U.S. patent number 3,912,592 [Application Number 05/467,175] was granted by the patent office on 1975-10-14 for method for producing l-sorbosone.
This patent grant is currently assigned to Hoffmann-La Roche Inc.. Invention is credited to Shraga Makover, David Louis Pruess.
United States Patent |
3,912,592 |
Makover , et al. |
October 14, 1975 |
Method for producing L-sorbosone
Abstract
The present invention relates to a microbiological method for
producing L-sorbosone, an important intermediate in the production
of vitamin C, from L-sorbosone.
Inventors: |
Makover; Shraga (Verona,
NJ), Pruess; David Louis (Passaic, NJ) |
Assignee: |
Hoffmann-La Roche Inc. (Nutley,
NJ)
|
Family
ID: |
23854678 |
Appl.
No.: |
05/467,175 |
Filed: |
May 6, 1974 |
Current U.S.
Class: |
435/105; 435/828;
435/866; 435/874; 435/880; 435/886; 435/822; 435/832; 435/877;
435/881 |
Current CPC
Class: |
C07H
3/08 (20130101); C12P 19/02 (20130101); Y10S
435/832 (20130101); Y10S 435/828 (20130101); Y10S
435/866 (20130101); Y10S 435/822 (20130101); Y10S
435/874 (20130101); Y10S 435/88 (20130101); Y10S
435/886 (20130101); Y10S 435/877 (20130101); Y10S
435/881 (20130101) |
Current International
Class: |
C07H
3/08 (20060101); C07H 3/00 (20060101); C12P
19/00 (20060101); C12P 19/02 (20060101); C12D
001/00 () |
Field of
Search: |
;195/36,47,49,32,31R,65,43,96,81 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Chem. Abstracts, 73:R86467z. .
Chem. Abstracts, 77:112489n. .
Chem. Abstracts, 78:27851a..
|
Primary Examiner: Monacell; A. Louis
Assistant Examiner: Wiseman; Thomas G.
Attorney, Agent or Firm: Welt; Samuel L. Saxe; Jon S.
Epstein; William H.
Claims
We claim:
1. A method for producing L-sorbosone from L-sorbose which
comprises pre-culturing under submerged aerobic conditions at a
temperature of from 20.degree. to 45.degree.C. and a pH of from 5
to 9, a microorganism selected from the group consisting of:
Paecilomyces;
Mycobacterium;
Pseudomonas;
Serratia;
Bacillus;
Aerobacter;
Streptomyces;
Gluconobacter;
Sarcina; and
Acinetobacter,
in an aqueous medium to produce cells containing an enzyme system
capable of converting L-sorbose to L-sorbosone, incubating under
submerged aerobic conditions at a temperature of from 20.degree. to
45.degree.C. and a pH of from 5 to 9, said cells with a medium
containing L-sorbose to convert the L-sorbose to L-sorbosone and
recovering L-sorbosone from said media.
2. The process of claim 1 wherein said microorganism is Pseudomonas
putida.
3. The process of claim 1 wherein said microorganism is Aeorbacter
aerogenes.
4. A process for the preparation of L-sorbosone which comprises
pre-culturing, under submerged aerobic conditions at a temperature
of from 20.degree. to 45.degree.C. and a pH of from 5 to 9, a
microorganism selected from the group consisting of:
pseudomonas;
Serratia;
Bacillus;
Aerobacter;
Streptomyces;
Sarcina;
Mycobacterium;
Acinetobacter;
Gluconobacter; and
Paecilomyces,
in an aqueous medium to produce cells containing an enzyme system
capable of converting L-sorbose to L-sorbosone, extracting said
enzyme system from said cells and incubating this system, under
submerged aerobic conditions at a temperature of from 20.degree. to
45.degree.C. and a pH of from 5 to 9, with a L-sorbose containing
medium to convert the L-sorbose in the medium to L-sorbosone.
5. The process of claim 4 wherein said microorganism is Pseudomonas
putida.
6. The process of claim 4 wherein said microorganism is Aerobacter
aergenes.
Description
CROSS REFERENCE TO RELATED APPLICATION
This application is related to copending application Ser. No.
285,265, filed Aug. 31, 1972, Makover and Pruess.
BACKGROUND OF THE INVENTION
The compound L-sorbosone, which has the formula: ##EQU1## is
disclosed in U.S. patent application Ser. No. 285,265, filed Aug.
31, 1972, as an intermediate for 2-keto-L-gulonic acid and can be
converted to 2-keto-L-gulonic acid by various enzymatic means.
Kondo and Ameyama have disclosed in Reports of Department of
Agriculture, Shizueka University, volume 7, page 139 (1957) a
pathway from L-sorbose to L-sorbosone utilizing Acetobacter
suboxidans. However, it has long been desired to utilize other
microorganisms for producing L-sorbosone.
SUMMARY OF THE INVENTION
In accordance with this invention, it has been found that
L-sorbosone can be produced from L-sorbose by the microbiological
oxidation of L-sorbose from a microorganism selected of the
following genera:
Gluconobacter;
Pseudomonas;
Acinetobacter;
Bacillus;
Sarcina;
Streptomyces;
Serratia;
Aerobacter;
Mycobacterium; and
Paecilomyces.
DETAILED DESCRIPTION
The novel process of this invention involves the one step
microbiological conversion of L-sorbose to L-sorbosone.
Any microorganism of the aforementioned genera can be utilized in
accordance with this invention. Among the preferred strains are
included: Acinetobacter calcoaceticus (ATCC 10153) Bacillus sp.
strain TA (ATCC 27860)
Streptomyces cellulocae (ATCC 3313), (Serratia sp. (ATCC 93),
Serratia marcescens (ATCC 27857), Aerobacter aerogenes (ATCC
27858), Sarcina lutea (ATCC 9341), Pseudomonas putida (ATCC 21812),
Gluconobacter melanogenus (IFO 3293), Mycobacterium phlei (ATCC
355); and Paecilomyces varioti (ATCC 26820).
The strains of these species are publicly availabe in culture
collections, both in the United States and abroad at ATCC (American
Type Culture Collection) Washington, D.C. and IFO (Institute for
Fermentation) Osaka, Japan. Mutant strains of the above mentioned
microorganisms have superior ability relative to the former wild
strains to transform L-sorbose to L-sorbosone. These mutant strains
can be produced from the wild strains by mutagenic treatments. Such
mutation can be caused by treating a wild strain with a mutagen
such as ultra-violet irradiation, X-ray irradiation or contact with
nitrous acid, or by isolating a clone occurring by spontaneous
mutation. These means for inducing the desired mutation on a wild
type strain may be effected in any of the ways per se well known
for this purpose by one skilled in the art. Many of these methods
have been described in various publications, for example, "Methods
in Medical Research" Vol. 3, edited by R.W. Gerard, published by
the Year Book Publishers, Inc., Chicago, U.S.A., in 1950, and
"Nature" Vol. 183, p. 1829 (1959) by F. Kaudewitz.
The production of L-sorbose is effected by the cultivation of one
of the above genera of microorganisms in an aerated deep tank,
i.e., under submerged fermentation. The fermentation should be
conducted at pH values of from about 5 to about 9 with pH values of
from about 6.5 to about 7.5 being preferred. It is particularly
preferred to carry out the process of this invention at a pH of
from 7 to 7.2. Although the temperature is not critical, best
results are usually obtained utilizing temperatures of from
20.degree. to 45.degree.C., with temperatures of from about
25.degree. to 35.degree.C. being particularly preferred. In
general, about one to ten days are required to obtain the best
results and from about 1 to 2 days are found most suitable.
The method of the present invention can be carried out by culturing
the microorganism in a medium containing appropriate nutrients and
L-sorbose. On the other hand, the process of this invention can be
carried out by culturing the microorganisms and then, after
culturing, bringing the whole cells or the cell free extract
prepared from the culture into contact with L-sorbose.
In the case where the microorganism is cultured in a medium
containing L-sorbose and appropriate nutrients, the microorganism
may be cultured in an aqueous medium in an aerated fermentor. The
cultivation should be conducted at pH values of from about 5 to
about 9, with pH's of from about 6.5 to 7.5 being preferred.
Especially preferred is utilizing a pH of about 7.0 to 7.2. A
preferred temperature range for carrying out this cultivation is
from about 20.degree. to about 45.degree. C. with temperatures of
from about 25.degree. to 30.degree. C. being especially preferred.
While the time for cultivation varies, with the kind of
microorganisms and nutrient medium to be used, about 1 to 10 days
cultivation usually brings about most preferable results.
Concentration of L-sorbose in the media varies with the kind of
microorganism, is generally desirable to be about 1 to 150
grams/liter, most preferably from about 2 to about 70
grams/liter.
It is usually required that the culture medium contains such
nutrients for the microorganism as assimiable carbon sources,
digestible nitrogen sources and preferably inorganic substances,
vitamins, trace elements, other growth promoting factors, etc.
L-sorbose per se can serve as the carbon source, but it is
preferred to utilize other substances as carbon sources. Among the
substances which can be utilized as carbon sources are included
starch, cane sugar, lactose, dextrin, glycerol, maltose, etc. They
can be employed at a concentration of from about 1 g./l. to about
10 g./l. As the nitrogen sources, there may be used various organic
or inorganic substances such as soybean meal, meat extracts,
peptone, casein, yeast extracts, corn steep liquor, urea, nitrates,
ammonium salts, etc. As the inorganic nutrients, for example,
potassium phosphates, magnesium sulfate, ferrous and ferric
chlorides, calcium carbonate, etc., are usually employed. As the
constituents of the medium vary also with the kind of
microorganisms to be employed. it is preferable to choose a proper
medium case by case.
In the case where after cultivation, the whole culture, i.e., the
cells collected from the culture are brought into contact with the
L-sorbose cultivation of the microorganisms is carried out under
similar conditions described above. Substances mentioned above can,
if desired, also be used for nutrients for this cultivation.
On the other hand, no nutrients need be present in cultivating the
microorganisms. The whole grown culture is then utilized to convert
L-sorbose to L-sorbosone. This conversion can be simply carried out
in an aqueous medium under submerged conditions utilizing a pH of
from about 5 to 9. In this conversion, no additional nutrients need
be present.
Generally, from about 1 to 3 days culture is preferable for
obtaining the most effective cells for the conversion of L-sorbose
to L-sorbosone. In one case, L-sorbose or its aqueous solution is
added to the cultured medium to make its final concentration from
about 1 g./l. to about 200 g./l. The mixed solution may be
incubated for about 1 to 10 days under the same conditions as
discussed above. In another case, the cells may be collected by
centrifugation from the cultured broth and resuspended in an
aqueous medium at a pH of from about 5 to 9. Then L-sorbose is
added in the same way as mentioned above. The succeeding incubation
may be effected under similar conditions as those described
above.
On the other hand, when cell free extracts from the whole grown
culture are utilized, these cell free extracts can be utilized to
convert L-sorbose to L-sorbosone by treating L-sorbose with the
cell free extracts in an aqueous medium at a pH of from 5 to 9. In
this case, no nutrients need be present.
Where the whole cells or the cell free extract are utilized to
convert L-sorbose to L-sorbosone, this conversion is generally
carried out with no additional nutrients being present. However, if
desired, any of the conventional nutrients such as those mentioned
hereinbefore may be added to the aqueous fermentation medium
containing either the whole grown cells or the cell free extract.
Furthermore, in carrying out the microbiological conversion of
L-sorbose with whole grown cells or with cell free extract,
temperatures are not critical. However, it is generally preferred
to utilize incubating temperatures of from 20.degree. to
40.degree.C. Generally, this microbiological conversion is carried
out at a pH of from 5 to 9.
The reaction can be stopped by freezing the reaction medium, i.e.,
cooling the reaction medium to a temperature of 0.degree.C. or
below to convert. Any temperature of 0.degree.C. or below can be
utilized to stop the reaction. Generally, temperatures of from
-5.degree. to -50.degree.C. are utilized to stop the reaction. If
desired, the L-sorbosone can be isolated by separating it from the
reaction medium. Any coventional separation means can be utilized
to carry out this isolation procedure. Among the preferred methods
of isolation is by chromatography such as paper chromatography, or
column chromatography.
On the other hand, the L-sorbosone in the reaction medium need not
be isolated but can be converted directly to 2-keto-gulonic acid in
accordance with the disclosure in Ser. No. 285,265, filed Aug. 31,
1972.
The following examples are illustrative of the invention. All
temperatures are in degrees centigrade.
EXAMPLE 1
Enzyme prepared from Pseudomonas putida
Pseudomonas putida 190B (ATCC 21812) (20 ml. inoculum) were
inoculated in 6 liter flasks containing 2 liters of a medium
containing the following:
g./l. of distilled water ______________________________________
Ammonium sulfate 0.28 Sodium sulfate 2.5 K.sub.2 HPO.sub.4 10
KH.sub.2 PO.sub.4 5 Sodium citrate 5 CaCl.sub.2 . 6H.sub.2 O 0.025
FeCl.sub.3 . 6H.sub.2 O 1 .times. 10.sup..sup.-3 ZnCl.sub.2 5
.times. 10.sup..sup.-4 CuCl.sub.2 . 2H.sub.2 O 5 .times.
10.sup..sup.-4 MnCl.sub.2 . 4H.sub.2 O 5 .times. 10.sup..sup.-4
MgCl.sub.2 . 6H.sub.2 O 0.2 L-sorbose 4 Glycerol 5
______________________________________
The pH of the medium was adjusted to 7 and the medium was then
autoclaved for 15 minutes at 15 p.s.i. and a temperature of
151.degree.C. After autoclaving the cultures were grown in the
flasks by aeration on a shaker at approximately 280 RPM at
28.degree.C. for 8 hours. Cell-pellets were obtained by
centrifugation for 15 minutes at approximately 10,000 x gravity.
The cell-pellets were pooled and resuspended in 500 ml. of cold
0.02M aqueous sodium phosphate buffer pH 7.0 and the suspension
centrifuged again. The precipitates obtained were pooled and
resuspended in 50 ml. of 0.02M aqueous sodium phosphate buffer pH
7.0 and processed twice through a French press 14,000 lb. per sq.
inch. The resulting cell-extracts were centrifuged at approximately
27,000 .times. gravity for 30 minutes. The supernatents obtained
after removal of debris were dialyzed twice against 0.02M aqueous
sodium phosphate pH 7.0 containing 1 .times. 10.sup..sup.-3 M
ethylenediamine tetraacetic acid (EDTA) and then dialyzed against
buffer without EDTA. The resulting preparation designated dialyzed
fraction 1, was used as given below.
Reaction mixtures were prepared containing in a total volume of 1.0
ml. of following:
Sodium phosphate, pH 7.0 40 .mu. mole L-sorbose.sup.1 2.95 .times.
10.sup..sup.-2 .mu. mole Dialyzed fraction 1 15 mg. (protein)
.spsp.1.sup.14 C-labelled L-sorbose (1 .mu.Ci; sp. act: 34
mCi/mM).?
To each of these reaction mixtures, there was added L-sorbosone at
different concentrations as given in the table below. The reaction
mixture was incubated aerobically for 6 hours at 30.degree.C.
Analysis for .sup.14 C-labelled L-sorbosone was carried out by
paper chromatography utilizing the following method.
A 50 .mu.1 portion of each of the incubated reaction mixtures were
applied to Whatman 3 MM paper (ascending) and developed for 18
hours with the following solvents: ethyl acetate, pyridine, water,
acetic acid 5/5/3/1 parts by volume. L-sorbosone (R.sub.f = 0.33)
and L-sorbosone (R.sub.f = 0.50) are clearly separated by this
system. The developed strips (50 .times. 5 cm) were air dried and
then cut along the width into 1 cm segments. The folded segments
were placed in vials for radioassays using a scintillation counter.
Authentic samples of L-sorbosone and L-sorbose used as markers were
located on the developed chromatograms by spraying with a silver
nitrate solution or by location of the radioactive authentic
markers through radioassays counted in a scintillation counter.
From the counts, the weight percent of L-sorbose converted to
L-sorbosone were determined as given in the table below:
Table 1 ______________________________________ L-sorbosone weight
of L-sorbosone added to the synthesized per reaction medium weight
of L-sorbose (.mu. moles) present in reaction medium (%)
______________________________________ 2.13 .times. 10.sup..sup.-5
1.46 2.13 .times. 10.sup..sup.-2 1.37 2.13 1.14
______________________________________
EXAMPLES 2 through 5
The organisms listed in Table 2 were grown in ether Medium 1 or
Medium 2. The composition of Medium 1, which was adjusted to a pH
of 7, was as follows:
Grams/Liters of Distilled Water
______________________________________ Glucose 5 Yeast Extract 5
Beef Extract 5 Difco bacto Peptone.sup.2 5
______________________________________
The composition of Medium 2 was as follows:
Grams/Liters of Distilled Water
______________________________________ Difco bacto Peptone.sup.2 6
Glucose 1 Yeast Autolysete 3 Beef Extract 1.5 N-Z Amine A.sup.3 4
______________________________________ .sup.2 A mixture of amino
acids and peptides. .sup.3 A mixture of amino acids and peptides
formed by the enzymatic hydrolysis of casein.
Innoculations were perfomred by transferring a loopfull of each
culture from agar slants into 100 ml. of one of the two media in
500 ml. flasks. All cultures were grown with aeration on shakers
(approximately 250 RPM) for 24 hours at 35.degree. C. with the
exception that Serratia marcescens was grown at 28.degree. C. for
48 hours. After the growth period, each culture was split into
equal parts and centrifuged at approximately 10,000 times gravity
for 15 minutes, at 4.degree.C. Each of the precipitates of the
organisms was washed once with a buffer, i.e., either cold 0.02M
aqueous sodium phosphate (pH 7.2) or cold 0.02M aqueous potassium
phsophate (pH 7.2). The resulting buffered mixtures were then
centrifuged as above. The precipitates were resuspended in 5 ml. of
either of the buffers. Cell-extracts were prepared from each of the
suspensions as in Example 1 except that only one passage through
the French press was utilized.
In addition, all extracts so prepared were dialyzed at 4.degree.C.
either twice against 0.02M aqueous sodium phosphate buffer (pH 7.2)
containing 1 .times. 10.sup..sup.-3 M ethylene diamine tetraacetic
acid EDTA followed by twice against the same buffer devoid of EDTA
or twice against 0.02M potassium phosphate buffer containing 1
.sub.3/8 10.sup..sup.-3 M EDTA buffer containing 1 .times.
10.sup..sup.-3 M EDTA which was followed by twice against the last
buffer devoid of EDTA to obtain the resulting cell-free extract
preparation. The use of potassium phosphate or sodium phosphate is
indicated in the following table:
Reaction mixtures containing each of the cell-free extracts
prepared above were prepared with a volume of 1 ml. and a pH of 7.0
and containing the following ingredients:
L-sorbose.sup.4 1 .mu. Ci Cell-free Extract 0.5 ml. Aqueous sodium
or potassium phosphate 50 .mu.moles .spsp.4.sup.14 C -Labelled
L-sorbose (72 mCi/mMole;)
To these reaction mixtures there was added L-sorbosone at
difficient concentrations as given in the table below. The reaction
mixtures were incubated aerobically for 6 hours at 30.degree.C. The
reactions were terminated by freezing at -20.degree.C. Analysis for
L-sorbosone was carried out by the method given in Example 1. The
results are given in Table 2. In this table Bacillus s.p. was the
strain TA.
Table 2
__________________________________________________________________________
Conversion of L-Sorbose to L-Sorbosone Example Organism No. Grown
Ion In Percent Conversion of L-Sorbose in Media Cell-Free to
L-Sorbosone based upon Extract L-Sorbosone added reaction medium 0
2.2 .times. 10.sup..sup.-3
__________________________________________________________________________
* 1 Serratia s.p. ATCC 93 1 Potassium 1.9 1.4 2 Serratia s.p. ATCC
93 2 Sodium 1.0 0.6 3 Bacillus s.p. ATCC 27860 1 Potassium 0.5 -- 4
Bacillus s.p. ATCC 27860 2 Potassium 0.2 0.6 5 Sarcina lutea ATCC
9349 2 Potassium 0.4 0.4 6 Streptomyces ATCC 3313 2 Sodium 0.3 0.3
cellulosae 7 Serratia ATCC 27857 2 Sodium 0.3 0.1 marcescens 8
Aerobacter ATCC 27858 2 Potassium 1.1 1.0 aerogenes 9 Aerobacter
ATCC 27858 2 Sodium 3.3 3.7 aerogenes 10 Aerobacter ATCC 37858 1
Sodium 2.9 1.5 aerogenes
__________________________________________________________________________
*.mu.moles of L-Sorbosone added to the reaction mixture.
EXAMPLE 6
Pseudomonas putida (ATCC 21812) (20 ml. inoculum) were inoculated
in 6 liter flasks containing 2 liters of a medium which contains
the ingredients and the amounts set forth in Example 1.
The pH of the medium was adjusted to 7 and the medium was then
autoclaved for 15 minutes at 15 p.s.i. and a temperature of
151.degree.C. After autoclaving, the cultures were grown in the
flasks by aeration on a shaker at approximately 250 RPM at
28.degree. C. for 18 hours. Cells were harvested by centrifugation
for 15 minutes at approximately 10,000 .times. gravity. The cells
were pooled and resuspended in 500 ml of cold 0.02M aqueous sodium
phosphate buffer pH 7.0 and the suspension centrifuged again. The
washed cells obtained were pooled and resuspended in 50 ml. of
0.02M aqueous sodium phosphate buffer pH 7.0 and processed twice
through a French press 14,000 lb. per sq. and extracts centrifuged
at 27,000 .times. gravity for 30 minutes. The resulting
supernatent, i.e. cell free extract was centrifuged at
approximately 160,000 .times. gravity for 90 minutes. The
supernatent obtained was designed as the "soluble fraction"; it was
centrifuged again at high speed and the precipitate was
discarded.
Reaction mixtures were prepared containing in a total volume of 1.0
ml. the following:
Sodium phosphate, pH 7.0 50 .mu. mole L-sorbose.sup.5 1.4 .times.
10.sup..sup.-2 .mu. mole Soluble fraction 9.4 mg. .spsp.5.sup.14
C-labelled L-sorbose (72 MCi/mM). In some of the reaction mixtures
there was placed NAD.sup.+ [nicotinamide-adenine dinucleotide
(oxidized)] as a cofactor. In other reaction mixtures there was
placed NADP.sup.+ [nicotinamide-adenine dinucleotide phosphate
(oxidized)] as a cofactor. On the other hand, reaction mixtures
were prepared without a cofactor. Where the cofactor was present,
it was present in an amount of 1.4 .mu. mole per 1.0 ml. of
reaction mixture.
The reaction mixture was incubated aerobically for 7 hours at
30.degree.C. An analysis for .sup.14 C-L-sorbosone was carried out
by paper chromatography utilizing the method described in Example
1. From the counts, the moles of L-sorbose converted to L-sorbosone
was determined as given in the table below:
Table 3 ______________________________________ Reactions Cofactor %
of total counts in .sup.14 C-L-sorbosone
______________________________________ I None 0.3 II NAD.sup.+ 0.5
III NADP.sup.+ 0.2 ______________________________________
EXAMPLE 7
By the procedure given in Example 2 through 5, utilizing
Paecilomyces varioti (ATCC 26820) and Mycobacterium phlei (ATCC
355), L-sorbose was converted to L-sorbosone.
In the case of Mycobacterium phlei, the medium 2 was utilized, the
cation was potassium and 2.2 .mu. moles of L-sorbosone was added to
the reaction mixture. The percent conversion of L-sorbose to
L-sorbosone was 0.3%.
In the case of Paecilomyces varioti, the medium 1 was utilized, the
cation was poptassium and 2.2 .mu. moles of L-sorbosone was added
to the reaction medium. The percent conversion of L-sorbose to
L-sorbosone was 0.3%.
* * * * *