Purification and characterization of an intracellular chymotrypsin-like serine protease from Thermoplasma volcanium

Article abstract of DOI:10.1271/bbb.70.126

An intracellular serine protease produced by Thermoplasma (Tp.) volcanium was purified using a combination of ammonium sulfate fractionation, ion exchange, and α-casein agarose affinity chromatography. This enzyme exhibited the highest activity and stabil

Full text of DOI:10.1271/bbb.70.126

Biosci. Biotechnol. Biochem., 70 (1), 126–134, 2006
Purification and Characterization of an Intracellular
Chymotrypsin-Like Serine Protease from Thermoplasma volcanium
y
˙
¨
Semra KOCABIYIK and Inci OZDEM˙IR
Middle East Technical University, Department of Biological Sciences, 06531 Ankara, Turkey
Received July 20, 2005; Accepted October 4, 2005
An intracellular serine protease produced by Ther-
moplasma (Tp.) volcanium was purified using a combi-
nation of ammonium sulfate fractionation, ion ex-
change, and ꢀ-casein agarose affinity chromatography.
This enzyme exhibited the highest activity and stability
bioremediation processes.4) Presently, due to their
critical position in the physiological and industrial
fields, there is growing interest among researchers in
the identification and molecular characterization of SP
family enzymes from diverse sources, including extrem-
ophiles. Several thermophilic and hyperthermophilic
archaea produce significant levels of intra- and extrac-
ellular proteolytic enzymes with high intrinsic molecular
stability. Most of these enzymes, which are recognized
as the serine type, have been detected in the genera
ꢀ
at pH 7.0, and at 50 C. The purifed enzyme hydrolyzed
synthetic peptides preferentially at the carboxy termi-
nus of phenylalanine or leucine and was almost
completely inhibited by PMSF, TPCK, and chymostatin,
similarly to a chymotrypsin-like serine protease. Kinetic
analysis of the Tp. volcanium protease reaction per-
formed using N-succinyl-L-phenylalanine-p-nitroanilide
as substrate revealed a Km value of 2.2 mM and a Vmax
5
,6)
Pyrococcus (Pyrolysin and PfpI),
(Archaelysin), Sulfolobus,
Desulforococcus
11–13)
and Thermococcus.
7
)
8–10)
Genome sequence data revealed even more expansive
proteolytic genotypes of these organisms than can be
value of 0.045 ꢀmol 1 ml min . Peptide hydrolyzing
ꢁ
ꢁ1
ꢁ1
1
4)
activity was enhanced by >2-fold in the presence of
inferred from biochemical analyses. A majority of
these organisms are capable of growth on proteinaceous
Ca2 and Mg at 2–12 mM concentration. The serine
þ
2þ
1
5,16)
protease is a monomer with a molecular weight of
4
substrates.
Therefore, the SP enzyme family in
2 kDa as estimated by sodium dodecyl sulfate–poly-
acrylamide gel electrophoresis and zymogram activity
staining.
archaea might be involved in generating a pool of
intracellular peptides and amino acids. But the role of
proteolysis in the metabolisms of thermophilic archaea
and the mechanisms through which peptide-based
substrates are utilized are less clear, and this is a
challenge to continued research from fundamental
perspectives. In this study, for the first time we report
the purification and biochemical characterization of an
intracellular SP from the thermoacidophilic archaeon
Thermoplasma volcanium GSS1 (DSM 4299). This
organism was originally isolated from submarine and
Key words: Thermoplasma volcanium; serine protease;
chymotrpsin-like protease; Archaea; ther-
mophilic
Serine proteases (SPs) are a large class of proteolytic
enzymes that have a reactive serine residue (nucleo-
phile), as a part of catalytic triad, in the active center to
hydrolyze peptide bonds. SPs execute a diverse array of
functions including primary metabolic and regulatory
events that extend from the normal physiology of the
cells to abnormal patho-physiological conditions, which
are most often implicated in protein turn-over, digestion,
fibrinolysis, blood coagulation, sporulation, fertilization,
immune response, homeostasis, and pathogenesis. Their
importance in conducting these essential functions is
evident from the ubiquitous occurrence of SPs in all
three forms of living organisms, as reviewed in detail by
1
7)
continental solfataras at Vulcano Island in Italy.
ꢀ
Tp. volcanium, which grows optimally at 60 C and
pH 2.0, is a facultatively anaerobic heterotroph.
Materials and Methods
Archaeal strain and growth. Thermoplasma (Tp.)
volcanium GSS1 (type strain 4299) was purchased from
DSMZ (Deutsche Sammlung von Microorganismen und
Zellkulturen, Braunschweig, Germany). Culture of the
Kalisz,1 Nduwimana et al., and Rao et al. On the
other hand, the SP family constitutes an important
enzymatic group of biotechnological relevance world
wide, and has been used extensively in the detergent,
leather, and pharmaceutical industries, and in food and
)
2)
3)
organism was grown at 60 C in liquid Volcanium
ꢀ
1
8)
Medium (pH 2.7), supplemented with glucose (0.5%,
w/v) and yeast extract (0.1%, w/v). Renewal of the
culture was achieved by subculturing once a week
through 1% (v/v) inoculum transfer into fresh medium.
y
To whom correspondence should be addressed. Tel: +90-312-210-31-03; Fax: +90-312-210-12-89; E-mail: biosemra@metu.edu.tr

A New Intracellular Serine Protease from Thermophilic Archaebacterium Thermoplasma volcanium
127
Preparation of cell-free extract. Tp. volcanium cells
from 48 h cultures were harvested by centrifugation at
increase in A280 equal to 0.01 OD under the above-
mentioned assay conditions.
ꢀ
7
;000 ꢁ g for 15 min at 4 C. The pellet was washed in
unsupplemented growth medium and resuspendend in
/5 volume (v/v) protease assay buffer (50 mM Tris–
Purification of serine protease by column chromatog-
raphy. Solid ammonium sulfate was added to the culture
1
ꢀ
HCl, pH 7.5, 5 mM CaCl2). The cells were disrupted by
sonication (Sonicator VC 100, Sonics and Materials,
Hartford, CT), and the cell debris was removed by
supernatant to 80% (w/v) saturation and kept at 4 C
overnight through gentle stirring. The precipitate was
collected by centrifugation at 11;000 ꢁ g for 30 min,
followed by dissolution in 1/20 volume (v/v) protease
assay buffer, pH 7.5. The crude enzyme in this fraction
was filtered through a 0.45 mm Millipore, millex HV
filter (Billercia, Boston, MA), and then desalted and
concentrated by ultrafiltration using a 5,000 cut-off
membrane filter (Vivascience, Sartarious Group,
Glourestershire, UK). After exclusion of ammonium
sulfate, the enzyme sample was loaded to an Econo Pac
Q ion exchange cartridge (4 ꢁ 1:5 cm) (BioRad, Rich-
mond, VA), which was previously equilibrated with the
protease assay buffer. The column was washed with
50 ml of the same buffer, and elution was done with
100 ml of a salt gradient of 0–1.0 M NaCl in the same
ꢀ
centrifugation at 15;700 ꢁ g for 2 h at 4 C. The cell-free
ꢀ
extract was stored at ꢂ20 C until use.
Protease assays. SP activity was determined by
measuring p-nitroaniline release from free or N-pro-
tected aminoacyl- or peptidyl-p-nitroanilides (Sigma,
Chemical, St. Louis, MO). The assay mixture (total
volume 820 ml) contained 720 ml assay buffer and 50 ml
of enzyme solution. After a 5 min preincubation at
ꢀ
5
5 C, the reaction was started by adding 50 ml of 10 mM
of each oligopeptidyl-p-nitroanilide dissolved in di-
methylsulfoxide. The absorbance of liberated p-nitro-
aniline was measured at 410 nm, continuously using
a UV 1601 A spectrophotometer (Shimadzu, Kyoto,
ꢂ1
buffer at a flow rate of 2 mlꢃmin . Fractions containing
ꢀ
Japan) during a 5 min incubation period, at 55 C. One
activity towards N-succinyl L-phenylalanine-p-nitroani-
lide (N-Suc-Phe-pNA) were pooled, desalted, and con-
centrated by ultrafiltration (5,000 cut-off ultrafiltration
device). Affinity chromatography was performed on ꢀ-
casein agarose column (5 ꢁ 1:2 cm) (Sigma), which was
equilibrated with 20 mM Tris–HCl, pH 8.5, containing
2 mM CaCl2 (Buffer A). Desalted and concentrated
active fractions from the previous step were applied to
the column and washed with excess Buffer A. The
bound protein was eluted from the column in two steps
using assay Buffer A plus 1 M NaCl (Buffer B), fol-
lowed by 25% (v/v) isopropanol in Buffer B, each at a
unit of peptidyl-p-nitroanilide-hydrolysing activity was
defined as the amount of enzyme that catalyzed the
formation of 1 mmol of p-nitroaniline (" ¼ 8800
410
ꢂ1
ꢂ1
cm ) per min.
M
The caseinolytic activity of the purified enzyme was
determined using casein as substrate, according to the
1
9)
method of Anson. The reaction mixture contained
7
00 ml of 1% (w/v) casein solution (prepared in protease
assay buffer, pH 7.5) and 100 ml of purified enzyme. The
assay was performed at 55 C in a time-dependent
ꢀ
manner from 0 to 20 min, and terminated by the addition
of 900 ml of 15% (w/v) trichloroacetic acid (TCA),
which was incubated at room temperature for 15 min.
The precipitate was removed by centrifugation at
ꢂ1
flow rate of 0.6 mlꢃmin . Absorbance at 280 nm and
protease activity of the fractions (each 1 ml) were
determined. Active fractions were pooled and concen-
trated by ultrafiltration using Centriprep-Microconcen-
trators equipped with a 5,000 cut-off membrane, and
1
5;000 ꢁ g for 10 min, and the supernatant was filtered
through Whatman no. 1 filter paper (Whatman Interna-
tional, Maidstone, UK). The absorbance at 275 nm was
measured. A control lacking the enzyme was included in
each assay. One unit of casein hydrolyzing activity was
defined as the amount of enzyme required to produce an
increase in absorbance at 275 nm of 0.01 OD under the
above-mentioned assay conditions. The proteolytic
activity was also determined using bovine serum
albumin (BSA) as substrate, following the method
ꢀ
stored in small aliquots at ꢂ20 C until use.
The amount of protein in the samples was calculated
2
1)
using the method described by Whitaker and Granum,
according to the following equation: protein concen-
tration ðmg/mlÞ ¼ ðA235 ꢂ A280Þ=2:51.
Effect of temperature and pH on enzyme activity. The
optimum temperature for the enzyme activity was
determined by assaying peptidolytic activity at different
2
0)
described by Foltman et al. The enzyme reaction
was carried out in a mixture containing 1 ml of 1%
ꢀ
temperatures over a range from 40 to 80 C. The assays
(
w/v) BSA, prepared in protease assay buffer, pH 7.5,
were performed using N-Suc-Phe-pNA as substrate, and
residual activity was expressed as a percentage of the
highest enzyme activity measured.
and 100 ml enzyme solution. The assay was performed at
5
ꢀ
5 C in a time-dependent manner from 0 to 60 min, and
the reaction was terminated by the addition of 3 ml of
5% (w/v) TCA. The resulting precipitate was removed
by centrifugation at 3;000 ꢁ g for 20 min. The absorb-
ance was read at 280 nm using a Shimadzu UV-1601A
spectrophotometer. One unit (U) of enzyme activity was
defined as the amount of enzyme required to produce an
To determine the effect of pH on purified protease,
peptidolytic activity was measured at different pH
values, using N-Suc-Phe-pNA as substrate under stand-
ard assay conditions. The pH was adjusted using one
of the following buffers (50 mM): 3-(N-morpholino)pro-
panesulfonic acid (MOPS) (pH 6.0–7.0), Tris–HCl
1

128
S. KOCABIYIK and I˙ . O¨ ZDEM˙IR
(pH 7.5–9.0), and glycine (pH 9.5–12.0), each contain-
ing 2 mM CaCl . The residual activity was expressed as a
Makowski and Rampsy.22) After electrophoresis, the
gels were washed twice in 2.5% TritonX-100 solution
for 30 min to remove SDS. The proteolytic reaction was
carried out in zymogram development solution contain-
ing 45% methanol, 10% acetic acid, and 0.1% Coomas-
sie Brilliant Blue R-250. Clear zones against a blue
background were observed after several washings in the
destaining solution (40% v/v methanol, 10% v/v acetic
acid, and 50% distilled water). Sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS–PAGE) was
carried out on 10% (w/v) acrylamide gel containing
2
percentage of the highest enzyme activity. The pH
stability of protease enzyme was determined by pre-
ꢀ
incubating the purified protease at 25 C in different
buffers (pH 6.0–12.0, 50 mM buffers as above) for 1 h.
The residual protease activities were then measured as
described above.
Effect of protease inhibitors and metal ions on enzyme
activity. The effects of various protease inhibitors, such
as phenylmethyl sulphonyl fluoride (PMSF), chymosta-
tin and tosyl-L-phenylalanyl-chloromethane (TPCK),
ethylenediamine tetraacetic acid (EDTA), ethylene
2
3)
0.5% (w/v) SDS, as described by Laemmli.
Results and Discussion
0
0
glycol bis (ꢁ-aminoethylether)-N,N,N ,N -tetraacetic
acid (EGTA), and reducing agent, 1,4 dithiothreitol
Purification of serine protease
(
activity of the purified protease were assessed. The
DTT) (all from Sigma) on the peptide hydrolyzing
The purification steps of the serine protease from
Tp. volcanium are summarized in Table 1. The crude
extract (27 ml) from 1 liter culture was subjected to 80%
ammonium sulfate precipitation. The precipitated en-
zyme sample was dissolved in protease assay buffer and
after desalting, applied to a column of anion exchanger.
Elution by a linear gradient of NaCl yielded two distinct
protein peaks (Fig. 1a). Proteolytic activity against
N-Suc-Phe-pNA was detected in fractions 117 to 134
covering the second protein peak. In this step a
purification fold of 3.6 with a yield of 27.5% and a
specific activity of 0.0039 U/mg were achieved.
2þ
2þ
2þ
2þ
metal ions tested were Ca , Mg , Mn , Ni , and
Co
2þ
(in chloride salts) at 4 mM concentration. The
protease enzyme (5 mg) was incubated with inhibitors
and metal ions for 15 min at room temperature. The
residual activity was determined as described before, by
measuring the N-Suc-Phe-pNA hydrolyzing activity, and
expressed as the percentage of the activity measured in
the absence of inhibitor and metal ion. The effects of
2
þ
2þ
Ca and Mg ions at different concentrations (in a
range 0.5–12.5 mM) on the protease activity of the
enzyme were also determined, as described above.
As shown in Fig. 1b, substrate affinity chromatogra-
phy on ꢀ-casein agarose, the enzyme was efficiently
separated from other proteins. This step increased the
specific activity to 0.0065 U/mg. The overall purifica-
tion was approximately 6-fold and the yield was 24%
(Table 1). A single distinct peak of protease was eluted
from the column with 1 M NaCl only in the presence, not
in the absence, of isopropanol. Therefore, like some
Kinetic measurements. Initial velocity assays were
performed by continuous spectrophotometric assay us-
ing N-Suc-Phe-pNA as substrate. The Km and Vmax
values of the purified protease were determined from a
Lineweaver–Burk plot generated from the initial reac-
tion velocities obtained with substrate concentration in a
range of 0.12–1.2 mM. Each assay was carried out in
2
4,25)
Bacillus serine proteases,
the binding of Tp. volca-
ꢀ
duplicate at 55 C in protease assay buffer, pH 7.5. The
nium protease to ꢀ-casein agarose must be due to a
hydrophobic affinity interaction. This phenomenon
correlates well with findings from the substrate specific-
ity study, which indicated that the enzyme attacks
hydrophobic amino acids residues such as phenylalanine
and alanine of various oligopeptidyl p-nitroanilide
substrates (Table 2).
Enzyme purified to homogeneity by two sequential
chromatographies migrated as a single band on SDS–
PAGE with an apparent molecular mass of 42 kDa
change in absorbance at 410 nm was monitored con-
tinuously, and the initial velocity was used for calcu-
lation of the kinetic constants.
Electrophoretic analysis. For activity detection by
zymogram staining, purified samples were electro-
phoresed in zymogram-resolving gel containing 7.5%
acrylamide, 0.1% sodium dodecly sulfate (SDS), and
0
.15% (w/v) co-polymerized gelatin, as described by
Table 1. Summary of Purification of Intracellular Serine Protease from Tp. volcanium
Total
protein
Specific
activity ꢁ10
Total
activity ꢁ10
(U)
Yield
%)
Purification
(fold)
ꢂ3
ꢂ3
Purification step
(
(
mg)
(U/mg protein)
Crude extract
NH4)2SO4 precipitation
133.55
18.98
9.72
1.084
2.014
3.865
6.450
136.62
37.62
37.55
35.50
100.00
27.54
27.48
23.70
1.0
1.9
3.6
6.0
(
Ion exchange chromatography
Affinity chromatography
5.50
Unit (U), The amount of enzyme that liberated 1 mmol of p-nitroaniline per min.

A New Intracellular Serine Protease from Thermophilic Archaebacterium Thermoplasma volcanium
129
a
3
,5
2
12
10
8
2
1
0
1
0
,5
1
6
4
.5
,5
0
2
0
1
11 21 36 51 66 81 94 103 113 123 129 139 149 159 169 183
Fraction number
b
B
Fraction number
Fig. 1. Purification of the Serine Protease from. Tp. volcanium.
a) Anion exchange chromatography on Econo Pack Q ion exchange column; (b) Affinity chromatography on ꢀ-casein agarose column. Arrow
(
A, application of 20 mM TrisꢃHCl (pH 8.5)-2 mM CaCl2 containing 1 M NaCl; Arrow B, the same buffer with the addition of 25% isopropanol.
Symbols: , absorbance at 280 nm; , proteolytic activity based on N-succinyl-L-phenylalanine-p-nitroanilide hydrolysis: –ꢃ–ꢃ–, NaCl. U, the
amount of enzyme that catalyzed the formation of 1 mmol of p-nitroaniline per min.
(
Fig. 2a). Zymogram activity staining also revealed one
Table 2. Hydrolysis of Various Chromogenic Synthetic Peptides and
Protein Substrates by the Purified Protease from Tp. volcanium
clear zone of proteolytic activity against a blue back-
ground (Fig. 2b). These gel analyses of the purified
enzyme indicate that it is a monomer. The molecular
mass of the purified protease as estimated by the native
gel was 35 kDa, smaller than that determined by SDS–
PAGE. This might be due to more compact structure of
the native protease, which has been proposed as a means
of thermostabilization for some thermophilic proteases
through tight packing of hydrophobic residues at the
Chromogenic substrates
Relative activity (%)
N-Succinyl-Phe-p-nitroanilide
Ala-Ala-Phe p-nitroanilide
100
55
54
51
38
35
24
17
L-Leu-p-nitroanilide
N-Succinyl-Ala-Ala-Ala p-nitroanilide
N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide
N-Succinyl-Ala-Ala-Val p-nitroanilide
N-CBZ-Arg-p-nitroanilide
2
6)
core of the molecule.
N-Succinyl-Ala-Ala-Val-Ala p-nitroanilide
Although the zymogram gel contained 0.1% SDS, the
enzyme still displayed activity, indicating that the
Tp. volcanium protease was resistant to SDS denatura-
tion. As reported earlier, SDS resistance is a property
often associated with heat-stable proteases of thermo-
Proteins
Casein
BSA
Activity
1.800 U
0.026 U
ꢄ
ꢄꢄ
ꢄ
U (Unit), the amount of enzyme required to produce an increase in
absorbance at 275 nm of 0.01 OD under the assay conditions.
2
7)
stable archaea and bacteria.
The purified enzyme proved to be extremely labile.
ꢄꢄ
U
equal to 0.01 OD under the assay conditions.
(Unit), the amount of enzyme required to produce an increase in A280

130
S. KOCABIYIK and I˙ . O¨ ZDEM˙IR
a
Protein
size
1
M
marker
90.6 kDa
6
4
6 kDa
5 kDa
42 kDa
29 kDa
b
1
M
Protein
size
marker
4
5 kDa
35 kDa
2
9 kDa
Fig. 2. Purified Serine Protease Activity of Tp. volcanium Visualized on SDS–PAGE (a) and Gelatin Zymogram (b).
Lane 1, purified protease; lane M, molecular weight markers: Urease (90.67 kDa), BSA (66 kDa), egg albumin (45 kDa), carbonic anhydrase
(29 kDa).

A New Intracellular Serine Protease from Thermophilic Archaebacterium Thermoplasma volcanium
131
Activity decreased dramatically on storage on ice, even
ꢀ
Effects of pH and temperature on activity and stability
The optimum pH of Tp. volcanium protease was
measured using N-Suc-Phe-pNA as substrate, in a series
of 50 mM buffers, as explained in ‘‘Materials and
Methods’’. The effect of pH on protease activity is
shown in Fig. 4. The optimum pH of the purified
protease was 7.0. The results indicate that more than
80% of the activity was retained at pHs between 6.0 and
8.0. The enzyme was quite stable in the pH range 7.0–
9.5. Although Tp. volcanium is an thermoacidophilic
archaeon, the purified protease has a neutral pH
optimum and is stable over an alkaline range of pH.
However, the intracellular enzymes from acidophilic
and alkaliphilic microorganisms need not be adapted to
extreme growth conditions, since these organisms are
capable of maintaining a neutral pH internally.
at ꢂ20 C for 24 h, but the enzyme in crude preparation
or 80% ammonium sulfate precipitate was relatively
more stable. This might be due to susceptibility of the
purified enzyme to autodigestion, as experienced with
1
2,28,29)
the other microbial SPs.
When kinetic analysis of the purified Tp. volcanium
protease reaction was performed using N-Suc-Phe-pNA
as substrate, the enzyme showed the classical Michae-
lis–Menten kinetics. The following kinetic constants
were obtained from the double reciprocal plots of the
initial reaction rates and substrate concentrations be-
ꢂ1
tween 0.12–1.2 mM: Km 2.2 mM, Vmax 0.045 mmolꢃml
ꢃ
ꢂ1
min
(Fig. 3). The kcat and kcat=Km values for N-
Suc-Phe-pNA were 0.0014 s and 6.55 Mꢂ1ꢃsꢂ1 respec-
ꢂ1
tively.
0.25
0.2
0
.1
0
-2
-1
0
1
2
3
4
5
-1
1
/S (mM )
Fig. 3. Lineweaver–Burk Plot Generated from the Initial Reaction Rates Obtained with N-Succinyl-L-phenylalanine-p-nitroanilide Concentrations
in the Range 0.12–1.2 mM and 5 mg of purified Tp. volcanium SP Per Assay.
1
1
20
00
80
60
40
20
0
5
6
7
8
9
10
11
12
13
pH
Fig. 4. Effect of pH on Proteolytic Activity of Tp. volcanium Serine Protease.
Activity was assayed by the hydrolysis of N-succinyl-L-phenylalanine-p-nitroanilide in 50 mM MOPS ( ), 50 mM Tris–HCl ( ), and 50 mM
Glycine buffer ( ) under standard conditions.

132
S. KOCABIYIK and I˙ . O¨ ZDEM˙IR
The effect of temperature on protease activity is
shown in Fig. 5. The optimum temperature for N-Suc-
position, and only very low tryptic activity was detected
using N-CBZ-L-Arginine-p-nitroanilide. According to
these results, the purified protease preferentially cata-
lyzes the hydrolysis of substrates with non-polar amino
acid residues (like L-Ala, L-Leu, and L-Phe) at the
P1 site.
ꢀ
Phe-pNA hydrolysis was 50 C, and activity decreased
markedly below it. A significant loss (about 40%) in
ꢀ
activity was observed at 60 C, the temperature optimum
for the growth of Tp. volcanium. This indicates that in
thermophilic archaea, some form of stabilization (e.g.,
by extrinsic stabilization factors, such as salts or
polyamines) of labile enzymes must occur in vivo to
Our results also show that Tp. volcanium protease was
active on natural substrates, BSA, and casein. (Table 2).
The enzyme more efficiently hydrolyzed casein than
3
0)
ꢂ1
make possible cellular functions at high temperatures.
BSA. The initial reaction rates (ꢀAꢃmin ) were 1.8 and
0.026 respectively.
Substrate profile of the protease
A series of oligonucleotidyl-p-nitroanilide substrates
with various P1 residues was used to test the substrate
specificity of Tp. volcanium protease. Table 2 summa-
rizes the relative activities of trypsin, chymotrypsin, and
elastase with different substrates associated with
Tp. volcanium protease. Tp. volcanium protease hydro-
lyzed the synthetic peptides preferentially at the carboxy
terminus of Phe or Leu, similarly to chymotrypsin-like
serine proteases. The enzyme was most active on N-Suc-
Phe-pNA. Therefore, the activities were expressed
relative to that of N-Suc-Phe-pNA. The protease
displayed hydrolytic activity against other substrates,
N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide, Ala-Ala-
Phe p-nitroanilide, and L-Leucine-p-nitroanilide, which
are also specific for chymotrypsin-like activity.
On the other hand, fairly high catalytic activity was
detected on substrates having Ala at the P1 position, as
well. The enzyme efficiently hydrolyzed N-Succinyl-
Ala-Ala-Ala p-nitro-anilide, which is a typical elastase
substrate. The other elastase substrates, N-Succinyl-Ala-
Ala-Val p-nitroanilide and N-Succinyl-Ala-Ala-Val-Ala
p-nitroanilide, were also hydrolyzed, but with relatively
low efficiency. But the enzyme was not effective in
hydrolysis of the substrate with the basic residue at P1
Effects of inhibitors and divalent cations on protease
activity
Table 3 shows the effects of various protease inhib-
itors and divalent cations (at 4 mM) on the activity
Tp. volcanium protease. Enzyme activity towards N-
Suc-Phe-pNA was strongly inhibited by the SP inhib-
itors PMSF, TPCK, and chymostatin. The reducing
2
þ
agent DTT and the Mg chelator, EGTA had no effect
on the enzyme activity, but 25% of the activity was lost
in the presence of the divalent cation chelator EDTA.
The fact that EDTA up to a concentration of 10 mM did
not lead to complete inhibition of enzyme activity means
that this enzyme is not a metalloprotease.
2
þ
2þ
The presence of Ca and Mg in the assay mixture
enhanced the peptide hydrolyzing activity by 2.6- and
2þ
2þ
2.3-fold respectively. Similarly, Mn and Co were
also shown to have a stimulatory effect on activity, but
Ni ions exhibited an inhibitory effect, which resulted
in a 27% decrease in activity. The stimulatory effects of
2þ
2þ
2þ
Ca and Mg were concentration-dependent, and over
a range of 2–12 mM the highest level of enzyme activity
was observed (Fig. 6). This result show that Tp. volca-
2
þ
nium SP requires Ca ion for activity as other microbial
3,31–33)
1
SPs.
Ions chelated by subtilases at either high or
1
1
20
00
8
6
4
2
0
0
0
0
0
20
30
40
50
60
70
o
Temperature ( C)
Fig. 5. Effect of Temperature on Proteolytic Activity of Tp. volcanium Serine Protease.
Activity was based on N-succinyl-L-phenylalanine-p-nitroanilide hydrolysis.

A New Intracellular Serine Protease from Thermophilic Archaebacterium Thermoplasma volcanium
133
4
3
2
1
00
00
00
00
0
0
2
4
6
8
10
12
14
Concentrations (mM)
Fig. 6. The Effect Ca2
þ
(
) and Mg
2þ
(
The enzyme assays were carried out in the presence of different concentrations of Ca and Mg , and the activity in the absence of Ca and
) Concentration on Proteolytic Activity.
2þ
2þ
2þ
Mg2 was taken as 100%.
þ
Our research on the possible association of the serine
protease we have isolated with predicted serine pro-
teases of Tp. volcanium is still in progress.
Table 3. Effect of Proteinase Inhibitors and Divalent Cations on the
Activity of Tp. volcanium Serine Protease
Remaining
Concentration
activity (%)
Acknowledgments
No Inhibitor
0
100.0
This work was supported by grants from the Turkish
Scientific and Technical Research Council (TBAG-
Inhibitors:
TPCK
1 mM
400 mM
2 mM
0
4.5
1
927/100T038) and the Graduate School of Natural and
Applied Sciences of Middle East Technical University
AFP-2000-07-02-00-17).
Chymostatin
PMSF
EDTA
5.5
10 mM
10 mM
75.0
94.0
(
EGTA
Reducing agent:
DTT
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