Note: Descriptions are shown in the official language in which they were submitted.
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NUCLEASE ENZYME PREPARATION HAVING HIGH RESISTANCE TO HEAT
BACKGROUND OF THE INVENTION:
The present inventlon relates to an enzyme preparation
comprised of highly heat-resistant nuclease active fractions
present in the product of fungi. More specifically, the
present invention relates to an enzyme preparation comprised
of nuclease active fractions that will not lose their activ-
ity even after heating at 100C for 30 minutes.
Various enzyme preparations comprised of cellulase
produced by fungi are commercially available, among which are
Cellulase Onozuka~ (derived from Trichoderma and manufactured
by Kinki Yakult Co., Ltd.), Cellulase AP (derived from
Aspergillus and manufactured by Amano Seiyaku Co., Ltd.) and
Toyo Cellulase~ (derived from Fusarium and manufactured by
Toyo Jozo Co., Ltd.). These cellulose preparations are known
to contain various enzymes that decompose polysaccharides or
proteins. Some of these enzymes have already been isolated
and their properties have been reviewed. Because of their
nature, these cellulose preparations are used extensively for
decomposing polysaccharides and proteins. However, no
attempt has been made to review the action these cellulase
preparations and the products of fungi will exert on DNA. No
knowledge has been obtained as to whether they have nuclease
activity.
While a great number of enzymes have been known, most
of them are labile to heat and their activity will decrease
so greatly upon heating as to suffer a substantial loss in
practical value. In particular, those enzymes which are
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1 335084
capable of maintaining their activity even if they are heated
at 100C for 30 minutes and longer are almost nil. A need
has, therefore, arisen for the development of enzyme prepara-
tions that are capable of maintaining their activity even if
they are exposed to prolonged heating at elevated temperatures.
SUMMARY OF THE INVENTION:
One object of the present invention is to provide an
enzyme preparation comprised of highly heat-resistant nuclease
active fractions that occur in the products of fungi.
Another ob~ect of the present invention is to provide
a nuclease enzyme preparation that has such high heat resis-
tance that its enzymatic activity will not be lost upon
heating at 100C for 30 minutes and longer.
The present invention has been accomplished on the
basis of the finding by the present inventors of the fact
that nuclease activity occurred in the products of fungi.
~tated more specifically, the present invention has been
accomplished on the basis of the first discovery by the
present inventors of the fact that fungal products such as
Cellulase Onozuka~ derived from Trichoderma, Cellulase AP
derived from Aspergillus and Toyo Cellulase~ derived from
Fusarium have nuclease activity (see Example 1 to be
described hereinafter). In accordance with the present
invention, nuclease active fractions are isolated from the
product of fungi and used as the active ingredient of a
nuclease enzyme preparation.
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To this end, in one of its aspects, the invention
provides an enzyme composition for decomposing proteins
containing a heat-stable nuclease produced from fungus which
continues to exhibit nuclease activity following heating at
100C for 30 minutes.
In yet another of its aspects, the invention
provides a process for preparing a heat-stable nuclease
comprising the steps of culturing a fungus of the genus
Trichoderma, Aspergillus or Fusarium in a culture medium to
produce a nuclease-containing solution, heating the solution
produced in step (a) to a temperature of 80C, removing the
insoluble products of heat denaturation, and recovering the
heat-stable nuclease.
In another of its aspects, the invention provides
a process of producing a heat-stable nuclease comprising
heating a cellulase and nuclease-containing composition
produced by culturing Trichoderma, Aspergillus, or Fusarium
at about 80C to inactivate the cellulase present to produce
an enzymatically active, heat-stable nuclease.
In yet another aspect, the present invention
provides an enzyme composition for decomposing proteins
containing a heat-stable nuclease which continues to exhibit
nuclease activity following heating at 100C for 30 minutes,
said heat-stable nuclease being prepared by the steps of
(a) culturing a fungus of the genus Trichoderma,
Aspergillus or Fusarium in a culture medium to produce a
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,,
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nuclease-containing solution; (b) heating the solution
produced in step (a) to a temperature of about 80C; (c)
removing the insoluble products of heat denaturation; and
(d) recovering the heat-stable nuclease.
In yet another aspect, the present invention
provides a method of decomposing nucleic acids comprising
subjecting a nucleic acid to a heat stable nuclease which
remains active after heating at 100C for 30 minutes,
produced by Trichoderma or Fusarium.
BRIEF DESCRIPTION OF THE DRAWINGS:
Fig. 1 shows the DNA decomposing activity of a
cellulase enzyme preparation;
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Fig. 2 shows the relationship between fractions in a
DEAE-Sephadex column chromatogram and optical density at
280 nm;
Fig. 3 shows the ~DNA decomposing activities of
various nuclease active fractions;
Fig. 4 shows the changes in nuclease activity that
accompany treatments at elevated temperatures;
Fig. 5 shows the heat resistance of nuclease active
fractions and the profile of change in their activity with
temperature;
Fig. 6 shows the profile of change in the activity of
nuclease active fractions with temperature;
Fig. 7 shows the relationship between time and the
decomposition of ~DNA by nuclease active fractions; and
Fig. 8 shows the substrate specificity of nuclease
active fractions.
DETAILED DESCRIPTION OF THE INVENTION:
The nuclease active fractions of the present invention
may be obtained from the products of fungi by the following
methods but it should be understood that the nuclease active
fractions that can be used in the present invention are not
limited to those which are obtained by these methods.
According to one method for obtaining the nuclease active
fractions of the present invention, a solution (pH 7.0) of a
cellulase preparation derived from fungi such as Cellulase
Onozuka~, Cellulase AP~ or Toyo Cellulase~ is heated at 80C
for 10 minutes and after removing the resulting insoluble
product of heat denaturation, the solution is charged in a
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DEAE-Sephadex chromatographic column, which is then eluted
with 0.5 M NaCl (see Examples 2 - 4). Alternatively, a
solution of cellulase preparation not heated to 80C may be
directly subjected to DEAE-Sephadex column chromatography
and the fractions not adsorbed at pH of 7.0 are recovered
(see Examples 5 - 7). If desired, a fungus of a genus such
as Trichoderma, Aspergillus or Fusarium is cultured in a
medium such as a potato dextrose medium or Sabouraud's agar
medium, ethanol is added to the culture obtained, the super-
natant is separated, and the separated supernatant is treatedby one of the methods described above to obtain nuclease
active fractions.
Investigations of nuclease activity against ~DNA
showed that the activity was detected in the fractions of the
present invention obtained by the methods described above,
but not at all in other fractions (see Example 8). This made
it clear that the nuclease activity found in the product of
fungi was due solely to the fractions of the present inven-
tion. It is therefore anticipated that the concentrate of
such active fractions that are selectively recovered from the
product of fungi or cellulase enzyme preparations derived
therefrom will have higher levels of nuclease activity than
that inherently present in the products of fungi or enzyme
preparations derived therefrom. Furthermore, said concen-
trate has the potential to exhibit higher selectivity.
The active fractions of the present invention have avery high level of heat resistance (see Examples 9 and lO).
The active fractions of the present invention exhibit
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nuclease activity in a high temperature range of 60 - 100C
and particularly high activities are exhibited by active
fractions derived from Aspergillus or Fusarium. The active
fractions of the present invention have a marked advantage
over the conventional enzymes in that they will not lose
their activity even if they are heated at 100C for 30
minutes. Active fractions derived from Trichoderma retained
their activity even after heating at 100C for 45 minutes
(see Example lO). The enzyme preparation of the present
invention which is comprised of such highly heat-resistant
fractions will be effectively used in various applications
such as where it is necessary to decompose nucleic acids at
high temperatures. An optimum temperature for the decomposi-
tion of ~DNA is 45C for active fractions derived from
Trichorderma (see Example ll).
The nuclease active fractions of the present invention
have another characteristic feature in that they are capable
of yielding DNA decomposition products of uniform length.
When DNA is decomposed with an enzyme, decomposition products
of various lengths will normally result. However, if one
uses the nuclease active fractions of the present invention,
he can obtain decomposition products of a fairly uniform
length. For instance, if active fractions of the present
invention are allowed to act on ~DNA, the lengths of decompo-
sition products will become substantially uniform in 30 - 45
minutes after the reaction is started (see Example 12).
Another advantage of the nuclease active fractions of
the present invention is that they have a sufficiently low
1 335084
level of substrate specificity to be used extensively in
decomposing various kinds of nucleic acids. For example,
these fractions exhibit satisfactory activity against a broad
spectrum of substrates including ~DNA, heat-denatured ~DNA,
bovine thymus DNA, heat-denatured bovine thymus DNA, herring
sperm DNA, heat-denatured herring sperm DNA, Ml3 DNA, yeast
RNA and calf liver DNA (see Example 13).
The processes for preparing the nuclease active frac-
tions of the present invention, as well as their activities
are described below in greater detail.
Example l
The nuclease activities of cellulase enzyme prepara-
tions were investigated by the following methods.
Solutions having an enzyme concentration of 20 mg/ml
(0.05 M phosphate buffer, pH 7.0) were prepared from each of
the following five cellulase preparations: Cellulase
Onozuka~ (product of Kinki Yakult Co., Ltd.; sample l),
Dorimelase (product of Kyowa Hakko Kogyo Co., Ltd.; sample
2), Nagase (product of Nagase & Company, Ltd.; sample 3),
Toyo Cellulase (product of Toyo Jozo Co., Ltd.; sample 4),
and Cellulase AP (product of Amano Seiyaku Co., Ltd.; sample
5). These solutions were allowed to act on ~DNA for l hour
at 35C, 45C or 55C, and the mixture were subjected to
electrophoresis through agarose gel at a current of 38 mA for
l hour. The resulting profiles are shown in Fig. l, in which
(a), (b) and (c) refer to the profiles obtained at 35C, 45C
and 55C, respectively. Symbols A to G respectively corre-
spond to the following: ~DNA, sample l, sample 2, sample 3,
1 335084
sample 4, sample 5 and the marker prepared by treating ~DNA
with HindIII.
Example 2
Nuclease active fractions of the present invention
were obtained by the following method.
A 2% solution of Cellulase Onozuka 3S (product of
Kinki Yakult Co., Ltd.) whose pH was held at 7.0 with 0.05 M
phosphate buffer was heated at 80C for lO minutes. The
insoluble product of heat denaturation that formed upon
heating was removed by centrifugation and the supernatant was
charged into a DEAE-Sephadex A-50 chromatographic column and
fractions obtained by elution with 0.5 M NaCl were recovered.
Example 3
Nuclease active fractions were obtained by repeating
the procedures of Example 2 except that Cellulase Onozuka
(product of Kinki Yakult Co., Ltd.) was replaced by Cellulase
AP (product of Amano Seiyaku Co., Ltd.)
Example 4
Nuclease active fractions were obtained by repeating
the procedures of Example 2 except that Cellulase Onozuka~
(product of Kinki Yakult Co., Ltd.) was replaced by Toyo
Cellulase (product of Toyo Jozo Co., Ltd.)
Example 5
Nuclease active fractions of the present invention
were obtained by the following method.
A 2% solution of Cellulase Onozuka 3S (product of
Kinki Yakult Co., Ltd.) having its pll held at 7.0 with 0.05 M
phosphate buffer was prepared. This solution was loaded on a
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DEAE-Sephadex A-50 column and unabsorbed fractions were
recovered.
Example 6
Nuclease active fractions were obtained by repeating
the procedures of Example 5 except that Cellulase Onozuka
(product of Kinki Yakult Co., Ltd.) was replaced by Cellulase
AP (product of Amano Seiyaku Co., Ltd.)
Example 7
Nuclease active fractions were obtained by repeating
the procedures of Example 5 except that Cellulase Onozuka
(product of Kinki Yakult Co., Ltd.) was replaced by Toyo
Cellulase (product of Toyo Jozo Co., Ltd.)
Example 8
The nuclease activities of fractions prepared in
accordance with the present invention were compared with
those of other fractions.
A 2% solution of Cellulase Onozuka 3S~ (product of
Kinki Yakult Co., Ltd.) having its pH held at 7.0 with 0.05 M
phosphate buffer was prepared. This solution was loaded on a
DEAE-Sephadex A-50 column and fractions unabsorbed at pH of
7.0 (the first group of fractions) were obtained.
Thereafter, with the concentration of NaCl being gradually
increased from O to 2 moles, the second, third and fourth
group of fractions were eluted. The individual fractions
were separated by observing the optical density at 280 nm
(see Fig. 2).
The activity for decomposition of ~DNA was investi-
gated by observing the profiles of electrophoresis through
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agarose gel. The results were as shown in Fig. 3 for the
first group of fractions, and in the upper part of Fig. 2 for
the second, third and fourth group of fractions. The profile
of ~DNA per se was as shown at the left end of Fig. 3.
As is clear from Figs. 2 and 3, a peak of decomposi-
tion activity centering at fraction No. 31 was observed for
the first group of fractions. However, no decomposition
activity was observed for the second, third and fourth group
of fractions.
Example 9
The heat resistance of active fractions prepared in
accordance with the present invention was examined by the
following method.
The active fractions obtained in Examples 5, 6 and 7
were allowed to act on ~DNA after their enzyme concentration
was adjusted to 20 mg/ml.
Condition 1 : reaction temperature, 60C; reaction time,
10 minutes
Condition 2 : reaction temperature, 70C; reaction time,
10 minutes
Condition 3 : reaction temperature, 80C; reaction time,
10 minutes
Condition 4 : reaction temperature, 100C; reaction time,
10 minutes
Condition 5 : reaction temperature, 100C; reaction time,
30 minutes
Condition 6 : reaction temperature, 100C; reaction time,
60 minutes.
1 335084
The mixtures were subjected to electrophoresis at a
current of 38 mA through agarose gel for 1 hour. The result-
ing profiles are shown in Fig. 4, in which (a), (b) and (c)
refer to the profiles for the active fractions obtained in
Example 5, 6 and 7, respectively. Symbols A - I denote the
following: ~DNA left intact (A); ~DNA treated with the
reaction solution from which active fractions were yet to be
isolated (B); ~DNA treated with active fractions under
condition 1 (C); ~DNA treated with active fractions under
condition 2 (D); ~DNA treated with active fractions under
condition 3 (E); ~DNA treated with active fractions under
condition 4 (F); ~DNA treated with active fractions under
condition 5 (G); ~DNA treated with active fractions under
condition 6 (H); and ~DNA treated with HindIII (I).
Example 10
The heat resistance of active fractions prepared in
accordance with the present invention was investigated by the
following method.
The first group of fractions obtained in Example 5 in
accordance with the present invention were heated at 100C
for different periods of time, i.e. 0, 10, 20, 30, 45 and 60
minutes, and their activities in decomposing ~DNA were inves-
tigated in terms of profiles of electrophoresis through
agarose gel (see the upper part of Fig. 5). Compared to 0-
minute heating, 10-, 20- and 30-minùte heatings caused a
gradual decrease in activity but the fractions yet retained
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substantially high levels of activity. They were consider-
ably attenuated by heating for 45 minutes but they still had
detectable levels of activity.
Example 11
The relationship between temperature and the activity
of active fractions prepared in accordance with the present
invention was investigated.
The active fractions were heated for 10 minutes at
varying temperatures of 0, 40, 50, 60, 70, 80, 90 and 100C
and their activities in decomposing ~DNA were investigated in
terms of profiles of electrophoresis through agarose gel (see
the lower portion of Fig. 5). Between 0 and 50C, no
substantial difference in activity was observed, but the
activities of the fractions decreased at 60C and were not
detectable at all at 70C. Nevertheless, uniform activities
were observed again at 80 - 100C.
Detailed activity investigations conducted at respec-
tive temperatures of 20, 30, 40, 45, 50, 60 and 70C showed
that an optimal temperature for the first group of fractions
was 45C (Fig. 6).
Example 12
The relationship between time and the decomposition of
~DNA by active fractions prepared in accordance with the
present invention was investigated.
To the first group of fractions obtained in Example 5,
~DNA was added and the mixtures were subjected to electro-
phoresis through agarose gel for 0, 3, 5, 10, 15, 30, 45 and
60 minutes. The resulting electrophoretic profiles are shown
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in Fig. 7, from which one can see that the lengths of decom-
position products became uniform 30 - 45 minutes after the
addition of ~DNA. Their length was calculated to be 500 bp
for 0.6% agarose and 400 bp for 1.2% agarose.
Example 13
The substrate specificity of active fractions prepared
in accordance with the present invention was investigated.
The second group of fractions obtained in Example 5
were reacted with ~DNA (A), heat-denatured ~DNA (B), bovine
thymus DNA (C), heat-denatured bovine thymus DNA (D), herring
sperm DNA (E), heat-denatured herring sperm DNA (F), Ml3 DNA
(G), yeast RNA (H), and calf liver DNA (I) and the mixtures
were subjected to electrophoresis through agarose gel. The
resulting electrophoretic profiles are shown in Fig. 8, from
which one can see that each of the substrates tested
decomposed, indicating the low substrate specificity of the
active fractions prepared in accordance with the present
invention.
