JP2007143467A - Glutamine synthase gene, bacillus natto enhanced in expression of the gene, and natto of reduced ammonia content which is produced using the bacillus natto - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing high-quality natto of reduced ammonia odor through sufficient fermentation, using Bacillus natto that has been bred and developed so as to produce such natto of slight ammonia odor with decreased ammonia productivity. <P>SOLUTION: The method for producing the objective natto having reduced ammonia odor and an ammonia content of less than 50 ppm is provided, involving using the Bacillus natto with glutamine synthase activity enhanced to 0.01 unit/mg protein or greater that has been developed and obtained by isolating and identifying Bacillus natto's glutamine synthase gene and enhancing the expression of the gene. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a low-ammonia productive natto bacterium that is a causative substance of the odor of ammonia in natto and natto with a low ammonia odor produced using the bacterium. More specifically, the present invention relates to a low-ammonia-producing natto bacterium with enhanced glutamine synthase activity, and a low-ammonia odor natto produced using the natto bacterium.

  Natto is produced by fermenting with natto bacteria using soybean as a raw material, and is characterized by its unique odor along with the sticky material produced by natto bacteria.

  Natto contains various volatile components such as pyrazines, acetoin, diacetyl, acetic acid, propionic acid, short-chain branched fatty acids (isobutyric acid, isovaleric acid, and 2-methylbutyric acid), and ammonia. Although it is known (for example, refer nonpatent literature 1), pyrazines, diacetyl, etc. of these are said to be the main components of what is called a natto odor preferred by consumers who like natto.

  On the other hand, so-called ammonia odor and stuffy odor are said to be typical unpleasant odors. If natto with a low content of substances causing these unpleasant odors can be developed as much as possible, the quality of natto will be improved. It can be expected that

  From such a viewpoint, as a method of suppressing the Ammonian odor, which is one of the unpleasant odors, we developed an improved natto bacterium that does not produce much ammonia during fermentation and storage, and uses the natto bacterium. Many attempts have been made to produce natto, but most of these have been methods for producing natto by developing natto bacteria with reduced protease activity inherent in natto (for example, patent literature). 1).

  However, the protease activity of Bacillus natto plays an important role in the natto fermentation process, and when Bacillus natto with reduced protease activity is used, sufficient fermentation is not carried out, leading to immature natto. was there.

  In addition, in order to remedy such drawbacks, Bacillus natto has been developed that can exhibit sufficient protease activity during fermentation and can reduce protease activity only after the latter half of the fermentation. Even when used, there is a tendency that it is difficult to sufficiently reduce the amount of ammonia in natto and the ammonia odor cannot be sufficiently reduced (see, for example, Patent Document 2).

In other words, there has been a demand for the development of Bacillus natto that can produce natto with sufficient fermentation of natto, but with less ammonia odor.
Journal of Japanese Society of Food Industry, Volume 31, p. 587-595, 1984 JP-A-6-269281 JP-A-8-275772

  The present invention develops and develops natto bacteria that can sufficiently ferment natto and produce natto of good quality, and that can produce natto with reduced ammonia productivity and low odor of ammonia. An object of the present invention is to provide a method for producing natto of good quality with reduced ammonia odor by using natto to produce.

  As a result of intensive studies on the ammonia production mechanism of Bacillus natto, the present inventors have focused on the fact that the ammonia content in natto can be reduced by consuming the ammonia once produced, and the glutamine synthetase gene of Bacillus natto As a result, it was found that the target natto bacteria can be developed by enhancing the expression of the gene to increase the activity of glutamine synthetase.

  That is, the present invention described in claim 1 relates to a DNA comprising a base sequence encoding a glutamine synthetase consisting of a base sequence of base numbers 61 to 1395 out of the base sequence set forth in SEQ ID NO: 1 in the sequence listing.

  The present invention described in claim 2 relates to a glutamine synthetase having the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing.

The present invention according to claim 3 relates to Bacillus natto characterized by enhanced expression of a glutamine synthetase gene.
Furthermore, the present invention described in claim 4 is a nucleotide sequence wherein the glutamine synthetase gene encodes a glutamine synthetase consisting of the nucleotide sequences of base numbers 61 to 1395 among the base sequences set forth in SEQ ID NO: 1 of the sequence listing. It is related with the Bacillus natto of Claim 3 characterized by the above-mentioned.

  Furthermore, the present invention according to claim 5 is characterized in that the Bacillus natto according to any one of claims 3 to 4 is Bacillus subtilis glnAh2 (FERM BP-10446). It relates to natto bacteria described in the same paragraph.

  Furthermore, the present invention according to claim 6 relates to natto bacteria according to any one of claims 3 to 5, wherein the glutamine synthetase activity is 0.01 units / mg protein or more. .

  Furthermore, the present invention described in claim 7 relates to natto characterized by being manufactured using the natto bacteria described in any one of claims 3 to 6.

  Furthermore, the present invention according to claim 8 relates to natto according to claim 7, characterized in that the ammonia content is less than 50 ppm.

  INDUSTRIAL APPLICABILITY According to the present invention, there is provided a method for breeding and developing a natto bacterium having a very low production amount of ammonia, which is a causative substance of ammonia odor which is one of the unpleasant odors of natto. By producing natto using natto, it becomes possible to produce natto with a very low ammonia content and extremely low ammonia odor.

  Hereinafter, the present invention will be described in detail.

  There is no particular limitation on the original natto bacteria used for breeding improvement in the present invention, but usually, natto bacteria with excellent fermentation ability used in natto industry, natto bacteria isolated and obtained from nature, and further improvements It is desirable to use superior natto bacteria.

  Bacillus natto is classified as Bacillus subtilis, but it can ferment beans such as soybeans and create the characteristics of natto such as sticky material (stringent material). It is classified as Bacillus natto (Bacillus natto) or as a variant of Bacillus subtilis because it has the characteristics that it is required to biotin for growth. . Natto or Bacillus subtilis (natto) may be distinguished from Bacillus subtilis.

  Examples of Bacillus natto include Bacillus Natto IFO3009, Bacillus subtilis IFO3335, IFO3336, IFO3936, and IFO13169, and various other Bacillus natto can be widely used.

Specific examples include the O-2 strain isolated from commercial natto and the r22 strain (see, for example, Japanese Patent Application Laid-Open No. 2000-224982). by Takahashi bacteria (T ₃ strains, TUA culture collection chamber) various Bacillus natto such or Miyagino bacteria (Miyagino natto Seisakusho) can be appropriately used.

  The present invention obtains a Bacillus natto that does not produce ammonia by breeding Bacillus natto having increased glutamine synthetase activity, and one of the breeding methods includes a glutamine synthetase gene (hereinafter referred to as glnA). There is also a method by a genetic recombination method in which it is linked to a multi-copy number vector and introduced into Bacillus natto.

  In addition, in order to use such a gene recombination method with Bacillus natto, a transformation system for gene introduction into Bacillus natto is required. In the present invention, Bacillus natto with improved transformation ability is used. Using the practical level of transformation system, the plasmid vector was efficiently introduced into Bacillus natto and the target breeding was achieved.

  Further, as one of the gene recombination systems of Bacillus natto, a transduction method using a phage vector (for example, “Appl. Environ. Microbiol.”), Vol. 63, p. 4087-4089 (see 1997) has been developed, and this method is also used in the present invention.

  Further, for example, after the glutamine synthase gene is ligated to a high copy number plasmid vector such as pHY300PLK, an existing transformation method (for example, “FEMS Microbiol. Lett.”, 236). The method of increasing glutamine synthetase activity by introducing into the Bacillus natto using the volume, p.13-20, 2004) and making the glutamine synthetase highly expressed is also effective.

  Furthermore, in the present invention, it is also effective to increase the expression of the promoter region in the upstream region of the glutamine synthetase gene by genetic recombination or natural mutation.

  It should be noted that other than the above-described gene recombination techniques, that is, so-called screening methods for selecting naturally occurring Bacillus natto having already enhanced target gene expression and increased enzyme activity, drug mutation methods, and ultraviolet irradiation. Breeding is also possible by other methods such as those conventionally performed, such as increasing the expression of these genes by mutation treatment such as mutation methods.

  The use of the natto bacteria thus developed for natto production is not particularly limited as long as conventional methods are employed.

  For example, natto is generally soy natto manufactured using whole soybeans as raw material, but there is also some ground natto using raw soybeans as a raw material.

The method of manufacturing whole soybean natto is to immerse the whole soybean, which is a raw material, in cold water for more than ten hours, and then use a steaming vessel with pressurized steam (1.5-2 Kg / cm ² · 128-133 ° C.). ), And inoculated and mixed with natto bacteria at a high temperature (70 to 100 ° C.), filled in a predetermined container, and then brought into a fermentation chamber to be heated to a relatively high temperature (40 Generally, it is fermented for about a predetermined time (about 12 to 48 hours) at a temperature of about ~ 55 ° C, and then refrigerated at about 5 ° C (about 12 to 72 hours) for completion.

  Further, in the case of ground natto, it is produced in the same manner as in the case of ordinary round soybean natto, except that the previously ground soybean is soaked in water.

  In such a conventional method for producing natto, in the present invention, natto bacteria used in the fermentation process are used in place of the low-ammonia productive natto bacteria that are breed-improved by the above method.

  When comparing natto produced using low-ammonia-producing Bacillus natto and natto using conventional natto-bacteria used in the past, It is confirmed that there is almost no so-called ammonia odor.

  Note that the threshold concentration for sensing the ammonia odor varies depending on the individual and cannot be determined. However, when the concentration at which the ammonia odor is felt is investigated using a panelist sensitive to the odor of natto, the ammonia concentration clearly exceeds 50 ppm. Since the result of feeling odor was obtained, it can be said that it is about 50 ppm.

  Therefore, it is desirable that the ammonia concentration be less than 50 ppm, and in order to make the ammonia concentration less than 50 ppm, the present invention requires natto bacteria having significantly increased glutamine synthase activity compared to ordinary natto bacteria. Therefore, it is necessary to develop a natto bacterium that can detect a glutamine synthetase activity that is about 10 times or more that of a normal bacterium.

  The present invention has been made to achieve the above object, and according to the present invention, for example, as described in the Examples, the glutamine synthase gene itself (structural gene itself) is obtained by gene recombination technology. A new gene with enhanced expression was created and introduced into Bacillus natto, which succeeded in breeding a new Bacillus natto with enhanced expression of the glutamine synthase gene, one of which is Bacillus subtilis. (Bacillus subtilis) It is named glnAh2, and it has just been deposited internationally as FERM BP-10446 at the (Germany) Patent Organism Depositary.

  The novel natto bacterium bred for the first time according to the present invention has a glutamine synthetase activity of 0.01 unit / mg protein or more, and 0.5-1.5 unit / mg protein is also possible. A 2.6 unit / mg protein is obtained, and a 3 unit / mg protein or more is sufficiently possible.

  And by using such a new natto bacterium, it becomes possible to produce natto having an ammonia content of less than 50 ppm, and a product with a content of 30 ppm or less, for example, 28 ppm is obtained (Example), and a product with a content of 20 ppm or less. Of course, those below 10 ppm are also possible.

  The ammonia concentration in the present invention can be defined as a value measured by the following method. That is, first, about 30 g of a natto specimen is taken in a petri dish and placed on a disk-shaped ground glass having a diameter of 18 cm and a thickness of 4 mm. Put a bell-shaped sealing device called (see Figure 1). At this time, petrolatum is applied to the contact surface between the ground glass and the bell jar to maintain airtightness. Then, after standing for 30 minutes at room temperature to sufficiently volatilize ammonia, the air inside the bell jar is sucked through a suction tube attached to the top of the bell jar, and the concentration of volatile ammonia in the air inside the bell jar is measured with an ammonia gas detector tube ( (Measured by Gastec). For suction, 100 ml of air in the bell jar is collected using a gas sampling device (manufactured by Gastec), and the reaction time of the ammonia gas detection tube is 1 minute.

  Hereinafter, the present invention will be specifically described with reference to examples and the like.

Example 1
(1) Strains used, etc. Natto bacillus r22 is a mutant strain with improved transformation ability of the parent strain O-2 isolated from commercial natto, and the parent strain O-2 was chemically treated using nitrosoguanidine (NTG). It is a strain obtained by carrying out a mutation treatment (see, for example, JP 2000-224982 A).

  E. coli JM109, Bacillus subtilis vector pHY300PLK, and E. coli vector pUC19 were manufactured by Takara Shuzo.

  As the culture medium, a broth medium, a spore formation medium, an NP regeneration medium, or the like described in a natto test method (see, for example, “Natto Test Method”, Kotsu Publishing, p. 85-97, 1990) was used. However, when necessary, tetracycline (2 μg / ml) was added.

(2) Preparation of glutamine synthetase gene-enhanced strain The glutamine synthetase-enhanced strain was obtained by converting the approximately 1.3 kb fragment of the full-length glutamine synthetase gene (glnA) obtained by the following method into the promoter sequence of the alkaline protease gene (aprE). A vector carrying DNA linked to 0.3 kb was constructed and prepared by introducing the vector into Bacillus natto by the following method.

i) Vector construction Since the base sequence of the structural gene of the glutamine synthetase gene (glnA) differs depending on the strain even if it belongs to the same Bacillus subtilis, the base of the glutamine synthetase gene (glnA) of r22 strain The sequence was newly determined by the following method.

  That is, based on the base sequence (registration number D00854) including the periphery of the Bacillus subtilis glutamine synthetase gene registered in the DNA sequence database GenBank, the glutamine synthetase gene is amplified including the upstream and downstream thereof. Two types of oligo DNAs, 5′-TGTCCCGCTTTCTTCATT-3 ′ (described in SEQ ID NO: 3 (FIG. 5)) and 5′-AAAAATCCCCCTGACACCA-3 ′ (described in SEQ ID NO: 4 (FIG. 6)) Was prepared.

  Using these as primers and using the total DNA of the r22 strain as a template, PCR was performed as usual to amplify the glutamine synthetase gene to include upstream and downstream. The nucleotide sequence of the obtained DNA fragment was determined by a conventional method (described in SEQ ID NO: 1 (FIG. 2) in the Sequence Listing). It became clear that it corresponds to the base number 61-1395). (In the DNA sequence of GenBank registration number D00854, the 1379th, 1399th, and 1402nd bases are t, a, and c, whereas in SEQ ID NO: 1 in the sequence listing, they are g, t, and a. Although there are obvious differences and the detailed mechanism must be further studied in the future, this difference is considered to be one factor that enhances the expression of the glutamine synthetase gene in the present invention. And a DNA of a gene encoding a glutamine synthetase comprising the base sequence of base numbers 61 to 1395 of the base sequence of SEQ ID NO: 1 or comprising the base sequence).

  It was also clarified that the glutamine synthetase gene encodes a protein having the amino acid sequence shown in SEQ ID NO: 2 (FIGS. 3 to 4) in the sequence listing (the third and tenth amino acids in the GenBank accession number D00854). The amino acid at the 11th and 11th is lysine, valine, and lysine, whereas in the sequence listing, SEQ ID NO: 2 is asparagine, glutamic acid, and threonine, and there is a clear difference between them. Therefore, the present invention relates to a glutamine synthetase consisting of the amino acid sequence shown in SEQ ID NO: 2 (FIGS. 3 to 4) or a protein having glutamine synthetase activity containing the amino acid sequence. It is to provide.

Subsequently, based on the determined base sequence, 5′-GGAATTC CATATG GCAAATTATACTAGAGGAAG-3 ′ (described in SEQ ID NO: 5 (FIG. 7)) and 5′-CG GGATCC TTAATATGGACACATATAC-3 ′ (SEQ ID NO: 6 (described in FIG. 8), and using these as primers, using the total DNA of the r22 strain as a template, PCR was performed in the usual manner, and the glutamine synthetase gene open reading was performed. The entire frame length (1335 bases) was amplified, and recognition sites for restriction enzymes NdeI and BamHI were introduced at the positions indicated by underlined parts. The obtained DNA fragment was cleaved with restriction enzymes NdeI and BamHI to obtain an NdeI-BamHI fragment containing a glutamine synthetase gene.

Furthermore, based on the base sequence of the alkaline protease gene of Bacillus subtilis registered in the DNA sequence database GenBank (registration number Y14083), 5′-GC TCTAGA GACAGGAATTTGCCCGACTAGGG-3 ′ (SEQ ID NO: 7 in the sequence listing (FIG. 9)) 2) and 5′-GGAATTC CATATG TTACCCTCTCCATTTAAAAAA-3 (described in SEQ ID NO: 8 (FIG. 10) in the Sequence Listing), and using these as primers, using the total DNA of the r22 strain as a template In this method, PCR is carried out as usual to amplify the promoter region (274 bases) of the alkaline protease gene and introduce recognition sites for restriction enzymes XbaI and NdeI at the positions indicated by the underlined parts. It was.

  The obtained DNA fragment was cleaved with restriction enzymes XbaI and NdeI to obtain an XbaI-NdeI fragment containing the promoter region of the alkaline protease gene.

  Subsequently, plasmid pGLNA1 was obtained by simultaneously ligating the above two kinds of DNA fragments and a total of 3 fragments of plasmid pUC19 cleaved with XbaI and BamHI.

  After cleaving pGLNA1 with restriction enzymes XbaI and BamHI, the shorter band (about 1.8 kb) of the two bands generated by agarose electrophoresis was recovered from agarose and digested with XbaI and BamHI. It was inserted into a fungus shuttle vector pHY300PLK. The resulting plasmid was named pGLNA2 and was used for the following experiment.

ii) Transformation Plasmid pGLNA2 prepared using Escherichia coli JM109 as a host was transformed into Bacillus natto r22 strain by a protoplast method as usual.

  The transformant was selected using tetracycline resistance as an index. Of the obtained transformants, two strains were selected and the plasmids were recovered to confirm that pGLNA2 was retained, and named glnAh1 and glnAh2 strains.

  Among these transformed strains, the glnAh2 strain, which has a better ability to suppress the odor of ammonia in natto, was named Bacillus subtilis glnAh2, and this was designated as FERM BP-10446 as an independent administrative agency, National Institute of Advanced Industrial Science and Technology patent microorganisms. Deposited internationally at the center.

iii) Enzymatic activity of glutamine synthase In a Sakaguchi flask containing 100 ml of soybean hydrolyzate medium (each containing 1.5% glucose, sodium glutamate, and soybean hydrolysate) And cultured at 37 ° C. with shaking.

  After overnight culture, Bacillus natto was collected by a conventional method, suspended in 5 ml of 50 mM imidazole-hydrochloric acid buffer (pH 7.0, containing 0.2 mM ethylenediaminetetraacetic acid, 1 mM β-mercaptoethanol), and then added with 100 μg of lysozyme. And treated at 37 ° C. for 1 hour. Thereafter, the lysozyme-treated cell solution was crushed with ultrasonic waves for 30 seconds and used as a crude enzyme solution.

  The enzyme activity was measured according to a conventional method (for example, see “Methods in Enzymology”, Vol. 11, p. 337-342, 1955). The results are shown in Table 1 (enzyme activity of glutamine synthetase).

  In the r22 strain as a control, only weak activity was detected, whereas in the glnAh2 strain, about 1700 times higher activity was detected, confirming that the glnAh2 strain was a glutamine synthase highly active strain. It was.

iv) Natto production Since the odor of ammonia in natto is due to ammonia present in the gas phase, it is desirable to measure the amount of ammonia volatilized. For this reason, a new method for measuring the amount of volatile ammonia described below was devised, and the concentration of gaseous ammonia generated from natto in the air was measured.

  About 30 g of each natto specimen produced as a prototype according to a conventional method was placed in a petri dish at 18 hours and 24 hours after the start of fermentation, and placed on a disc-shaped ground glass having a diameter of 18 cm and a thickness of 4 mm. A bell-shaped sealing device called a bell jar with a glass volume of 2.5 liters (see FIG. 1) was placed on the ground glass on which natto was placed. At this time, petrolatum was applied to the contact surface between the ground glass and the bell jar to maintain airtightness. After leaving to stand at room temperature for 30 minutes to sufficiently volatilize ammonia, 100 ml of air inside the bell jar is sucked using a gas sampler (made by Gastec) through a suction tube attached to the top of the bell jar, and the volatile ammonia concentration is reduced to ammonia. Measured with a gas detector tube (manufactured by Gastec). The results are shown in Table 2 (Volatile ammonia concentration).

  In natto produced using the glnAh2 strain, the volatile ammonia concentration was significantly reduced compared to natto produced using the r22 strain as a control.

  The quality of the natto produced using the glnAh2 strain, such as the appearance and stringing strength, was the same as that of the control prepared using the r22 strain as a result of a sensory test by a specialized panelist. As for the scent, a unique ammonia odor was suppressed.

It is a figure which shows the outline of the bell jar used at the time of the ammonia concentration measurement of this invention. The base sequence containing the base sequence (base numbers 61 to 1395) of the DNA of the gene encoding glutamine synthetase is shown. The amino acid sequence of glutamine synthetase is shown. The same as above. 3 shows the base sequence of the oligo DNA represented by SEQ ID NO: 3. The base sequence of the oligo DNA indicated by base number 4 is shown. This shows the base sequence of the oligo DNA represented by SEQ ID NO: 5. This shows the base sequence of the oligo DNA represented by SEQ ID NO: 6. The base sequence of the oligo DNA represented by SEQ ID NO: 7 is shown. This shows the base sequence of the oligo DNA represented by SEQ ID NO: 8.

Claims (8)

  DNA of a gene encoding a glutamine synthetase consisting of a base sequence of base numbers 61 to 1395 in the base sequence set forth in SEQ ID NO: 1 in the Sequence Listing.   A glutamine synthetase having the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing.   A natto bacterium characterized by enhanced expression of a glutamine synthetase gene.   The glutamine synthetase gene is a base sequence encoding a glutamine synthetase consisting of the base sequences of base numbers 61 to 1395 among the base sequences set forth in SEQ ID NO: 1 of the sequence listing. The natto bacteria described.   Bacillus subtilis (Bacillus subtils) glnAh2 (FERM BP-10446), Bacillus natto according to claim 3 or 4,   6. The natto bacterium according to any one of claims 3 to 5, wherein glutamine synthetase activity is 0.01 unit / mg protein or more.   A natto produced using the natto bacterium according to any one of claims 3 to 6.   The natto according to claim 7, wherein the ammonia content is less than 50 ppm.

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