salmonicida EndA shows typical cold-adapted features such as lower unfolding temperature, lower temperature optimum for activity, and higher specific activity than V.. Enzyme properties T
Trang 1Vibrio salmonicida and mesophilic Vibrio cholerae
Bjørn Altermark1, Laila Niiranen2, Nils P Willassen1,2, Arne O Smala˚s1and Elin Moe1
1 Norwegian Structural Biology Centre, Faculty of Science, University of Tromsø, Norway
2 Department of Molecular Biotechnology, Faculty of Medicine, University of Tromsø, Norway
The marine and estuarine environment harbors a vast
diversity of bacteria Some of the most extensively
studied marine or estuarine bacteria belong to the
genus Vibrio, with Vibrio cholerae being the most
notorious species as it is the cause of cholera in
humans V cholerae is found in tropical and
temper-ate areas, and can be classified as a mesophilic
bac-terium with growth optimum around 37C It prefers
estuarine waters, is halotolerant, and does not require
NaCl for growth [1,2] The bacterium with one of the
lowest growth optimum temperatures found in the
genus Vibrio is the fish pathogen Vibrio salmonicida
It has an optimal growth temperature of 15 C and
requires NaCl for growth [3] It can therefore be
classified as a psychrophilic and mildly halophilic
bacterium
A living cell can be considered as a chemical factory which produces many substances The speed of pro-duction is limited by reaction rates The reaction rates are in turn limited by, among other things, environ-mental factors such as pH, salinity, pressure and tem-perature Temperature is a very important factor for growth and proliferation of the cells At high tempera-tures, at which thermophiles thrive, chemical reaction rates are very high, and the main challenge for cells is
to adapt their enzymes, membranes and molecules to cope with the heat At low temperatures, the chemical reaction rates are lower, and hence, in order to be competitive and grow fast at low temperatures, evolu-tionary pressure favors enzymes that are more efficient than their high-temperature counterparts This higher efficiency at low temperatures is believed to be caused
Keywords
cold adaptation; endonuclease I;
psychrophilic enzymes; salt adaptation;
stability
Correspondence
E Moe, Norwegian Structural Biology
Centre, Faculty of Science, University of
Tromsø, N-9037 Tromsø, Norway
Fax: +47 77644765
Tel: +47 77646473
E-mail: elin.moe@chem.uit.no
(Received 14 September 2006, revised
2 November 2006, accepted 9 November
2006)
doi:10.1111/j.1742-4658.2006.05580.x
Endonuclease I is a periplasmic or extracellular enzyme present in many different Proteobacteria The endA gene encoding endonuclease I from the psychrophilic and mildly halophilic bacterium Vibrio salmonicida and from the mesophilic brackish water bacterium Vibrio cholerae have been cloned, over-expressed in Escherichia coli, and purified A comparison of the enzy-matic properties shows large differences in NaCl requirements, optimum
pH, temperature stability and catalytic efficiency of the two proteins The
V salmonicida EndA shows typical cold-adapted features such as lower unfolding temperature, lower temperature optimum for activity, and higher specific activity than V cholerae EndA The thermodynamic activation parameters confirm the psychrophilic nature of V salmonicida EndA with
a much lower activation enthalpy The optimal conditions for enzymatic activity coincide well with the corresponding optimal requirements for growth of the organisms, and the enzymes function predominantly as DNases at physiological concentrations of NaCl The periplasmic or extra-cellular localization of the enzymes, which renders them constantly exposed
to the outer environment of the cell, may explain this fine-tuning of bio-chemical properties
Abbreviations
DSC, differential scanning calorimetry; VcEndA, recombinant Vibrio cholerae endonuclease I; VsEndA, recombinant Vibrio salmonicida endonuclease I.
Trang 2by a more flexible structure, and the increased
flexi-bility is thought to be the reason for the lower
thermo-stability of cold-adapted enzymes [4]
Endonuclease I is a periplasmic or extracellular
enzyme known to cleave both RNA and DNA at
unspecific internal (endo) sites It also cleaves plasmids
and single-stranded DNA [5] The enzyme cleaves at
the 3¢ side of the phosphodiester bond, leaving
prod-ucts with 5¢ phosphate ends A histidine functions as a
general base, which activates a water molecule for an
in-line attack on the scissile phosphate in the DNA
substrate The role of the active-site magnesium ion is
to stabilize the phosphoanion transition state and
make a proton available for the 3¢-oxygen leaving
group, via a bound water molecule An arginine is
believed to stabilize the product via a hydrogen bond
to the phosphate, which also decelerates the reverse
reaction [6] A chloride atom is found buried in the
structure of V cholerae endonuclease I and probably
also in the Vibrio vulnificus endonuclease I structure
[7]
Orthologues of endonuclease I from many bacterial
species are described in the literature [5,8–13], but
there seems to be an uncertainty about the main
func-tion of this enzyme in vivo It is well known for its
ability to reduce the level of transformation [14–16],
but appears to have no effect on conjugation [5] The
enzyme is shown not to be involved in the
patho-genicity of V cholerae [15], V vulnificus [5] or Erwinia
chrysanthemi[17] Most of the bacteria that harbor the
gene live in close contact with eukaryotic hosts, which
may provide nutritious DNA and RNA through their
mucus barriers The mucus itself becomes less viscous
if the DNA is broken down, and this may facilitate the
movement of the bacterium through the mucus layer
[15] The enzyme is reported to be constitutively
expressed in V vulnificus [5] and Erwinia chrysanthemi
[18]
Here we report the cloning, expression and purifica-tion of the endonuclease I enzymes from the psychro-phile V salmonicida (VsEndA) and the mesopsychro-phile
V cholerae (VcEndA) The two orthologous enzymes have been biochemically and biophysically character-ized to reveal possible differences related to environ-mental adaptation
Results Sequence similarity and composition VsEndA and VcEndA show 71% identity and 80% similarity (Blosum62) at the amino acid level, when the active enzymes are compared without their N-terminal signal peptide A sequence alignment of VcEndA and VsEndA is shown in Fig 1 The first two amino acids
at the N-terminus (Thr and Met) are encoded by the expression vector
An analysis of the amino-acid composition shows that VsEndA contains 13 more lysines and two fewer arginines than VcEndA, resulting in a very high R⁄ K ratio for the mesophilic enzyme (1.6 versus 0.6, respectively) In addition VsEndA contains two less
D + E than VcEndA However all the cysteines involved in disulfide bridge formation in VcEndA are also found in the sequences of VsEndA (Fig 1) The theoretical pI was 9.61 for VsEndA and 8.62 for VcEndA
Expression and purification From 7 L Escherichia coli culture, a total of 24 and
50 mg pure recombinant VsEndA and VcEndA pro-teins, respectively, were obtained (Fig 2) The final NaCl concentration after cation-exchange chromato-graphy was estimated to 0.8 m for VsEndA and 0.65 m for VcEndA
Fig 1 Sequence alignment showing the amino acids of VsEndA and VcEndA Numbers indicate cysteines involved in disulfide bridges; stars indicate Mg 2+ -co-ordinating residues, triangles indicate the catalytically important His80 and Arg99, and squares indicate Cl)-co-ordinating residues The sequence numbering is according to the structure of V vulnificus endonuclease I in complex with a DNA octamer, PDB id 1OUP [6].
Trang 3The calculated molecular masses were 25.0 and
24.7 kDa for VsEndA and VcEndA, respectively,
which is in agreement with the results from the
SDS⁄ PAGE analysis shown in Fig 2
Enzyme properties
To find the optimal buffer conditions for the
biochemi-cal characterization of the enzymes, we carried out an
analysis of the NaCl requirements and pH optimum
of VsEndA and VcEndA The optimum NaCl
concen-trations for activity were found to be 425 mm for
VsEndA and 175 mm for VcEndA, respectively
(Fig 3)
The optimum pH for activity of VsEndA and VcEndA was 8.5–9.0 and 7.5–8.0, respectively, when measured in Tris⁄ HCl and diethanolamine ⁄ HCl buffers
as shown in Fig 4 The pH optimum was unaffected
by the NaCl concentration in the buffers When tested
in glycine buffer at pH 9.0, the enzymes showed very low activity compared with that in diethanolamine and Tris buffers at the same pH, indicating that glycine inhibits the enzymes VcEndA activity decreases stee-ply below pH 6.5 (measured in Bis-Tris buffer, data not shown)
The optimum temperature for activity was deter-mined using a modified Kunitz assay The results showed optimum activity at 45 C for VsEndA and
50C for VcEndA, as shown in Fig 5
Kinetic constants for VsEndA and VcEndA were measured by incubating the enzymes in the presence of substrate with different concentrations and at different temperatures The kinetic constants for the two enzymes at 5, 15, 25, 30 and 37C are shown in
200 116.3 97.4 66.3 55.4
36.5 31.0 21.5 14.4
6.0 3.5
Fig 2 SDS ⁄ PAGE Lane 1, Mark12 MW ladder; lane 2, 5 lg
VcEndA; lane 3, 5 lg VsEndA The relative molecular masses of
the standard are shown on the left.
0
25
50
75
VcEndA
[NaCl] (m M )
Fig 3 Optimum NaCl concentration for DNase activity DNaseAlert
was used as substrate, and activity was measured in increasing
amounts of NaCl Each replicate is plotted and the mean values are
drawn.
pH optimum VsEndA
A
0.0 0.5 1.0 1.5 2.0 2.5
3.0
Tris DEA
pH
1.0 1.5 2.0 2.5
DEA
pH
Fig 4 Optimum pH for activity (A) VsEndA; (B) VcEndA Buffers used are 75 m M Tris ⁄ HCl, pH 7–9, and 75 m M diethanolamine ⁄ HCl,
pH 8–10 DNaseAlert was used as a substrate in the assay Each replicate is plotted and the mean values are drawn.
Trang 4Table 1 VsEndA possesses a higher kcatthan VcEndA
at all temperatures, and the Km values of VcEndA are
slightly lower than for VsEndA at all temperatures
The physiological efficiency is highest for VsEndA, but the difference decreased with concomitant increase in temperature
As determined from Arrhenius plots, the energy
of activation (Ea) is 35.7 kJÆmol)1 for VsEndA and 76.3 kJÆmol)1 for VcEndA The calculations of the enthalpy (DH#) and entropy (DS#) of activation revealed much lower values for VsEndA as shown in Table 2 Also note that TDS#values for VsEndA were negative, whereas those from VcEndA were positive (Table 2) Temperature stability was analyzed by evaluating thermal unfolding using differential scanning calorime-try (DSC) The results revealed a Tm of 44.8C for VsEndA and 52.8C for VcEndA as shown in Fig 6 The calorimetric enthalpy (area under the transition) is also much lower for VsEndA (328 kJÆmol)1) than for VcEndA (480 kJÆmol)1)
The rate of irreversible unfolding was analyzed by incubating both enzymes at 70C Samples were removed after 10 min and incubated for 1 h on ice
0 10 20 30 40 50 60
0
25
50
75
VcEndA
Temperature ( °C)
Fig 5 Optimum temperature for activity The enzymes were
assayed using the modified Kunitz assay Each replicate is plotted
and the mean values are drawn.
Table 1 Kinetic constants for VsEndA and VcEndA at 5, 15, 25, 30 and 37 C.
Table 2 Activation energy parameters were calculated (kJÆmol)1) for the psychrophilic VsEndA (p) and mesophilic VcEndA (m) The differ-ences in values (p ) m) is also shown.
T
Trang 5before being assayed Figure 7 shows that the rate
of irreversible unfolding for VsEndA is higher than
for VcEndA, with a half-life of 13 and 33 min,
respectively
Substrate specificity analysis
An analysis of the substrate specificity for DNA of the
enzymes shows that they both cleave plasmid DNA,
dsDNA and ssDNA as shown in Fig 8
To test the RNA specificity of the enzymes, we used
the RNaseAlert assay and compared the results with
those obtained using the DNaseAlert assay VsEndA
has over 900-fold higher preference for DNA than
RNA when measured in buffer with NaCl
concentra-tion optimal for DNase activity The RNase activity is inhibited in the presence of NaCl as shown in Fig 9, and at 425 mm NaCl the VsEndA is predominantly a DNase The VcEndA shows the same trend, with very low RNase activity at NaCl concentration optimal for DNase activity
Discussion The choices of enzyme orthologues, and their phylo-genetic relationship, which has been investigated in order to elucidate the cold-adapted properties of the enzymes, have previously been criticized [19] Here, orthologue monomeric enzymes from species within the same genus are studied to minimize other adapta-tional strategies that may have affected these enzymes differently When the amino acid compositions of VsEndA and VcEndA are compared, a remarkably low R⁄ K ratio in VsEndA is found In addition, there
is a slight decrease in D + E The difference in pI reflects this substitution of charged residues, by being one unit higher for VsEndA VsEndA also binds much more strongly to the SP Sepharose column because of its higher positive charge compared with VcEndA The primary structure of VsEndA also contains an extra lysine (Lys52a) which creates a gap in the alignment in Fig 1 The differences in charge between the two enzymes may be involved in temperature adaptation; however, two properties, which are not related to tem-perature adaptation, clearly distinguish these enzymes The two enzymes respond notably differently to varia-tions in both NaCl concentration and pH
A notable increase in activity against the DNase-Alert substrate was observed for the two enzymes when NaCl was added to the assay buffer The optimal NaCl concentrations coincide with the salinities encountered by the bacteria in their natural habitats Seawater at 3.5% salinity is composed of about
470 mm Na+ ions and 540 mm Cl– ions [20] The
0
10
20
30
40
50
60
VsEndA VcEndA
Temperature ( °C)
-1 K
-1 )
Fig 6 DSC endotherms of VsEndA and VcEndA Baseline
subtrac-ted data have been normalized for protein concentration.
1
10
100
VsEndA
VcEndA
Time (min)
Fig 7 Kinetic stability of VsEndA and VcEndA Enzyme was
incu-bated at 70 C Samples were removed after 10 min and incubated
for 1 h on ice before being assayed using the DNaseAlert QC
sys-tem kit Each replicate is plotted and the mean values are drawn.
Fig 8 Cleavage of plasmid, dsDNA and ssDNA (A) 14 n M VcEndA incubated at 23 C for 5 min with plasmid (lane 2), dsDNA (lane 4) and ssDNA (lane 6) Substrate incubated without enzyme is in lanes 1, 3 and 5, respectively (B) Substrate incubated with and without 14 n M VsEndA, as explained for VcEndA.
Trang 6optimum NaCl concentration found for VsEndA
cor-responds quite well to this V cholerae resides in more
brackish water with lower salinity, and the lower
[NaCl] optimum of VcEndA reflects this The optimal
salt concentrations were measured in a 75 mm Tris
buffer The optima may be higher in a Tris buffer of
lower ionic strength, but this was not tested Two
terrestrial orthologous endonucleases, one from the
plant pathogen Erwinia chrysanthemi and one from the
ruminal bacterium Fibrobacter succinogenes, are also
described in the literature [11,21] The optimum
concentrations of NaCl for these enzymes are 0–
75 mm and 10 mm, respectively, with DNA as sub-strate It seems that the salt optima of the enzymes are fine-tuned to match the salinity of their environment The outer membrane and cell wall of Gram-negative bacteria do not restrict passage of ions, and the peri-plasmic proteins are, like the extracellular proteins, constantly exposed to the salinity of the surrounding water Knowledge on cold adaptation is in many cases based on marine secreted enzymes Detailed data
on salt adaptation of marine cold-adapted secreted enzymes is lacking and may be a source of error in the conclusions drawn [22] For the endonuclease I enzymes studied here, the effect of NaCl is very prom-inent and underlines the need to dissect the different adaptational strategies in future studies The differ-ences observed in the number of charged residues, especially lysine, are probably related to adaptation to both salinity and temperature The Km of VsEndA is higher than that of VcEndA; therefore, the more posit-ive surface of VsEndA does not seem to significantly increase the affinity for the negatively charged sub-strate, and is apparently not a factor that aids VsEndA
in improving its catalytic efficiency It is possible that the Kmis highly affected by the NaCl concentration in the buffer, but this is not tested Halophilic enzymes have been reported to be more enriched in negatively charged amino acids than their nonhalophilic counter-parts [23,24] This is the opposite to that found for the enzymes studied here, in which the number of posi-tively charged amino acids is increased The chloride atoms probably position themselves around the posit-ive charges and make electrostatic interactions between surface amino acids and between surface amino acids and the substrate weaker To counteract this, the VsEndA may have developed a more positively charged surface It is possible that the surface charges
of the two enzymes are similar at their respective phy-siological salt concentrations The higher number of lysines seen in VsEndA may result in increased flexibil-ity, if the extra lysines repel other parts of the enzyme and do not form stabilizing salt bridges or hydrogen bonds This may also lower the stability of the enzyme The Na+ ions may affect the solvation of the phos-phate groups in the DNA substrate, and it is possible that the enzymes also have adapted strategies to remove Na+ around the phosphates of DNA before catalysis can take place It seems clear that the salt-adapted and cold-salt-adapted properties of VsEndA are intertwined
The differences in optimum pH for activity were
0.5–1 unit between the two enzymes as shown in Fig 4, with the optimum for VsEndA being shifted to
VsEndA
A
0 125 250 375 500 0.0
0.3
0.6
0.9
1.2
1.5
1.8
RNase DNase
[NaCl] (m M )
0 50 100 150 200 0.0
0.3
0.6
0.9
1.2
1.5
1.8
DNase RNase
[NaCl] (m M )
Fig 9 DNase and RNase activity with increasing amounts of NaCl.
(A) VsEndA; (B) VcEndA Enzyme was assayed using the
DNase-Alert and RNaseDNase-Alert QC system kits Each replicate is plotted and
the mean values are drawn.
Trang 7a higher pH The pH optima for endonuclease activity
show a similar trend to that for growth of the
corres-ponding organisms Activity in glycine buffer was very
low compared with that in Tris buffer at the same pH
Citrate buffer has been shown to be inhibitory to
Pro-teus mirabilis endonuclease I [13] Citrate and perhaps
also glycine may act as chelators that bind Mg2+ and
thereby inhibit enzymatic activity, similarly to EDTA
The carboxy group of the small amino-acid, glycine,
may also replace water molecules, which are bound
around the Mg2+ ion of the enzyme and thereby
inhi-bit activity
The kinetic analysis performed under optimal
condi-tions for each enzyme (Table 2) shows that VsEndA is
a better catalyst than VcEndA at all temperatures, and
the differences in catalytic efficiency (kcat⁄ Km) increase
with concomitant decrease in temperature Km values
for VcEndA are lower than for VsEndA, indicating
that the former has slightly greater affinity for the
sub-strate However, kcat is very different for the two
enzymes, especially at low temperatures, being 9 times
higher for VsEndA than for VcEndA It is clear that
VsEndA adapts to lower temperatures by increasing
the kcat The similar Km values of the two enzymes
may indicate that VsEndA is meant to function at high
substrate concentrations, at which the increase in kcat
is more important for adaptation to low temperatures
[25] The kcat values associated with both VsEndA
and VcEndA increase exponentially at temperatures
between 5C and 37 C in accordance with the
Arrhenius equation:
According to Eqn (1), there is an exponential decrease
in reaction rates (k) with decreasing temperature (T),
and the extent of this decrease depends on the
activa-tion energy, Ea The less steep slope for VsEndA when
the temperature is lowered in the temperature
opti-mum curve shown in Fig 5 is a direct consequence of
the lower energy of activation
Results from the thermodynamic calculations
(Table 2) reveal that there is a slight difference in the
free energy of activation between the two enzymes
ori-ginating from both the lower activation enthalpy
(DH#) and activation entropy (TDS#) of VsEndA The
TDS# values for VcEndA are positive, and, if we
assume that VcEndA is more rigid than VsEndA,
binding of substrate will not decrease the entropy of
activation to the same extent as in the psychrophilic
(and flexible) VsEndA However, the method of
calcu-lation, especially for DS#, must be carefully interpreted
as stated by Cornish-Bowden [26] Enthalpy
calcula-tions based on the experimentally determined values of
Ea give more precise information, and it is clear that VsEndA has adapted to low temperatures by lowering the enthalpy of activation
DSC measurements show that VsEndA is less ther-mostable than VcEndA with an unfolding temperature that is 8C lower This is in agreement with results from stability analysis of other cold-adapted enzymes, which show reduced temperature stability compared with their mesophilic homologues [27,28] The results support the theory of increased structural flexibility leading to lower thermostability in cold-adapted enzymes The NaCl concentrations in which the ther-mal scans were performed mimic the physiological con-ditions that each of the enzymes face in their natural environments Thermal scans of VcEndA at [NaCl] optimal for VsEndA (425 mm) revealed a higher Tm, and a thermal scan performed on VsEndA at [NaCl] optimal for VcEndA (175 mm) revealed a lower Tm than those found in optimal buffers (data not shown) This highlights again that it is crucial to perform the comparative analysis under physiological conditions for each enzyme, as salt interferes with both the activ-ity and stabilactiv-ity of enzymes Reversibilactiv-ity could be detected by DSC, but the signal was very weak for both proteins, probably because of aggregation and destruction caused by the relatively long period at elevated temperatures As shown in Fig 7, VsEndA transforms into an irreversible unfolded state much faster than VcEndA However, a half-life of 13 min at
70C for VsEndA is substantially higher than that of other cold-adapted enzymes [22] Endonuclease I is located in the periplasmic or extracellular space, and the selective pressure to maintain stability must there-fore be high It would be a waste of energy to secrete enzymes that denature quickly, so it is in the bacter-ium’s interest for the secreted enzymes to be long lived However, it seems that, in order to achieve appropriate activity at low temperatures, the enzyme must sacrifice some of its stability It has previously been suggested that the lower thermal stability of cold-adapted enzymes is simply a consequence of the lack of select-ive pressure for stability [29] A lack of selectselect-ive pres-sure for stability is not the case for this periplasmic⁄ extracellular protein, and our results indi-cate that in order for it to be active at low tempera-tures, its stability must be reduced
The enzymes did not show any apparent difference
in ability to degrade plasmid DNA, dsDNA or ssDNA However, both VsEndA and VcEndA dis-played decreasing activity against the RNaseAlert sub-strate with concomitant increase in [NaCl], as shown
in Fig 9 At physiological NaCl concentration, the two enzymes have extremely low RNase activity and may be
Trang 8considered solely as DNases The highest RNase activity
is in buffer without added NaCl, but it is still 3.5 times
(VsEndA) and 14 times (VcEndA) lower than the
DNase activity in the same buffer The opposite effect
that NaCl addition seems to have on the RNase activity
of the enzymes may be linked to an increase in Na+
around the phosphate groups and the 2¢-OH, which
reduces the negative charge, and hence the affinity of the
enzyme decreases with increasing NaCl concentration
However, it seems clear that both VsEndA and VcEndA
are intended to function purely as DNases in vivo
Conclusion
Endonuclease I from the psychrophilic bacterium
V salmonicida is an enzyme that shows cold-adapted
features, such as lower thermal stability, lower
tem-perature optimum, and higher catalytic efficiency,
when compared with the corresponding enzyme from
the related mesophilic bacterium V cholerae The
peri-plasmic or extracellular localization of these enzymes
means that they are constantly exposed to the external
environment of the bacterium Their differences in
enzymatic properties, such as pH optimum, salt
opti-mum and catalytic efficiency, seem to be fine-tuned to
match their respective environments The salt-sensitive
and relatively low RNase activity of the enzymes
indi-cates that their physiological substrate is DNA To our
knowledge, VsEndA is the first endonuclease described
that displays more than 90% activity against DNA in
0.5 m NaCl This unique property in combination with
high activity at low temperatures and low RNase
acti-vity may be advantageous for future commercial
exploitation Determination of the crystal structure of
VsEndA is in progress and will facilitate a detailed
explanation of the mechanisms behind the observed
cold-adapted properties, in addition to interesting
dif-ferences in pH and salt optima
Experimental procedures
Bacterial strains and molecular biology materials
Genomic DNA from V cholerae ATCC14035 and V
DNA Purification kit from Promega (Madison, WI, USA)
according to the manufacturer’s protocol for
chem-ically competent E coli TOP10 cells were purchased from
Invitrogen (Carlsbad, CA, USA) Oligonucleotide primers
(Table 3) were purchased from Invitrogen and
Sigma-Aldrich Co (St Louis, MO, USA) Phusion DNA
polym-erase from Finnzymes (Espoo, Finland) and Vent and Taq
polymerase from Promega were used in the PCRs Restric-tion enzymes NcoI and SalI were purchased from New England Biolabs (Ipswich, MA, USA), and T4 DNA ligase
Inc (Austin, TX, USA) and Integrated DNA Technologies (Coralville, IA, USA)
Construction of the expression plasmids The nucleotide sequences of VsEndA and VcEndA have the
respectively
gIII b vector, a restriction site for SalI was first removed by point mutation using the overlap extension procedure [30] PCR was conducted using primers 3 +4 and 1 +4 (Table 3), with genomic DNA from V salmonicida as a template In a 0.2-mL PCR tube, a total of 50 lL reaction mix containing
(10 lm), 1 lL template, and 1 U Vent polymerase was sub-jected to PCR using a DNA Engine (PTC-200) Peltier Ther-mal Cycler from Bio-Rad (Hercules, CA, USA) TherTher-mal
254-bp product when run on a 1% agarose gel The 656-254-bp frag-ment was used as a template in a second PCR conducted under the same conditions as above, but with primers 3 +2 This PCR yielded a product of 423 bp Purified 254-bp and 423-bp fragments were then used as a template in a third PCR using primers 3 +4 Thermal cycle conditions were the
and use of 1 U Taq polymerase instead of Vent polymerase The two primers 3 +4 contain restriction sites for SalI and NcoI, respectively, and the primers were created so that the gene would be amplified without the native N-terminal peri-plasmic signal Instead, the periperi-plasmic signal incorporated
recombinant enzyme into the periplasmic space The final PCR product was analyzed on an agarose gel and purified using the Qiaquick gel extraction kit from Qiagen (Hilden,
Table 3 List of PCR primers Restriction sites are underlined.
Trang 9Germany) The DNA fragment and the pBAD⁄ gIII b vector
were then digested with SalI and NcoI The insert and vector
were purified from a 1% agarose gel using the Qiaquick gel
extraction kit Vector and insert were ligated overnight at
col-onies resistant to ampicillin were selected and used for
expression The VcEndA was cloned using the same
proce-dure as for VsEndA, but no mutation was necessary The
clo-ning primers for VcEndA are listed in Table 3 The plasmids
were thereafter sequenced using the PE Biosystems BigDye
Terminator Cycle Sequencing kit, ABI 377 Genetic Analyzer
and ABI Sequence Analysis software according to the
proto-col supplied by Applied Biosystems (Foster City, CA, USA)
Enzyme expression and purification
A Chemap CF 3000 fermentor (Chemap AG,
Switzerland) was used for production of the recombinant
supplemen-ted with 60 mL 20% glucose was inoculasupplemen-ted with a 200-mL
pro-duction was induced by adding 50 mL 14.5% l-arabinose
when the glucose was depleted The pH was held constant
levels were automatically adjusted by increasing agitation
speed when the level went below 20% of maximum The
cells were harvested 7 h after induction by centrifugation at
separ-ate the periplasmic fraction containing the recombinant
protein Harvested cells were resuspended in 800 mL of a
fractionation buffer containing 20% sucrose, 1 mm EDTA
tempera-ture After centrifugation at 8281 g for 20 min, the
superna-tant was collected as the periplasmic fraction and frozen at
)80 C The thawed periplasmic fraction was centrifuged at
13 180 g for 20 min before application on a SP Sepharose
Uppsala,
eluted using a linear gradient from 0 to 100% buffer B
activity were pooled and concentrated using Centriprep
Centrifugal Filter Units (molecular mass cut off, 10 kDa)
Enzyme analysis
The enzyme purity was analyzed by applying 5 lg protein
(In-vitrogen) The gel was stained with Simply Blue Safe Stain
(Invitrogen) according to the manufacturer’s protocol The
protein concentration was determined using Bio-Rad Pro-tein Assay based on the method of Bradford [32] and according to the microtiter plate protocol described by the manufacturer using BSA as standard N-Terminal signal sequence cleavage sites were predicted using the SignalP server [33] Sequence alignment was performed using BioEdit [34], and the alignment was visualized using the ESPript server [35] Theoretical isoelectric point, molecular mass and sequence composition were calculated using the
Enzyme assay
deter-mination of kinetic constants, pH optimum and optimum NaCl concentration of the two enzymes The
end and a dark quencher on the other end In all reactions, except for the kinetic measurements, 200 nm substrate was used The reaction volumes were adjusted to 90 lL with nuclease-free water Reactions were started by pipetting
10 lL of the diluted enzyme solution into eight wells with a multichannel pipette to a total reaction volume of 100 lL Non-binding 1.5-mL tubes from Eppendorf (Hamburg, Germany) were used for enzyme dilution New dilutions were made before each measurement because of the sticky nature of the enzymes Black 96-well, low-protein-binding trays from Corning (Corning, NY, USA) were used in com-bination with a Spectramax Gemini fluorimeter from Molecular Devices (Sunnyvale, CA, USA) to detect the
calculated from a minimum of three linear readings on the time versus fluorescence curve using the program softmax
auto-mix for 1 s before the first read A minimum of two parallel
[NaCl] optimum, pH optimum, temperature optimum
The optimum concentration of NaCl was measured in
75 mm Tris buffer with various concentrations of NaCl (0–
750 mm) The pH optimum was measured in 75 mm
addition, 175 and 425 mm NaCl were added to the solution when VcEndA and VsEndA, respectively, were assayed
A modified Kunitz DNase assay was used for measuring optimum endonuclease activity of the two enzymes at dif-ferent temperatures In all reactions, 200 lg calf thymus DNA (Sigma) dissolved in diluted TE buffer (1 mm
Reactions were performed in assay buffers that were
Trang 10Tris⁄ HCl (pH 8.5)⁄ 5 mm MgCl2; VcEndA, 175 mm
buffer pH was adjusted at the respective assay
tempera-tures The total reaction volume was 1 mL Reaction
mix-tures were preincubated for 5–10 min at the respective
assay temperatures before the addition of enzyme The
same amount of enzyme (VsEndA 1.5 ng, VcEndA 4.2 ng)
was used at each temperature Reactions were allowed to
proceed for 20 min and then stopped by adding 0.5 mL
ice-cold 12% perchloric acid For blank reactions, enzyme was
added after the addition of perchloric acid Quenched assay
solutions were incubated on ice for 20 min, centrifuged for
supernatants in triplicate
Enzyme kinetic measurements
Fixed amounts of enzyme were incubated at seven different
substrate concentrations ranging from 23 to 1470 nm at 5,
The amounts of VsEndA enzyme used were 0.69, 0.44,
respect-ively For VcEndA the amounts used at these temperatures
were 4.2, 0.21, 0.56, 0.31 and 0.14 ng, respectively Assay
buffer was optimal for each enzyme [VsEndA, 425 mm
assay temperatures The initial velocities were recorded and
the program sigma plot (Systat
each enzyme by fitting the velocity data to the Michaelis–
Menten equation using nonlinear regression All
measure-ments were performed in triplicate for each substrate
per nmol substrate was calculated from a standard curve
obtained by measuring the maximum fluorescence emitted
as a function of various substrate concentrations By using
this linear standard curve (slope, 0.88; intercept, 21.8),
Thermodynamic activation parameters were calculated as
was extracted from the slope of the linear regression curve
Stability measurements
DSC measurements were performed using the
Nano-Differ-ential Scanning Calorimeter III, model CSC6300
(Calori-metry Sciences Corp., Lindon, UT, USA) The IUPAC
(International Union of Pure and Applied Chemistry)
recommendations for DSC measurements and analysis [38] were used as a guideline The scan rate was set to
at a constant pressure of 304 kPa All samples were dia-lyzed overnight against 50 mm Hepes, pH 8.0, containing
The dialysates were used in the reference cell and for buffer baseline determination The thermograms obtained were analyzed using the computer program cpcalc (Calorimetry
the maximum of the peak) was extracted The exact protein
measured before DSC analysis Reversibility of unfolding
fol-lowed by a second scan The molecular masses used to con-vert the DSC data to molar heat capacity are as described above Kinetic stability was determined by incubating equal amounts of enzyme (dissolved in optimal buffer for activity
Samples were removed after 10 min and incubated for 1 h
on ice before being assayed using the DNaseAlert QC Sys-tem kit Samples incubated for 1 h on ice only served as the 100% activity reference
Measurement of substrate specificity Enzyme specificity towards dsDNA, ssDNA and plasmid
linea-rized and denatured plasmid and intact plasmid The
and then kept on ice Approximately 300 ng of the various substrates was mixed with 30 ng enzyme in a total volume
diethanolam-ine buffer with optimal [NaCl] and pH for each enzyme
stopped by the addition of 5 lL 0.5 m EDTA The samples were analyzed on a 1% agarose gel for 1 h at 90 V and visualized by ethidium bromide staining The substrates were also incubated without enzyme as a reference
Activity towards RNA was measured using the RNase-Alert QC System kit with the same instrumental set up as for the DNaseAlert system mentioned above, except that
64 s for 20 min The effect of [NaCl] on the RNase activity
VsEndA and pH 8.0 for VcEndA with increasing concen-trations of NaCl (0–425 mm for VsEndA, 0–175 mm for
mix-ture The maximum fluorescence obtained with 200 nm RNaseAlert and DNaseAlert was measured by adding 5 lL
after the initial measurements and 2 lL undiluted VcEndA
to wells with DNaseAlert substrate The initial velocities