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

Sequence analysis suggests that the peptidase consists of a prepro region, a catalytic domain and two C-terminal domains.. In the present work, the Serratia peptidase SPRK is compared wi

proteinase K-like enzyme from a psychrotrophic Serratia sp. Atle Noralf Larsen1, Elin Moe1,2, Ronny Helland2, Dag Rune Gjellesvik3and Nils Peder Willassen1,2

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 kcat at the tem-perature range 12–37
C, but a fivefold higher Km results in a slightly lower catalytic efficiency (kcat⁄ Km) of SPRK compared to PRK

Abbreviations

AQUI, aqualysin I; PMSF, phenylmethylsulphonyl fluoride; PRK, proteinase K; SPRK, Serratia sp peptidase; VPRK, Vibrio sp PA44

peptidase.

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

thermophiluscells [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 temperatempera-ture rarely exceeds 4
C Enzymes from microorganisms living in such harsh environment show in general a higher catalytic effi-ciency (kcat⁄ Km) 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 (kcat) might be a more valid approach for adaptation to cold than optimization of kcat⁄ Kmsince the contribution of

Kmbecomes 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

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 WalkerTM 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.

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.

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 sugbrid-ges-

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 coliTOP10 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)

Fig 3 SDS ⁄ PAGE (4–12% Bis-Tris) of purified SPRK Lanes 1 and 4: SeeBluestandard (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).

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

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
C and 50
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.

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 kcat (cat-alytic activity) and kcat⁄ Km (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
C and

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)

kinetic parameters of SPRK and PRK are shown in

Table 3 Serratia sp peptidase had a 3.5–4.5 fold

higher kcat at all temperatures tested On the other

hand, SPRK had a fivefold higher Km (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

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 Atlimport-antic 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 disuldisul-fide 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

kcat Km kcat⁄ K m kcat Km kcat⁄ K m

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]

Serratiasp 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, kcat

(catalytic activity) and Km (substrate binding),

between the two peptidases were observed Serratia

sp peptidase had a much higher kcat (3.5–4.5 fold)

than PRK at the moderate temperatures tested

(12
C, 22
C and 37
C), and the difference in kcat

between the two enzymes increased slightly with

increasing temperature Serratia sp peptidase

exhib-ited a much higher Km 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 kcat than its mesophilic homologue subtilisin Carlsberg, but has on the other hand a higher Km, 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 kcat and kcat⁄ Km 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 kcat and

Km 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 WalkerTMkit 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

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

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

WalkerTM 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:

OP6,

5¢-GATGAAAATCCTAACCTCTCCCCCGCACAG-3¢; OP7, 5¢-ACTGCACCTACGGCGGGTCGTTGGTACG

TG-3¢; NP4, 5¢-GACACCGTAGGTTGAGCCGCCAATC

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 (NH4)2SO4,

2 mm MgSO4, 0.1% Triton X-100, 0.1 mgÆmL)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 (NH4)2SO4,

2 mm MgSO4, 0.1% Triton X-100, 0.1 mgÆmL)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, ALFexpressTM DNA Sequencer and ALFwin Sequence Analyser version 2.10 according to the manufacturer’s instructions Gels were made with ReprogelTM 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 mm 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 (OD600)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