Indian Journal of Biotechnology
Vol. 4, April 2005, pp 232-240
Translocation of cytoplasmic β-galactosidase across the inner membranes
of Kluyveromyces lactis
Vivek D Farkade and Aniruddha B Pandit*
Chemical Engineering Division, Institute of Chemical Technology, University of Mumbai, Matunga, Mumbai 400 019, India
Received 17 December 2003; revised 11 May 2004; accepted 26 May 2004
The translocation behaviour of cytoplasmic β-galactosidase to periplasmic space and through the outside cell wall across
the inner membranes of Kluveromyces lactis has been investigated to optimize the cell disruption process by ultrasonication
for the production and separation of intracellular target biomolecule i.e. β-galactosidase. The translocation of
β-galactosidase in the cells was judged by a concept of location factor (LF), which allows the location of the enzymes to be
judged within the cell and has been determined using the relative rates of the enzyme and protein during the cell disruption
process. The temperature was found to be useful external stimuli for the translocation of target enzyme (LF could be
increased to one or more). The LF values were maximum when cells were subjected to heat stress between 45-50°C for a
specified time. The enzyme activity was also found to decrease with an increase in the temperature. Maximum enzyme
activity was found to be at 45°C of the heat treatment process for translocation. The kinetics of translocation of the target
enzyme across the inner membrane has been reported on the basis of the variation in the location LF.
Keywords: translocation, β-galactosidase, Kluyveromyces lactis, location factor, cytoplasm, periplasm
IPC Code: Int. Cl.7 A01N63/02; C12N9/38
Introduction
The distinct proteins synthesized in the cytoplasm
are exported to the three layers of cell envelope: the
inner and the outer membrane and the intervening
peiplasmic space1.
The translocation of proteins across the biological
membrane is a key step in the intracellular sorting and
secretion of proteins. Most protein translocation
across hydrophobic membrane occurs through an
evolutionarily conserved proteinaceous complex,
which forms the pore, through which proteins pass on
their way out of the cytoplasm2,3. The energy required
for protein translocation is provided by different
mechanisms in various organisms as discussed in
detail by Watts and Depont4. The need for
translocation arises as all the cells transport, specific
subsets of their own proteins across the membranes in
order to segregate the metabolic pathways to
communicate with the other cells and to build new
membranes5.
Translocation is driven by the chain elongation,
which in turn is powered by the hydrolysis of highenergy phosphate bonds. The import of protein across
__________
*Author for correspondence:
Tel: 91-22-24145616; Fax: 91-22-24145614
E-mail: [email protected]
the inner membrane requires an electrochemical
potential across the membrane5. The efficient transfer
of proteins through the membrane during the process
of secretion requires polypeptide that has not adopted
the stable folded structure of the mature species6.
Ellis7 proposed the term ‘molecular chaperones’ to
describe the class of cellular proteins whose function
is to ensure that the folding of certain other
polypeoptide chains and their assembly into
oligomeric structures occur correctly. Molecular
chaperones would prevent the formation of improper
structures. These proteins disassemble the aggregated
structures either that are no longer required or that are
formed during stresses such as heat shock7. The
‘molten globule’ state is involved in the translocation
of proteins across the membrane as an intermediate
state8,9. The formation of transient molten globule
state occurs early on the pathway of folding of all
globular proteins under the conditions of pH,
temperature and intermediate concentrations of strong
denaturants. Synthesis of heat shock proteins is a
conserved response to the environmental stress that is
found in all organisms. The heat shock related
proteins stimulate protein translocation into
microsomes10,11. The synthesis and sedimentation of a
subset of 15-kDa heat shock proteins in Escherichia
FARKADE & PANDIT: TRANSLOCATION OF CYTOPLASMIC β-GALACTOSIDASE USING HEAT TREATMENT
coli cells recovering from the sublethal heat stress
have been studied12. Singer13 has reviewed the
molecular organization of membranes, their proteins
and fluidity, membrane asymmetry and transmembrane transport mechanism. Recently, the heat
induced translocation of cytoplasmic β-galactosidase
across the phospholipid bilayer membrane as a model
system has been investigated14. The translocation of
proteins is thought to be a triggered interaction of
binding sites among the targeted protein; lipid
membrane and chaperones and thus controlling the
hydrophobic interaction that controls protein
translocation. To date, most of the data on protein
translocation have been obtained on the natural
membrane proteins in reconstituted membrane
systems8,9,15.
Through successful applications of recombinant
DNA technology, it is now feasible to produce desired
proteins in large amounts and destined them for
secretion to the extracellular sites of using appropriate
gene expression systems. The high level extracellular
production of penicillin acylase from genetically
engineered E. coli has been investigated16.
This suggested that genetic engineering could be
used to translocate the enzyme to a desired location in
the cell. Genetic manipulation needs large amount of
capital cost as well as most sophisticated instruments
to modify the organism with a particular gene. The
genetic analysis of protein export in E. coli has also
been reviewed17. The cytoplasmic protein produced in
E. coli cells has been shown to be translocated in vivo
by exposing the cells to heat stress18,19. Balasundaram
and Pandit20 have recently studied the translocation
of alcohol dehydrogenase in the baker’s yeast
(Saccharomyces cerevisiae).
The objective of present study was to select the
external stimulus for translocation of enzyme across
the inner membranes and to develop the kinetics for
the same, besides application of this process for the
optimization of cell disruption.
Materials and Methods
The microorganism, Kluyveromyces lactis NCIM
3566 obtained from National Chemical Laboratory,
Pune was used as the source of β-galactosidase
enzyme. The media components were procured from
Himedia Ltd. Mumbai. All the chemicals used in
the experiment were of analytical grade. ONPG
(o-nitro Phenyl β-D-galactopyranoside) substrate for
β-galactosidase was purchased from SRL, Mumbai.
233
Cultivation of Microorganism
Stock cultures were maintained on the YMPD
medium containing yeast extract, 0.3; malt extract,
0.3; peptone, 0.5 and dextrose, 1% at 4°C. A 48-hrold culture was used to inoculate the seed culture
medium (YMPD medium) in 100 ml conical flask
with a working volume of 20 ml and incubated at
room temperature for 24 hrs. 5 ml of seed culture was
used to inoculate 100 ml of medium in 250 ml of
conical flask. Fermentation medium contained:
lactose, 2.5; urea, 0.25; potassium dihydrogen
phosphate, 0.25, MgSO4.7H2O, 0.06; yeast extract,
0.25; and malt extract, 0.25%. Traces were added as
CaCl2 (0.01g), ZnSO4.7H2O (0.1 mg), MnSO4. H2O
(0.05 mg) and CuSO4.5H2O (0.025 mg) in 100 ml of
medium21. Fermentation was carried out in shake
flask (250 ml) with working volume of 100 ml at
room temperature on a rotary shaker (175 rpm) for 18
hrs. The pH of the medium was adjusted to 5.5 by
using 1N NaOH21. Cultivated cells were centrifuged
from the medium using Remi Research Centrifuge
operating at 8000 rpm for 20 min at room
temperature. Cells were then washed with phosphate
buffer of pH 6.6 (0.05 M) and resuspended in the
same buffer to study the location and translocation
behaviour of β-galactosidase across the inner
membrane of K. lactis.
Selection of Enzyme Translocation Stimulus
The selection of stimulus to translocate
enzyme/protein to periplasmic space depends upon of
various factors such as membrane fluidity, nature and
structure of protein, permeability, chemical composition of the membrane, molecular weight of the
protein, diffusivity of protein through the membrane,
type of external stimulus, folding behaviour of
protein, hydrophobic interaction of the binding sites
among the targeted protein, and response of the
enzyme to signal of stimulus factors. It should be
optimum for maximum rate of enzyme translocation
without deactivation and should not affect other functions of the cells. The heat treatment has been found
to be the external stimulus for translocation of
cytoplasmic β-galactosidase to periplasmic space and
in some cases up to the extracellular medium19.
Heat Stress Treatment
Heat treatment was carried out in serological water
bath (Hexatech, Mumbai). 4% of 50 ml cell
suspension (wet weight) was prepared in 100 ml
234
INDIAN J BIOTECHNOL, APRIL 2005
beaker and maintained at 15°C. Bath was maintained
at a particular temperature for specified treatment.
Prechilled cell suspension at 15°C was put in the bath
(at a specified temperature) in a glass beaker. The
temperature of cell suspension was monitored
continuously and heat treatment (maintained at any
specified temperature) was given for different time
intervals. The zero time of the treatment was that
when the cell suspension reached specified treatment
temperature (from 15°C, initial temperature). The
heat-treated cell suspension was gradually cooled to
room temperature and stored at 4°C for 15 min and
then centrifuged at 8000 rpm for 20 min. Supernatant
was removed and analyzed for the enzyme and
protein. Cells were resuspended in 50 ml phosphate
buffer and stored at 4°C for half an hour to chill the
cell suspension, which was then used for cell
disruption.
Cell Disruption by Ultrasonication
All the cell disruption studies were carried out
using ultrasonic bath (Dakshin Ltd). The choice of
ultrasonic bath was due to its simplicity and having
higher energy efficiency for the low intensity
irradiation. The ultrasonic bath had an operating
frequency of 22.5 KHz with a power rating of 120W.
The internal body of bath (15 cm length, 15 cm
breadth, and 15 cm height) made up of stainless steel
and the bottom is fitted with three piezoelectric
transducers arranged in triangular fashion and bonded
externally to the base. A drainage valve has also been
provided at the side of the bath. A processor provides
energy to the transducer, which is a separate unit. The
experimental set up is shown in Fig. 1. The cell
disruption was carried out using 250 ml beaker (6 cm
diam and 11.5 cm length) kept at the centre position at
a height of 7.5 cm from the bottom with working
volume of 50 ml of cell suspension after filling the
bath with water up to a level of 14 cm. The pressure
intensity was found to be highest at this position as
studied by Puthli22. The temperature of water in the
bath was maintained at 10°C throughout the
disruption process by periodically replacing water in
the bath. Samples of the disrupted cell suspension
were drawn at regular intervals and centrifuged at
room temperature in Remi Research Centrifuge with
Eppendorf tube rotor for 20 min to remove the cell
debris. The clear supernatant was collected and
analyzed for the enzyme β-galactosidase and other
proteins.
Fig.1⎯Ultrasonic bath
Analytical Methods
β-Galactosidase Activity
Enzyme acts on its substrate ONPG and converts it
into o-nitro phenol. This assay was based on the
15 min hydrolysis of ONPG to o-nitro phenol at 30°C
and pH 6.6. Thus, the amount of β-galactosidase
formed could be estimated from the amount of the
absorbance of o-nitro phenol at 420 nm. One enzyme
unit equals the production of 1 nmol of o-nitro phenol
min/ml under the given conditions. The molar
extinction coefficient of o-nitro phenol under these
conditions was 4.5 × 103 m-1 cm-1 as reported by
Dickson and Markin23.
Total Protein Estimation
Proteins have a characteristic absorbance around
275-280 nm due to the presence of tyrosine and
tryptophan. Each type of protein has a specific
extinction coefficient. Absorbance varies with the
amount of tyrosine and tryptophan in the sample. This
method is relatively fast, easily automated, nondestructive and reasonably sensitive24. Bovine serum
albumin (BSA) was used as a standard. Various
dilutions of BSA were prepared in the phosphate
buffer and absorbances were measured by UV
spectroscopy at 280 nm. A plot of absorbance versus
concentration of BSA was generated and used as a
calibration curve. The amount of protein in the sample
was estimated by extrapolating the absorbance of the
sample on concentration axis.
Measurement of Translocation
The extent of translocation can be judged by the
concept of LF and also from the reduction in time
required to release the enzyme by ultrasonication as
the enzyme release depends on the location of enzyme
within the cell. The LF can be derived from the
enzyme and the protein release rate ratios during the
mechanical cell disruption process. First order
FARKADE & PANDIT: TRANSLOCATION OF CYTOPLASMIC β-GALACTOSIDASE USING HEAT TREATMENT
kinetics is reported to be applicable to the cell
disruption process25-29. Rate constant for the enzyme
release (k1) and protein release (k1) is given by the
expression of the form.
⎡ Rlm ⎤
1
In ⎢
⎥ = In = Kt × t
R
−
R
D
1⎦
⎣ lm
… (1)
or T and R1
where i=I or T and R1 and R1 is the released amount of
total protein and enzymes (I=1,2,3) in time ‘t’ and
R1,m is the maximum amount of total protein and
enzymes (I=T) obtainable from the cells. Thus, the In
values were plotted against time to get the release rate
of the enzyme and protein. The LF values obtained by
taking a ratio of the release rate constant (k1 and kt) of
enzyme to protein19.
LF =
k1
kt
… (2)
The proteins are located throughout the cell and
mostly evenly distributed, but the enzyme is confined
in most cases to either periplasm or cytoplasm.
Hence, when the release rate of enzyme (k1) is greater
than or equal to that of protein (kt), the ratio becomes
greater than unity. This happens when the enzyme is
periplasmic. When the enzyme is cytoplasmic, the
release rate of enzyme would be less than or equal to
that of protein and hence, the LF value will be less
than one. Thus, the above concept, based on the
relative rates of release of enzyme to protein, has been
successfully used by Balasundaram and Pandit20, to
measure the rate of translocation of the enzyme
during heat treatment.
235
2. When the enzyme structure partially unfolds,
hydrophobic amino acids appear on the limited
area of the enzyme surface.
3. Heat induces the disappearance of local
hydrophobic moieties on the enzyme surface and
makes the enzyme surface fully unfolded.
4. The enzyme also losses some activity with an
increase in the temperature30.
5. Heat stress increases the permeability of the
membrane and results in an increase in the
diffusivity of the enzyme across the cytoplasmic
membrane within the cell.
6. When the aqueous solutions of the functional
cytoplasmic proteins are heated, they undergo a
thermally induced change in their conformation
from highly organized, fairly compact, ‘globular’
structure to a relatively disorganized, extended,
‘random coil like’ arrangement, accompanied by
the loss of their physiological functions15.
The above changes in the enzyme structure and its
behaviour due to heat stress showed that enzyme can
be translocated from the cytoplasmic space to the
periplasmic space and possibly to extracellular
medium as reported in this work in terms of the
values of the LF. The protein translocation may
represent an adaptive response to an altered
environment and enabling the cell to respond to heat
stress by stabilizing its outer membrane. The value of
LF corresponds well with the subcellular distribution
of intracellular soluble proteins27. The effect of heat
treatment time and temperature on the subcellular
distribution of enzymes in terms of LF has been
discussed in subsequent sections and is shown in the
Fig. 2.
Results and Discussion
Effect of Ttemperature on Enzyme and Protein
Under normal conditions (28-30°C), the
hydrophobic amino acids of the enzyme are tightly
packed inside the enzyme molecule, so the enzyme
maintains its hydrophilic nature. As temperature
increases, the following effects are known to occur in
the enzyme:
1. Structure of the enzyme is partly destroyed and
then some hydrophobic amino acids, which are
contained inside, are exposed to the enzyme
surface.
Fig.2⎯Effect of heat treatment time on LF at different
temperatures
236
INDIAN J BIOTECHNOL, APRIL 2005
Enzyme Translocation
Effect of Heat Treatment Ttime (40°C)
At 40°C of the treatment temperature, the LF
values increased from 0.59 after 0 min to 0.795 after 4
min to 0.941 after 6 min. 1.091 after 8 min and 1.27
after 10 min of treatment. The LF values increased
linearly at 40°C indicating the onset of translocation.
From Fig. 2, it has been observed that the initial rate
of translocation was higher as the values of LF
increase from 0.236 for control to 0.590 during the
heating up time of 8 min to achieve the treatment
temperature of 40°C initial translocation rate (rate of
change of LF) was higher and then reduced gradually.
This could also be due to reduced enzyme
concentration as most of the enzyme has already been
translocated. This confirms that variable stress was
more effective than constant stress.
Effect of Heat Treatment Time (45°C)
At 45°C of the treatment temperature, the LF
values increased from 0.372 after 0 min to 0.606 after
1 min, 0.816 after 2 min, 0.854 after 3 min, 1.5 after 5
min, 1.244 after 8 min and 2.094 after 10 min of
treatment. The LF values increasing linearly at 45°C
clearly indicated the continued translocation of βgalactosidase across the inner membranes as LF
values give the subcellular distribution of enzymes
within the cell. From Fig. 2 it can be observed that
rate of translocation of enzyme increased as LF value
changed from 0.372 for 0 min to 0.606 for 1 min of
heat treatment time and 0.816 for 2 min of heat
treatment. Then there was a decrease in the rate of
translocation as LF value increased from 0.816 to
0.854 for 2 min to 3 min of heat treatment. Again
there was increase in the rate of translocation with
continued heat treatment as LF value increased from
0.854 to 1.5 with an increase in the heat treatment
time by 2 min (total time of 5 min) and 2.094 for 10
min of treatment. The value of LF at 45°C after
achieving the temperature was 0.372 (for 0 min heat
treatment), which was very nearer to the LF value of
the control (0.236).
Effect of Heat Treatment Time (50°C)
At 50°C of the treatment temperature, the LF
values increased from 0.329 after 0 min of treatment
to 0.569 after 2 min, 0.814 after 4 min, 1.613 after 6
min, 2.086 after 8 min and then decreased to 0.444
after 10 min of treatment. The translocation rate of
enzyme was first linear as the value LF increased
from 0.329 to 0.569 for 2 min of treatment. The
translocation rate again increased after 2 min of heat
treatment as LF value changed from 0.569 to 0.814
for 4 min. The rate of translocation further increased
with an increase in the treatment time as LF value
changed from 0.814 to 1.613 after 6 min of treatment
and then continued upto 8 min of treatment. The heat
treatment at 50°C resulted into LF values increasing
from 0.329 to 2.086 for 0 min to 8 min of heat
treatment and with further increasing the treatment
time by only 2 min (total 10 min) LF values decreased
to 0.444. This indicated that at higher temperature (≥
50°C) the enzyme was getting deactivated. Therefore,
at 50°C for 10 min of heat treatment, LF values does
not give the information about the subcellular location
of enzyme but showd that there was an inactivation of
enzyme at higher treatment temperature. As LF values
were derived on the basis of relative enzyme and the
protein release rates, the deactivation of the enzyme
resulted into reduced values of the enzyme
concentration and hence the LF. Thus, LF concept
cannot be used for the estimation of translocation rate
in such a case of enzyme.
Comparison of Variation of LF Value at Different Heat
Treatment Temperature
From the LF value of 0 min of treatment at 40°C
(0.59), 45°C (0.372) and 50°C (0.329) as compared to
the control suggest that the deactivation of enzyme
was more at 50°C resulting in the reduction in the LF
value. If compared for 0 min of heat treatment time
(initial heat up period to achieve the desired
temperature) at different temperature, it was observed
that LF value decreases with an increase in the
treatment temperature. This could be due to the
deactivation of enzyme. On comparing the LF values
for 10 min (Fig. 2), LF value increased from 1.27 at
40°C to 2.094 at 45°C and then decreased to 0.444 at
50°C.
Effect of Heat Treatment Temperature for constant Heat
Treatment time of 8 min
From the experimental values of LF it can be
observed that minimum 8 min of treatment time was
required to achieve the LF = 1, equivalent to a
complete translocation of the β-galactosidase to the
periplasmic space or just beyond the membrane of the
cytoplasmic space. So the LF value was compared for
8 minutes of treatment time and the graphical
representation shown in Fig. 3.
FARKADE & PANDIT: TRANSLOCATION OF CYTOPLASMIC β-GALACTOSIDASE USING HEAT TREATMENT
From Fig. 3, it has been observed that LF value
increased with an increase in the treatment
temperature. It has also been observed that with an
increase in the treatment temperature from 40 to 45°C
the increase in the LF value was not very significant.
This also suggested that the translocation rate of
enzyme was not much higher with an increase in the
temperature from 40 to 45°C. But with an increase in
the treatment temperature from 45 to 50°C, there was
steep rise in the LF value. This indicated that
translocation rate of enzyme with an increase in the
treatment temperature from 45 to 50°C was much
higher.
Kinetics of Translocation
As the value of LF increased with an increase in the
treatment temperature and heat treatment time, it
indicated that the translocation was dependent on the
treatment temperature and the time of treatment. Heat
stress has also been reported to induce the
conformational change in intracellular proteins and
there was a change in the hydrophobicity of the
enzyme and cytoplasmic membrane, which resulted in
an increased interaction between the two and resulted
in the translocation of cytoplasmic enzyme to the
periplasm. Thus, there could be a different migration
rate for the enzyme at different treatment temperatures. Thus, the translocation rate, corresponds to the
rate of increasing of the LF. An attempt has been
made to correlate LF as a function of temperature and
time in the form of following equations.
At 40°C, LF = 0.0648 T + 0.5603
At 45°C, LF = 0.1739 T + 0.4315
At 50°C, LF = 0.2279 T + 0.1706
… (3)
… (4)
… (5)
where T is the heat treatment time (min).
Differentiating the above equation with respect to
T, the translocation rate was calculated. The value of
rate constant at 40, 45 and 50°C was 0.064, 0.173 and
0.227 min-1. This indicated that the translocation rate
was higher at 45°C and the value of rate constant was
0.173 min-1, which was nearly three times greater than
the rate constant at 40°C (0.064 min-1) and 1.3 times
lesser than rate of translocation at 50°C (0.227 min-1).
From the kinetics of translocation, it has been
observed that to obtain a value of LF = 1 corresponding to the complete translocation of the enzyme into
the periplasmic space, time required at 40, 45 and
50°C was 6.78, 3.26 and 3.63 min, respectively.
237
Relationship between Translocation Rate and Temperature
The relationship between the temperature and the
translocation rate is shown in Fig. 4 and it has been
observed that translocation rate increased with an
increase in the temperature. The relationship obtained
by fitting the data as shown in Fig. 4 is,
… (6)
dLF/dT = 0.163-0.5784
where (-) = temperature (°C).
This relationship could be used to optimize the heat
treatment protocol for the translocation of the target
enzymes across the inner membranes (cytoplasmic) of
the cell and to decide the optimum parameters such as
irradiation intensity and time for the cell disruption
process.
Application of Translocation of Enzyme for the Optimization
of Cell Disruption Process
Balasundaram and Pandit20 have studied the
significance of the location of the enzymes on their
release rate during the microbial cell disruption.
Fig.3⎯Effect of temperature on LF for 8 minutes of heat
treatment time
Temp θ[°C]
Fig.4⎯Relationship
temperature
between
the
translocation
rate
and
238
INDIAN J BIOTECHNOL, APRIL 2005
Earlier studies27 have indicated that more energy was
required to release the cytoplasmic enzymes than the
periplasmic enzymes. This showed that the amount of
enzyme released and energy requirements for cell
disruption will depend on the location of enzyme
within the cell and if the location of the enzyme can
be changed from cytoplasmic to periplasmic space, a
significant energy saving can be achieved. This means
that the enzyme in the periplasmic space will come
out earlier in the leached liquor than the cytoplasmic
enzyme and the time required for the release of the
same quantity of enzyme with the ultrasonic
disruption will also decrease. This will also make the
subsequent purification steps easier, as the
translocation may provide the selective release of the
enzyme. As discussed earlier, location of the enzyme
is an important factor, which will decide the release
rate of the enzyme during the cell disruption process.
The energy required for the release of enzyme from
the specified site within the cell is directly
proportional to the sonication time. If the sonication
time increases, energy required is also higher. This
also confirms that translocation of enzyme leads to
the reduction of energy requirements for the cell
disruption process. The results demonstrated in terms
of LF as discussed above show that translocation of
enzyme is possible from the cytoplasmic to
periplasmic space by the heat treatment of the cell
suspension. The experiments carried out with a
constant sonication time of 50 min showed that the
enzyme activity increased from 21 l to 59 U/ml for
untreated to heat-treated cells, a 3-fold increase in the
activity. It has also been observed that initial rate of
enzyme release during the cell disruption process was
more for heat-treated cells than the untreated cells.
Fig. 5 clearly depicts the different locations of the
enzyme in the cell. From the figure, it has been
observed that to recover the enzyme from the position
one (Fig. 5-a), more energy will be required to break
the cell wall and also the inner cytoplasmic
membrane. But due to the translocation of the enzyme
to the periplasmic space (Fig. 5-b & c), lesser energy
will be required, to break only the outer cell wall and
there will be saving in the energy required to break
the inner cytoplasmic membrane, as it is no longer
necessary. So it can safely be concluded that
translocation of enzyme can be used to optimize the
cell disruption process.
Fig.5⎯Schematic representation of different locations of βgalactosidase in Kluyveromyces lactis [(a) cytoplasmic location;
(b) translocation across the cytoplasmic membrane (c)
translocation from cytoplasmic space]
Translocation of β-Galactosidase to Extracellular Locations
The efficient release of the enzyme by the addition
of glycine and subsequent thermal treatment has been
studied31. The effect of heat and chemical treatment
on the release kinetics of the enzyme has been
investigated32. Kula and Vogels33 have explained that
the physical treatment weakens the cell wall structure
or affects the function of membrane as barriers.
Therefore, possibility of the enzyme release for it to
become extracellular is more but it was found that,
short pretreatment was not sufficient to lead to an
extensive release of the enzyme to the extracellular
suspension medium. However, it was found to
improve the efficiency of a subsequent mechanical
cell disruption. This resulted in the savings in the
energy requirement for the cell disruption and also
reduction in the processing time.
FARKADE & PANDIT: TRANSLOCATION OF CYTOPLASMIC β-GALACTOSIDASE USING HEAT TREATMENT
239
References
1
2
3
4
5
6
Fig.6⎯Variation in the extracellular enzyme activity in the
suspension with the heat treatment time
The amount of enzyme released in the extracellular
medium with heat treatment time at different
temperatures is shown in Fig. 6. The maximum
amount of enzyme, which could be made to become
extracellular, was at treatment temperature of 50°C.
Maximum activity (20 U/ml) was found to be for 8
min of heat treatment at 50°C, which is still less than
2 times the maximum enzyme activity (59 U/ml)
obtained after the heat treatment at 45°C and
subsequent ultrasonic disruption. It was also observed
that at 50°C, the enzyme deactivates faster, thus, even
though by heat treatment it is possible to make
enzyme extracellular but is not practical.
Conclusions
The heat stress was found to induce translocation of
the target enzyme (β-galactosidase) and also other
proteins and resulted in the formation of insoluble
protein aggregates, which could be removed during
centrifugation. The LF concept can be successfully
used to study the translocation of enzyme. The
translocation rate was found to be different at
different temperature conditions. Though at and above
50°C, an increase in the treatment temperature,
significant enzyme deactivation was observed. The
LF concept has been successfully applied to acquire
the information about translocation (enzyme
migration) rates. The stress condition for the selective
recovery of cytoplasmic β-galactosidase from
Kluveromyces lactis has been optimized. The
translocation of the enzyme can be used to optimize
the cell disruption process and minimize the energy
requirements of the cell disruption process and to
recover the intracellular products.
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