“Model Manuscript (IJBSANS)”
Partial purification and Insilico analysis of Bst1 from Bacillus
stearothermophilus
xxxxxxxa*, xxxxxxxxb*, xxxxxxc, xxxxxxd, xxxxxe, and xxxxxxxxxd#.
a
xxxxxxx china
xxxxxxx India.
c
xxxxxxxxxxxxxxxxxxxx USA.
d
xxxxxxxxxxx Italy.
e
xxxxxxxx Germany
b
* These authors are co-first authors
# Corresponding author
xxxxxxxx
xxxxxxxx
Email: xxxxxxxxxx
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Abstract:
There are more than 3000 restriction endonucleases which cut the DNA in their
respective sites. In the present study, the thermostable restriction enzyme BstI was partially
purified from Bacillus stearothermophilus. The phosphocellulose column purified Bst1 elutes
was standardized to digest E.coli, plasmid and λ DNA. Further, an in silico analysis was done
using NETCUTTER, a bioinformatics tool to generate restriction map to match the fragment
sizes we obtained in the agarose gel electrophoresis.
Keywords: Bacillus stearothermophilus, BstI, phosphocellulose, DNA, insilico
Introduction
The discovery of the enzymes which led to Nobel Prize for Artbar and Nathans in 1978
was one of the key breaks through in the development of genetic engineering. By definition,
restriction endonucleases are part of restriction modification systems (RM), which comprise an
endoculease and methyl transferase activity (Pingound and Jeltsch, 2001). Type of RM system
have been found and were classified according to their subunit composition, co-factor
requirements and mode of action (Simth and Nathans, 1973) the distinction between type I,II and
III system is still useful that there are intermediate cases. Most characterized enzymes fall into
type II class that is further divided into many subclasses have been characterized primarily with
respect to their recognition sequence and cleavage specificity rather than their proteins
properties. All restriction endonuclases cleave their DNA substrate to form 5’Phosphate and
3’Hydroxyl termini on each strand. The first thermostable restriction endouclases was reported
was TaqI from the North American T. aquaticus type stains YT (Sato et al., 1977). All structure
of restriction endonuclease show a common structural are comprising four β-stands and one αhelix (Pingound and Jeltsh, 2001).
Site specific endonuclease is currently indispensable tools in the studies of DNA structure
and other molecular genetic studies. There are about more than 3000 type II restriction enzymes
available at the market now. The range of available enzymes should be expanded to provide
further progress in this research field. The quality of enzymes is an important as the range of
their choice. Therefore the development of effective technologies of preparation of restriction
endonuclease and also to prepare thermophilic restriction enzymes was urgent problem. The goal
of the present study was to develop a small scale production and purification of BstI restriction
endonuclease from Bacillus stearothermophilus.
2. Materials and Methods:
2.1 Strains
Bacillus stearothermophilus (MTCC 1521) strain was purchased from MTCC,
Chandigarh, India.
2.2 Production media for Bacillus Sterostermophilius
Bacillus sterostermophilius were grown in a modified SLBH medium contains [for
100ml: 1.1g- tryptone, 2.25g - yeast extract, 5.1ml- (1M) K2PHO4, 1.57(1M) - KH2PO4, 0.4mlglycerol] this medium kept in 60°C h in a shaker.
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2.3 Production of restriction enzymes
Culture grown till late log phase was centrifuged at 6000rpm 15 min at 4°C. The
supernatant was discarded and the pellet was collected and stored at -20°C for 2 hours. Forzen
cell pellets were thawed and suspended in 10ml of buffer A (10mM potassium sulfonylfloride
pH 7.0, 1mM EDTA, 1mM 2-mercaptoethanol) containing 2.5mg phenylmethyl sulfonylfluoride
(PMSF) and 0.4M NaCl. Sometimes it can be effective without proteolysis inhibitor (PMSF).
However this was generally added as a precaution. The cell suspension was sonicated with the
cell suspension on ice was carried out in 30’-90’ pulse; the temperature was monitored and kept
below10°C strains which were resistant to sonication. These were stirred on ice with the addition
of 10µl/ml lysozyme for 15-30 min before sonication. They were sonicated for cell repture for
50cycle of 2 min each with a time gap of 5 min between each cycle. The sonicated cell
suspension was centrifuged for 1 hour at 12,000 rpm in 4°C. The supernatant was collected as
crude of restriction enzyme.
2.4 Purification of restriction endonucleases
2.4.1 Ammonium Sulphate Preparation
The crude enzymes were precipitated using the standard salting out procedure using solid
ammonium sulphate using 80% saturation and stirred well for 2h at 4°C and centrifuged at
20,000rmp for 1h. The supernatant was removed and the precipitated formed was dissolved in
buffer A contains 0.1M NaCl.
2.4.2 Dialysis
The sample from ammonium sulphate precipitation was transferred to in dialysis
membrane bag with 1000MV cut off dialysis tubing and dialyzed against buffer A containing
0.1M NaCl at 4°C for 18h. The buffer was changed every 4 h.
2.4.3 Phosphocellulose chromatography
Careful preparation of phosphocellulose is essential to the success of this purification
method. 1g of phosphocellulose were suspended in 32ml of 0.2N HCl diluted 1:1 with 95%
ethanol and stirred gently for 30min at room temperature and filtered solution using Whatemann
filter paper. The slurry was allowed to settle and the supernatant aspirated or decanted to remove
fine and other particulate matter. The collected resin was washed 2 to 3 times by suspension in
20ml of distilled water. The pH was adjusted to near neutral with 1M NaOH and the resin was
suspended in 50ml 0.1N NaOH stirred for 30min at room temperature, collected by filtration and
suspended in 50ml of 1mM EDTA. It was stirred for 30min at room temperature and washed 3
times with distilled water. The pH was adjusted to near neutral with 1M HCl and the resin was
suspended in extract buffer plus 0.2M NaCl. The pH was carefully adjusted to pH 7.0 before
pouring a column since many column volume of dilute buffer and required for equilibration of
the pH in a packed column.
The phosphocellulose column (1.2X15cm) is sufficient for cell. It should be washed with
several volumes of extract buffer-A prior to loading. The extract was loaded directly on to the
column and the column washed with several column volumes of extract. The restriction enzyme
was eluted with the buffer-A containing 0.4M NaCl. Ten fractions of 1ml were collected and
assayed for endonucleae activity and protein estimation.
2.5 Genomic and plasmid DNA isolation from bacteria
A single colony of Escherichia coli DH5 α cell containing pGEM 7Z f+ was inoculated
in to 3ml of LB medium containing ampicillin (100µg/ml) and was grown with constant shaking
at 37°C for 16h. The plasmid DNA was isolated according to standard protocol Sambrook, et al.,
(1989).
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2.6 Restriction Assay with λ and E.coli Genomic DNA
Two vials were used for E.coil and λ DNA Restriction assay. The reaction was set in 30µl
of reaction mixture containing 3µl of assay buffer (100mM Tric HCl pH 7.0, 10mM MgCl2,
10mM NaCl), 1µl of DNA (E.coli DNA in one tube and λ DNA in another tube) and 21µl of
crude, dialyzed and purified restriction enzyme BstI. The reaction mixture was incubated 24h at
37°C and the reaction was stopped after incubation time. The fragments were analyzed using
1.0% agarose gel electrophoresis containing ethidium bromide (2µl/ml). The bands were
visualized under UV illuminator and were documented using a gel documentation system.
2.7 Restriction assays for plasmid DNA
Plasmids isolated from Staphylococcus aureus were digested using BstI and 24 h at 37°C.
The fragments were analyzed using 1% agarose gel electrophoresis.
2.8 In Silico analysis
Using bioinformatics tool, NET CUTTER, restriction map was generated for Bst1 with λ
and E.coli DNA to compare with size of bands of BstI observed in agarose gel electrophoresis.
3. Results
3.1 Production of Restriction enzymes
Bacillus stearothermophilus grown in SLBH medium at 65°C for 6h showed maximum
activity for BstI.
Similarly, Salleh, et al. (1977) isolated proteases from Bacillus
stearothermophilus which is stable at 60°C and Razak, et al., (1993) reported that Bacillus sp.
has produced a thermostable protease that has an optimum activity at 60°C.
3.2 Purification of BstI
Phosphocellulose column was used for the purification of BstI. The purification profile of
BstI showed 72.72% yield and 514 fold increase in purification. Highest specific activity was
obtained in third fraction of phosphocellulose column and the specific activity of 10 fractions
was shown in Figure 1. Catterall and Welker, (1977) investigated the small scale purification of
class II-endonuclease (BstI) from strain NCA 1503.
3.4 Restriction analysis of lambda, E.coli, and plasmid DNA
Restriction assay of BstI with λ and E.coli DNA showed 22 sites and 20 sites respectively
(Fig 2). This result was cross checked using bioinformatics analysis (Table 1). In order to
confirm the cutting efficacy of purified BstI, we analyzed this enzyme to restrict plasmids
isolated independently by one of our colleague. This restriction analysis showed BstI indeed cut
in its recognition sites as observed by many bands in the agarose gel electrophoresis (Fig.2).
4. Discussion
Similarly, Salleh, et al. (1977) isolated proteases from Bacillus stearothermophilus which
is stable at 60°C and Razak, et al., (1993) reported that Bacillus sp. has produced a thermostable
protease that has an optimum activity at 60°C. Catterall and Welker, (1977) investigated the
small scale purification of class II-endonuclease (BstI) from strain NCA 1503. This result was
cross checked using bioinformatics analysis In order to confirm the cutting efficacy of purified
BstI, we analyzed this enzyme to restrict plasmids isolated independently by one of our
colleague. This restriction analysis showed BstI indeed cut in its recognition sites as observed by
many bands in the agarose gel electrophoresis.
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5. Conclusion
In conclusion, restriction enzymes are indispensable tools in the molecular biology
studies the standardization of the procedure for isolating and obtaining high purify enzymes are
very important. Bacillus stearothermophilus (MTCC 1521) was tested for the production of
restriction enzymes. Ammonium sulphate precipitation and dialysis were used for the partial
purification of the restriction enzymes preparations. Phosphocellulose column which is not only
an anion exchange but also an affinity column for DNA binding proteins was used for the final
level of purification. The BstI was partially purified and fractions were standardized to assay
with E.coli, λ DNA and plasmid DNA and restriction sites were confirmed using insilico
analysis. BstI can be used as an alternative for Sau3AI and MboI because of its same recognition
sequence.
5. REFERENCE:
1. Alfered Pingound and Albert Jeltsch, (2001). Structure and function of Type II
Restriction enzymes. Nucleic Acid Research, 29, 18, 3705-3727.
2. Simth, H.O. and Nathans, D. (1973). A suggested ammenclature for bacterar hast
medification and restriction system and other enzymes. Journal of Molecular Biology, 18,
419-423.
3. Sato S and Hutchison CA, et al., (1977). A thermostable sequence-specific endonuclease
from Thermus aquaticus. Proc Natl Acad Sci. 74:542–546.
4. Sambrook,J and Fritsch EF, et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd
edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
5. Salleh AB and Basri , et al., (1977). The effect of temperature on the protease from
Bacillus stearothermophilus strain F1. Mal. J. Biochem. Mol. Biol. 2, 37-41.
6. Razak, C and Samad, M, et al., (1993). Thermostable extracellular protease by B.
stearothermophilus. World J. Microbiol. Biotechnol. 10, 260–263.
7. Catterall, JF and Welker NE, (1977) Isolation and properties of a thermostable restriction
endonuclease (ENDO R-Bst1503)J. Bacteriol. 129, 1110-1120.
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Figure1: Specificity Activity of different fraction of BstI
500
A c tiv it y
400
300
200
100
0
1
2
3
4
5
6
F r a c t io n s
6
7
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Figure 2: Restriction of BstI with E. coli DNA (a) lambda DNA (b)
Line 1: Marker DNA
Lane 2: BstI with lambda DNA
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Table 1: Analysis of restriction site
S.NO
Site Bst
Target
1
AGCTC
2
2
ACGTCAG
3
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