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Tài liệu Báo cáo khoa học: Characterization of a recombinantly expressed proteinase K-like enzyme from a psychrotrophic Serratia sp. ppt

Characterization of a recombinantly expressed
proteinase K-like enzyme from a psychrotrophic Serratia sp.
Atle Noralf Larsen
1
, Elin Moe
1,2
, Ronny Helland
2
, Dag Rune Gjellesvik
3
and Nils Peder Willassen
1,2
1 Department of Molecular Biotechnology, Institute of Medical Biology, Faculty of Medicine, University of Tromsø, Norway
2 The Norwegian Structural Biology Centre, University of Tromsø, Norway
3 Biotec Pharmacon ASA, Tromsø, Norway
Serine endo- and exo- peptidases are widespread in
nature and found in viruses, archaea, bacteria and euk-
aryotes. The biological importance of peptidases are
clearly indicated by the fact that 2% of all genes
encode peptidases (or their homologues) in all kinds of
organisms [1]. Extracellular peptidases hydrolyse large

proteins into smaller peptides for absorption by the
cell, whereas intracellular peptidases play a major role
in regulation of metabolism [2].
The families of chymo(trypsin) (S1) and subtilisin (S8)
are regarded as the largest families of serine peptidases
[1]. The two families share a similar arrangement of the
catalytic triad, the Asp, His and Ser residues, but display
a totally different protein fold where the subtilisin clan
has an a ⁄ b-fold and the (chymo)trypsin clan a b ⁄ b-fold.
More than 600 members of the subtilisin-superfamily
(S8 family) are currently known according to the MER-
OPS peptidase database (http://merops.sanger.ac.uk/).
Siezen and Leunissen (1997) subdivided the subtilisin-
like serine peptidases or subtilases into six families based
on sequence homology, where the subtilisin and protein-
ase K are examples of family representatives.
Keywords
Bioprospecting; proteinase K like;
psychrotrophic; Serratia sp; stability
Correspondence
N. P. Willassen, Department of Molecular
Biotechnology, Institute of Medical Biology,
University of Tromsø, N-9037 Tromsø,
Norway
Tel: +47 77 64 46 51
Fax: +47 77 64 53 50
E-mail: nilspw@fagmed.uit.no
(Received 8 September 2005, revised 26
October 2005, accepted 31 October 2005)
doi:10.1111/j.1742-4658.2005.05044.x
The gene encoding a peptidase that belongs to the proteinase K family of
serine peptidases has been identified from a psychrotrophic Serratia sp.,
and cloned and expressed in Escherichia coli. The gene has 1890 base pairs
and encodes a precursor protein of 629 amino acids with a theoretical
molecular mass of 65.5 kDa. Sequence analysis suggests that the peptidase
consists of a prepro region, a catalytic domain and two C-terminal
domains. The enzyme is recombinantly expressed as an active  56 kDa
peptidase and includes both C-terminal domains. Purified enzyme is con-
verted to the  34 kDa form by autolytic cleavage when incubated at
50 °C for 30 min, but retains full activity. In the present work, the Serratia
peptidase (SPRK) is compared with the family representative proteinase K
(PRK) from Tritirachium album Limber. Both enzymes show a relatively
high thermal stability and a broad pH stability profile. SPRK possess
superior stability towards SDS at 50 °C compared to PRK. On the other
hand, SPRK is considerably more labile to removal of calcium ions. The
activity profiles against temperature and pH differ for the two enzymes.
SPRK shows both a broader pH optimum as well as a higher temperature
optimum than PRK. Analysis of the catalytic properties of SPRK and
PRK using the synthetic peptide succinyl-Ala-Ala-Pro-Phe-pNA as sub-
strate showed that SPRK possesses a 3.5–4.5-fold higher k
cat
at the tem-
perature range 12–37 °C, but a fivefold higher K
m
results in a slightly
lower catalytic efficiency (k
cat
⁄ K
m
) of SPRK compared to PRK.
Abbreviations
AQUI, aqualysin I; PMSF, phenylmethylsulphonyl fluoride; PRK, proteinase K; SPRK, Serratia sp. peptidase; VPRK, Vibrio sp. PA44
peptidase.
FEBS Journal 273 (2006) 47–60 ª 2005 The Authors Journal compilation ª 2005 FEBS 47
The proteinase K family is a large family of secreted
endopeptidases found in fungi, yeast and Gram-negative
bacteria, where especially the bacterial enzymes show
a high degree of sequence identity (> 55%) [3]. The
bacterial endopeptidases in this family are probably
synthesized as prepro enzymes along with a C-terminal
extension beyond the catalytic domain as reported for
some of these enzymes [4–6]. Proteinase K from the
fungus Tritirachium album Limber (PRK) is also pro-
duced as a prepro enzyme but lacks the C-terminal
extension [7]. The prepeptide functions as a signal pep-
tide and is cleaved off after translocation of the protein
through the membrane [8–11]. The pro-peptide probably
functions as an intramolecular chaperone to ensure
the proper folding of the enzyme and is cleaved off
by autolysis to give the fully active enzyme [12]. The
C-terminal extension might be involved in extracellular
secretion as reported for aqualysin I (AQUI) in Thermus
thermophilus cells [13,14].
Two 3D structures of peptidases have been deter-
mined from the proteinase K family, and includes PRK
[15] and a peptidase from a psychrotrophic Vibrio sp.
PA44 (VPRK) [16]. Disulfide bridges may contribute
to the overall stability of proteins, and both PRK and
AQUI of this family have been described to contain two
disulfide bridges in different positions [15,17]. VPRK
contains three disulfide bridges according to the struc-
ture, where the two first disulfide bridges are located in
the same position as suggested for AQUI. The third
disulfide bridge present in the VPRK structure is located
in the C-terminal part of the enzyme.
The subtilisin-like peptidases are dependent on cal-
cium to maintain their stability, and PRK contains
two calcium binding sites, one strong (Ca1) site and
one weak (Ca2) site [15]. VPRK possesses three cal-
cium binding sites, where one corresponds to Ca1 in
PRK, one corresponds to the medium site in thermi-
tase [18] whereas the third site is new and not identi-
fied in other subtilases so far [16].
PRK possesses a broad substrate specificity, but pre-
fers to cleave peptide bonds after aliphatic and aroma-
tic amino acids [19,20]. PRK is reported to be very
stable even in presence of denaturants like urea and
SDS. Cleavage of protein substrates by PRK is in fact
stimulated by SDS [21]. The enhanced activity in the
presence of SDS is probably due to denaturation of
the protein substrate which in turn leads to increased
accessibility for cleavage. Because of these features,
PRK is typically used in procedures for inactivation of
RNases and DNases during nucleic acid extraction
[22,23].
Bioprospecting has become increasingly important in
order to search for interesting genes, biomolecules and
organisms from the marine environment with features
that might be of commercial interest. The polar marine
regions are characterized by their stabile low tempera-
ture where the sea temperature rarely exceeds 4 °C.
Enzymes from microorganisms living in such harsh
environment show in general a higher catalytic effi-
ciency (k
cat
⁄ K
m
) and lower stability against tempera-
ture or pH than enzymes from microorganisms
adapted to warmer climate. For enzymes that are
secreted, and often submitted to high substrate concen-
tration, an optimization of the catalytic activity (k
cat
)
might be a more valid approach for adaptation to cold
than optimization of k
cat
⁄ K
m
since the contribution of
K
m
becomes negligible at high substrate concentrations
[24]. VPRK is the only peptidase from the proteinase
K family that has been isolated and characterized from
a psychrotrophic or psychrophilic source [4,25]. This
peptidase showed the typical characteristics of enzymes
adapted to cold by having an increased catalytic effi-
ciency (and catalytic activity) and lower thermal stabil-
ity compared to related mesophilic and thermophilic
counterparts.
Bioprospecting of marine microorganisms in coastal
seawater in Northern Norway resulted in a large col-
lection of diverse cold adapted bacteria that serves as
a basis for exploration of different enzymatic activities
for industrial or biotechnological use. In this paper we
present a serine peptidase of the proteinase K family
isolated from a psychrotrophic bacterium originating
from this bioprospecting, and we report some of its
properties compared to the commercially available and
mesophilic PRK.
Results
Bioprospecting in coastal waters in Northern Norway
resulted in a large collection of cold adapted (psychro-
philic and psychrotrophic) bacteria. The bacterial
strains were isolated and cultivated at 4 °C, and the
API ZYM system (BioMerieux, Paris, France) was
chosen in order to study the enzymatic activities
originating from these strains (unpublished data).
One of the marine bacteria showing peptidase activ-
ity was closely related to Serratia proteamaculans of
the Serratia genus belonging to the Enterobacteriaceae
based on 16S rDNA analysis. The bacterium does not
grow at 37 °C, but grows well below 30 °C indicating
psychrotrophic nature.
Identification and analysis of the peptidase gene
Degenerate primers were constructed on the basis of
multiple sequence alignment of proteinase K-like
Characterization of a Serratia proteinase K-like enzyme A. N. Larsen et al.
48 FEBS Journal 273 (2006) 47–60 ª 2005 The Authors Journal compilation ª 2005 FEBS
enzymes from Gram-negative bacterial sources, and
the codon usage in the sequences from Vibrio alginolyt-
icus [26] and Alteromonas sp.O7 [5] were taken into
account. A  200-bp fragment was generated by PCR
and the sequence of this fragment was used for con-
struction of PCR primers for genome walking (Gen-
ome Walker
TM
Kit, Clontech, Palo Alto, CA, USA).
By using several different primers described in Experi-
mental procedures and genome walking on the differ-
ent restriction enzyme ‘libraries’, the full length
sequence of the Serratia sp. peptidase (SPRK) gene
was identified and found to be 1890 bp long, encoding
a protein of 629 amino acids with a theoretical
molecular mass of 65.5 kDa. The nucleotide sequence
and deduced amino acid sequence is shown in Fig. 1.
The peptidase sequence can be divided into a 22-resi-
due presequence, a  100–105 residue pro-sequence, a
catalytic domain of  280 residues and two C-terminal
Fig. 1. Nucleotide sequence and deduced
amino acid sequence of the Serratia sp.
gene encoding the precursor form of the
peptidase. The catalytic residues Asp (D),
His (H) and Ser (S) are bolded; N-terminal
residues of the catalytic domain are under-
lined. The preregion is indicated in red, the
pro-region in black, catalytic domain in blue
and the C-terminal domains in violet. The
assumed start of the second C-terminal
domain is indicated with a black arrow.
A. N. Larsen et al. Characterization of a Serratia proteinase K-like enzyme
FEBS Journal 273 (2006) 47–60 ª 2005 The Authors Journal compilation ª 2005 FEBS 49
domains (repeated sequences) that are  220–225 resi-
dues long (including linker-region between the catalytic
and C-terminal domains) as indicated in Fig. 1. Data-
base searches revealed that the deduced amino acid
sequence showed high identity to other enzymes in the
proteinase K family of serine peptidases, especially
with sequences from Gram-negative bacterial sources.
Sequences from cold adapted as well as sequences of
mesophilic and thermophilic origin are included.
Figure 2A shows a multiple sequence alignment gener-
ated by clustalx [27] of some of these sequences
belonging to the proteinase K family, and the number-
ing in this alignment is used throughout the Results
and Discussion. In addition to the mesophilic family
representative, PRK from the fungus T. album [7],
sequences from Alteromonas sp. O7 [5], V. alginolyticus
[26] and V. cholera [28] (mesophilic), T. aquaticus [6]
(thermophilic), Pseudoalteromonas sp. AS11 (Genebank
Fig. 2. (A) Multiple alignment of the full length peptidase sequences from Serratia sp. (SPRK), Pseudoalteromonas sp. AS-11, Alteromonas
sp. O-7, Vibrio sp. PA44, V. alginolyticus, V. cholera, Thermus aquaticus aqualysin I (AQUI) and Tritirachium album proteinase K (PRK). Blue
is 100% sequence identity, red is 80–99% while green is 60–79% sequence identity. The catalytic domain from position 145 to 429. (B)
Multiple alignment of the C-terminal sequences from Serratia sp. (SPRK), Pseudoalteromonas sp. AS-11, Alteromonas sp. O-7, Vibrio sp.
PA44, V. alginolyticus, V. cholera, T. aquaticus (AQUI) belonging to the proteinase K family of serine peptidases. In addition, C-terminal
sequences of zinc metolloproteases from V. cholera S01, Helicobacter pylori, V. anguillarum, V. vulnifucus and V. parahaemolyticus are
included. Blue is 100% sequence identity, red is 80–99% while green is 60–79% sequence identity. Both alignments are generated using
ClustalX.
Characterization of a Serratia proteinase K-like enzyme A. N. Larsen et al.
50 FEBS Journal 273 (2006) 47–60 ª 2005 The Authors Journal compilation ª 2005 FEBS
accession number: BAB61726) and Vibrio sp. PA44 [4]
(cold-adapted) are included.
The catalytic domain is well conserved, especially
the sequences around the catalytic triad (D183, H216
and S373). There are three disulfide bridges present in
the VPRK structure [16]. The two first disulfide brid-
ges observed in VPRK are in agreement with sugges-
tions made for AQUI [17], and are formed between
C213-C245 and C314-C345. Serratia sp. peptidase pos-
sesses cysteines in the equivalent sequence positions as
VPRK and AQUI, hence these disulfide bridges are
probably present in SPRK. PRK has its disulfide brid-
ges positioned elsewhere (C178-C270, C325-C399) [15].
Based on Fig. 2A, one or two extended C-terminal
region(s) of the peptidase sequences are common
within the bacterial subgroup of the proteinase K fam-
ily. Database search on the second C-terminal domain
(CII) of SPRK revealed that this region shows > 43%
sequence identity with C-terminal region(s) in most of
the other sequences in this alignment. The exception
is the sequence from T. aquaticus which only shows
about 15% identity. In addition, several sequences of
metallopeptidases originating from pathogenic organ-
isms have a C-terminal region showing > 43%
sequence identity with CII of SPRK. Figure 2B shows
a multiple alignment of the C-terminal sequences ori-
ginating from peptidase sequences of the proteinase K
family along with some of the metallopeptidase
sequences.
Expression and purification
The gene encoding SPRK was cloned into the pBAD ⁄
gIII vector (Invitrogen) for recombinant expression in
Escherichia coli TOP10. The presequence of SPRK was
not included in the construct as a signal sequence is
provided in this vector. Small-scale expression was
compared at 37 °C, 30 °C and 22 °C, but peptidase
activity could only be detected at 22 °C. Large-scale
expression was therefore performed at 22 °C.
The purification of SPRK includes ion exchange,
hydrophobic interaction chromatography and gel filtra-
tion and the scheme is summarized in Table 1. Serratia
sp. peptidase was purified approximately sixfold with
a total yield of  0.7 mg. Serratia sp. peptidase is
expressed as a  56-kDa protein, but after purification
five bands at 56, 45, 34, 28.5 and 22 kDa appear when
analysing the purified sample by SDS ⁄ PAGE as shown
in Fig. 3 (lane 3). The purified sample was incubated
with 1 mm (final concentration) phenylmethylsulfonyl
fluoride (PMSF) to inhibit autolytic degradation prior
to analysis on SDS ⁄ PAGE. If the peptidase sample was
not treated with PMSF during preparation for electro-
phoresis, the major band observed in the gel corres-
ponds to the 34-kDa protein (Fig. 3, lane 2). No
proteins above this size could be observed, although
some weak degradation products could be detected.
Molecular characteristics
Some characterized bacterial enzymes in the proteinase
K family that have a C-terminal extension have pre-
viously been shown to include several bands on a
SDS ⁄ PAGE gel after purification [5,25], as seen with
SPRK (Fig. 3, lane 3). Conversion of the enzyme
sample from the  56-kDa protein to the 34-kDa
protein readily took place when incubating the enzyme
at 50 °C (Fig. 4). No decrease in enzyme activity,
Table 1. Purification scheme of SPRK expressed in E. coli.
Step
Volume
(ml)
Activity
(U ⁄ ml)
Protein concentration
(mg ⁄ mL)
Total
activity (U)
Total
protein (mg)
Specific activity
(U ⁄ mg)
Yield
(%)
Purification
(fold)
Periplasmic fraction 435 1.23 0.08 534 34.4 15.5 100 1
Q-sepharose 160 2.52 0.15 403 23.8 16.9 75 1.1
Phenyl-seph. 45 5.61 0.26 252 11.7 21.5 47 1.4
Source 15Q 22.5 8.82 0.16 198 3.7 54 37 3.5
Superdex 75 1.5 41.33 0.46 62 0.7 88.6 12 5.7
Fig. 3. SDS ⁄ PAGE (4–12% Bis-Tris) of purified SPRK. Lanes 1 and
4: SeeBlue
Ò
standard (Invitrogen); Lane 2: Purified SPRK without
addition of PMSF prior to SDS ⁄ PAGE analysis; Lane 3: Purified
SPRK (PMSF inhibited); Lane 5: Heat treated (50 °C) and purified
SPRK (PMSF inhibited).
A. N. Larsen et al. Characterization of a Serratia proteinase K-like enzyme
FEBS Journal 273 (2006) 47–60 ª 2005 The Authors Journal compilation ª 2005 FEBS 51
however, was observed during incubation (results not
shown). Based on the results shown in Fig. 4, together
with analysis of other enzymes in the same family;
Alteromonas sp. O-7 [5], Vibrio sp. PA44 [4,25] and
T. aquaticus [29], we suggest that the bands at
 56 kDa and  45 kDa refer to a peptidase form
including two C-terminal domains and one C-terminal
domain, respectively. The protein band at  34 kDa
refers to the ‘mature’ peptidase containing the catalytic
domain only.
To verify the experiment shown in Fig. 4, and to
obtain the ‘mature’ form of the peptidase, a periplas-
mic extract of SPRK was submitted to the same purifi-
cation procedure as described previously with one
major exception: the concentrated sample (3 mL) was
heated to 50 °C for 30 min before application on a
Superdex 75 (2.6 ⁄ 60) column. Figure 3, lane 5 shows
the SDS ⁄ PAGE after gel filtration (the sample was
treated with PMSF as described previously). One
single band corresponding to a protein of  34 kDa
was present in the gel. As conversion to the 34-kDa
protein or ‘mature’ form readily took place at 50 °C,
only the ‘mature’ form of SPRK was used during
further characterization experiments.
Stability
The pH stability of SPRK and PRK was compared by
preincubating the enzymes for 24 h at 22 °C in buffers
of different pH. PRK was stable in the pH range from
pH 4 to 12, while SPRK had optimal stability in the
range from pH 5.5 to 9.5 (Fig. 5). Temperature stabil-
ity was measured by preincubating SPRK and PRK at
temperatures ranging from 4 to 80 °C in 15 min. PRK
was slightly more stable than SPRK, and had a half-
life of 30 min at 70 °C while SPRK had a half-life of
19 min at this temperature. Stability of SPRK and
PRK towards SDS was measured by preincubating the
enzymes with various concentrations of SDS (0.1, 0.25,
0.5 and 1.0%) at 37 °C and 50 °C for 30 min, and the
results are shown in Fig. 6. At 37 °C there were no sig-
nificant difference between the two enzymes, both hav-
ing  90% residual activity even in presence of 1%
SDS. Significant differences in stability between the
two enzymes appeared at 50 °C. Serratia sp. peptidase
still had 90% residual activity in the presence of 1%
SDS, while PRK only had  19%. Stability of SPRK
and PRK towards EDTA was tested by preincubating
the enzymes at 37 °C and 50 °C for 120 min, and
the results are shown in Fig. 7. At 37 °C, PRK is
Fig. 4. Processing of the purified SPRK. SDS ⁄ PAGE (4–12% Bis-
Tris) showing the effect of incubation at 50 °C on the apparent
molecular mass. PMSF is added to a final concentration of 1 m
M at
each time point to inhibit enzyme activity. Lane 1: SeeBlue
Ò
stand-
ard; Lane 2–8: Enzyme sample heated to 50 °C in time intervals
ranging from 0 to 45 min.
Fig. 5. pH stability of SPRK and PRK. Enzymes were preincubated
for 24 h at 22 °C at various pHs. One hundred percent activity
refers to the pH value with highest activity. (r), SPRK; (n), PRK.
Fig. 6. Stability of SPRK and PRK towards SDS at 37 °Cand50°C.
The enzymes were preincubated for 30 min in buffer containing
0.1%, 0.25%, 0.5% and 1% SDS. One hundred percent activity
refers to enzyme samples incubated at the selected temperatures
during the experiments without SDS present. (
), SPRK 37 °C; ( ),
PRK 37 °C; (
), SPRK 50 °C; ( ), PRK 50 °C.
Characterization of a Serratia proteinase K-like enzyme A. N. Larsen et al.
52 FEBS Journal 273 (2006) 47–60 ª 2005 The Authors Journal compilation ª 2005 FEBS
unaffected by the presence of EDTA, while SPRK had
 60% residual activity. At 50 °C, SPRK was totally
inactivated after 120 min, while PRK retained  50%
residual activity.
pH and temperature optimum
The pH optimum for activity of SPRK and PRK was
determined by measuring the enzyme activity towards
suc-Ala-Ala-Pro-Phe-pNA at different pH values. Ser-
ratia sp. peptidase had a broad pH optimum with the
highest activity in the range pH 8–11, and an apparent
optimum at pH 10.5; PRK had the highest activity in
the range pH 8–9.5, and an apparent optimum at pH
8 (Fig. 8).
The temperature optimum was determined to be
70 °C for SPRK, and 55 °C for PRK (Fig. 9). Protein-
ase K exhibits a broad optimum with > 90% activity
in the temperature range 40–70 °C.
Effect of SDS and EDTA on activity
The effect of SDS on activity of SPRK and PRK was
measured by addition of 0.1, 0.25, 0.5 and 1.0% SDS
(final concentrations) in the standard assay buffer.
Both enzymes were inhibited by addition of SDS dur-
ing activity measurements, and showed  30% of the
maximum activity in presence of 1% SDS (Table 2).
The effect of EDTA on activity was measured by
including EDTA (10 mm) in a calcium-free assay buf-
fer. EDTA had no inhibitory effect on the activity of
the enzymes (Table 2).
Kinetics
To investigate if there were any differences in k
cat
(cat-
alytic activity) and k
cat
⁄ K
m
(catalytic efficiency)
between SPRK and the mesophilic PRK, kinetic
experiments using the synthetic substrate suc-Ala-Ala-
Pro-Phe-pNA was performed at 12, 22 and 37 °C. The
Fig. 7. Stability of SPRK and PRK towards EDTA at 37 °Cand
50 °C. Enzymes were incubated at the selected temperatures in
calcium free buffers containing 10 m
M EDTA, and sampled after
15, 30, 45, 60, 90 and 120 min. One hundred percent (0 min) resid-
ual activity refers to enzyme sample incubated on ice. (r), SPRK
37 °C; (n), PRK 37 °C; (d), SPRK 50 °C; (m), PRK 50 °C.
Fig. 8. pH optimum of SPRK and PRK. Enzyme assay was per-
formed at 22 °C in different buffers from pH 5.5–11, and activity
towards Suc-Ala-Ala-Pro-Phe-pNA was measured. One hundred per-
cent activity refers to the pH value with the highest activity. (r),
SPRK; (n), PRK.
Fig. 9. Temperature optimum for activity of SPRK and PRK.
Enzyme assay was performed in the temperature range of
20–75 °C. One hundred percent activity refers to the temperature
value with the highest activity. (r), SPRK; (n), PRK.
Table 2. Effect of SDS and EDTA on activity for SPRK and PRK at
22 °C.
Inhibitor Concentration
SPRK (% relative
activity)
PRK (% relative
activity)
0.10% 85 87
SDS 0.25% 67 77
0.50% 49 56
1.00% 30 32
EDTA 10 m
M 100 100
A. N. Larsen et al. Characterization of a Serratia proteinase K-like enzyme
FEBS Journal 273 (2006) 47–60 ª 2005 The Authors Journal compilation ª 2005 FEBS 53
kinetic parameters of SPRK and PRK are shown in
Table 3. Serratia sp. peptidase had a 3.5–4.5 fold
higher k
cat
at all temperatures tested. On the other
hand, SPRK had a fivefold higher K
m
(lower binding
affinity) leading to a slightly lower catalytic efficiency
at the selected temperatures compared to PRK.
Discussion
Based on 16S rDNA sequencing, the gene encoding
a PRK-like serine peptidase was isolated from a bac-
terial strain most closely related to S. proteamaculans
of the Serratia genus. The gene was found to be
1890 bp long, encoding a precursor protein of 629
amino acids with a theoretical molecular mass of
65.5 kDa. The deduced amino acid sequence of the
peptidase gene revealed that the peptidase consists of
an N-terminal prepro sequence, a catalytic domain
and two C-terminal domains (repeated sequences).
The presequence (22 residues) helps to guide the pro-
tein into the periplasmic space [10], while the pro-
sequence ( 100–105 residues) assist the peptidase to
achieve its correct folding [12]. The catalytic domain
consists of  280 residues. The function of the C-ter-
minal domains ( 220–225 residues) in SPRK is
unknown, but may be necessary for extracellular
secretion as reported for AQUI in T. thermophilus
cells [13,14]. It has also been suggested that the
C-terminal pro-sequence may play a role in translo-
cation across both the cytoplasmic and outer mem-
branes [30]. In the case of the psychrotrophic Vibrio
peptidase (VPRK), the wild-type enzyme was secre-
ted into the medium as a 47-kDa peptidase with the
C-terminal domain intact, and was converted to the
36-kDa ‘mature’ form by mild heat treatment [25].
Furthermore, VPRK has also been recombinantly
expressed in E. coli and showed similar molecular
characteristics to those of the wild-type enzyme [4].
The C-terminal region (CII) of SPRK shows > 43%
sequence identity compared to the corresponding
region of the bacterial members in the PRK family
compared here (Fig. 2B). The only exception is the
C-terminal region of AQUI which has  15%
identity with the other sequences, although its cata-
lytic domain has 60% sequence identity. Database
searches using CII from SPRK revealed an interest-
ing feature as several metallopeptidase also showed
> 43% sequence identity with CII of SPRK.
Recently, it has been shown that a metallopeptidase
from V. anguillarum with a similar C-terminal region
(C-terminal sequence is shown in Fig. 2B) is import-
ant for virulence in Atlantic salmon [31]. In addition,
the C-terminal domain of a metallopeptidase from
V. vulnificus with > 50% sequence identity to CII of
SPRK, is shown to be essential for efficient attach-
ment to protein substrates or erythrocyte membranes
[32]. The question arises why peptidases from the
bacterial subgroup of the PRK family and the metal-
loproteases have one similar C-terminal domain or
two (repeated) domains as seen for peptidase sequen-
ces from Alteromonas sp. O7, Pseudoalteromonas sp.
AS11 and the Serratia sp.? From the information
discussed above one might speculate that the C-ter-
minal domains of SPRK could have an additional
function than that reported for AQUI, and may
function in attaching the peptidase to cellular surfa-
ces or protein substrates.
Disulfide bridges may contribute to the overall sta-
bility of proteins, and some peptidases of this family
are known to contain cysteine residues involved in
disulfide bridges. Proteinase K and AQUI are both
described to have two disulfide bridges, but at differ-
ent positions in the structure [15,17], whereas the
VPRK structure revealed the presence of three disul-
fide bridges [16]. One or more of the disulfide bonds
is shown to be essential for maintaining the active
conformation of VPRK, since cleavage of the disul-
fides lead to inactivation of the enzyme [25]. The
first two disulfide bonds in VPRK are the same as
suggested for AQUI and should also be present in
SPRK. Based on the sequence alignment in Fig. 2A,
it seems that all members of the bacterial subgroup
contain these two disulfide bonds. Attempts to stabil-
ize the cysteine-free subtilisin BPN¢ by introducing
disulfide bridges in structurally analogous positions
to those in PRK showed no stabilizing effect [33].
However, stabilizing subtilisin E by introducing an
S–S bond (positioning C213–C245 compared to
Fig. 2A) was successfully performed using AQUI as
a template molecule [34]. This introduction did not
affect the catalytic efficiency of the enzyme, and may
therefore be a suitable target for site-directed muta-
genesis in order to create a more temperature labile
SPRK.
The two peptidases PRK and SPRK possess both
high thermal and pH stability. Proteinase K was stable
Table 3. Kinetic parameters for the hydrolysis of suc-AAPF-pNA at
12 °C, 22 °C and 37 °C for SPRK and PRK.
Substrate
SPRK PRK
k
cat
K
m
k
cat
⁄ K
m
k
cat
K
m
k
cat
⁄ K
m
S-AAPF-pNA
12
°C 175 2,36 74 51 0,48 106
S-AAPF-pNA
22
°C 364 2,46 148 88 0,46 191
S-AAPF-pNA
37
°C 827 2,72 304 180 0,52 346
Characterization of a Serratia proteinase K-like enzyme A. N. Larsen et al.
54 FEBS Journal 273 (2006) 47–60 ª 2005 The Authors Journal compilation ª 2005 FEBS
over the whole pH range tested from pH 4–12 and had
a half-life of 30 min at 70 °C, while SPRK possessed
highest stability from pH 5.5)9.5 and had a half-life of
19 min at 70 °C. An interesting feature was the differ-
ence in stability between the two peptidases toward
SDS and EDTA (Fig. 6 and 7). Serratia sp. peptidase
was clearly more stable against SDS at 50 °C, but
showed stability similar to that of PRK at 37 °C. Pro-
teinase K is, on the other hand, significantly more sta-
ble towards EDTA at both 37 °C and 50 °C,
indicating that SPRK is more dependent on calcium
for stability.
It was difficult to get reproducible pH optimum
measurements for PRK in different 0.1 m buffers. Nev-
ertheless, the results indicate that SPRK possesses a
broader (and higher) pH optimum for activity than
PRK (Fig. 8). Interestingly, SPRK also showed a
higher temperature optimum for activity (Fig. 9). Pro-
teinase K has a broad temperature optimum with only
minor difference in activity in the temperature range
from 40–70 °C. PRK has also previously been des-
cribed to have a broad temperature optimum profile
with more than 80% of the maximum activity in the
range of 20–60 °C (with an apparent optimum at
37 °C) [35]. Serratia sp. peptidase shows the same tem-
perature and similar pH optimum for activity as repor-
ted for the peptidase from Alteromonas sp. O-7 [5].
Serratia sp. peptidase and PRK were unaffected by the
presence of EDTA, while the activity of both were
inhibited in the presence of SDS (Table 2). Protein-
ase K has previously been demonstrated to exhibit
similar effects of EDTA and SDS on activity when act-
ing on small substrates [20,21].
No significant differences in pH or temperature sta-
bility ⁄ optimum were found between purified samples
of the unprocessed (56 kDa) and processed (34 kDa)
SPRK (data not shown); this is in accordance with
analysis performed with the peptidase from the Vibrio
sp. PA44 [25].
Significant differences in the kinetic parameters, k
cat
(catalytic activity) and K
m
(substrate binding),
between the two peptidases were observed. Serratia
sp. peptidase had a much higher k
cat
(3.5–4.5 fold)
than PRK at the moderate temperatures tested
(12 °C, 22 °C and 37 °C), and the difference in k
cat
between the two enzymes increased slightly with
increasing temperature. Serratia sp. peptidase exhib-
ited a much higher K
m
at the same temperatures
(fivefold), leading to a slightly lower catalytic effi-
ciency in SPRK. Similar effects have been observed
in subtilisin S39 from the psychrophilic Antarctic
Bacillus TA39 when hydrolysing the substrate suc-
FAAF-pNA. The psychrophilic enzyme shows
twofold higher k
cat
than its mesophilic homologue
subtilisin Carlsberg, but has on the other hand a
higher K
m
, leading to a more or less preserved cata-
lytic efficiency [36]. These results differ somewhat
from the characterization of the psychrotrophic
VPRK that possesses both higher k
cat
and k
cat
⁄ K
m
ratio at moderate (15–45 °C) temperatures compared
to mesophilic (PRK) and thermophilic (AQUI) coun-
terparts [25].
To elucidate the differences in stability and activity
between SPRK and PRK, a high-resolution structure
of SPRK is needed. The catalytic domain of SPRK
has been crystallized and the crystal structure was
compared with the already known structure of PRK
and will be published in an accompanying paper in
FEBS [37].
Since there were significant differences in k
cat
and
K
m
between the two enzymes, kinetic studies to iden-
tify possible differences in the substrate-binding region
will be initiated. This knowledge will further be used
in redesign of SPRK to yield an enzyme with higher
catalytic efficiency and lower temperature stability.
Experimental procedures
Materials
The Genome Walker
TM
kit was from Clontech (Palo Alto,
CA, USA). Restriction enzyme NcoI was from New Eng-
land Biolabs (Beverly, MA, USA). Escherichia coli TOP10
[F- mcrA n(mrr-hsdRMS-mcrBC) u80lacZnM15 nlacX74
deoR recA1 araD139 n(araAleu)7697 galU galK rpsL
endA1 nupG] and expression vector pBAD ⁄ gIII were from
Invitrogen (Carlsbad, CA, USA). Q-Sepharose FF, Phenyl
sepharose FF, Hi-Prep Desalting, Source 15Q and Super-
dex 75 were from Amersham Biosciences (Uppsala,
Sweden). Suc-Ala-Ala-Pro-Phe-pNA and PRK were from
Sigma Aldrich (St. Louis, MO, USA) and Finnzymes
(Espoo, Finland), respectively.
16SrDNA sequencing
Bacterial genomic DNA was purified by using Qiaquick
DNA purification kit (Qiagen, Germany) according to
manufacturer’s protocol. Polymerase chain reaction was
performed with 50 ng template DNA, 0.2 mm dATP,
dCTP, dGTP and dTTP, 0.2 lm upstream primer (5¢-AGA
GTTTGATCMTGGCTCAG-3¢) and downstream primer
(5¢-GGTTACCTTGTTACGACTT-3¢) and 1 U Taq poly-
merase (Promega). PCR amplification was carried out at
95 °C for 5 min, 30 cycles of 95 °C for 30 s, 53 °C for 30 s
and 72 °C for 1 min, and a final extension step of 72 °C for
7 min.
A. N. Larsen et al. Characterization of a Serratia proteinase K-like enzyme
FEBS Journal 273 (2006) 47–60 ª 2005 The Authors Journal compilation ª 2005 FEBS 55
Isolation of genomic DNA from Serratia sp.
Genomic DNA was isolated as described by Chen and Kuo
[38] for use in identification of the peptidase gene.
Generation of an  200-bp fragment of the
peptidase gene
Polymerase chain reaction was carried out in a final vol-
ume of 50 lL containing 1 ng of bacterial genomic DNA
as template, 10 mm Tris ⁄ HCl pH 9.0 (25 °C), 50 mm
KCl, 0.1% Triton X-100, 0.2 mm dATP, dCTP, dGTP
and dTTP, 0.4 lm upstream primer (5¢-GACTGTAA
CGGTCATGGYACMAYGT-3¢) and downstream primer
(5¢-CCGCCACCCAAACTCATRTTRGC-3¢) and 1.5 U
Taq-polymerase (Promega). PCR-amplification was per-
formed at 94 °C for 7 min, 30 cycles at 94 °C for 30 s,
60 °C for 80 s and 2 min at 72 °C, and a final extension
step at 72 °C for 5 min.
Full length gene identification
Genomic DNA was treated according to the Genome
Walker
TM
kit manual (Clontech) with four different blunt
end restriction enzymes; EcoRV, DraI, PvuII and SspI each
giving rise to a genome walking ‘library’. The following gene
specific primers were used to obtain the full length sequence:
OP5, 5¢-GACTGTAACGGTCATGGYACMAYGT-3¢;
OP6, 5¢-GATGAAAATCCTAACCTCTCCCCCGCACAG-
3¢; OP7, 5¢-ACTGCACCTACGGCGGGTCGTTGGTACG
TG-3¢; NP4, 5¢-GACACCGTAGGTTGAGCCGCCAATC
GTCCC-3¢; NP5, 5¢-CTTTAACTTGTTGGGCACTGG
CATTG-3¢; NP6, 5¢-TTGATCGATTCTGTCTATGCCC
CA-3¢ along with the adaptor primers: AP1 (5¢-GTAATAC
GACTCACTATAGGGC-3¢) and AP2 (5¢-ACTATAGGG
CACGCGTGGT-3¢).
Nested PCR was carried out in a final volume of 50 lL
containing 1 lL of a genome walking ‘library’ in 20 mm
Tris ⁄ HCl pH 8.8 (25 °C), 10 mm KCl, 10 mm (NH
4
)
2
SO
4
,
2mm MgSO
4
, 0.1% Triton X-100, 0.1 mgÆ mL
)1
nuclease
free BSA, 0.2 mm dATP, dCTP, dGTP and dTTP, 0.2 lm
gene specific primer and adaptor primer and 1 U Pfu-poly-
merase (Promega). PCR-amplification was done at 94 °C for
2 min, 7 cycles at 94 °C for 30 s, 55 °C for 30 s and 4 min at
72 °C, 30 cycles at 94 °C for 30 s, 50 °C for 30 s and 4 min
at 72 °C and a final extension step at 72 °C for 5 min. The
final product of this first PCR reaction (1 lL) was used as
template in a secondary or nested PCR reaction in 20 mm
Tris ⁄ HCl pH 8.8 (25 °C), 10 mm KCl, 10 mm (NH
4
)
2
SO
4
,
2mm MgSO
4
, 0.1% Triton X-100, 0.1 mgÆ mL
)1
nuclease
free BSA, 0.2 mm dATP, dCTP, dGTP and dTTP, 0.2 lm
gene specific primer and adaptor primer and 1 U Pfu-poly-
merase (Promega) and 94 °C for 2 min, 30 cycles of 94 °C
for 30 s, 55 °C for 1 min and 4 min at 72 °C and a final
extension step of 72 °C for 5 min.
Construction of expression vector
The peptidase gene lacking the first 66 bp (encoding the pre-
sequence) was cloned into pBAD ⁄ gIII B expression vector
(Invitrogen). PCR was performed in 50 lL containing 1 ng
of genomic DNA as template, 0.2 mm dATP, dCTP, dGTP
and dTTP, 0.2 lm of upstream primer (OP17: 5¢-GA
AAAACCATGGTGAATGAATACCAAGCGACT-3¢ ) and
downstream primer (NP7: 5¢-CAATCTCCATGGCTAG
TAGCTTGCACTCAG-3¢) containing a NcoI restriction site
and 1 U of Pfu-polymerase. PCR amplification was carried
out at 94 °C for 5 min, 30 cycles at 94 °C for 30 s, 60 °C for
1 min and 3 min at 72 °C and a final extension step at 72 °C
for 5 min. PCR products were purified using Qiaquick PCR
Purification Kit (Qiagen), digested with 10 U NcoI (New
England Biolabs), ligated into NcoI digested pBAD ⁄ gIII B
expression vector using T4-DNA-ligase and transformed into
competent TOP10 E. coli cells.
DNA sequencing
DNA sequencing was performed with the Amersham Phar-
macia Biotech Thermo Sequenase Cy5 Dye Terminator Kit,
ALFexpress
TM
DNA Sequencer and ALFwin Sequence
Analyser version 2.10 according to the manufacturer’s
instructions. Gels were made with Reprogel
TM
Long Read
and Reproset UV-polymerizer. All items were from Amer-
sham Biosciences (Uppsala, Sweden).
Expression and fermentation of SPRK in E. coli
Small-scale expression was performed at 37, 30 and 22 °C
in 1-L baffled shake flasks containing 100 mL Luria–
Bertani (LB) medium with 20 m m glucose and 50 lgÆmL
)1
ampicillin. A 10-mL preculture of E. coli TOP10 pBAD ⁄
gIIIB containing the SPRK gene was used as inoculum,
and induced with 0.1% arabinose. Fermentation was per-
formed in a 15-L Chemap CF 3000 fermentor (Switzer-
land). A 200-mL preculture of E. coli TOP10 pBAD ⁄ gIIIB
containing the SPRK gene was inoculated to 7 L of 2· LB-
medium supplemented with 20 mm glucose and 50 lgÆmL
)1
ampicillin. Cells were grown until no glucose could be
detected (OD
600
)2.5). Gene expression was induced by
0.1% arabinose and cells were grown further for 12 h at
22 °C. Cells were harvested and centrifuged at 5000 g for
15 min at 4 °C.
Purification of SPRK
Bacterial cell pellet was resuspended in 10% of the ori-
ginal volume (700 mL from 7 L culture) in 20% sucrose,
0.1 m Hepes, 1 mm EDTA. Freshly made lysozyme was
added to a final concentration of 0.5 mgÆmL
)1
. The solu-
tion was incubated 30 min at 22 °C, and centrifuged for
Characterization of a Serratia proteinase K-like enzyme A. N. Larsen et al.
56 FEBS Journal 273 (2006) 47–60 ª 2005 The Authors Journal compilation ª 2005 FEBS
30 min at 5000 g at 4 °C. The supernatant contained the
periplasmic fraction of the proteins. CaCl
2
was added to
a final concentration of 10 mm before freezing at )20 °C.
Periplasmic fraction (450 mL) was filtrated through glass-
wool, and then applied to a Q-Sepharose FF (5.0 ⁄ 10,
50 mm in diameter and 10 cm in height) equlibrated in
buffer A (25 mm Hepes pH 8.0, 1% glycerol, 10 mm
CaCl
2
). The column was washed with three column vols
(CV) of buffer A and the proteins were eluted over four
CV isocratic 30% buffer B (buffer A + 1.0 m NaCl) and
isocratic 100% buffer B over two CV. Fractions contain-
ing peptidase activity were pooled. Ammonium sulphate
(80 mL, 3 m) was added to the pooled Q-Sepharose frac-
tion (160 mL), and applied to a phenyl sepharose FF
(high substituted, 1.6 ⁄ 10) column. The column was equli-
brated with buffer C (25 mm Hepes pH 8.0, 10 mm
CaCl
2
, 1% glycerol, 1 m ammonium sulphate). The col-
umn was washed with three CV of buffer C, and pro-
teins were eluted using two isocratic steps; 25% buffer A
over seven CV and 100% buffer A over seven CV. The
peptidase containing fractions collected in the last step
were pooled. The pooled fraction (45 mL) after phenyl
sepharose was applied to a Hi-Prep Desalting (26 ⁄ 10)
column equlibrated in buffer A. The protein fraction
after the desalting step (62.5 mL) was applied to a
Source 15Q (2.6 ⁄ 3.5) equlibrated with buffer A. The
column was washed with three CV of buffer A, and
bound proteins were eluted with a linear gradient of
0–100% buffer B over 10 CV. Fractions containing pepti-
dase activity were pooled. Using an Amicon Ultra
(15 mL, Millipore) the Source 15Q fraction (22.5 mL)
were concentrated to 2.2 mL, and applied to the Super-
dex 75 (2.6 ⁄ 60) column equlibrated with buffer A +15%
buffer B. Fractions containing peptidase activity were
pooled.
Protein determination
Protein concentrations were determined with Bio-Rad Pro-
tein Assay based on the method of Bradford [39] and
according to the microtiter plate protocol as described by
the manufacturer using BSA as standard.
Standard enzyme assay
SPRK activity was routinely measured using Suc-Ala-Ala-
Pro-Phe-pNA (Sigma Aldrich) as substrate. The peptidase
assay was carried out in a total volume of 250 lL, contain-
ing 10 lL enzyme solution and 240 lL of standard assay
buffer (0.1 m Tris ⁄ HCl pH 8.0, 10 mm CaCl
2
, 1% DMSO,
1mm substrate) for 2 min at 22 °C. Activity was measured
by increase in absorbance at 405 nm (e ¼ 8480 m
)1
Æcm
)1
)
using the V
max
kinetic microplate reader (Molecuar Devices
Co., CA). One unit of enzyme hydrolyses 1 lmol of sub-
strate per minute at 22 °C.
SDS ⁄ PAGE
SDS ⁄ PAGE was performed using The Nupage
Ò
Pre-Cast
gel system (Invitrogen) according to manufacturer’s proto-
col. Gels were stained with SimplyBlue
TM
SafeStain (Invi-
trogen). The standard used is SeeBlue
Ò
Pre-Stained Protein
Standard (Invitrogen). Enzyme samples were treated with
1mm (final concentration) of the serine peptidase inhibitor
phenylmethylsulphonyl fluoride (PMSF) for 30 min at
room temperature prior to analysis on 4–12% NuPAGE
Ò
Novex Bis-Tris Gels. The effect of temperature on auto-
catalytic cleavage was performed using purified SPRK incu-
bated at 50 °C. Samples were taken after 1, 5, 10, 15, 30
and 45 min and analysed by SDS ⁄ PAGE. Approx. 3 lgof
protein sample was added to each well.
pH and temperature stability
The effect of pH on stability was determined by preincubat-
ing SPRK (5 lgÆmL
)1
) and proteinase K (PRK,
10 lgÆmL
)1
) for 24 h at 22 °C in the following buffers
containing 10 mm CaCl
2
:25mm sodium acetate (pH 4.0–
5.5), 25 mm Mes ⁄ NaOH (pH 5.5–6.5), 25 mm Mops ⁄ -
NaOH (pH 6.5–7.5), 25 mm Tris ⁄ HCl (pH 7.5–8.5), 25 mm
diethanolamine ⁄ HCl (pH 8.5–9.5), 25 mm piperazine ⁄ HCl
(pH 9.5–10), 25 mm glycine ⁄ NaOH (pH 10–12). The effect
of temperature on stability was determined by incubating
SPRK (5 lgÆmL
)1
) and PRK (10 lgÆmL
)1
) at tempera-
tures ranging from 4 to 80 °C for 15 min in 25 mm Hepes
pH 8.0, 10 mm CaCl
2
. The samples were kept on ice prior
to and after incubation. Temperature stability at fixed
temperature (70 °C) was also performed to estimate the
half-life. Residual activities towards Suc-AAPF-pNA were
determined under standard assay conditions. One hundred
percent activity refers to samples kept on ice during the
whole experiment.
Stability against SDS and EDTA
The effect of SDS on the stability of SPRK (3 lgÆmL
)1
)
and PRK (7.5 lgÆmL
)1
) was determined by incubating
the enzymes in 25 mm Hepes, 1 mm CaCl
2
pH 8.0 at 37 °C
and 50 °C for 30 min containing various SDS concentra-
tions (0.1, 0.25, 0.5 and 1%). Residual activities towards
Suc-AAPF-pNA were determined using standard assay con-
ditions. One hundred percent activity refers to enzyme sam-
ples incubated at the selected temperatures without SDS
present. The effect of EDTA on the stability of SPRK
(3 lgÆmL
)1
) and PRK (7.5 lgÆmL
)1
) was determined by
incubating the enzymes in 25 mm Hepes pH 8.0, 10 mm
EDTA at 37 °C and 50 °C and samples were collected after
15, 30, 60, 90 and 120 min. At each time point, the sample
was incubated on ice for 5 min and remaining activity
toward Suc-AAPF-pNA was determined. One hundred
percent activity (0 min) is sample incubated on ice for
A. N. Larsen et al. Characterization of a Serratia proteinase K-like enzyme
FEBS Journal 273 (2006) 47–60 ª 2005 The Authors Journal compilation ª 2005 FEBS 57
5 min before measuring activity under standard assay
conditions.
pH and temperature optimum
The effect of pH on the activity of SPRK (3.75 lgÆmL
)1
)
and PRK (7 lgÆmL
)1
) towards 1 mm Suc-AAPF-pNA
was determined at 22 °C using the following buffers con-
taining 1% (v ⁄ v) DMSO and 10 mm CaCl
2
: 0.1 m sodium
acetate ⁄ HCl (pH 4.0–5.5), 0.1 m Mes ⁄ NaOH (pH 5.5–6.5),
0.1 m Mops ⁄ NaOH (pH 6.5–7.5), 0.1 m Tris ⁄ HCl (pH 7.5–
8.5), 0.1 m diethanolamine ⁄ HCl (pH 8.5–9.5), 0.1 m pipera-
zine ⁄ HCl (pH 9.5–10), 0.1 m glycine ⁄ NaOH (pH 10–12).
One hundred percent activity refers to the pH value with
highest measured activity. Temperature optimum was per-
formed using a thermostatted Perkin Elmer Lamda15
uv ⁄ vis Spectrophotometer. The peptidase assay was carried
out in a total volume of 500 lL, containing 20 lL diluted
enzyme solution (in 25 mm Hepes, 10 mm CaCl
2
pH 8.0)
and 480 lL of 0.1 m Tris ⁄ HCl, 0.0005% Triton X-100,
10 mm CaCl
2
and 0.5% DMSO (pH adjusted to 8.0 for all
temperatures). The temperature range used was 20–75 °C,
and the activity of SPRK (0.75 lgÆmL
)1
) and PRK
(1 lgÆmL
)1
) towards 0.5 mm and 0.1 mm Suc-AAPF-
pNA, respectively, was measured at the selected tempera-
tures.
Effect of SDS and EDTA on activity
The effect of SDS on the activity of the two peptidases
(3 lgÆ mL
)1
SPRK; 7.5 lgÆmL
)1
PRK) was determined
using 0.1, 0.25, 0.5 and 1.0% SDS (final concentrations)
in the standard assay buffer, and activity towards Suc-
AAPF-pNA was measured using standard assay condi-
tions. One hundred percent activity refers to enzyme
samples assayed using standard conditions without SDS.
The effect of EDTA on activity was determined using
10 mm EDTA in the standard assay buffer without CaCl
2
present. Enzyme samples were diluted 2500-fold in 25 mm
Hepes pH 8.0 without CaCl
2
to a concentration of
3 lgÆmL
)1
(SPRK) and 7.5 lgÆmL
)1
(PRK) before activity
towards Suc-AAPF-pNA was measured. Enzyme diluted
in Hepes buffer containing 1 mm CaCl
2
and standard
assay conditions without EDTA was used as controls,
and refers to 100% activity.
Kinetic studies
Hydrolysis of the p-nitroanilide derivative Suc-AAPF-pNA
was determined at 405 nm. All assays were performed in
0.1 m Tris ⁄ HCl pH 8.0, 0.0005% (v ⁄ v) Triton X-100, 5%
(v ⁄ v) DMSO at 12, 22 and 37 °C (pH adjusted for each
temperature). Substrate concentration was in the range
from 0.05 mm to 5 mm with minimum eight different
concentrations. The parameters K
m
and k
cat
were estimated
by nonlinear regression analysis to fit the Michaelis–
Menton equation by using the SigmaPlot Kinetic module
(Systat Software Inc., Richmond, CA). When K
m
values
reached the upper limit for the substrate concentrations,
also manual linear plots such as Eadiee–Hofstee and
Hanes–Woolf were used as verification of the nonlinear
regression analysis.
Database searches and prediction
blast (http://www.ncbi.nlm.nih.gov/blast/) was used to
search for related protein sequences of SPRK. N-terminal
presequence was predicted by Signal P [10].
Acknowledgements
The present study was supported by the national
Functional Genomics Programme (FUGE) in The
Research Council of Norway and Biotec Pharmacon
ASA. We wish to thank Jonas Jakobsen and Sigmund
Sperstad for technical assistance.
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