. The absence of aggregation at higher pressures
suggested that aggregation observed in ALP at 206 MPa was
reversible and at pressures above 206 MPa, aggregates
dissociated. In general, when the percentage of polydispersity
(%Pd) of a solution is below 15%, the solution is likely to
crystallize. A solution having polydispersity below 30% has
a moderate polydispersity. When the Pd% is higher than 30%,
the solution is highly polydispersed and therefore the
quaternary structure of biomolecules in this solution can be
analyzed (31). In this study, the %Pd of ALP was below
30% except at 515 MPa, where it was 33.1%. This suggests
the pressure-treated samples are relatively stable and likely
to crystallize except at 515 MPa, where the likehood of
crystallization is reduced possibly because of low enzyme
concentration or hazing in the enzyme solution (31, 32).
Figure 5 shows the mass distribution histogram of DLS
measurements of LDH samples after HPP treatments and the
characteristics of the samples are shown in Table 4. Before
HPP treatments, LDH is stable in the buffer as indicated by
the presence of a single component of Rh 4.4 and MW 149.2
kDa attributable to native LDH. From 206 to 515 MPa, the
enzyme exhibited a bimodal distribution with two compo-
nents, one of which was attributable to LDH in its native
state on the basis of the molecular weight, with the other
components being an aggregate. The sizes of the aggregates
varied with pressure and the Rh ranged from 31.2 to 39.7
nm, with MW between 1220.77 and 21173 kDa. Such an
aggregate would have involved as many as 150 molecules
that assembled to form a massive cluster of molecules. To
estimate the magnitude of LDH aggregation, it is necessary
to take into account the formation of larger aggregates, which
are eventually removed through filtration. Table 5 shows the
concentrations of ALP and LDH remaining after centrifuga-
tion and filtration. The concentration of ALP changed only
from 0.20 to 0.17 mg/mL in the 206–482 MPa range, which
corresponded to about 15% protein loss. Increasing pressure
treatments up to 620 MPa induced a further reduction in the
concentration of the enzyme to 45% of the initial concentra-
tion. However, the concentration of LDH was gradually
reduced from 0.3 and 0.1% as pressure increased from 515
to 620 MPa, respectively. This reduction of the LDH
concentration is attributable to denaturation as well as
aggregation, as suggested by the DLS results. The relative
stability in the concentration of ALP is consistent with the
stability in the activity and the secondary structure of this
enzyme on the basis of the results of CD spectroscopy
analysis. On the other hand, the disappearance of LDH upon
HPP treatments as result of denaturation and aggregation is
in accordance with the drastic loss of activity and change in
the secondary structure of this enzyme.
pressure
(MPa)
peak area
(%)
distribution
Rh (nm)
ALP
MW (kDA)
RSD %
untreated
206
monomodal
bimodal
4.4
4.5
37.6
4.3
4.3
4.2
4.3
4.3
143.1
142.1
17429.9
145.1
141.5
141.5
144.4
143.2
21.9
22.5
25.5
25.1
16
26.3
33.1
26.7
100
99.5
0.5
100
100
100
100
100
345
415
482
515
620
monomodal
monomodal
monomodal
monomodal
monomodal
LDH
untreated
206
monomodal
bimodal
4.4
4.5
39.8
4.5
39.9
4.2
37.4
4.1
31.2
4.3
33.4
4.4
145.1
149.2
21173.9
148.4
1220.8
145.5
11224.4
91.5
11224.4
143.1
13134.9
146.8
19.4
27.9
31.3
16.7
30
100
12.3
87.7
99
1
99.3
0.7
98.9
1.1
98.8
1.2
345
415
482
515
620
bimodal
bimodal
bimodal
bimodal
monomodal
16.6
20
17.3
20.3
21.9
29.3
25.6
100
k and each fraction of the secondary structure of ALP and
LDH are presented in Table 3. The Pearson correlation
coefficient r between k and the R-helix, ꢀ-sheet, and other
fractions of ALP after treatment for 6 min at 206–620 MPa
were 0.772, -0.674, and -0.804, respectively. These values
indicate a poor correlation between secondary structure and
enzyme activity. The poor correlation could be explained by
the fact that HPP did not induce changes when samples were
treated for 6 min. However, For LDH, r values between k
and R-helix, ꢀ-sheet, and other fractions of LDH after 6 min
at 206–620 MPa were 0.888, 0.304, and -0, 628, respec-
tively. This suggests a strong correlation between LDH
activity and R-helix component; however, good correlation
was also found between LDH activity and its ꢀ-sheet, and
other fractions. The relationship between the R-helix com-
ponent and the activity supports the idea that the secondary
structure plays a crucial role in enzyme activity.
Dynamic Light Scattering. ALP samples did not precipitate
and appeared stable after all HPP treatments. In contrast,
extensive precipitation was visually observed in LDH samples
at pressures of 482 MPa and higher. The amount of precipitation
increased with increasing pressure; however, the precipitates
were not visible after centrifugation and filtration. The mass
distribution of ALP obtained from DLS is shown in Figure 4.
The hydrodynamic radius (Rh) is the radius of enzyme in
solution, and the relative standard (RSD) or polydispersity is
indicative of the distribution in the peak or subpeak; the peak
area refers to the proportion of each particle in the sample. The
mass distribution of untreated samples is characterized by the
presence of a single component of hydrodynamic radius (Rh)
4.4 with a molecular weight (MW) of 143 kDA and 100% peak
area (Table 4). These characteristics suggest that this component
corresponded to ALP.
ALP owes its stability to its relative resistance to aggregation
and the stability of its secondary structure. The results obtained
on LDH agree with those of Chapleau et al. (29), who
demonstrated that pressures above 300 MPa induced denatur-
ation of myofibrillar proteins by destabilizing the secondary
structure and causing irreversible aggregation.
At 206 MPa, the enzyme solution showed a bimodal
distribution as illustrated by the occurrence of two peaks in
the mass distribution histogram (Figure 4). The Rh and the
molecular weight (MW) of this component were 4.46 nm
and 142.14 kDa, respectively, which were consistent with
native ALP, whereas the second peak with Rh 36.7 nm and
Conclusions. In this study, we have shown that ALP exhibited
resistance to inactivation and its secondary and quaternary structure
were preserved after 6 min at 206–620 MPa. The study also

9528 J. Agric. Food Chem., Vol. 55, No. 23, 2007
Kouassi et al.
Figure 5. Mass distribution histogram of LDH obtained from DLS measurements after 6 min hold at 206, 345, 415, 482, 515, and 620 MPa. The arrows
represent formation of aggregates.
revealed that increasing HPP from 6 to 12 min at 620 MPa reduced
ALP activity without causing complete inactivation of the enzyme.
Alkaline phosphatase likely owes its pressure resistance to the
stability of its secondary structure, demonstrated by the preservation
of its R-helix content and the limited amount of aggregates formed
upon various pressure treatments. In contrast, LDH underwent
extensive loss of activity, which was in accordance with the
alteration of its secondary structure and the concomitant aggregation
observed via DLS. This study has demonstrated that HPP can
inactivate an enzyme by altering the stability of its secondary
structure and inducing aggregation of enzyme molecules. In turns,
aggregation impacts enzyme activity by reducing the concentration
of active enzyme in the reaction medium. Future studies might
investigate LDH crystal formation following HPP. The information
obtained from this study is essential to understanding the mecha-
nism(s) of HPP inactivation of enzyme activity. Such information
is necessary to understand the inactivation of both microorganisms
and enzymes that affect the safety and quality of foods processed
using high pressure.
ACKNOWLEDGMENT
We thank Dr. Neela Yennawar for advice and technical
assistance with DLS.

High-Pressure Processing and Enzyme Structure and Activity
J. Agric. Food Chem., Vol. 55, No. 23, 2007 9529
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Received for review May 23, 2007. Revised manuscript received August
29, 2007. Accepted August 30, 2007. This work was funded by a USDA
Milk Safety Grant to The Pennsylvania State University.
JF071518Q