Process Biochemistry 35 (2000) 1037 – 1043

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The stability of enzymes after sonication

Belma O8 zbek a,*, Kutlu O8 . U8 lgen b
a
Department of Chemical Engineering, Yıldız Technical Uni6ersity, Şişli Campus, Şişli 80270, Istanbul, Turkey
b
Boğaziçi Uni6ersity, Department of Chemical Engineering, Bebek 80815, Istanbul, Turkey

Received 15 October 1999; received in revised form 24 December 1999; accepted 29 January 2000

Abstract

The effects of operating conditions of sonication on the stability of some commercially purified enzyme preparations were
investigated. Buffered solutions of six enzymes, alcohol dehydrogenase (ADH), malate dehydrogenase (MDH), glucose-6-phos-
phate dehydrogenase (G6PDH), L-lactic dehydrogenase (LDH), alkaline phosphatase (AP) and b-galactosidase (bG)were sonified
over a range of power outputs up to 40 W. The enzymes had variable stabilities with complete stability for AP, and over 70%
inactivation for G6PDH. Some inactivation models were tested for an understanding of the relation between sonification intensity
and enzyme stability. Sonication processing times also affected the inactivation rate of ADH and MDH. The stability of sonified
ADH was decreased with time when compared with unsonified controls. Increasing the viscosity of process fluid with glycerol gave
39% inactivation of ADH, while the control showed 15% inactivation for the operational conditions. The forces involved in the
fluid must therefore have a significant role to play in the inactivation process. © 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Enzyme stability; Inactivation; Acoustic cavitation; Wave duty cycle

1. Introduction caused by mechanical effects, i.e. shear stress developed

The majority of enzymes produced by microorgan-
isms are intracellular and some of these have been
successfully produced on an industrial scale. The ex-
ploitation of intracellular microbial products for indus-
trial use, mainly in food and medicine, is continuing to
increase with the new developments in genetic engineer-
ing and recombinant DNA technology. In order to
isolate intracellular microbial products, it is necessary
to use methods that are capable of breaking down the
cell wall, but at the same time not to cause inactivation
of the biological substances. Sonication, homogenisa-
tion, bead milling and nebulisation are some of the
processing methods available for cell disruption and
protein release from cells [1 – 3]. Among these effective
methods, sonication offers many advantages [3,4], it has
low operating costs and it does not require sophisti-
cated equipment or extensive technical training. It has
the easy cleaning in place feature. However, inactiva-
tion of released products by ultrasonication can be
* Corresponding author. Fax: +90-212-2244968.
E-mail address: bozbek@yildiz.edu.tr (B. O8 zbek)
by eddies arising from shock waves.
Many studies on cell disruption have been carried
out using the aforementioned methods and a range of
microorganisms [4–9]. Different models are proposed
for cell disintegration as well as for the fast release of
intracellular protein, either native or recombinant. The
enzyme release process is mostly reported to follow
first-order kinetics [5,7,10]. However, the rate of release
of an enzyme depends on its location within the cell
[11]. For example, alkaline phosphatase is located in
cell membrane and therefore released slowly, while
L-lactic dehydrogenase and alcohol dehydrogenase are
mostly located in the cytoplasm and their release during
cell disruption is instantaneous [5,6]. Therefore, it is
likely that they are highly affected by the disruption
method.
The severity of the disruption conditions can have an
impact on the yield of active protein/enzyme recovered
in the process. In the present study, the effect of
processes on maximal recovery efficiency of active en-
zyme was investigated using one of the recently pre-
ferred mechanical disruption methods, i.e.
ultrasonication. Disruption with ultrasonic energy at

0032-9592/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 2 - 9 5 9 2 ( 0 0 ) 0 0 1 4 1 - 2
1038 B. O8 zbek, K.O8 . U8 lgen / Process Biochemistry 35 (2000) 1037–1043
sufficiently high acoustic power occurs due to the phe-
nomenon of cavitation. However, cavitation causes
chemical effects such as formation of free radicals that
can be harmful to labile molecules released from the
cells [10]. In order to study the stability (inactivation
kinetics) of the biological substances, six commercial
enzyme preparations from different microbial and
mammalian sources were chosen: alcohol dehydroge-
nase (ADH), malate dehydrogenase (MDH), glucose-6-
phosphate dehydrogenase (G6PDH), L-lactic
dehydrogenase (LDH), alkaline phosphatase (AP) and
b-galactosidase (bG). The dependence of enzyme stabil-
ity on power, wave duty cycle, temperature, viscosity
and processing time was determined. The process
parameters of ultrasonication were optimised, and the
correlation equations between enzyme activity and op-
erating variables derived.
2. Materials and methods
2.1. Sonication of the enzymes in the disrupter
The ultrasonication experiments were carried out at
20 kHz on a Branson 450 Sonifier equipped with a horn
of 9 mm diameter. The tip of the horn was immersed
about 1 cm into 10 ml solution to be processed. The
acoustic power was controlled manually between 7 and
40 W. The ultrasonic energy was pulsed using the Duty
Cycle Control in order to reduce the formation of free
radicals. In pulsed mode, ultrasonic vibrations are
transmitted to the solution at a rate of one pulse per
second. The solution was processed with the sonication
horn for 2 ×30 s (with 30 s still time between the
operations) unless otherwise stated. The temperature
inside the solutions was intermittently checked and the
temperature difference was kept below 5°C by the use
of an outer ice jacket. The treated solutions were
analysed by the described techniques to assess the de-
gree of stability.
2.2. Enzymes used
Purified enzymes were obtained from Sigma Chemi-
cal Co. (Munich, Germany): ADH from Bakers Yeast
(Product No. A7011; EC 1.1.1.1), MDH from Porcine
heart (mitochondrial) (Product No. M2634; EC
1.1.1.37), G6PDH from Bakers Yeast, (Product No.
G7750; EC 1.1.1.49), LDH from Rabbit muscle
(Product No. L2500; EC 1.1.1.27), AP from Bovine
intestinal mucosa (Product No. P6774; EC 3.1.3.1) and
bG from Escherichia coli (Product No. G2513; EC
3.2.1.23).
Enzyme solutions were prepared by suspending them
in 20 mM phosphate buffer at pH 7.5. These enzyme
solutions contained approximately 1100 – 7500 U/l de-
pending on the enzyme tested.
Typically, a 10 ml enzyme solution was sonified in
the disrupter. Once the samples had been sonified, they
were immediately stored on ice. Activities were then
determined according to the assays given elsewhere
[3,12–14]. At least three measurements were made for
each condition and the data recorded below is an
average of these data. An original sample was kept
without sonification as a control activity measurement.
3. Computational work
The software package MATLAB 5.0 was used for
numerical calculations. The parameters were evaluated
by the non-linear least-squares method of Marquardt–
Levenberg until minimal error was achieved between
experimental and calculated values. The residual (SSR)
is defined as the sum of the squares of the differences
between experimental and calculated data, given by
Nd
SSR= % (C obs calc 2
m −Cm )
m=1
where m is observation number and Nd is total number
of observations. The estimated variance of the error
(population variance) is calculated by the SSR at its
minimum divided by its degrees of freedom:
s 2 : s 2(SSR)min/(m − p)
where p is the number of parameters and s 2 is the
variance. The standard error, s, (the estimated standard
deviation) is calculated by taking the square root of the
estimated variance of the error.
4. Results and discussion
The enzymes were suspended in 20 mM phosphate
buffer at pH 7.5 and were ultrasonicated under various
process conditions. The acoustic power (W), processing
time (s), wave duty cycle (%), viscosity (cps) and tem-
perature (°C) were chosen as the key elements in influ-
encing the enzyme stability during ultrasonication.
Enzyme activities prior to ultrasonication were also
determined and used as controls. In calculations, Amax
was denoted as the enzyme activity without disruption,
i.e. prior to sonication. This value was then considered
as 100% activity. Activity at any operational condition
(A) was then recorded in terms of percentages of the
undisrupted control.
4.1. The effect of temperature during the sonication
process
The violent collapse of cavitation bubbles in an ultra-
sonic field causes extremely high local temperatures [9].
The temperature change of ultrasonicates (10 ml solu-
 B. O8 zbek, K.O8 . U8 lgen / Process Biochemistry 35 (2000) 1037–1043 1039

tions) was investigated at two different wave duty cy- Pulsing the ultrasonic energy retarded the rate of tem-
cles, i.e. 10 and 90%, and at various power outputs (i.e. perature increase.
15 – 40 W) with processing time 2 ×30 s. The results are
summarised in Table 1. At 27 W and 90% duty cycle 4.2. The effect of acoustic power on enzyme stability
(DC), the temperature increased from ambient (23.7°C)
to 39.5°C in 2× 30 s. Therefore, strict control over the In order to investigate the effect of ultrasonic inten-
temperature was needed, maintained by the use of an sity, the enzyme solutions were sonicated at different
ice jacket. At 27 W and 90% DC with an ice jacket, the acoustic powers ranging from 7 to 40 W for 2×30 s at
temperature difference was only 4.4°C. If the processing wave duty cycles of 10 and 90%, respectively. Data
time was increased further, i.e. 3×30 s, the tempera- shown in Fig. 1a,b indicate the relative activity (A/
ture difference (DT) also increased to 36°C at 27 W and Amax) versus acoustic power profiles for six commercial
90% DC. However, on ice, DT remained at 4.9°C. enzyme preparations. Apart from AP, all enzymes
At 10% wave duty cycle, the temperature differences showed some inactivation with increasing power. The
profile and the degree of degradation was dependent on
were smaller than those obtained at 90% wave duty
the enzyme (see Fig. 1a,b). The order of stability
cycle, e.g. at comparable outputs, 7.1° versus 20.5°C.
throughout the power range tested at duty cycle 90%
(Fig. 1a) was as follows:
Table 1
Temperature change during the sonication process without using an AP\ LDH\ bG \ G6PDH\ MDH\ ADH
ice jacket
For first-order dependence of activity on power, the
Acoustic power (W) Duty cycle (%) Time (s) DT (°C)
activity versus acoustic power profiles in a semilogarith-
15 10 2x30 3.6 mic plot were expected to yield straight lines. However,
32 10 2x30 7.1 simple first-order kinetics were clearly not applicable to
27 90 2x30 15.8 the inactivation of the enzymes at 90% DC. The linear
40 90 2x30 20.5 regression coefficients, R 2, are 0.7803, 0.8089, 0.9740,
0.9693 and 0.9378 for G6PDH, LDH, bG, MDH and
ADH, respectively. The deviation from first-order de-
pendence was less pronounced at 10% DC and the
linear regression coefficients, R 2, were above 0.9436.
The presence of two types of enzyme subunits has
been reported for ADH and some other enzymes [6].
Thus, a two-component, first-order inactivation model
was used to fit the data at both 10 and 90% wave duty
cycles. The following activity–power expression given
by Skerker and Clark [7] was used:
A/Amax = a1 exp(− k1DP)+a2 exp(− k2DP) (1)
where k1D, and k2D, are the degradation coefficients
dependent on the acoustic power output (W − 1 and
W − 2, respectively), and a1 and a2 are the initial frac-
tions of enzyme components 1 and 2, respectively. The
parameters in Eq. (1), a1, a2, k1D, and k2D, were esti-
mated using the Marquardt–Levenberg optimisation
routine of the MATLAB 5.0 software. However, the
program either estimated negative values or resulted in
inconsistent values.
For some enzymes, it has been reported that the
single-step inactivation leads to a final state that does
exhibit some residual activity [15].
kD
E“ E a11
In this reaction, E and E1 are enzyme states of different
Fig. 1. (a) Percent activities of enzymes at various acoustic powers
specific activities and are homogeneous, a1 is the ratio
(W) at duty cycle 90%. (b) Percent activities of enzymes at various of the specific activity of the final state to the initial
acoustic powers (W) at duty cycle 10% state, and kD is the degradation coefficient (W − 1).
1040 B. O8 zbek, K.O8 . U8 lgen / Process Biochemistry 35 (2000) 1037–1043

Table 2 the enzymes bG, ADH and MDH resulted in meaning-

Estimated parameters of the single-step model (Eq. (2))
ful values of these parameters. The degradation coeffi-
Enzyme a1 kD SSR Standard error cients of enzymes, kD, increased in the order of
(W−1) (s) decreasing stability.
A single-step non-first-order model (Eq. (2)) simu-
bG (at 90% 0.0107 0.0145 0.0044 0.0271 lated very effectively the inactivation data of bG, MDH
DC)
and ADH enzymes at both 10 and 90% DC (Fig. 1a,b).
bG (at 10% 0.0056 0.0054 0.0141 0.0484
DC) The data of LDH and G6PDH, on the other hand,
MDH (at 90% 0.0060 0.0275 0.0289 0.0694 produced quadratic fits. After evaluation of the data of
DC) LDH enzyme, Eqs. (3) and (4) were derived for 90 and
MDH (at 10% 0.3656 0.0232 0.0005 0.0091 10% duty cycles, respectively.
DC)
ADH (at 90% 0.2018 0.0317 0.0014 0.0153 ALDH (90%) = 99.61+0.2570P −0.0352P 2 (3)
DC)
ADH (at 10% 0.5666 0.0585 0.0009 0.0122 ALDH (10%) = 100.42− 0.1655P − 0.0141P 2 (4)
DC)
Again, after evaluation of the data of G6PDH enzyme,
Eqs. (5) and (6) were derived for 90 and 10% duty
Table 3 cycles, respectively.
Estimated parameters of quadratic fit
AG6PDH (90%) = 101.48− 0.2481P −0.0415P 2 (5)
2
Enzyme kD1 kD2 R Statistic Standard AG6PDH (10%) = 99.35+0.1677P − 0.0436P 2
(6)
(W−1) (W−2) error (s)
where ALDH and AG6PDH are residual enzyme activities
LDH (at 0.2570 0.0352 0.9926 0.0213 (percentage values after disruption), P is the acoustic
90% DC)
LDH (at 0.1655 0.0141 0.9972 0.0059
power output value (W), and kD1 and kD2 are the
10% DC) degradation coefficients dependent on the acoustic
G6PDH (at 0.2481 0.0415 0.9913 0.0368 power output (W − 1 and W − 2, respectively). The
90% DC) parameters, kD1 and kD2, were estimated using the
G6PDH (at 0.1677 0.0436 0.9913 0.0235 Marquardt–Levenberg optimisation routine of MAT-
10% DC)
LAB 5.0 software and given in Table 3.

4.3. The effect of processing time on ADH and MDH

enzymes

The time of exposure to ultrasound is a critical

Fig. 2. Percent activities of ADH and MDH enzymes at various
processing times (s).
The activity–power relationship [15] is then as
follows:
A/Amax =(1−a1) exp( −kDP) + a1
Eq. (2) was used to model the inactivation data of
enzymes at both wave duty cycles of 10 and 90%. The
parameters, a1 and kD, were estimated using the Mar-
quardt–Levenberg optimisation routine of the MATLAB
5.0 software and are presented in Table 2. The data of
parameter responsible for cell disruption as well as for
enzyme stability. Since the sonifier is a constant-ampli-
tude device, i.e. the oscillating frequency and the ampli-
tude of oscillation are fixed, the rate of energy input is
fixed at the chosen output and duty cycle control level.
The rate of change in activity with time was investi-
gated using two enzymes from different sources, i.e.
microbial and mammalian. Fig. 2 shows the activity
versus time profiles of ADH (from Baker’s yeast) and
MDH (from porcine heart mitochondria) under process
conditions of 15 W acoustic power and 10% wave duty
cycle, as well as at 27 W acoustic power and 90% wave
duty cycle. Steady decreases in activities of ADH and
MDH were observed with time of exposure (Fig. 2).
The following relationship was used to represent the
(2) data of ADH and MDH enzymes:
A= Amax − k0·t (7)
−1
where k0 is the zero order inactivation coefficient (s ).
The zero-order rate constants are given in Table 4.
ADH was then used to investigate some of the
sonifier parameters.
 B. O8 zbek, K.O8 . U8 lgen / Process Biochemistry 35 (2000) 1037–1043 1041

Table 4
The effect of processing time on inactivation of ADH and MDH enzymes

Enzyme Duty cycle (%) and acoustic power (W) k0 (s−1) R 2 Statistic Standard error (s)

ADH 10 and 15 0.2872 0.998 0.0081

ADH 90 and 27 0.8193 0.998 0.0173
MDH 10 and 15 0.2089 0.995 0.0121
MDH 90 and 27 0.8267 0.997 0.0217

4.4. The effect of wa6e duty cycle on the acti6ity of 4.6. The effect of the 6iscosity on the acti6ity of the
the ADH enzyme ADH enzyme

ADH enzyme in 10 ml of 20 mM phosphate buffer Another factor known to affect the inactivation of
was subjected to sonication for 2 ×30 s. The ultrasonic enzymes in high shear disruption medium is the viscos-
energy was pulsed using duty cycle control settings
from 10 to 90, which changes the duration of the pulse
from 10 to 90% of each second, respectively. At 10 and
20% wave duty cycles, the power output recorded 15
W, and from 30% DC onwards it recorded 27 W. The
data are shown in Fig. 3. No linear relationship was
obtained from these results. The data were then fitted
to a single-step unimolecular non-first-order enzyme
inactivation model. The a1 and kD values were esti-
mated as 0.5913 and 0.0365 W − 1, respectively. The
residual (SSR) was 0.0067. Compared with the results
of ADH obtained at 27 W (see Table 2), the parame-
ters, a1 and the degradation coefficient, kD, with respect
to change in wave duty cycle have the same order of Fig. 3. Percent activity of ADH enzyme at various wave duty cycles
magnitude. (%).

4.5. The stability of ADH enzyme after disruption

Enzyme inactivation is, to some extent, reversible

[16 – 18]. In a further experiment, ADH was sonicated
at different intensities from 7 to 32 W at 10% DC for
2 × 30 s and the fractions assayed for enzyme activity.
The ultrasonicates were then incubated at 20°C for 5 h
and activities determined periodically. No recovery in
enzyme activity was observed. The activities continued
to decline with incubation time in a first-order decay
(Fig. 4a). The acoustic power appeared to increase the
susceptibility of the ADH enzyme over the control.
Fig. 4b shows the effect of the acoustic power in the
range 0–32 W at 10% DC on the inactivation rate
constant, kI, of ADH enzyme. The inactivation rate
constant dependent on time, kI, increased linearly with
increasing power intensity in the 5 h following the
sonication process. As can be seen in Fig. 4b, a linear
relationship was observed from these results:

ln A =ln Amax − kI·t (8)

The half-life of ADH enzyme was determined using

these data and linear regression analysis (Table 5). The Fig. 4. (a) ln(activity of ADH) enzyme at duty cycle 10% versus time
half-life of ADH at 20°C decreased from 12.7 to 8.1 h (h). (b) Inactivation rate constant (kI, h − 1) of ADH enzyme versus
at 32 W and 10% wave duty cycle. acoustic power (W) at duty cycle 10%.
1042 B. O8 zbek, K.O8 . U8 lgen / Process Biochemistry 35 (2000) 1037–1043

Table 5
Half-life of the ADH enzyme at various acoustic power outputs

Acoustic power (W) Inactivation rate constant, kI (h−1) Half-life, t (h) R 2 Statistic Standard error (s)

0 0.0546 12.7 0.9933 0.0084

7 0.0614 11.3 0.9958 0.0075
12 0.0672 10.3 0.9949 0.0089
15 0.0689 10.1 0.9918 0.0117
18.5 0.0731 9.5 0.9948 0.0098
25 0.0792 8.8 0.9923 0.0130
30 0.0818 8.5 0.9783 0.0215
32 0.0862 8.1 0.9838 0.0207

ity of the solution. Glycerol solutions at 10, 20, 30, 40
and 50% were prepared in 20 mM phosphate buffer at
pH 7.5. These give viscosities [19] of 1.311 –6.050
cPoise. ADH was then added to the 20 mM phosphate
buffer containing 10 – 50% glycerol. The effect of glyc-
erol on the enzyme activity was investigated both prior
to and after the sonication process (at 15 W acoustic
power, 10% duty cycle and 2× 30 s processing time),
and it was shown that the enzyme activity reduced in
proportion to glycerol concentration. The reduction in
activity was calculated as a percentage reduction
against an undisrupted control at the specified glycerol
concentration. Increasing the viscosity of the process
fluid from 1 to 6 cps with glycerol gave 39% inactiva-
tion of ADH, while the control showed 15% inactiva-
tion for the operational conditions.
No linear relationship was obtained from these re-
sults (Fig. 5). The data were then fitted to a single-step
unimolecular non-first-order enzyme inactivation model
(Eq. (2)). The a1 and kDV (inactivation rate constant
dependent on viscosity) values were estimated as 0.5990
and 0.4891 (cps − 1), respectively. The residual (SSR)
and standard error (s) values were 0.0006 and 0.0122,
respectively.
4.7. Comparison of the energy efficiencies of ca6itation
processes
The energy efficiencies of cavitation processes (acous-
tic versus hydrodynamic) were compared in order to see
the energy requirements. The power consumption of
the ultrasonic horn was between 7 and 40 W (J/s). The
quantity of the suspension treated was 10 ml. The
energy requirement per millilitre of suspension is calcu-
lated for the lowest (7 W, 2 ×30 s) and highest (40 W,
8 ×30 s) cases as follows.
Compared with hydrodynamic cavitation [8] of 21
J/ml, the acoustic cavitation (ultrasonication) equip-
ment required higher amounts of energy in order to
treat the same quantity of suspension.
5. Conclusions
An evaluation of the experimental and theoretical
data showed that many process factors are involved in
the inactivation of enzymes after disruption by sonifica-
tion. The order of stability was obtained as AP
(100%)\ bG (85%)\ LDH (81%)\ MDH (66%)\
ADH (64%)\ G6PDH (52%) and AP (100%)\bG
(55%)\ LDH (54%)\ MDH (33%)\ ADH (44%)\
G6PDH (29%) at acoustic powers 32 W with 10% DC
and 40 W with 90% DC, respectively. As the acoustic
power (ultrasonic intensity) increased, the loss of en-
zyme activity also increased.
The inactivation mechanisms with each enzyme stud-
ied were different, and was specific to the enzyme.
Various kinetic parameters dependent on the sonifica-
tion intensity and enzyme stability were estimated. The
modelling studies showed that a single-step model with
non-zero activity of the final enzyme state accurately
represented the inactivation data of enzymes bG, ADH
and MDH. The data of LDH and G6PDH, on the
other hand, resulted in quadratic fits.

(7 J/s) (60 s)/(10 ml) = 42 J/ml

(required minimum energy)

(40 J/s) (240 s)/(10 ml) =960 J/ml

Fig. 5. Percent activity of ADH enzyme versus viscosity of the
(required maximum energy) solution (cps).
 B. O8 zbek, K.O8 . U8 lgen / Process Biochemistry 35 (2000) 1037–1043
Sonication processing time also affected the relative
activities of the enzymes. There was a zero-order inacti-
vation in acoustic cavitation for the enzymes ADH and
MDH.
From the studies on the activity of ADH enzyme at
various duty cycles, it was observed that 30% wave duty
cycle was a threshhold value causing maximum degra-
dation. A significant decrease in relative activity was
not observed in the range 30 – 90% wave duty cycles
(DC).
The effect of acoustic power output over the long-
term stability for ADH enzyme after disruption was
also investigated. This showed that ADH progressively
lost its activity with time and that the decay rates were
related to the operating acoustic power of the sonifier.
The effect of the fluid viscosity was investigated on
ADH enzyme activity. Further deactivation of the en-
zyme was observed in glycerol solutions with an in-
crease in the viscosity. The physical nature of the fluid
and the forces generated within the fluid, especially
those associated with shear field, are known to be of
importance in generating conditions for inactivation.
Finally, the energy requirements were calculated for
both hydrodynamic cavitation and acoustic cavitation
(ultrasonication) equipment. The maximum energy re-
quirement for acoustic cavitation was approximately 45
times higher than that for hydrodynamic cavitation in
treating the same quantity of suspension. Ultrasonica-
tion could be an uneconomical process for large-scale
use due to this higher energy requirement. Enzyme
degradation should also be compared and its potential
kept in mind.
The forces involved in the sonified fluid have a
significant role to play in the inactivation process. The
most likely are those associated with the shear field
within the fluid while sonification occurs. The stability
behaviour observed was different for each enzyme. This
could be related to location in the cell (i.e. in the cell
membrane or in the cytoplasm etc.), their molecular
weights or their sources (i.e. microbial or mammalian).
Thus, the operational parameters of the sonifier should
be optimised specifically for each enzyme, as parame-
ters such as time and intensity could cause degradation
of the released enzyme during the disruption of cells.
Acknowledgements
This work was supported by the Yildiz Technical
University Research Fund (Project Number 98-A-07-
1043
01-03) and the Bogaziçi University Research Fund
(Project Number 99HA502D).
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