Heat sensitive bacterial alkaline phosphatase

This invention discloses a newly discovered enzyme and a method of producing said enzyme wherein said enzyme is completely inactivated by treatment with temperatures of 50.degree. or higher for 10 minutes or longer. The alkaline phosphatase is isolated from the psychrophilic bacteria, particularly bacteria from certain areas of the oceans surrounding Antarctica. The preferred alkaline phosphatase is derived from the bacteria HK-47 (ATCC 39469).

FIELD OF THE INVENTION 
The present invention relates to the field of nucleic acid research, and 
more particularly to an enzyme useful in performing radioactive 
end-labeling of nucleic acids. 
BACKGROUND OF THE INVENTION 
Alkaline phosphatase (APase) is an enzyme which catalyzes the hydrolysis of 
an ester bond of terminal phosphate groups (PO.sub.4.sup.-). This reaction 
is useful as a research tool particularly for radioactive end-labeling of 
nucleic acids using T4 polynucleotide kinase to study nucleic acid 
structure and function. 
In the procedure for radioactive end-labeling, the substrate, such as DNA, 
RNA or an oligonucleotide, is first dephosphorylated with APase prior to 
the labeling step. In the next step, the APase activity must then be 
eliminated to avoid both degradation of ATP and loss of label from the 
nucleotide substrates. Finally, polynucleotide kinase (PNK) is used to 
catalyze the phosphorylation of the substrate, wherein the phosphate donor 
is typically a gamma-P.sup.32 ATP. As noted above, residual APase activity 
in the reaction vessel during or subsequent to the phosphorylation step, 
can result in a loss of label from the ATP and polynucleotide substrate, 
thereby destroying the results of the radioactive end-labeling reaction. 
Most researchers are using APase isolated from the microorganism E. coli, 
which is available commercially, to cleave the terminal phosphates from 
the nucleotide substrate. The disadvantage of this enzyme is its great 
stability, especially its heat stability, which makes inactivation of the 
APase particularly difficult. 
Severaly methods are currently employed to remove or inactivate E. coli 
APase from the phosphorylation reaction vessel prior to the labeling step. 
The most effective method is phenol extraction; however, this method is 
time-consuming, results in poor recovery of nucleic acids and is 
inappropriate for processing large numbers of samples. Methods of 
treatment with NaOH, HCl, boiling, or nitriliatriacetic acid are not 
suitable because the APase is not completely inactivated thereby. One 
other method is the use of inorganic phosphate (Pi) to inhibit APase 
activity. This method is disadvantageous in that Pi also inhibits 
polynucleotide kinase activity, so that the phosphate labeling of highly 
structured polynucleotide substrate, such as DNA or RNA, is also greatly 
reduced. The general problem with the above methods is that E. coli APase 
is highly stable making removal or inactivation thereof difficult. 
One solution to the above-noted problems of the stability of E. coli APase 
in end-labeling experiments is to use heat sensitive APase. Some heat 
sensitive APases are disclosed in Alkaline Phosphatase, R. B. McComb, et. 
al. (1979), Plenum Press, N.Y. p. 404 in "Table of Thermal Denaturation 
Rates of Selected Microbial Alkaline Phosphatases." As stated therein, the 
APases which are most heat sensitive include Bacillus megaterium with a 
half-life of 4 minutes at 55.degree. C., and Sacharomyces cerevisiae with 
a half-life of 2.5 minutes at 60.degree. C. However, the report of these 
temperature sensitive APases does not disclose what treatment is necessary 
for complete APase inactivation, and there is no necessarily direct 
relationship between half-life (50% inactivation of enzyme activity) and 
complete inactivation of the enzyme. Moreover, effective phosphorylation 
of the polynucleotide substrate can only occur if the APase is completely 
inactivated. 
One particularly significant problem with using temperature treatment to 
inactivate APase is that double stranded polynucleotides are somewhat heat 
labile. This problem is caused by the fact that double stranded DNA, RNA 
and oligonucleotides are held together by hydrogen bonding which can be 
broken at elevated temperatures. Shorter polynucleotide double stranded 
chains and those polynucleotides containing a large percentage of 
adenine-thymine base pairs are examples of particularly temperature 
sensitive polynucleotides. In fact, double stranded DNA can be completely 
denatured by heat treatment at 65.degree. C. Thus, it is preferable for 
radioactive end-labeling procedures, where the integrity of the 
polynucleotide substrate may be important that the temperature of heat 
treatment of the polynucleotide substrate be as low as possible, and the 
duration of such treatment be short. The present invention obviates the 
need for higher elevated temperatures or other extreme conditions for 
removing or inactivating APase. 
SUMMARY OF THE INVENTION 
The present invention comprises a new form of the enzyme alkaline 
phosphatase, isolated from microorganisms collected from Antarctica. These 
microorganisms were collected from a number of sources such as the sea 
ice, sea water and sediment around the McMurdo Sound in Antarctica. 
Twenty-two of the 150 strains of bacteria tested produced heat sensitive 
alkaline phosphatase which was completely inactivated by heat treatment at 
65.degree. C. for 10 minutes. The alkaline phosphatase from one strain, 
HK-47 (ATCC 39469), which has a maximum growing temperature of less than 
25.degree. C., showed the highest activity among the heat sensitive APase 
producers. It was determined that this strain produced an APase which was 
inactivated substantially instantaneously at 50.degree. C. The half-life 
at 40.degree. C. was determined to be 2.0 minutes. 
The advantage to this invention over the prior art is that the alkaline 
phosphatase disclosed herein may be inactivated completely by a simple, 
safe and quick procedure. In the case of end-labeling experiments, 
nucleotides may be dephosphorylated using the alkaline phosphatase 
disclosed herein. The reaction vessel is then heat treated at 50.degree. 
C. for 10 minutes to completely inactivate the APase. Polynucleotide 
Kinase and radioactive Adenosine Triphosphate (gamma-.sup.32p -ATP) may 
then be added directly to the original reaction vessel, thereby labeling 
the dephosphorylated nucleotide, and thus avoiding the inconvenience and 
potential loss of yields through multiple transfers of the reaction 
mixture. Temperature treatment of nucleic acids at 50.degree. C. for 10 
minutes which is more than sufficient to inactivate the invented APase, 
and does not generally permit denaturation of RNA, DNA or oligonucleotide 
substrates on which the end-labeling is performed. 
Whereas 21 strains of bacteria have been found to produce heat sensitive 
enzymes of alkaline phosphatase, it is anticipated that other strains of 
bacteria, yet undiscovered but existing under similar environmental 
conditions, will be found to be useful in practicing the present invention 
without departing from the scope thereof.

DETAILED DESCRIPTION OF THE SPECIFIC EMBODIMENT 
1. Isolation of Antarctic Bacteria and Screening of Heat Labile APase 
It is generally agreed that psychrophilic bacteria are found in permanently 
cold environments. Most of the well characterized psychrophilic bacteria 
were isolated from the ocean. Since more than 90% of the ocean is at a 
temperature of 5.degree. C. or below, the ocean is a natural habitat for 
psychrophilic bacteria. It is expected that psychrophilic bacteria can be 
isolated relatively easily below the thermocline of the ocean, 
particularly from polar regions. For this reason, we decided to isolate 
psychrophilic bacteria from the antarctic. 
Various samples of sea ice, sea water, sea sediments, and sea animals were 
obtained from two areas of Antarctica (McMurdo Sound and New Harbour). The 
frozen samples of seawater and sea ice were melted at 0.degree. C. and 0.1 
ml of serially diluted samples was spread onto agar plates containing 
2216E medium. Animal samples were homogenized before spreading onto the 
plates for bacterial colonies. 
One hundred and sixty-six colonies appeared after two to twelve weeks 
incubation at 0.degree. C. Among these 41 strains (24%) were found to be 
psychrophiles. Twenty-two strains (including 3 psychrophiles) produced 
heat labile APase which is completely inactivated after incubating at 
65.degree. C. for 10 minutes. The APase from strain HK-47 yielded the 
highest activity among the 22 heat labile APase producers. HK-47 showed 
the maximum growth rate in medium containing 60% sea water and no growth 
over three days in media containing no seawater. HK-47 is a psychrophilic 
bacterium having an optimal growth temperature of about 15.degree. C., a 
maximal growth temperature of 22.degree.-24.degree. C., and a minimal 
growth temperature of 0.degree. C. or lower. The HK-47 strain is on 
deposit with the ATCC (No. 39469). Pursuant to the applicant's contract 
with the ATCC, the micro-organism identified as HK-47, shall be maintained 
on deposit for at least 30 years and will be available to the public, 
without restriction, after the patent issued for at least as long as the 
life of the patent. 
2. Purification and Characterization of HK-47 APase 
All purification procedures were carried out at 0.degree.-4.degree. C. 
Marine bacteria HK-47 was grown at 0.degree.-4.degree. C. in HK medium, 
containing 60% aged seawater, 1% Bacto-tryptone, and 0.2% yeast extract. 
The medium was buffered at pH 7.6 with HCl before inoculation of the 
bacteria. The cells grown to late exponential phase in 5.4 liters were 
harvested by centrifugation at 10,000 xg for 15 minutes at 0.degree. C. 
APase activity appears to be localized in the periplasmic space outside the 
cytoplasmic membrane of all gram-negative bacteria. Therefore, the first 
step in the purification procedure is to treat cells by osmotic shock. 
Sixty percent (60%) of the total APase is successfully released by a 
modified osmotic shock procedure as described by Unemoto, (1973 Biochem. 
Biophys. Acta, 315, 83-93), whereas only 6% of the total protein was 
solubilized from HK-47 cells under the same conditions. 
The harvested cells were washed by centrifugation in 5.4 liters of 1M NaCl 
in 50 mM Tris-HCl buffer, pH 7.4, suspended in 350 ml of hypertonic medium 
containing 1.0M NaCl, 1.0M sucrose, and 50 mM Tris-HCl buffer pH 7.4, and 
then stirred for 15 min at 0.degree. C. The cell suspension was 
centrifuged and the pellet was rapidly suspended in 54 ml of cold shock 
buffer containing 50 mM NaCl, 10 mM MgCl.sub.2, and 50 mM Tris-HCl pH 8.0. 
After stirring for 15 min at 0.degree. C., the mixture was centrifuged and 
the supernatant was collected. The pellet was further extracted with 27 ml 
of the shock buffer, and two supernatants were combined. The osmotic shock 
fluid was dialyzed against 50 mM Tris-HCl buffer pH 8.4 containing 5 mM 
NaCl, and 10% glycerol (T buffer) and centrigued at 100,000 xg for 30 
minutes. 
The supernatant was applied to affinity chromatograph column (0.8 cm.sup.2 
.times.12 cm) equilibrated with 10 mM Tris-HCl buffer pH 8.4. The affinity 
chromatography was prepared by coupling diazonium salt of 
4-(p-aminophenylazo) phenyl arsenic acid to a tryaminyl-Sepharose as 
described by Brenna et al. (1975 Biochem. J., Vol. 151, p. 291). The 
column was washed with, first, 5 mM NaCl in 10 mM Tris-HCl pH 8.5, and 
then 5 mM NaCl in 100 mM Tris-HCl buffer pH 8.4. The APase was eluted by a 
linear gradient from 20 to 100 mM sodium phosphate in 150 mM Tris-HCl 
buffer pH 8.4, and 5 mM NaCl (FIG. 1). The APase peak fraction was pooled 
and dialyzed against T buffer. 
The dialyzate was applied on DEAE-Sephacel column (0.5 cm.sup.2 .times.10 
cm) equalibrated with T buffer, and the APase was eluted by a linear 
gradient from 0 to 0.4 m NaCl in T buffer. (FIG. 2) The pooled fraction of 
the enzyme was dialyzed against, first, T buffer and second, 50% glycerol 
in 10 mM Tris, pH 8.4. The purified enzyme was stored at -20.degree. C. 
A summary of purification scheme is presented in Table 1 (below). 
TABLE 1 
______________________________________ 
Summary of purification scheme for HK-47 APase. 
Specific 
Total Total activity 
Total 
Purification protein units U/mg recovery 
step mg U protein 
% 
______________________________________ 
Osmotic shock fluid 
110.0 668.5 5.8 100 
Affinity chromatography 
4.5 138.7 30.8 21 
DEAE-Sephacel 0.08 72.5 906.3 11 
______________________________________ 
More than 90% of the total proteins in the osmotic shock extract were not 
absorbed by the affinity column, and APase activity was eluted at 
approximately 60 mM sodium phosphate (FIG. 1). As shown in FIG. 2, APase 
of the pooled fraction from the affinity column appeared at 190 mM as a 
single peak on DEAE Sephacel column. In separate experiments, when osmotic 
shock extract was directly applied onto DEAE Sephacel column, APase 
activity was found in two peaks. 
The pooled fraction of DEAE Sephacel chromatography was analyzed by SDS gel 
electrophoresis. One major band (69,000) and three minor bands were seen 
after silver staining. The molecular weight of the native enzyme was found 
to be 67,000 by P200 gel filtration, indicating that native HK-47 APase is 
uniquely monomeric. The specific activity of the APase obtained after DEAE 
Sephacel is among the highest so far reported for the bacterial APases. 
Although the final specific activity varied from one experiment to 
another, a specific activity of 800-1,600 units per mg protein were 
generally obtained. 
The HK-47 APase does not require the four major cations in seawater 
(Na.sup.+, K.sup.+, Ca.sup.2+, and Mg.sup.2+) for the manifestation of its 
activity, although Ca.sup.2+ is required for maximum activity. In the 
presence of 10 mM Ca.sup.2+, the activity of HK-47 APase was 6 times 
higher than the activity seen without adding any cations in the assay 
mixture. 
HK-47 APase activity was inhibited 50% by as little as 0.1 mM EDTA and 
almost 100% at 1 mM EDTA. In contrast, 50% of the inactivation of E. coli 
APase occurs at 10 mM, so that HK-47 APase is 100-fold more sensitive to 
EDTA. HK-47 APase is also more sensitive to lower pH, activity thereof 
being lost irreversibly when said APase is treated at pH 4.5 for 10 min at 
4.degree. C. 
The enzymatic activity of HK-47 phosphatase is particularly sensitive to 
temperature. The optimum temperature for the activity is 25.degree. C. 
(FIG. 3a). The enzyme is still active at 0.degree. C. and 17% of the 
maximum activity was seen at this temperature. As the assay temperature 
was raised, the APase activity rapidly decreased, and at 50.degree. C. 
virtually no activity was detected. 
Further, the APase is rapidly inactivated during incubation at higher 
temperature. HK-47 APase was incubated for 10 minutes in 50 mM Tris-HCl 
buffer at the temperatures indicated in FIG. 3b. Little loss of the 
activity was seen at 10.degree. C. incubation. However, even at 15.degree. 
C., 40% of the activity was lost during the incubation. Very little 
activity remained above 50.degree. C. incubation. 
The enzyme was found to be stable within a range of pH 7.0 to 9.5 when it 
was incubated at 0.degree. C. for 10 minutes at such pH (FIG. 4b). 
However, the enzyme was active only at pH 8.5 to pH 10.0; and its maximum 
enzyme activity was found at pH 9.5 (FIG. 4a.) The half-life time of HK-47 
APase was 40.degree. C. at 2 min. Thermal inactivation of the APase was 
detectable even at 15.degree. C. and the APase was completely inactivated 
by heat treatment of 50.degree. C. for 10 minutes. 
3. Analysis of Enzyme Activity 
APase activity was routinely assayed at 25.degree. C. for 30 min in 100 
.mu.l reaction mixture containing 2 mM p-nitrophenyl phosphate, 5 mM 
CaCl.sub.2, 0.1M CAPS-NaOH buffer pH 9.5. The reaction was stopped by 
adding 300 .mu.l of 13% (W/V) EDTA-1N NaOH to the reaction mixture. 
Control mixtures were similarly prepared, except that 13% EDTA-1N NaOH was 
added before the enzyme activity was assayed. APase activity was measured 
as the difference in absorbance at 410 nm between samples and control. One 
unit of APase activity is expressed as 1 micromole of p-nitrophenol (pNp) 
liberated per minute at 25.degree. C. under the assay conditions. Protein 
concentration was determined by the Bio-Rad protein assay kit. Bovine 
serum albumin (BSA) was used as the standard. Homogeneity of the APase was 
examined by polyacrylamide gel electrophoresis and Bio-Rad (Bio-Rad 
laboratories, Richmond, CA.) silver stain. Molecular weight was determined 
by Bio-gel P200 gel filtration (FIG. 5a) and SDS gel electrophoresis (FIG. 
5b) to be approximately 67,000 daltons. 
4. Radioactive End-Labeling Using HK-47 APase 
An autoradiograph of the end-labeled Hinf-I DNA fragments with serial 
treatments of HK-47 APase and polynucleotide Kinase is illustrated in FIG. 
6. Five micrograms of pBR322 DNA were digested by 250 units of Hinf-I 
restriction enzyme in 100 .mu.l reaction mixture of 10 mM Tris-HCl, pH 
7.5, 50 mM NaCl, 10 mM MgCl.sub.2, 1 mM beta-mercaptoethanol, and 15 
micrograms of BSA at 37.degree. C. for 16 hr. The terminal phosphate 
groups of the Hinf-I treated DNA fragments were removed by either HK-47 or 
E. coli APase. Using HK-47 APase, 0.2 micrograms of Hinf-I fragments were 
incubated in 10 .mu.l of 100 mM CAPS-NaOH pH 9.5, 5 mM CaCl.sub.2, and 
0.04 units of HK-47 APase at 25.degree. C. for 1 hr. Another sample of a 
0.2 micrograms of Hinf I fragments were also dephosphorylated in 10 .mu.l 
of 100 mM Tris-HCl pH 8.0, and 0.04.mu. of E. coli APase at 37.degree. C. 
for 1 hr. Both reaction mixtures were incubated at 60.degree. C. for 10 
minutes, and then brought to 0.degree. C. Phosphorlyation was carried out 
by adding 10 microliters of a mixture containing 25 mM Hepes-NaOH, pH 7.5, 
10 mM dithiothreitol, 10 mM MgCl.sub.2, 0.36 micromoles of [gamma-.sup.32p 
] ATP (3,000 Ci/mmole), and 1.6 units of polynucleotide kinase. Reaction 
mixtures were incubated at 37.degree. C. for 30 min, and then chilled on 
ice. One microliter of the kinase reaction mixture was mixed with 4 
microliters of dye solution containing 0.05% xylene cyanol. 0.05% 
bromophenol blue (BPB), and 8.3M urea in TBE buffer (2.5 mM EDTA, 39 mM 
boric acid, and 89 mM Trizma base). Samples were loaded onto 10% 
polyacrylamide slab gel and electrophoresed in TBE buffer at 400 volts 
until the BPB dye reached to 12 cm from the top of the gel. The .sup.32p 
-DNA fragments were visualized by exposing the gel to Kodak XAR-5 ray film 
for 90 min at -70.degree. C. (FIG. 6). Lanes 1 and 6 contain .sup.32p 
-labeled pBR322 Hinf-I fragment controls treated with E. coli bacterial 
alkaline phosphatase (BAP) was inactivated using a phenol extraction 
technique well known in the art. Lanes 2 and 4 contain 32p-labelled pBR322 
Hinf-I fragments treated with HK-47 and E. coli APase, respectively, and 
heat treated at 60.degree. C. as described above. Lanes 3 and 5 contain 
32pBR322 Hinf-I fragments treated wtih HK-47 and E. coli Apase, 
respectively, and kept at 0.degree. C. for 10 minutes, rather than 
receiving heat treatment.