JP2779079B2 - Pentose selective fermentation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for selective fermentation of pentose, and more particularly to a method for selective fermentation of pentose in which the fermentation rate of pentose is increased by improving microorganisms.

[0002]

2. Description of the Related Art When two or more substrates are subject to metabolism in a certain microorganism, the phenomenon that a substrate which is easily used by the microorganism inhibits the induction of an enzyme which controls the catabolism of another substrate is caused by catabolite lyophilization. Compression (Catabolite Rep
Hartwell L. and B. Magasanik (Hartwell L. and B. Magasanik)
, Journal of Molecular Biology
(J. Mol. Biol), Volume 7, Pages 401-420 (1963),
Monod J., Recherches-sur-la-Croissance-de-Courture-Bactaenne, Rezzes sur la croissance des cult
ures bacteriennes, thesis), Armand (Harman),
Paris, p. 145 (1942)].

[0003] This catabolite repression includes not only metabolic systems such as sugars, amino acids and organic acids, which are substrates that serve as an energy source for bacteria, but also degradation systems for polymer substrates such as nucleic acids, polysaccharides and proteins. This is generally known. Therefore, in general, when an easily available substrate such as glucose, mannose, or fructose is present, the enzyme biosynthesis of the metabolic system of the other substrate is suppressed, and the other substrate is not available or the desired enzyme is not used. There are many cases where production becomes impossible.

[0004] Among bacteria belonging to the genus Pediococcus, for example, Pediococ halophilus (Pediococ)
The above phenomenon occurs when C. halophilus is cultured in a medium containing two or more substrates that the fungus can use as an energy source. That is, catabolite repression is one of the very powerful metabolic regulation mechanisms even in the genus Pediococcus, and only in the case of selective fermentation of a preferential lower substrate in a mixture of multiple substrates, for example, pentose. In addition, even in the case of the production of some enzymes and other secondary metabolites, it has become a major problem, and usually escaping this inhibitory effect,
Almost impossible. Even if you can do some in special cases,
It is necessary to cultivate for a very long time or to specifically remove the substrate in the medium which is an obstacle for the selective fermentation. This requires extra cost or makes it difficult to use a natural medium, which is an inexpensive and excellent but complex mixture, and the solution is strongly desired.

[0005] Pentose is one of the sugars involved in the browning reaction. For example, in a brewed product, an effective method for eliminating the pentose is desired in order to prevent coloring. Therefore, the present inventors have conducted various studies to release the catabolite repression of a microorganism belonging to Pediococcus halophilus, for example, pentoses such as arabinose and xylose. Pilebate dependent sugar: Phosphotransferase system (Phosphoenolpyruvate dependent Sugar: Phosphotr
ansferase system (hereinafter abbreviated as PTS), and the presence and function of the PTS, phosphofructokinase (hereinafter abbreviated as PFK) and glucokinase (hereinafter abbreviated as GK). ) Was found to be able to release this catabolite repression by triple deletion.

[0006] PTS stands for enteric bacteria (ente bacteria).
ricbacteria), a mechanism of sugar transport that has been studied in this regard, and Kundich W. et al.
igW. et al.,), Proceedings National Academy of Sciences (Proc. Natl. Acad. Sc)
i.), United States (USA), Volume 52, pp. 1067-1074 (1964
Years), PW Postma e
t al.,), Microbiological Review (Microb
iol. Rev.), Vol. 49, pp. 232-269 (1985) .The presence of PTS in microorganisms belonging to the genus Pediococcus was previously obtained by the present inventors. This is new knowledge.

[0007] Based on these findings, the present inventors have obtained triple-deficient mutants of PTS, PFK, and GK from microorganisms belonging to Pediococcus halophilus, and catabolism using these mutants has been achieved. A method for selectively fermenting a substrate to be inhibited, particularly pentose, has been developed (see, for example, Japanese Patent Application No. 3-28969). However, when fructose is contained in the substrate, the selective fermentability of pentose, particularly xylose, is inhibited, and these methods are not necessarily suitable depending on the purpose of use.

[0008]

DISCLOSURE OF THE INVENTION The present invention provides a microorganism capable of fermenting pentose even when glucose, mannose, fructose and the like, which are higher priority substrates, coexist and obtains a pentose by using the microorganism. The purpose of the present invention is to provide a selective fermentation method for pentose with a higher fermentation rate.

[0009]

Means for Solving the Problems The present inventors have conducted intensive studies to achieve the above object, and as a result, PTS isolated and isolated from microorganisms belonging to Pediococcus halophilus and having an arginine deiminase pathway. The new mutant strain deficient in cytoplasmic phosphotransferase is different from its parent strain, for example, arabinose, all PTS sugars except pentoses such as xylose (e.g., glucose, mannose, fructose, sucrose, lactose, sorbitol, It does not have the ability to ferment non-PTS sugars (e.g., maltose, glycerin, etc.) and non-PTS sugars.If this new mutant strain is used, catabolite lysate can be produced even in the presence of glucose, mannose, fructose, etc. Without receiving any impressions,
Pentose, in particular that xylose can be efficiently and selectively fermented, furthermore, by obtaining a new finding that the novel mutant has an arginine deiminase pathway, the fermentation rate of pentose can be further increased,
Based on this finding, the present invention has been completed.

That is, the present invention inoculates and cultures a pentose-containing microorganism with a microorganism belonging to the genus Pediococcus, having an arginine deiminase pathway, and lacking the cytoplasmic phosphotransferase of PTS, and having pentose fermentation ability. And selectively fermenting the pentose. Hereinafter, the present invention will be described in detail.

The microorganisms belonging to the genus Pediococcus of the present invention, having the arginine deiminase pathway, deficient in cytoplasmic phosphotransferase of PTS, and having pentose fermentation ability include any microorganisms having such properties. Any strain may be used as long as it is, for example, the following.
First, in general, PTS receives cytosolic phosphotransferase which receives and transfers phosphoric acid from cytosolic phosphoenolpile (hereinafter, abbreviated as PEP), and accepts phosphate to transfer sugar into cells. And Enzyme II (hereinafter abbreviated as EII) of the cell membrane which is transported to and phosphorylated in the cell. The cytoplasmic phosphotransferase receives cytoplasmic enzyme I (Enzyme I) (hereinafter abbreviated as EI), which first receives phosphate from cytoplasmic PEP, and then receives phosphate from phosphorylated EI. Cytoplasmic HP
r (transferred phosphate is transferred to EII) [for example, P. W. Postma et al., Microbiological Review, Vol. 49, pp. 232-269. (1985).

The microorganism deficient in the cytoplasmic phosphotransferase of the PTS of the present invention is a microorganism deficient in one or both of EI and HPr in the cytoplasm. By the way, the microorganism deficient in the cytoplasmic phosphotransferase of the PTS efficiently and selectively ferments pentose as described above, but does not utilize sugars such as glucose, mannose, and fructose, and therefore uses these. Energy supply to the cells is not sufficient compared to microorganisms.

The present invention utilizes the ATP regeneration system in the arginine deiminase pathway of the microorganism as an energy source for increasing the energy supply to the cells and further increasing the pentose fermentation rate. . This arginine deiminase pathway is, for example, the genus Lactococcus, Pseudomonas
onas), and is schematically shown in FIG. 1 [Raymond Cunin et al., Microbiol. Rev., Volume 50, No. 314-352
(See p. 1986)

[0014]

That is, in the arginine deiminase pathway, arginine is metabolized to citrulline and further to ornithine by arginine deiminase (ADI), while one of the formed carbamyl-phosphate is decomposed into NH ₃ and CO _2. The ATP regeneration system is involved in the process. The present inventors have found that this arginine deiminase pathway is present in some strains of Pediococcus halophilus.

The novel mutants of the present invention belonging to the genus Pediococcus, having the arginine deiminase pathway, lacking the cytoplasmic phosphotransferase of PTS and having the ability to ferment pentose include, for example, Pediococcus halophilus ( P
ediococcus halophilus) 5P-1 (hereinafter sometimes referred to as the present mutant strain). The Pediococcus halophilus 5P-1 is a mutant strain obtained by mutating a wild-type Pediococcus halophilus 5P having an arginine deiminase pathway and having a pentose fermentation ability.

And, Pediococcus halophilus
The mycological properties of 5P and Pediococcus halophilus 5P-1 are as follows. In the following mycological properties, the presence or absence of the arginine deiminase pathway was positive for "the ability to convert arginine to ornithine" (conversion rate was 90%).
% Or more is defined as positive. ) Is defined as “Yes”. In addition, the conversion rate was measured using the media shown in Table 1 below (hereinafter referred to as YP-XA-
Abbreviated as 1-5. ) And the conversion of arginine to ornithine was determined by quantification using a Hitachi high speed amino acid analyzer L-8500. Table 1 Yeast extract 0.3 g / 100 ml Polypeptone 0.5 Dipotassium hydrogen phosphate 0.5 Sodium thioglycolate 0.1 NaCl 5.0 Xylose 0.23 Arginine (hydrochloride) 0.32 Mycological properties of Pediococcus halophilus 5P Bacteria of Pediococcus halophilus 5P Biological properties, among the mycological properties of Pediococcus halophilus I (FERM BP-1302) (see JP-A-63-219368),
(1) Generation of acid and gas from sugars ”and“ (2)
Except that "the presence or absence of the arginine deiminase pathway" is as follows, it is exactly the same as the mycological properties of Pediococcus halophilus I.

(1) Whether or not acid and gas are generated from saccharides (2) Presence or absence of arginine deiminase pathway: Yes Possibility of converting arginine to ornithine (conversion rate
98%) Mycological properties of Pediococcus halophilus 5P-1 Mycological properties of Pediococcus halophilus 5P-1
Among the bacteriological properties of Pediococcus halophilus 5P, "(1) Presence or absence of production of acid and gas from saccharides""(2)
Arginine Deiminase Pathway ”and“ (3) PTS
Cell membrane E II, cytoplasmic phosphotransferase EI + HPr and HP
r, PFK and GK activities (relative activities) are exactly the same as the mycological properties of Pediococcus halophilus 5P except as described below.

(1) Whether or not acid and gas are generated from saccharides (2) Presence or absence of arginine deiminase pathway: Yes Possibility of converting arginine to ornithine (conversion rate
96%) (3) PTS cell membrane E II, cytoplasmic phosphotransferase EI +
Activity of HPr and HPr, PFK and GK (relative activity)

[0020]

Various enzyme activities in Table 2 were measured by the following methods. Cell membrane EII and cytoplasmic EI + HPr:
Chessy BM and Jay Thompson
BM and Thompson) [Journal of Bacteriol., 154, 1195-120]
3 and 1204-1214 (1983)] and Abe and Uchida (A
be K. and K. Uchida) [Archives of
Microbiology (Arch. Microbiol.), No. 155
Volume, pp. 517-520 (1991)] Cytoplasmic HPr: EI above
For the cytoplasmic fraction used for measuring the activity of + HPr, Hengstenberg W. et al.
Method [Journal of Bacteriology (J. Bac
teriol.), Vol. 99, pp. 383-388 (1969)] PFK and G
K: Abe K. and K. Uchida's method [Journal of Bacteriol.
1, 1793-1800 (1989)] From Table 2, it is found that Pediococcus halophilus 5P of the present invention.
-1 indicates that there is no defect in EII, PFK and GK, and that cytoplasmic phosphotransferase is deficient,
The values of I + HPr and HPr indicate that EI is missing.

These strains were obtained from the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology by Pediococcus halophilus 5P as Microbiological Research Bacteria No. 12418 (FERM P-12418) and Pediococcus halophilus 5P-1 by Koken Bacteria No. 12419 (FERM
P-12419). Examples of a method for treating the wild-type strain Pediococcus halophilus 5P to obtain the present mutant strain include N-methyl-N'-nitro-N-nitrosoguanidine (hereinafter abbreviated as MNNG) and ethyl methanesulfo. And methods for mutagenesis such as methylate, methylmethanesulfonate, ultraviolet irradiation, x-ray irradiation, irradiation treatment, spontaneous mutation, phage or plasmid transformation, transduction, conjugation, and the like.

The medium used for culturing the present mutant strain includes a medium used for culturing a strain belonging to general Pediococcus halophilus. The nitrogen source of the medium may be any available nitrogen compound or any containing nitrogen compounds, such as yeast extract, peptone, meat extract, gelatin, corn steep liquor, amino acids, leachate of soybean or wheat koji. One or more nitrogen sources are used. One or more suitable inorganic salts such as manganese, phosphoric acid, potassium, magnesium and calcium are appropriately added to the nitrogen source, and carbon sources necessary for the growth of the bacterium, if necessary, for example, sugars, various organic substances, inorganic substances, vitamins A medium to which sodium chloride is added is suitably used as a medium, and a medium containing 2 to 17% of sodium chloride is preferable. In addition, moromi sap at the initial stage of preparation in a normal soy sauce production method is appropriately diluted to prepare a salt solution of about 15%.

The present mutant strain is preferably a liquid culture method, and is preferably cultured under static or anaerobic conditions. The culture temperature is 15 to 50 ° C, preferably 20 to 40 ° C. The cultivation time varies greatly depending on the salt concentration during cultivation.
-10 days, and the pH during cultivation is determined by the pH of the synthetic medium (eg, MYP medium, YPG medium) and moromi sap medium.
3 to 9 are preferred.

Next, any means may be used to collect the mutant cells from the culture thus obtained.
Separation is carried out according to a usual operation method such as centrifugation, filtration and the like, and if necessary, washing is performed to obtain the present mutant strain. And, in the present invention, the present mutant strain is inoculated into a pentose-containing substance, cultured,
Pentose is selectively fermented, and the pentose-containing substance needs to contain arginine in order to utilize the ATP regeneration system in the arginine deiminase pathway of the present mutant strain, and the amount thereof is required. It is 1 to 500 mM, preferably 5 to 100 mM.

Examples of the pentose include arabinose, xylose, ribose and the like. As the pentose-containing substance, any substance containing pentose may be used. For example, glucose, of course, may contain a higher priority substrate such as mannose, and, unlike the conventional case, may contain fructose.If the present mutant strain is used, pentose by the coexistence of this fructose There is no inhibition of fermentability. Further, as the pentose-containing material, for example, soy sauce moromi,
Wood hydrolyzate, cereal hydrolyzate, and the like, or those obtained by adding appropriate components such as nutrients to these, as necessary.

It goes without saying that the present mutant strain can selectively ferment pentose in a pentose-free substance containing no fructose. The inoculum amount of the present mutant strain is, for example, 10 ² −10 ⁹ cells / g, or
l, preferably 10 ⁴ -10 ⁶ cells / g, or number / ml.
The fermentation conditions may be any conditions as long as fermentation is achieved, and may be, for example, the same as the above-described culture conditions of the present mutant strain.

[0028]

EFFECT OF THE INVENTION The present mutant strain comprises glucose, mannose,
It does not have the ability to ferment saccharides such as fructose, but is extremely unique in that it has the ability to ferment pentose, and has an arginine deiminase pathway. The cell energy is increased by the ATP regeneration system, and as a result, the pentose fermentation rate is 1.2 to 1.2 compared to the case where a strain having no such pathway is used.
It is extremely remarkably increased to about twice. Then, if the present mutant strain is inoculated and cultured in a pentose-containing substance, for example, pentoses involved in coloring substances in soy sauce moromi, etc., especially xylose, which is particularly difficult to ferment, are extremely short even in the presence of fructose. The present invention is industrially very useful because it can be selectively fermented in time.

[0029]

The present invention will be described below in more detail with reference to examples. However, these examples do not limit the technical scope of the present invention.

[0030]

Example 1 PTS having an arginine deiminase pathway
Mutagenesis of Pentococcus halophilus 5P-1 Mutant with Pentose Fermentation Ability Deleting Cytoplasmic Phosphotransferase from Pediococcus in 5 ml of the medium shown in Table 3 (hereinafter abbreviated as YPX-1-5) -Halofilus 5P (FERM P-12418) was inoculated and cultured at 30 ° C for 4 days to obtain a culture.
100 μl of the culture was inoculated into 5 ml of a medium (YPDGX) prepared by adding 150 mM 2-deoxyglucose (2DG) to YPX-1-5, and cultured at 30 ° C. for 4 days. 2 that has grown on this medium
DG resistant bacteria were cultured in the medium shown in Table 4 (MYPX-1-5CaCO ₃ plate)
Were diluted and inoculated to form about 100 colonies. The colonies that grew on the MYPX-1-5CaCO ₃ plate were cultured in the medium shown in Table 5 (MYPG-1-5CaCO ₃ plate) and in Table 6
Replicate to the described medium (MYPFS-1-5CaCO ₃ plate),
While growing in MYPX-1-5CaCO ₃ plates and colonies were selected that can not be grown in MYPG-1-5CaCO ₃ plates and MYPFS-1-5CaCO ₃ plates.

Table 3 Yeast extract 0.3 g / 100 ml polypeptone 0.5 dipotassium hydrogen phosphate 0.5 sodium thioglycolate 0.1 NaCl 5.0 xylose 1.0 Table 4 Meat extract 0.5 g / 100 ml yeast extract 0.5 polypeptone 0.5 sodium thioglycolate 0.1 calcium carbonate 0.1 agar 1.5 NaCl 5.0 xylose 1.0 Table 5 Meat extract 0.5 g / 100 ml Yeast extract 0.5 Polypeptone 0.5 Sodium thioglycolate 0.1 Calcium carbonate 0.1 Agar 1.5 NaCl 5.0 Glucose 1.0 Table 6 Meat extract 0.5 g / 100 ml Yeast extract 0.5 Polypeptone 0.5 Sodium thioglycolate 0.1 Calcium carbonate 0.1 agar 1.5 NaCl 5.0 fructose 1.0 sucrose 1.0 EII, EI + HP
r, HPr, PFK and GK activities were measured to determine the novel mutant Pediococcus halophilus as shown in Table 2 above.
5P-1 (FERM P-12419) was obtained.

[0032]

Example 2 Novel mutant strain Pediococcus halophilus 5P-1 (FERM P-12419) and parent strain Pediococcus
Halofilus 5P (FERM P-12418) was replaced with YP described in Example 1.
One loopful of platinum loop was inoculated into 5 ml of X-1-5 medium and cultured at 30 ° C. for 4 days to obtain a culture. Next, the culture was subjected to a conventional method,
The cells were obtained by centrifugation at 18,000 rpm for 10 minutes, and the cells were washed with 5 ml of a 5% aqueous NaCl solution, dried by a conventional method, and used to produce Pediococcus halophilus 5P-1 and Pediococcus halophilus 5P. 0.9 dried cells each
mg and 0.93 mg were obtained.

[0033]

Example 3 Pseudococcus halophilus 5P-1 (RERM P-12419) cells of this mutant strain obtained in Example 2 and arginine obtained by the following method and having the following microbiological properties: Pentose fermentation mutant Pediococcus halophilus S-1 (FERM P-12417), which lacks deiminase pathway and lacks PTS cytosolic phosphotransferase
Was cultured in the same manner as in Example 2,
-MES-XAG medium was inoculated at a cell count of 1 × 10 ⁶ cells / ml, and cultured at 30 ° C. for 60 hours.

Table 7 Yeast extract 0.3 g / 100 ml polypeptone 0.5 g dipotassium hydrogen phosphate 0.5 sodium thioglycolate 0.1 NaCl 5.0 MES buffer (pH 7) 200 mM xylose 11 mM glucose 5 mM arginine 11 mM This fermentation rate (sugar Consumption rate), bacterial growth (growth)
1 and 2 show the results of the examination.

FIG. 1 shows the results for Pediococcus halophilus 5P-1, and FIG. 2 shows the results for Pediococcus halophilus S-1. In each figure, △ indicates xylose, □ indicates arginine, and ○ indicates growth. Furthermore,
The quantification of sugar was determined by the method of Sinner M. and Plus J. (J. Chromatogr., Vol. 156, p. 197 (1978).
Year)].

The quantification of arginine was carried out using the above-mentioned Hitachi high-speed amino acid analyzer L-8500. Growth was based on absorbance (OD) at 530 nm. From FIG. 1 and FIG.
Fermentation of 11 mM xylose at a rate 1.4 times higher than that of the mutant strain Pediococcus halophilus S-1 (consumed in 35 hours). You can see that it is.

That is, the Pentococcus halophilus 5P-1, which has an arginine deiminase pathway and lacks the cytoplasmic phosphotransferase of PTS and has pentose fermentation ability, has no arginine deiminase pathway. Pentococcus halophilus S-1 deficient in PTS cytosolic phosphotransferase and capable of pentose fermentation
It can be seen that the fermentation rate of xylose is further increased as compared to

The above Pediococcus halophilus S-1 was obtained by the following method. The above-mentioned Pediococcus halophilus I (FERM BP-1302) (for mycological properties, see JP-A-63-219368) was used as a parent strain, and the mutation was induced in the same manner as in Example 1.

Also, Pediococcus halophilus S-
1 is characterized by Pediococcus halophilus I
"(1) Presence or absence of acid and gas generation from saccharides" and "(2) Presence or absence of arginine deiminase pathway"
And (3) PTS cell membrane E II, cytoplasmic phosphotransferase E
Activity of I + HPr and HPr, PFK and GK (relative activity) "
Is exactly the same as the mycological properties of Pediococcus halophilus I except that

(1) Whether or not acid and gas are generated from saccharides (2) Presence or absence of the arginine deiminase pathway: No conversion ability from arginine to ornithine negative (conversion rate 3%) (3) PTS cell membrane E II, cytoplasmic phosphotransferase EI +
Activity of HPr and HPr, PFK and GK (relative activity)

[0041]

It should be noted that Pediococcus halophilus S-
1 is a microbial laboratory
Deposit No. 12417 (FERM P-12417).

[0043]

Example 4 100 kg of defatted soybeans were steam-denatured and 105 kg of wheat crushed in a roasted mushroom were mixed, inoculated with seed koji, and subjected to 42 hours of ventilation koji making to obtain soy sauce koji. 360 L of salted water containing 90 kg of sodium chloride and cooled to 15 ° C. was added thereto, and the mixture was charged into a 600 L closed container charging tank.

Next, the mutant Pediococcus halophilus 5P-1 (FERM P-1) obtained by culturing in the same manner as in Example 2 was used.
2419), parent strain Pediococcus halophilus 5P (FERM
P-12418) and Pediococcus halophilus S-1 (FE
RM P-12417) was aseptically filtered from a soy sauce moromi three weeks after preparation using a membrane filter with a pore size of 0.22 μm (manufactured by Nippon Millipore), and fructose was added to the moromi sap of about 0.5. %, Each loop was inoculated into 10 ml of the mixture, and cultured at a temperature of 30 ° C. for 130 hours to obtain respective cultures.

The arginine content in the moromi sap was 28 mM.
Met. Table 9 shows the results of comparison of the amount of residual sugar in the obtained culture. The quantification of the amount of residual sugar was measured by the method of Sinner M. and Plus J. described above.

[0046]

It should be noted that Pediococcus halophilus S-
When 1 strain was used, xylose was completely consumed (fermented) by culturing at 30 ° C. for 170 hours.
From Table 9, according to the method of the present invention, pentoses such as arabinose and xylose can be selectively and extremely fermented in a very short time in the presence of a substrate that inhibits the fermentation of pentoses such as glucose and fructose at a high concentration. It turns out that it gets.

[Brief description of the drawings]

FIG. 1 shows the mutant Pediococcus in Example 3
The graph which shows the fermentation rate at the time of using Halofilus 5P-1, the cell growth rate, etc.

FIG. 2 is a graph showing a fermentation rate, a cell growth rate, and the like when using Pediococcus halophilus S-1 in Example 3.

Continuation of the front page (51) Int.Cl. ⁶ identification code FI (C12S 13/00 C12R 1:01) (58) Investigated field (Int.Cl. ⁶ , DB name) A23L 1/238 C12S 13/00 C12N 1/20 BIOSIS (DIALOG) WPI (DIALOG)

Claims (1)

(57) [Claims] The present invention belongs to the genus Pediococcus, has an arginine deiminase pathway, and has a pentose fermentation ability lacking the cytosolic phosphotransferase of the phosphoenol pileate-dependent sugar: phosphotransferase system. A selective pentose fermentation method, comprising inoculating a microorganism with a pentose-containing substance, culturing the resultant, and selectively fermenting the pentose.