U.S. patent application number 10/859827 was filed with the patent office on 2005-01-27 for cellulose produced by filamentous fungi.
This patent application is currently assigned to Incorporated Administrative Agency National Agriculture and bio-oriented Research Organization. Invention is credited to Hoshino, Tamotsu, Kawakami, Akira, Kuriki, Midori, Morita, Naoki, Okuyama, Hidetoshi, Sawada, Michiko, Terami, Fumihiro.
Application Number | 20050019876 10/859827 |
Document ID | / |
Family ID | 26554178 |
Filed Date | 2005-01-27 |
United States Patent
Application |
20050019876 |
Kind Code |
A1 |
Morita, Naoki ; et
al. |
January 27, 2005 |
Cellulose produced by filamentous fungi
Abstract
The present invention relates to a method of producing cellulose
comprising the steps of: (i) culturing filamentous fungus having
the ability to produce cellulose; and (ii) recovering cellulose
from a culture obtained by the step (i) and cellulose produced by
filamentous fungi. The present invention provides technology to
produce cellulose from filamentous fungi. Further, the present
invention provides novel cellulose, which is produced by
filamentous fungi and can be utilized as various industrial
materials, additives, and the like.
Inventors: |
Morita, Naoki; (Hokkaido,
JP) ; Hoshino, Tamotsu; (Hokkaido, JP) ;
Sawada, Michiko; (Hokkaido, JP) ; Okuyama,
Hidetoshi; (Hokkaido, JP) ; Kuriki, Midori;
(Hokkaido, JP) ; Kawakami, Akira; (Hokkaido,
JP) ; Terami, Fumihiro; (Hokkaido, JP) |
Correspondence
Address: |
FISH & RICHARDSON, PC
12390 EL CAMINO REAL
SAN DIEGO
CA
92130-2081
US
|
Assignee: |
Incorporated Administrative Agency
National Agriculture and bio-oriented Research Organization
|
Family ID: |
26554178 |
Appl. No.: |
10/859827 |
Filed: |
June 3, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10859827 |
Jun 3, 2004 |
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10369327 |
Feb 14, 2003 |
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10369327 |
Feb 14, 2003 |
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09676054 |
Sep 29, 2000 |
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Current U.S.
Class: |
435/101 ;
435/254.1; 536/56 |
Current CPC
Class: |
C08L 1/02 20130101; C12P
19/04 20130101 |
Class at
Publication: |
435/101 ;
435/254.1; 536/056 |
International
Class: |
C12P 019/04; C12N
001/14; C08B 001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 1, 1999 |
JP |
281425/1999 |
Sep 11, 2000 |
JP |
275211/2000 |
Claims
1-25. (cancelled)
26. A method of producing cellulose comprising: (i) culturing
filamentous fungus belonging to a genus Microdochium having the
ability to produce cellulose; and (ii) recovering cellulose from a
culture obtained in (i).
27. The method of claim 26, wherein the filamentous fungus
belonging to a genus Microdochium having the ability to produce
cellulose is a Microdochium nivale.
28. The method of claim 27, wherein the Microdochium nivale is a
Michrochochium nivale deposited as FERM BP-7298.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to microbial production of
cellulose, in particular, to a method of producing cellulose using
cellulose-producing fungi belonging to psychrophilic filamentous
fungi and cellulose obtained by this production method.
BACKGROUND OF THE INVENTION
[0002] Cellulose is a major constituent of the cell wall of plants.
Most cellulose for use as paper is derived from plants, such as
woods.
[0003] In addition to plants, some bacteria, prokaryotic organisms,
produce extracellular cellulose, called bacterial cellulose, in the
culture medium during cultivation. Because of its superior property
of dispersing in water, the bacterial cellulose has been utilized
as an additive for food and cosmetics. Since it has physical
properties different from those of cellulose produced from a wood
pulp and the like, it has been applied as various industrial
materials, for example, to an acoustic oscillation board in an
audio speaker.
[0004] On the other hand, filamentous fungi, which are eukaryotes,
are known to produce extracellular polysaccharides. Among
polysaccharides produced by such filamentous fungi,
polysaccharides, which have been reported so far to consist of
glucose, similar to cellulose, are .alpha.-glucan including
pullulan and elsinan, and .beta.-glucan including
.beta.-(1.fwdarw.3)-glucan with branches or side chains of
.beta.-(1.fwdarw.3).multidot.(1.fwdarw.6)-glucan and
.beta.-(1.fwdarw.6).multidot..beta.-(1.fwdarw.44) glucan.
[0005] It has not been reported that filamentous fungi produce
cellulose which is .beta.-(1.fwdarw.4)-glucan.
SUMMARY OF THE INVENTION
[0006] The object of this invention is to provide a method of
producing cellulose using filamentous fungi and cellulose produced
by the filamentous fungi.
[0007] As a result of diligent research to solve the above
problems, the inventors have completed the invention by finding
that Microdochium nivale belonging to psychrophilic filamentous
fungus produces insoluble polysaccharide in a large quantity in the
culture medium during cultivation of the organism, and produced
polysasccharide is cellulose.
[0008] The present invention encompasses the following
inventions.
[0009] (1) A method of producing cellulose comprising the steps
of:
[0010] (i) culturing filamentous fungus having the ability to
produce cellulose; and
[0011] (ii) recovering cellulose from a culture obtained by the
step (i).
[0012] (2) The method of (1), wherein the filamentous fungus
belongs to the genus Microdochium.
[0013] (3) The method of (1), wherein the filamentous fungus is
Microdochium nivale (FERM BP-7298).
[0014] (4) Cellulose produced by filamentous fungus.
[0015] (5) The cellulose of (4), wherein the filamentous fungus
belongs to the genus Microdochium.
[0016] (6) The cellulose of (4), wherein the filamentous fungus is
Microdochium nivale (FERM BP-7298).
[0017] (7) Cellulose which is obtained by the method of (1).
[0018] (8) Cellulose which has the physico-chemical properties
of:
[0019] (a) being completely hydrolyzed when it is treated in 2M
trifluoroacetic acid for 2 hours at 121.degree. C.; and
[0020] (b) showing no peak at 2 .theta.=5.0.degree. to 45.0.degree.
when it is lyophilized (freeze-dried) and subjected to X-ray
diffraction analysis.
[0021] This specification includes part or all of the contents as
disclosed in the specification and/or drawings of Japanese Patent
Application Nos. 1999-281425 and 2000-275211, which is a priority
document of the present application.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 is a graph showing (A) Changes with time in the
amount of extracellular insoluble polysaccharide produced by
Microdochium nivale while culturing the organisms at 4.degree. C.,
and (B) The relationship between the amount of extracellular
insoluble polysaccharide produced from Microdochium nivale and the
culture temperature. Symbols: .circle-solid. indicates the amount
of insoluble polysaccharides and .box-solid. indicates the amount
of Microdochium nivale cells.
[0023] FIG. 2 shows the results of constituent sugar analysis by
gas chromatography performed on insoluble polysaccharide produced
by Microdochium nivale.
[0024] FIG. 3 shows the results of FT-IR analysis on insoluble
polysaccharide produced by Microdochium nivale.
[0025] FIG. 4 shows the analytical results of the linking pattern
between glucose units in the insoluble polysaccharide produced by
Microdochium nivale.
[0026] FIG. 5 shows the results of gel filtration chromatography
performed on the insoluble polysaccharide produced by Microdochium
nivale.
[0027] FIG. 6 shows X-ray diffraction of (A) insoluble
polysaccharicde produced by Microdochium nivale, and (B) a filter
paper (Whatman) (vegetable cellulose)
DESCRIPTION OF THE PREFERED EMBODIMENTS
[0028] The present invention relates to cellulose produced by
filamentous fungi. Filamentous fungi used herein may be any
filamentous fungi having the ability to produce cellulose, without
other limitation. Preferred examples of the filamentous fungi are,
but are not limited to, those belonging to the genera Microdochium.
A particularly preferred filamentous fungus is a snow mold,
Microdochium nivale, which was deposited with National Institute of
Bioscience and Human Technology, Agency of Industrial Science and
Technology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken
305-0046, Japan) on Sep. 2, 1999 (Accession No. FERM BP-7298)
[0029] The present invention also relates to a method of producing
cellulose comprising the steps of (i) culturing filamentous fungus
having the ability to produce cellulose; and (ii) recovering
cellulose from a culture obtained by step (i). The filamentous
fungus used in this method is as described above. According to this
method, cellulose of the invention can be properly produced.
[0030] Media used for culturing the filamentous fungus may be any
media known to allow filamentous fungi to grow, without other
limitation. Either liquid or solid media maybe used. Preferably, a
liquid medium is employed. A particularly preferred medium used in
the invention is a liquid potato dextrose medium.
[0031] Culturing may be performed by any method known to those
skilled in the art, including shaking culture, stationary culture,
and the like. A preferred culturing method is shaking culture. In
liquid culture, the amount of cells added to a medium is not
specifically limited because it can be properly determined by those
skilled in the art. The amount of cells to be added per 200 ml of a
medium is preferably 0.01 g to 0.15 g, more preferably 0.04 g to
0.08 g.
[0032] When the amount of cells is represented by weight in this
specification, unless otherwise specified, the cell amount is
represented by the wet weight (weight of cells in wet conditions)
which is obtained by centrifuging cultured cells to remove the
supernatant fluid.
[0033] Further, in solid culture, the amount of cells added to a
medium is not specifically limited because it can be properly
determined by those skilled in the art.
[0034] Temperature for culturing is not specifically limited,
because it can be determined based on the results of examinations
for efficiency of cellulose production by filamentous fungi at
various temperatures. When Microdochium nivale (FERM BP-7298) is
used as filamentous fungus, culture temperature may be determined
according to graph B in FIG. 1. Temperature for culturing is not
specifically limited as described above. However in a temperature
range below 10.degree. C., a preferred temperature ranges from
2.degree. C. to 8.degree. C., more preferably 3.degree. C. to
6.degree. C., and the most preferably at about 4.degree. C. In a
temperature range above 10.degree. C., a preferred temperature
ranges from 12.degree. C. to 25.degree. C., more preferably
15.degree. C. to 22.degree. C., and the most preferably at about
20.degree. C.
[0035] The culturing period is not specifically limited, because it
can be determined based on the results of examinations for
efficiency of cellulose production by filamentous fungi for various
culturing periods. When Microdochium nivale (FERM BP-7298) is used
as filamentous fungus and cultured at 4.degree. C., a culturing
period may be determined according to graph A in FIG. 1. As
described above, the culturing period is not specifically limited.
A preferred culturing period is from 7 to 35 days, more preferably
10 to 20 days, and the most preferably about 14 days.
[0036] According to the preferred embodiment of this invention,
cellulose can be efficiently produced by inoculating and
shake-culturing Microdochium nivale (FERM BP-7298) in a potato
dextrose (2.4%) liquid medium (pH 5.1) for 14 days at 4.degree. C.
For example, when 0.06 g of Microdochium nivale is inoculated in
200 ml of the said liquid medium and cultured while turning the
medium placed in a 500 ml flask at 200 rpm, 4 g/l of cellulose can
be produced.
[0037] Thus produced intracellular or extracellular cellulose can
be recovered by known methods. When cellulose is produced
intracellularly, cellulose can be recovered by known methods from a
mixture obtained from the disrupted cells. When cellulose is
produced extracellularly, cellulose can be recovered by known
methods from portions other than the cells in the culture. For
example, cellulose produced in a liquid medium can be recovered by
centrifuging the liquid medium to collect the fraction containing
insoluble polysaccharide.
[0038] The recovered cellulose can be washed with distilled water
and the like. Moreover, the obtained cellulose can be subjected to
various processings or treatments according to its applications.
Such processings or treatments may be properly performed by those
skilled in the art.
[0039] It can be confirmed that the recovered insoluble
polysaccharide is cellulose, by chemical analysis or instrumental
analysis known to those skilled in the art. For example, the
insoluble polysaccharide can be confirmed to consist of D-glucose
by constituent sugar analysis, such as alditol acetate method.
Further, each glucose unit can be confirmed to be type .beta. (that
is, each three-dimensional configuration between glucose units is
type .beta.) according to an absorbance at 890 cm.sup.-1 by
infrared rays absorption analysis (IR). Furthermore, in constituent
sugar analysis using sodium borodeuteride (NaBD.sub.4) as a
reducing agent after complete methylation, each linking pattern
between glucose units is confirmed to be (1.fwdarw.4) linking. This
can also be confirmed by other methods, such as Smith degradation.
Chemical analysis and instrumental analysis as described above are
routinely employed in the art, thus they can be appropriately
performed by those skilled in the art.
[0040] The molecular weight of cellulose according to the invention
can be measured using methods known to those skilled in the art,
including gel chromatography using gel filtration, gel permeation
chromatography (GPC), and the like. Gel used for gel filtration is
not specifically limited, because those skilled in the art can
select a proper gel. A preferred gel is TSKgel HW-65F. Thus
determined molecular weight of cellulose according to the invention
is not specifically limited. A preferred molecular weight of the
cellulose ranges from 350,000 to 800,000, more preferably 500,000
to 700,000, and the most preferably about 600,000. The resultant
average molecular weight (Mw) is preferably 550,000 to 600,000,
more preferably about 5,750,000.
[0041] The crystal structure of the cellulose of the invention can
be determined by X-ray diffraction. Process for X-ray diffraction
is not specifically limited. For example, insoluble polysaccharide
(cellulose of the invention) obtained as described above is put
into a plastic container with a flat and smooth surfaced bottom,
and freeze-dried to prepare a film with a smooth surface (1 mm of
thickness, 14 mm.times.14 mm). X-ray diffraction analysis can be
performed by irradiating X-rays over this smooth surface. When
cellulose is analyzed by this concrete technique, the cellulose of
the invention shows no peak at 2 .theta.=5.0.degree. to
45.0.degree.. That is, the cellulose of the invention has no
definite crystal structure.
[0042] In the above constituent sugar analysis, when treated with
2M trifluoroacetic acid for 2 hours at 121.degree. C., the
cellulose of the invention is completely hydrolyzed to glucose, but
known vegetable celluloses resist against such hydrolysis and are
not completely hydrolyzed. Further, in X-ray diffraction as
described above, the cellulose of the invention shows no peak at 2
.theta.=5.0.degree. to 45.0.degree., but the known vegetable
celluloses show peaks at 2 .theta.=14.6.degree., 16.5.degree. and
22.7.degree.. This suggests that the cellulose of the invention has
no definite crystal structure while the known vegetable celluloses
have a crystal structure identical to understandings obtained so
far. Therefore, the cellulose of the invention is totally different
from known celluloses.
[0043] Accordingly, the present invention relates to cellulose
having the physico-chemical properties of:
[0044] (a) being completely hydrolyzed when it is treated in 2M
trifluoroacetic acid for 2 hours at 121.degree. C.; and
[0045] (b) showing no peak at 2 .theta.=5.0.degree. to 45.0.degree.
when it is lyophilized (freeze-dried) and subjected to X-ray
diffraction analysis.
[0046] The molecular weight of the cellulose, which is not
specifically limited, is preferably 350,000 to 800,000, more
preferably 500,000 to 700,000, and the most preferably about
600,000. The molecular weight is determined by dissolving 1 mg of
insoluble polysaccharide obtained as described above in 0.5 ml of
cadoxen (tris(ethylendiamine) cadmium hydroxide), subjected to gel
chromatography (TSKgel HW-65F, 15 cm.times.0.5 cm), fractionated
every 2 minutes (flow rate: 0.3 ml/minute), then analyzing the
sugar content of each fraction by the phenol-sulfuric acid method.
The resultant average molecular weight (Mw) is preferably 550,000
to 600,000, more preferably about 5,750,000.
Industrial Applicability
[0047] The present invention provides technology to produce
cellulose from filamentous fungi. Further, the present invention
provides novel cellulose, which is produced by filamentous fungi
and can be utilized as various industrial materials, additives, and
the like.
EXAMPLES
[0048] The present invention is further described in the following
examples. These examples are provided for illustrative purposes
only, and are not intended to limit the scope of the invention.
Example 1
Culture of Microdochium nivale and Recovery of Insoluble
Polysaccharide
[0049] 0.06 g of Microdochium nivale (FERM BP-7298) was inoculated
in 200 ml of a potato dextrose (2.4%) liquid medium (pH 5.1)
(manufactured by Difco) and cultured while turning the medium
placed in a 500 ml flask at 200 rpm. The culture medium was
centrifuged (15,000.times.g, 20 minutes) to collect jelly-like
substances in the upper layer of the precipitated cells. The
collected substances were washed with distilled water to obtain
insoluble polysaccharide.
[0050] The productivity of the insoluble polysaccharide was
examined in the above culturing process by varying the culture
temperature in the range of 4.degree. C. to 20.degree. C.
[0051] In FIG. 1B showing the results of this examination, each
value represents a dry weight per litter of the culture medium at a
point when the maximum amount of extracellular insoluble
polysaccharide was produced at each temperature (4.degree. C.: on
day 14, 10.degree. C.: on day 14, 15.degree. C.: on day 4,
20.degree. C.: on day 6). The symbol, ".circle-solid." indicates
the amount of insoluble polysaccharide and ".box-solid." indicates
the amount of filamentous fungus. The highest productivity was seen
in the culturing at 4.degree. C. according to FIG. 1B.
[0052] Next, changes with time in the amount of insoluble
polysaccharide produced in case of culturing Microdochium nivale at
4.degree. C. were examined. FIG. 1A shows the results of this
examination. The symbol ".circle-solid." indicates the amount of
insoluble polysaccharide and ".box-solid." indicates the amount of
filamentous fungus in FIG. 1A. Insoluble polysaccharide produced in
the culture fluid continued to increase until day 14 then it
started to decrease according to FIG. 1A.
[0053] Based on these results, 0.06 g of Microdochium nivale was
cultured for 14 days at 4.degree. C. Thus, insoluble
polysaccharide, 4 g per liter of the culture medium, was
obtained.
Example 2
Identification of Insoluble Polysaccharide Produced by Microdochium
nivale.
[0054] (1) Constituent Sugar Analysis
[0055] 0.5 ml of 2M trifluoroacetic acid was added to 1 mg of
insoluble polysaccharide obtained in Example 1, then the mixture
was hydrolyzed for 3 hours at 121.degree. C. Trifluoroacetic acid
was removed from the mixture using nitrogen gas. The resulting
monosaccharide was reduced with sodium borohydride. The hydroxyl
groups of the reduced monosaccharide were acethylated by Merkle and
Poppe's method (Merkle and Poppe, Methods Enzymol. 230, 1-15,
1994). Analysis with gas chromatography (column; Spelco SP-2330:
internal diameter 0.25 mm.times.length 15 m, temperature condition;
kept at 170.degree. C. for 4 minutes: elevated from 170.degree. C.
to 240.degree. C. at a rate of 8.degree. C./minute: kept at
240.degree. C. for 8 minutes) resulted in a peak only for
acethylated glucose. Therefore the insoluble polysaccharide was
confirmed to be polyglucan (FIG. 2).
[0056] (2) Determination of Three-Dimentional Configuration for
Carbon at Position 1 in Glucose
[0057] The insoluble polysaccharide obtained in Example 1 was
freeze-dried, and then ground down together with potassium bromide
and shaped into tablets. Subsequently the three-dimentional
configuration of carbon at position 1 in the glucose was examined
by Fourier transform infrared rays absorption analysis (FT-IR). As
a reslult, a peak was detected at 890 cm.sup.-1, suggesting that
this was type .beta. (FIG. 3).
[0058] (3) Determination of the Linking Pattern Between Glucose
Units
[0059] To examine the linking position of glucose in the insoluble
polysaccharide obtained in Example 1, hydroxyl groups existing in
the insoluble polysaccharide were completely methylated, and
subjected to constituent sugar analysis as described in (1) except
that sodium borodeuteride (NaBD.sub.4) was used for reduction.
[0060] The complete methylation was performed as follows. First, 10
ml of dimethyl sulfoxide (DMSO) was added to 80 mg of insoluble
polysaccharide, then the mixture was stirred in a flask with argon
gas sealed therein for 2 hours, followed by ultrasonication for 2
hours. The insoluble polysaccharide dissolved partially. To
completely dissolve the insoluble polysaccharide, an additional 15
ml of DMSO and 9 ml of sodium dimsyl (The sodium dimsyl had been
prepared by adding 10 ml of DMSO to 0.5 g of oleaginous sodium
hydride in a flask containing argon gas sealed therein) were added
to the mixture, then stirred for 2 minutes followed by
ultrasonication for 2 minutes. Then 1 ml of methyl iodide was added
to the mixture in ice and dialyzed against distilled water.
[0061] Thus obtained products were analyzed with a gas
chromatography mass spectrometer (GC/MS) (Column: Spelco SP-2330,
Internal diameter 0.25 mm.times.Length 15 m, Temperature condition:
maintained for 4 minutes at 190.degree. C.; elevated from 190 to
240.degree. C. at a rate of 4.degree. C./minute; maintained for 8
minutes at 240.degree. C.) A control was prepared by similarly
treating the standard sample of cellulose, whose 30% of hydroxyl
groups had already been methylated. FIG. 4 shows the results. In
FIG. 4, "A" indicates the gas chromatogram of the insoluble
polysaccharide; "C" indicates the mass spectrum of substances
eluted at about 12 minutes of the gas chromatogram of "A"; "B"
indicates the gas chromatogram of 30% methylated cellulose; and D
indicates the mass spectrum of substances eluted at about 12
minutes in the gas chromatogram of "C." As shown in FIG. 4, for the
insoluble polysaccharide, only glucose whose carbons at positions
2, 3 and 6 had been methylated was detected.
[0062] (4) Conclusion
[0063] The results obtained in (1) to (3) above suggest that the
insoluble polysaccharide consisted only of cellulose, which is
.beta.-(1.fwdarw.4)-glucan.
Example 3
Molecular Size of Cellulose
[0064] 1 mg of insoluble polysaccharide obtained in Example 1 was
dissolved in 0.5 ml of cadoxen (tris(ethylendiamine) cadmium
hydroxide), subjected to gel chromatography (TSKgel HW-65F, 15
cm.times.0.5 cm), and then fractionated every 2 minutes (flow rate:
0.3 ml/minute). Analysis of the sugar content of each fraction by
the phenol sulfuric acid method resulted in a single peak, as shown
in FIG. 5. Molecular weight was measured using dextran (molecular
weight: 12,000, 50,000 and 150,000) as, a standard sample. The
majority of the insoluble polysaccharide had molecular weight of
600,000. The average molecular weight (Mw: weight average molecular
weight) was 5,750,000.
Example 4
Crystal Structure Analysis
[0065] The insoluble polysaccharide obtained in Example 1 was put
into a plastic container with a flat and smooth bottom and
freeze-dried to prepare a film with a smooth surface (1 mm of
thickness, 7 mm.times.7 mm). Then X-ray diffraction analysis was
performed by irradiating X-ray over the smooth surface. This
analysis resulted in no definite peak. On the other hand, X-ray
diffraction analysis made for a filter paper (Whatman) as a control
resulted in peaks at 2 .theta.=14.6.degree., 16.5.degree., and
22.7.degree., which is specifically observed for the
three-dimensional structure of vegetable celluloses. Therefore, it
was shown that unlike known vegetable celluloses, the insoluble
polysaccharide has no definite crystal structure.
[0066] All publications, patents and patent applications cited
herein are incorporated herein by reference in their entirety.
* * * * *