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

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

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

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-

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-

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

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-

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

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

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

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

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

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

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

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

(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 (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

)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

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

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