JP2779078B2 - Pentose selective fermentation - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for selective fermentation of pentose, and more particularly to a method for selective fermentation of pentose 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. Magasani (Hartwell L. and B. Magasani
k), Journal of Molecular Biology (J. Mol. Biol), Vol. 7, pp. 401-420 (1963),
Monod J., Lucerche sur Sur
La Croissance de Courture Bactaenne, These (Recherches sur la croissance des cultur)
es bacteriennes, thesis), Harman, Paris, 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, Pediococcus halophilus (Pediococ)
The above phenomenon occurs when C. halophilus is cultured in a medium containing two or more types of substrates that can be used as an energy source by the fungus. 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 superior 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 cancel catabolite repression of pentoses such as arabinose and xylose of microorganisms belonging to Pediococcus halophilus, and as a result, the microorganisms were first identified as phosphoenol pileate.・ Dependent Sugar: Phosphotransferase System (Phosphoenolpyruvate dependent Sugar: Phosphot
ransferase system) (hereinafter abbreviated as PTS), and functioning, and 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.
ig W. et al.,), Proceedings National Academy of Sciences (Proc. Natl. Acad. Sci.)
, United States (USA), Volume 52, pp. 1067-1074 (1964
Years), PW Postma e
tal.,), Microbiological Review (Microbi
ol. 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] First, the present inventors have proposed that Pediococcus
A triple-deficient mutant of PTS, PFK, and GK was obtained from a microorganism belonging to Halofilus, and a method for selectively fermenting catabolite-inhibiting substrates, particularly pentose, using these mutants was developed (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]

SUMMARY 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 method for fermentation that is well selective.

[0009]

Means for Solving the Problems The present inventors have conducted intensive studies to achieve the above object, and as a result, they have been derived and isolated from microorganisms belonging to Pediococcus halophilus.
A novel mutant deficient in PTS cytosolic phosphotransferase
Different from its parent strain, all PTS sugars (eg, glucose, mannose, fructose, sucrose, lactose, sorbitol, mannitol, etc.) and non-PTS sugars (eg, maltose, glycerin, etc.) except for pentoses such as arabinose, xylose, etc. ), And if this novel mutant is used, even if glucose, mannose, fructose, etc. coexist, pentose, especially xylose, can be efficiently selected without catabolite repression. New knowledge that fermentation can be performed
The present invention has been completed based on this finding.

That is, the present invention provides a method for inoculating a pentose-containing microorganism with a pentose-fermenting microorganism, which belongs to the genus Pediococcus and lacks the cytoplasmic phosphotransferase of PTS, and cultures the pentose to selectively ferment pentose. This is a selective fermentation method for pentose. Hereinafter, the present invention will be described in detail.

First, as a microorganism belonging to the genus Pediococcus of the present invention and lacking the cytoplasmic phosphotransferase of PTS and having a pentose fermentation ability, any microorganism having such properties may be used. For example, the following are mentioned. That is, the PTS is generally
Cytosolic phosphoenol pileate (hereinafter referred to as PEP
Abbreviated. ) And Enzyme II (hereinafter, abbreviated as E II) of the cell membrane that accepts and transfers phosphate from the cytoplasm, and Enzyme II that accepts phosphate and transports and phosphorylates sugars into cells. )). The cytoplasmic phosphotransferase is a cytoplasmic enzyme I (hereinafter abbreviated as EI) which first receives phosphate from the cytoplasmic PEP, and then phosphorylated EI.
It is composed of two types of cytosolic enzyme, HPr, which is a cytoplasm that receives more phosphate (the received phosphate is transferred to EII) [for example, PW Postma et al., Microbiological Review, ed. Volume 49, Volumes 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. Examples of novel mutants belonging to the genus Pediococcus of the present invention and lacking the cytoplasmic phosphotransferase of PTS and having pentose fermentation ability include, for example, Pediococcus halophilus S-1
(Hereinafter, also referred to as the present mutant strain).

The Pediococcus halophilus (Ped)
iococcus halophilus) S-1 is a wild strain Pediococcus halophilus (Pediococcus
halophilus) I (FERM BP-1302). This Pediococcus halophilus
The bacteriological properties of S-1 are as shown below.
The mycological properties of Pediococcus halophilus I are described in JP-A-63-219368. Mycological properties of Pediococcus halophilus S-1 Mycological properties of Pediococcus halophilus S-1
Among the mycological properties of Pediococcus halophilus I, "(1) the presence or absence of acid and gas generation from saccharides" and "(2)
Pediococcus halophilus I except that the activities (relative activities) of PTS cell membrane E II, cytoplasmic phosphotransferase E I + HPr and HPr, PFK and GK are as follows:
Is exactly the same as the mycological properties of (1) Generation of acid and gas from sugars (2) PTS cell membrane E II, cytoplasmic phosphotransferase E I + HP
r and HPr, PFK and GK activities (relative activities) Pediococcus halophilus S-1 and wild-type Pediococcus halophilus I (FE
The activity (relative activity) of each enzyme of RM BP-1302) is as shown in Table 1.

[0014]

The activities of various enzymes in Table 1 were measured by the following methods. Cell membrane E II and cytoplasmic E I + HPr
: Chassie BM and Jay Thompson
y BM and J. Thompson) [Journal of
Bacteriology (J. Bacteriol.), Vol. 154, No. 1195
Pp. 1203 and 1204 to 1214 (1983)] and the method of Abe and U. Uchida (Arch. Microbiol., No. 15).
5, 517-520 (1991)] Cytoplasmic HPr: Measured for the cytoplasmic fraction used in the above-mentioned EI + HPr activity measurement. The method of Hengstenberg W. et. Al. [Journal of Bacteriology (J. Bacteriol.), Vol. 99, No. 383]
Pp. 388 (1969)] PFK and GK: Abe K. and K. Uchida's method [Journal of Bacterio (J. Bacterio)
l.), 171: 1793-1800 (1989)] Table 1 shows that the novel mutant strain of the present invention, Pediococcus halophilus S-1, has no defect in EII, PFK and GK. And the cytoplasmic phosphotransferase is deficient. In particular, the values of EI + HPr and HPr indicate that EI is deficient.

This Pediococcus halophilus S-1
Was sent to the Institute of Microbial Industry and Technology,
No. 417 (FERM P-12417). Wild strain Pediococcus halophilus I for obtaining the mutant strain
For example, N-methyl-N'-nitro-
Mutagenesis such as N-nitrosoguanidine (hereinafter abbreviated as MNNG), ethyl methanesulfonate, methyl methanesulfonate, ultraviolet irradiation, x-ray irradiation, irradiation treatment, spontaneous mutation, phage or plasmid traits Examples include methods such as conversion, transduction, and gene recombination by conjugation.

The medium used for culturing the present mutant strain includes a medium used for culturing a strain belonging to general Pediococcus halophilus. As the nitrogen source of the medium, any available nitrogen compounds or those containing the same may be used.For example, 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 cells of the present mutant strain from such a culture, for example, centrifugation, filtration and the like.
Wash the cells as necessary to obtain the cells. In the present invention, the mutant strain is inoculated into a pentose-containing substance, cultured, and the pentose is selectively fermented.

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 or mannose may contain a higher priority substrate as well as fructose differently from conventional ones.If this mutant is used, fermentation of pentose by coexistence of this fructose There is no sexual inhibition. further,
Examples of the pentose-containing substance include soy sauce moromi, wood hydrolyzate, cereal hydrolyzate, and the like, or those obtained by adding appropriate components such as nutrients to these as required.

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.

[0022]

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.
For example, in soy sauce moromi, etc., pentoses involved in coloring substances, especially xylose, which is particularly difficult to ferment, can be efficiently and selectively fermented even in the presence of fructose, so that the present invention is industrially extremely useful. is there.

[0023]

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.

[0024]

Example 1 Cytoplasmic phosphotransferase of PTS was deficient.
Mutagenesis of mutant strain Pediococcus halophilus S-1 having pentose fermentation ability Pediococcus halophilus I (FERM BP-1302) was added to 5 ml of the medium shown in Table 2 (hereinafter abbreviated as YPX-1-5). Was inoculated and allowed to stand still at a temperature of 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.
The cells were cultured at 30 ° C. for 4 days. The 2DG-resistant bacteria grown on this medium were added to the medium shown in Table 3 (MYPX-1-5CaCO ₃ plate).
The cells were diluted and inoculated to form about 00 colonies. Table 4 shows the colonies that grew on the MYPX-1-5CaCO ₃ plate.
Replicate to the described medium (MYPG-1-5CaCO ₃ plate) and the medium described in Table 5 (MYPFS-1-5CaCO ₃ plate)
Colonies that grew on the -1-5CaCO ₃ plate but were unable to grow on the MYPG-1-5CaCO ₃ plate and MYPFS-1-5CaCO ₃ plate were selected.

Table 2 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 3 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 4 Meat extract 0.5g / 100ml 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 5 Meat extract 0.5g / 100ml 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 1 above.
S-1 (FERM P-12417) was obtained.

[0026]

Example 2 The novel mutant strain Pediococcus halophilus S-1 (FERM P-12417) and the parent strain Pediococcus halophilus I (FERM BP-1302) were used in the YPX-1-5 medium described in Example 1. One loopful of loop was inoculated to 5 ml and allowed to stand still at 30 ° C. for 4 days to obtain a culture.

Next, the culture was subjected to 18,000 r.
The cells were centrifuged at pm for 10 minutes to obtain cells, and the cells were
After washing with 5 ml of aCl aqueous solution and drying by a conventional method, 0.78 mg of dried cells of Pediococcus halophilus S-1 and 0.78 mg of dried cells of Pediococcus halophilus I were added.
81 mg were obtained.

[0028]

Example 3 100 kg of defatted soybeans were steam-denatured and mixed with 105 kg of wheat which had been roasted and shredded, 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 S-1 (FERM P-12417) obtained in Example 2,
Parent strain Pediococcus halophilus I (FERM BP-130
2) From the soy sauce moromi 3 weeks after preparation, the pore size
0.22μm membrane filter (Nippon Millipore)
The moromi sap obtained by aseptic filtration using fructose was added to about 0.5% fructose to about 0.5%, and each loop was inoculated into 10 ml loops, and cultured at 30 ° C for 7 days to obtain each culture. Was.

Table 6 shows the results of comparison of the amount of sugar remaining in the obtained culture. The residual sugar content was determined by the method of Sinner M. and Plus J. [Journal of Chromatography (J. Chromium).
atogr.), vol. 156, p. 197 (1978)].

[0031]

From Table 6, it can be seen that according to the method of the present invention, pentoses such as arabinose and xylose can be selectively and completely fermented in the presence of a substrate that inhibits the fermentation of pentoses such as glucose and fructose at a high concentration. And that this mutant shows a high degree of selectivity for pentose as compared with other strains.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) A23L 1/238 C12S 13/00 C12N 1/20 BIOSIS (DIALOG) WPI (DIALOG)

Claims (1)

(57) [Claims] 1. A pentose-fermenting microorganism belonging to the genus Pediococcus and lacking the cytosolic phosphotransferase of the phosphoenol pileate-dependent sugar: phosphotransferase system, which is capable of fermenting pentose. Pentose selective fermentation method characterized by inoculating, culturing, and selectively fermenting pentose