ARTICLES
MB
STUDY OF CATALASE ENZYME IN METHYLOTROPHIC YEASTS
D. Koleva1, V. Petrova1, Tz. Hristozova2 and A. Kujumdzieva1
Sofia University “St. Kliment Ohridski”, Faculty of Biology, Soifia, Bulgaria1
Bulgarian Academy of Science, Institute of Microbiology, Plovdiv, Bulgaria2
Correspondence to: Anna Kujumdzieva
E-mail: [email protected]
ABSTRACT
Catalase enzyme was explored in three methylotrophic yeast strains Pichia pastoris X-33, Hansenula polymorpha CBS 4732 and
Pichia pini NBIMCC 8360 during batch-wise growth on different carbon sources. For investigation of compartmentalization
of the enzyme within yeast cells a mitochondrial fraction from each strain was isolated. Peroxisomal and mitochondrial import
of catalase enzyme was tested quantitatively by subcellular fractionation followed by enzyme activity assays. Efficient catalase
import into peroxisomes was observed when cells were cultivated under peroxisome-inducing conditions (i.e. growth on methanol),
whereas more significant catalase activity was measured in mitochondria when cells were grown on polyvalent alcohol glycerol
that favor respiration and ROS (reactive oxygen species) accumulation within this organelle. When yeast cells utilize glucose the
catalase enzyme is relatively equal distributed between the two compartments (peroxisomes and mitochondria). Results indicated
that mitochondrial matrix in methylotrophic yeasts possess in situ mechanism for scavenging of ROS represented by Mn SOD
and mitochondrial catalase.
Keywords: methylotrophic yeasts, mitochondria, catalase
Introduction
Methylotrophic yeasts possess a respiratory type of metabolism
and during growth an accumulation of potentially cytotoxic
species (O2-) and hydrogen peroxide (H2O2) takes place (13,
27). The catalase [E.C.1.11.1.6] and superoxide dismutase
[E.C.1.15.1.1] enzymes play a key role in cellular defense
against the reactive species (9).
The intracellular localization of catalase enzyme has been
under debate for many years. It is a topic wildly investigated
and the initial localization in peroxisomes has been determined
by a series of cytochemical and biochemical studies which
also describe its position in other cellular compartments. This
approach has been used by number of authors (8, 18, 20, 32).
Michailova et al. (20) have isolated a pure heavy mitochondrial
fraction from Candida boidinii and have provided evidences
for catalase activity.
At present it is well documented with Saccharomyces
cerevisiae yeast cells that mitochondria possess catalase
A enzyme (24). It has been shown that catalase A, although
primary considered as a peroxisomal protein, could also be
independently target to mitochondria (25).
Given this data, it is important to perform broader
investigations on catalase enzyme in methylotrophic yeasts. In
the present work, we have studied the mitochondrial localization
of catalase using three different strains methylotrophic yeasts:
Pichia pastoris, Pichia pini and Hansenula polymorpopha
and demonstrated that similarly to S. cerevisiae yeast,
methylotrophic ones possess constitutive transport of catalase
into both organelles.
762
Materials and Methods
Microorganisms and growth conditions
The yeasts used in this investigation were, as follows: Pichia
pastoris X-33 (Invitrogen), Hansenula polymorpha CBS 4732
and Pichia pini NBIMCC 8360. The strains were cultivated
in liquid YP medium (1% Yeast Extract, 1% Bacto-Peptone)
supplemented either with 2 % glucose (YPD), 1 % methanol
(YPM) or 1 % glycerol (YPG) at 30oC on a reciprocal shaker
(204 rpm).
Cell-free extract preparation
Cells from 6, 12, 20, 30, 48 and 72 h of cultivation were harvested
by centrifugation at 800 x g for 10 minutes and washed twice
with distilled H2O. Cell wall disruption was carried out by
spheroplasting according to the procedure of Defontaine et al.
(3). The cell debris was removed by centrifugation at 1000 g
and 4 ◦C for 10 min and the cell free extracts were triply frozen
and thawed to break open organelles, and centrifuged at 15 000
g and 4 ◦C for 10 min. The supernatants obtained were used for
enzymatic analyses.
Subcellular fractionation and Nycodenz gradients
For cell fractionation, yeast cells were grown 20 h to reach
end of logarithmic phase. Spheroplasts were generated by the
procedure of Defontaine et al. (3) and unlysed cells, nuclei and
cell debris were removed by centrifugation at 1000 g and 4
◦
C for 10 min. The supernatant containing the crude yeast cell
organelles was again centrifuged at 25 000 g and 4◦C for 20
min, and the crude organelle fraction was resuspended in a total
BIOTECHNOL. & BIOTECHNOL. EQ. 22/2008/2
volume of 5 mM Mes/KOH buffer (pH 6.0) containing 0.24 M
sucrose (to provide osmotic protection for the peroxisomes)
and 1 mM EDTA. Purity of such an organelle fraction was
routinely determined by Western blot analysis and activity
assays of marker enzymes, such as glucose-6-phosphate
dehydrogenase, hexokinase, succinate dehydrogenase, and
isocitrate lyase, indicating that the organelle fraction was highly
enriched for yeast mitochondria and peroxisomes (results not
shown). For the separation of cell organelles, in particular for
separating peroxisomes from mitochondria, the crude organelle
fraction was layered on top of a Nycodenz (ICN) step gradient
consisting of 17%, 25% and 35% Nycodenz in the same buffer,
essentially as described by Lee et al., (16). The Nycodenz
gradient was centrifuged at 133 000 g and 4 ◦C for 90 min in
a SW50Ti rotor (Beckman) and the gradient was subsequently
fractionated from the bottom. Each fraction (1 ml) was diluted
5-fold with Mes/KOH buffer (5 mM, pH 6.0, containing 0.24
M sucrose and 1 mM EDTA), and the Nycodenz was removed
by a final centrifugation step at 15 000 g and 4 ◦C for 20 min.
For enzyme assays, gradient purified organelles were ruptured
by the addition of Triton X-100 to a final concentration of
1%. Yeast mitoplasts were isolated from Nycodenz-purified
mitochondria by hypotonic swelling-shrinking treatment
according to Zinser and Daum (37).
Biochemical analyses
Catalase [EC: 1.11.1.6] activity was determined
spectrophotometrically according to Aebi, (1) and Upadhya et
al. (31).
D-amino acid oxidase [EC: 1.4.3.3] activity was measured,
as described by Lichtenberg and Wellner (17).
Fumarase [EC: 4.2.1.2] activity was assayed according to
the procedure of Kanarek and Hill (10).
Analysis of carbon source utilization
Glucose was determined by the method of Somogy (29)
and Nelson (23). Methanol was determined by the method
of Gonzalez-Rodriguez et al. (7). Glycerol was determined
according to the procedure described by Moller and Roomi
(22).
Cell dry weight estimation
Cell dry weight was determined gravimetrically after drying
washed cells to constant weight at 105ºC.
Determination of kinetic parameters
Specific growth rate (μ) and maximal cell yield (Ys) ware
calculated according to Monod kinetics as described by Pirt
(26).
Protein determination
Protein content was determined by the method of Lowry et al.
(19). Bovine serum albumin (Sigma St. Louis, MO, USA) was
used as a standard.
Electron microscopy
Electron microscopy was performed as described previously
in Michailova et al. (20). Whole yeast cells were field with 1.5
% potassium permanganate for 20 min at room temperature
washed and stained with 1 % (w/v) uranyl acetate for 1 h.
After dehydratationin graded ethanol series the material was
embedded in Epon 812 resins. The ultrathin sections of the
cells were obtained on an Ultracut Reichert apparatus and were
stained with uranyl acetate and lead citrate. The observations
were carried out on a Zeiss EM 10C electron microscope at 60
or 80 kV.
Results and Discussion
Influence of carbon source on growth, proliferation of
mitochondria and peroxisomes, and catalase activity
The catalase enzyme activity was investigated during batchwise cultivation of three methylotrophic yeast strains (Pichia
pastoris X-33, Hansenula polymorpha CBS 4732 and Pichia
pini NBIMCC 8360) on standard YPD medium. Cultivation of
the cells was performed for 72 h and samples were withdrawn
during different growth phases of the culture in order to
determine cell dry weight and glucose consumption (Fig.
1a). It is evident that a diauxic growth takes place as glucose
is exhausted at the 20th hour of cultivation and cells enter
stationary phase at 48th h. The specific growth rate and maximal
cell yield were calculated according to Monod Equation
(Table 1).Obtained results for μmax = 0.30 ÷ 0.32 h-1 and
YGlu = 0.27 ÷ 0.30 during growth on glucose are in accordance
with the data published from Sola et al. (28) for metabolic
state of some methylotrophic yeasts. Thus the chosen strains
for investigations of compartmentalization of catalase enzyme
could be considered as effectively growing yeasts.
TABLE 1
Kinetic parameters measured during growth of Pichia pastoris X-33, Pichia pini NBIMCC 8360 and Hansenula polymorpha
CBS 4732 on nutrient media, supplemented with different carbon sources: glucose, glycerol and methanol
Strain
Pichia pastoris X-33
Pichia pini NBIMCC 8360
Hansenula polymorpha CBS 4732
glucose
0.30 ±0.05
0.28 ± 0.01
0.27 ± 0.02
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Ys/c
glycerol
0.32 ±0.05
0.30 ±0.03
0.30 ± 0.05
methanol
0.49 ±0.06
0.48 ±0.09
0.47 ± 0.01
μ (h-1)
glucose
glycerol
methanol
0.32 ± 0. 015 0.149 ± 0.023 0.101 ±0.12
0.31 ± 0.011 0.150 ± 0.025 0.102 ± 0.06
0.30 ± 0.023 0.152 ± 0.22 0.104 ± 0.09
763
compounds supplemented in YPD media are metabolized
from yeast and other fungi through hydrogen producing
oxidases (33). So, correlation of the catalase activity with the
accumulation of its specific substrate (H2O2) obviously takes
place.
10
Cell dry weight, g l
8
6
4
0
0
6
12
20
30
48
72
Time, h
Pichia pastoris
a
Pichia pini
Hansenula polymorpha
30
25
20
15
10
5
0
8
6
4
2
0
6
12
20
30
48
72
Pichia pastoris
b
Pichia pini
Hansenula polymorpha
2
1
0
72
5
6
12
Fig. 1. Cell growth and utilization of carbon source
Growth curves of the three methylotrophic strains (Pichia pastoris X-33;
Pichia pini NBIMCC 8360; Hansenula polymorpha CBS4732) were followed
up after their cultivation on standard YP media supplemented either with
glucose (a), glycerol (b) or methanol (c)
Further, samples of biomass were processed and crude
enzyme extracts prepared. А spectrophotometrical analysis
of the total catalase activity at different hours of growth is
shown in Fig. 2. Due to the presence of glucose in the media
at the 6th h of cultivation, no catalase activity was detected
as it is subjected to glucose repression (14, 36). The highest
catalase activity was measured after 48 h of cultivation, which
coincided, with the late exponential – early stationary growth
phase of the cultures. This increase in catalase activity, after the
exhaustion of glucose, is consistent with the available data for
glucose repression in methylotrophic yeasts on alcohol oxidase
and catalase synthesis (14). Additionally, the different amino
20
30
48
Glu
72
Hansenula polymorpha CBS4732
40
35
30
25
20
15
10
5
0
Hansenula polymorpha
Met
10
0
Time, h
Pichai pini
Gly
15
Time (h)
catalase activity (U mg -1)
-1
Cell dry weight, g l
3
Pichia pastoris
Met
20
45
48
Gly
25
b
30
Met
0
4
20
72
Gly
30
5
12
Glu
35
50
6
48
40
6
0
30
45
0
Growth on methanol
20
Pichia pini NBIMCC 8360
50
Time, h
764
12
a
10
0
c
6
Time (h)
catalase activity (U mg -1)
-1
35
0
Growth on glycerol
Cell dry weight, g l
Pichia pastoris X-33
40
2
catalase activity (U mg -1)
-1
Growth on glucose
c
6
12
20
Time (h)
30
48
Glu
72
Fig. 2. Total catalase activity during growth on different carbon sources
Total catalase activity of Pichia pastoris X-33 (a), Pichia pini NBIMCC 8360
(b) and Hansenula polymorpha CBS4732 (c) was measured at different hours
of cultivation during growth on YP media supplemented either with glucose,
glycerol or methanol
In order to investigate the influence of different carbon
sources on proliferation of cellular substructures corresponding
to catalase localization, except glucose, both glycerol and
methanol were used as growth substrates. It is known that
the catabolic pathway of glycerol in yeasts involves passive
diffusion across the membrane, phosphorilation by glycerol
kinase in cytosol, and oxidation by mitochondrial glycerol
phosphate ubiquinone oxireductase (5). During growth on
glycerol, the yeast cells are highly enriched with mitochondria
and respiration rate is enhanced to maximum (6). The utilization
of methanol proceeds via ROS producing alcohol oxidase,
located into peroxisomes, thus leading to active peroxisomal
proliferation in yeast cells (15, 20).
BIOTECHNOL. & BIOTECHNOL. EQ. 22/2008/2
Hansenula polymorpha CBS4732
Pichia pini NBIMCC 8360
Hansenula polymorpha CBS4732
Pichia pini NBIMCC 8360
Hansenula polymorpha CBS4732
Pichia pini NBIMCC 8360
Fig. 3. Electron micrographs of Hansenula polymorpha CBS4732 and Pichia pini NBIMCC8362. Bar = 0.2 μm.
The ultrathin sections were obtained after growth of tested methylotrophic yeast strains in rich media supplemented with different carbon sources: (a) glucose,
(b) glycerol and (c) methanol
BIOTECHNOL. & BIOTECHNOL. EQ. 22/2008/2
765
From the growth curves shown on Fig. 1b and Fig. 1c
we calculated specific growth rate on glycerol and methanol
that were respectively 0.149 ÷ 0.152 h-1 and 0.101 ÷ 0.104 h-1.
Obtained cell yield of glycerol substrate is YGly = 0.30 ÷ 0.32
and of methanol - YMet = 0.47 ÷ 0.49, which demonstrate the
effective utilization of these substrates by all tested strains and
confirms the active metabolic status for targeted structures
under investigation.
Consequently obtained biomass, enriched in mitochondria
and peroxisomes, was subjected to enzymatic lyses and a
spectrophotometrical analysis of the total catalase activity was
performed (Fig. 2). We found that catalase enzyme is present
during the whole period of cultivation and its maximum
is measured at 48th h of cell growth, which coincides with
exponential phase. These data suggest full induction of catalase
when a respiratory carbon sources are used, and support the
results previously obtained in our laboratory for subsequent
induction of catalase synthesis after glucose depletion (20, 24,
36). Further, comparative analysis between catalase activity
in Hansenula polymorpha, Pichia pastoris and Pichia pini,
cultured on the three different carbon sources, was performed.
It was shown that maximal catalase synthesis appears when
cells are grown on methanol, lower in glycerol and the
lowest catalase activity was detected when glucose is used as
sole carbon source (average 38.16 > 31.72 > 26.88 U mg-1,
respectively).
The abundance of the cellular organelles (i.e. mitochondria
and peroxisomes) has been visualized by performance of
electron microscopy assay of the biomass of different samples
(Fig. 3). The study of strains grown in glucose containing
media revealed single small peroxisomal structures and few
under differentiated mitochondria. In YPG medium yeast
cells contained only few small microbodies, slightly increase
in size compared to the organelles present during growth of
cells on glucose, as well as numerous elongated or enlarged
mitochondria. However, in methanol-grown cells peroxisomal
proliferation was strongly induced and decrease of the number
of mitochondria was detected
Compartmentalization of catalase activity in
methylotrophic yeast cells
In our previous studies (24, 25) we have already demonstrated
that peroxisomal catalase A of Saccharomyces cerevisiae could
successfully be targeted both to peroxisomes and mitochondria.
In order to explore catalase distribution in yeast cells from Pichia
pastoris X-33, Pichia pini NBIMCC 8360 and Hansenula
polymorpha CBS4732 strains, the already isolated cell free
extracts were subjected to cell fractionation. The resulted
subcellular fractions were purified by ultracentrifugation
through a Nycodenz step gradient and subsequently analyzed
for protein content, D-amino acid oxidase and fumarase
activity. The results presented in Table 2 clearly indicate that
there were two peaks in protein distribution: in fraction 4 and
fraction 8. As the maximum of D-amino acid oxidase activity
is located in fraction 4 and the maximum of fumarase activity
766
was detected in fraction 8 it could be concluded that these
fractions are highly enriched in peroxisomes and mitochondria,
respectively. The observed sedimentation profile of catalase
enzyme in Nycodenz fractions (Fig. 4) indicates that sole
peroxisomal catalase enzyme detected in methylotrophic yeast
species, resembling peroxisomal catalase A of Saccharomyces
cerevisiae, probably also is efficiently co-targeted to two
different organelles, peroxisomes and mitochondria.
TABLE 2
Determination of purity of peroxisomal and mitochondrial
fractions obtained from the tested strains through measurement
of the specific activity of peroxisomal (DAAO) and
mitochondrial (fumarase) marker enzymes
Enzyme
Protein
activity
concentration
Fraction
[mg ml-1]
Number
1
0.25±0.02
2
0.41±0.03
3
0.50±0.12
4
1.43±0.05
5
0.32±0.05
6
0.25±0.02
7
1.38±0.02
8
2.51±0.11
9
0.92±0.01
10
0.91±0.04
11
0.68±0.03
12
0.19±0.01
13
0.15±0.01
14
0.17±0.02
DAAO
[U mg-1]
Fumarase
[U mg-1]
0.05±0.02
0.23±0.04
0.23±0.04
3.63±0.02
0.82±0.11
0.32±0.08
0.22±0.04
0.25±0.03
0.28±0.06
0.12±0.02
0.08±0.01
0.04±0.01
0.02±0.01
0.5±0.04
0.12±0.01
0.15±0.02
0.13±0.03
0.32±0.04
0.3±0.02
0.31±0.06
0.6±0.06
1.2±0.09
0.8±0.08
0.3±0.04
0.15±0.02
0.05±0.01
0.2±0.02
0.5±0.05
Next, the already isolated cell fractions were studied for
catalase activity in order to check its organelle distribution during
yeast growth on glucose, glycerol and methanol. The obtained
data (Fig. 4) indicated that during utilization of methanol,
catalase enzyme is present in both organelles as the majority
of activity was detected in peroxisomes. On the contrary, when
cells were cultivated on the polyvalent-alcoholic carbon source
glycerol, catalase enzyme was predominantly targeted to
mitochondria. When cells were grown in YPD media catalase
was relatively equal distributed between the two organelles.
Evidently, when glycerol is consumed from yeast cells and
active respiration is induced, catalase enzyme is strongly
required in mitochondria. Here, we determine that about 65
% of catalase activity was localized in the mitochondria. The
opposite phenomenon could be observed under peroxisomesinducing conditions. When methanol is added to the nutrient
media the majority of catalase protein (80 %) was located into
peroxisomes. In all cases, catalase was constitutively targeted to
both organelles and only the percentage of enzyme distribution
differs depending on growth conditions.
BIOTECHNOL. & BIOTECHNOL. EQ. 22/2008/2
16
Catalase (U/m g protein)
It is reasonable to suggest that future investigation with
this model system will provide not only clear vision for
processes of oxidation in eukaryotic cell but will help for better
understanding of mechanisms of cellular aging and apoptosis.
Pichia pastoris X-33
14
12
10
8
Acknowledgements
6
4
2
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Fra ction number
Glucose
Methanol
This work was supported by a grant from the National
Science Fund, Ministry of Education and Science, Project
No B1509/05-2451.
Glycerol
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Pichia pini NBIMCC 8360
Catalase (U/mg protein)
16
14
12
10
8
6
4
2
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Fraction number
Glucose
Glycerol
Hansenula polymorpha CBS4732
25
Catalase (U/m g protein)
Methanol
20
15
10
5
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Fra ction number
Glucose
Methanol
Glycerol
Fig 4. Activity and distribution of peroxisomal and mitochondrial catalase
during cell growth on different carbon sources
Conclusions
The data obtained from our investigations, for presence
of mitochondrial catalase activity in fermentative
(Saccharomyces), as well as in respiratory (Pichia and
Hansenula) type of yeasts, allow us to speculate that targeting
of catalase enzyme to mitochondria is a common feature to all
yeast microorganisms.
Along side our investigations, many different studies
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as Alzheimer, cancer, rheumatoid arthritis, arteriosclerosis,
and etc. (11, 34). It was shown also that targeting catalase
to the mitochondria provides better protection than cytosolic
expression against H2O2-induced injury (2, 30).
BIOTECHNOL. & BIOTECHNOL. EQ. 22/2008/2
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