JPH11137254A - Production of transglutaminase derived from microorganism belonging to genus bacillus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a transglutaminase derived from Bacillus subtilis in an active form by using a microbe belonging to the genus Escherichia. SOLUTION: A microbe belonging to the genus Escherichia having a DNA fragment of a transglutaminase gene derived from the microbe belonging to the genus Bacillus is cultivated, and the expression of the transglutaminase gene is induced in the period from the time deteriorating the logarithmic propagation of the microbes to the time reaching stationary phase of the growth of the microbes. The transglutaminase is accumulated in the microbes belonging to the genus Escherichia.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing transglutaminase, and more particularly, to a method for producing transglutaminase derived from a bacterium belonging to the genus Bacillus using a bacterium belonging to the genus Escherichia using a gene recombination technique.

[0002]

2. Description of the Related Art Transglutaminase (hereinafter referred to as "T
G ") is an enzyme that catalyzes an acyl transfer reaction using a γ-carboxamide group of a glutamine residue in a peptide chain as a substrate. In the reaction, when the ε-amino group of a lysine residue in the peptide chain becomes an acyl acceptor,
Ε- (γ-Glu)-within or between peptide molecules
A Lys cross-link (hereinafter abbreviated as “GL bond”) is formed. When water becomes the acyl acceptor, a deamidation reaction occurs in the glutamine residue, and the glutamine residue becomes a glutamic acid residue.

[0003] Various crosslinked polymerized products can be produced by using TG, and the thus produced crosslinked polymerized products are tofu, pudding, yogurt, cheese, surimi, kneaded products, It is used as food, such as meat products such as sausages, and cosmetics.

Conventionally, TG has been known to exist in many animal tissues. For example, guinea pig liver (Connel
lan et al., Journal of Biological Chemistry 246 1
093-1098 (1971)). Regarding TG of microorganisms, actinomycetes and Bacillus subtilis (MVRa)
manujam et al., FASEB J.4, A2321), slime mold (JDklei
n et al., J. Bacteriol. 174: 2599-2605). Industrially, TGs produced by actinomycetes have been put into practical use (Japanese Patent Publication No. 6-65280, Japanese Unexamined Patent Publication No. 1-27471), but actinomycetes have a slower growth rate than general bacteria. The cultivation time is prolonged, which leads to an increase in production costs.

On the other hand, when TG derived from Bacillus subtilis is applied to industry, the conventionally known TG derived from Bacillus subtilis contains 5 mM C
There was a problem that it could not be used in an actual food system because it was inhibited by a ²⁺ , but Bacillus subtilis producing TG which showed activity in the presence of 5 mM Ca ²⁺ was found, The above-mentioned application is possible (Japanese
9-131180). In addition, a technology for producing TG by using gene recombination technology using the TG gene derived from Bacillus subtilis has been developed.

[0006]

However, when a TG gene derived from Bacillus subtilis is introduced into a bacterium belonging to the genus Escherichia and the gene is expressed to produce TG, the TG protein associates with the inclusion body ( Inclusion body) is often formed. To obtain an active TG from this inclusion body, an operation of solubilizing the inclusion body and unwinding the TG protein is required, but obtaining an active TG protein from the inclusion body of the TG protein derived from Bacillus subtilis. It is difficult. In addition, production of TG derived from Bacillus subtilis in a sufficient amount in an active form has not been successful.

The object of the present invention is to provide a method for producing Bacillus subtilis-derived TG in an active form using a bacterium belonging to the genus Escherichia.

[0008]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventor has found that when a bacterium belonging to the genus Escherichia carrying a TG gene derived from a bacterium belonging to the genus Bacillus is cultured, TG is produced. Furthermore, they have found that TG can be efficiently produced in an active form by inducing the expression of the TG gene in a specific culture period, and the present invention has been completed.

That is, the present invention provides a method for producing TG by culturing a bacterium belonging to the genus Escherichia carrying a DNA fragment containing a TG gene derived from a bacterium belonging to the genus Bacillus, and expressing the gene. Is a method characterized by inducing the expression of the TG gene during a period from the time at which the growth of the bacteria slows down to the time at which the growth of the bacterium reaches a stationary phase.

[0010] As a preferred embodiment, the present invention provides a method as described above, wherein TG is accumulated in a bacterial cell belonging to the genus Escherichia.

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

<1> TG gene used in the present invention The TG gene derived from a bacterium belonging to the genus Bacillus used in the present invention contains DNA encoding TG derived from a bacterium belonging to the genus Bacillus, and has a desired culture period in a cell of the bacterium belonging to the genus Escherichia. Is a gene capable of inducing TG expression. Such a TG gene can be obtained by ligating a DNA encoding TG derived from Bacillus bacterium to a promoter capable of inducing expression in Escherichia bacterium cells.

The bacterium belonging to the genus Bacillus from which the DNA encoding TG is derived is not particularly limited as long as it is a bacterium belonging to the genus Bacillus which produces TG. licheniformis), Bacillus megaterium, Bacillus stearothermophilus, Bacillus brevis, Bacillus sphaericus, Bacillus polymyx, Bacillus polymyx (Bacillusalcalophilus) and the like.

Of these, Bacillus subtilis is preferred, and Bacillus subtilis AJ12866 and AJ13
07 is preferred. Bacillus subtilis AJ12866 has been deposited with the Ministry of International Trade and Industry at the National Institute of Advanced Industrial Science and Technology (hereinafter abbreviated as "Life Institute") on February 2, 1995, and its deposit number is FERM P- 14750. In addition, Bacillus subtilis AJ12866 was
It was transferred to an international deposit under the Budapest Treaty on February 4, and its international deposit number is FERM BP-5325. Bacillus subtilis AJ1307 is
Deposited on August 22, 1995, the deposit number is FERM P-15123. Also, Bacillus subtilis
AJ1307 has been transferred to an international deposit under the Budapest Treaty on January 18, 1996, and its international deposit number is FERM BP-5367.

A method for obtaining TG-encoding DNA from Bacillus bacterium as described above will be described (see JP-A-9-131180). First, the amino acid sequence of the purified TG is determined. Edman, P., Acta C
Amino acid sequence can be determined using hem. Scand. 4, 227 (1950)). The amino acid sequence can be determined using a sequencer manufactured by Applied Biosystems.

Based on the revealed amino acid sequence,
The nucleotide sequence of the DNA encoding this can be deduced. DN
To deduce the nucleotide sequence of A, a universal codon or a codon most frequently used in a gene of a bacterium belonging to the genus Bacillus is employed.

On the basis of the deduced nucleotide sequence, 30 to 5
A DNA molecule of about 0 base pairs is synthesized. A method for synthesizing the DNA molecule is described in Tetrahedron Letters, 22, 1859 (1981).
Is disclosed. The DNA molecule can be synthesized using a synthesizer manufactured by Applied Biosystems. The D
The NA molecule is D, which encodes TG derived from a bacterium belonging to the genus Bacillus.
It can be used as a probe when isolating the full length NA from a Bacillus bacterium chromosome gene library. Alternatively, DNA encoding TG derived from Bacillus bacteria is
It can be used as a primer when amplifying by the CR method.
However, since the DNA amplified using the PCR method may not include the full length DNA encoding TG derived from Bacillus bacterium, the DNA amplified using the PCR method may be omitted.
Is preferably used as a probe to isolate the full length DNA encoding TG derived from Bacillus bacterium from a Bacillus bacterium chromosome gene library.

For the operation of the PCR method, see White, TJ
et al., Trends Genet. 5, 185 (1989). For the method of preparing chromosomal DNA of Bacillus bacteria, see Molecular Biological Methods for Bacillu.
s, John Wiley & Sons Ltd (1990). For information on how to create a library of bacterial chromosomes such as Bacillus, see Molecular Biological Methos.
ds for Bacillus, John Wiley & Sons Ltd (1990). For a method of isolating a target DNA molecule from a gene library using a DNA molecule as a probe, see Molecular Cloning, 2nd edition, Co.
ld Spring Harbor press (1989).

A method for determining the nucleotide sequence of the DNA coding for the isolated TG is described in A Practical Guide to Molecul.
ar Cloning, John Wiley & Sons, Inc. (1985). The nucleotide sequence can be determined using a DNA sequencer manufactured by Applied Biosystems.

D encoding TG derived from Bacillus bacterium
One of the NAs is shown in SEQ ID NO: 1 in the Sequence Listing. The DNA was isolated from the chromosomal DNA of Bacillus subtilis AJ1307 strain. The DNA encoding TG derived from Bacillus bacteria is not limited to the DNA shown in SEQ ID NO: 1 in the Sequence Listing. That is, a difference in nucleotide sequence should be observed for each species and strain of bacteria belonging to the genus Bacillus.

In addition, a DNA encoding TG isolated from the chromosomal DNA of a bacterium belonging to the genus Bacillus is artificially mutated (for example, the encoded TG has one or more amino acid substitutions, deletions, insertions or additions). And mutations that maintain TG activity) to alter the nucleotide sequence. A frequently used artificial mutation method is Method. In Enzymol., 154 (198
There is a site-directed mutagenesis method described in 7).

TG derived from Bacillus subtilis AJ1307
Escherichia coli AJ13172, which has a recombinant DNA (pBSTG75-11) obtained by connecting a DNA encoding DNA and a vector DNA in a cell, was reported to the Institute of Life Sciences on December 20, 1995 based on the Budapest Treaty. Deposit number is FERM BP-5346. pBSTG75-11 is obtained by adding Bacillus subtilis AJ13 to pUC18.
Approximately 2 kb of DNA containing the sequence encoding TG from 07
This fragment is a plasmid into which a fragment has been inserted, in which DNA encoding TG is connected downstream of lacZ '(a sequence encoding a part of the N-terminal side of β-galactosidase),
It is designed to express a fusion protein in which a peptide consisting of 11 amino acid residues is added to the N-terminal side of TG under the control of a promoter (see JP-A-9-131180). Upon digestion of pBSTG75-11 with HindIII and BamHI, T
A DNA sequence encoding G is obtained.

As the promoter for expressing the DNA encoding TG, any promoter that is usually used for the production of a heterologous protein in Escherichia coli and that can be induced to be expressed in any culture period can be used. trc promoter, trp promoter, ta
Strong promoters such as c promoter, PL promoter, lac promoter and the like can be mentioned.

As a vector for introducing the TG gene into Escherichia coli, a so-called multicopy type vector is preferable, and a plasmid having a replication origin derived from Col E1, such as a pUC plasmid, a pBR322 plasmid, or a derivative thereof. Is mentioned. Further, in order to select a transformant, the vector preferably has a marker such as an ampicillin resistance gene. As such plasmids, expression vectors having a strong promoter are commercially available (pTrc99A (Pharmacia), pUC (Takara Shuzo), pPROK (Clontech), pKK233-2
(Clontech) and others).

A terminator which is a transcription termination sequence may be connected downstream of the TG gene. Examples of the terminator include a T7 terminator, an fd phage terminator, a T4 terminator, a terminator for a tetracycline resistance gene, and a terminator for an Escherichia coli trpA gene.

By transforming Escherichia coli with the above-described vector, a plasmid in which a promoter, a DNA encoding TG derived from Bacillus bacterium and, if necessary, a DNA fragment linked in the order of a terminator are inserted, Escherichia coli derived from Bacillus bacterium is obtained. An Escherichia bacterium having the TG gene is obtained. Some of the above-mentioned vectors contain a promoter suitable for expressing a DNA encoding TG. Using such a vector, TG derived from Bacillus bacterium is downstream of the promoter contained in the vector. Does not need to be separately inserted into the vector.

In order to introduce a vector containing the TG gene into a bacterium belonging to the genus Escherichia, the method of DM Morrison (Methods
in Enzymology 68, 326 (1979)) or a method of increasing DNA permeability by treating recipient cells with calcium chloride (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)).
And the like, for example, for the transformation of Escherichia bacteria.

The bacterium belonging to the genus Escherichia used in the present invention is not particularly limited as long as it is a microorganism belonging to the genus Escherichia such as Escherichia coli. Specifically, a book by Neidhardt et al. (Neidhardt, FC et.
li and Salmonella Typhimurium, American Society for
Microbiology, Washington DC, 1208, table 1) can be used. For example, Escherichia
Coli JM109 strain and MC1061 strain.

As a medium used for culturing the Escherichia bacterium having the TG gene obtained as described above, a 2 × YT medium (Bctotrypton 1.6
%, Yest extract 1.0%, NaCl 0.5%), or a medium usually used for culturing Escherichia coli, such as an M9-casamino acid medium and an LB medium. The culture conditions and production induction conditions are appropriately selected depending on the type of the vector marker, host bacterium and the like used.

In the present invention, when a bacterium belonging to the genus Escherichia carrying a DNA fragment containing a TG gene derived from a bacterium belonging to the genus Bacillus is cultivated, the growth of the bacterium at a stationary stage is started from the time when the logarithmic growth of the bacterium belonging to the genus Escherichia is slowed. TG gene expression during the period of Inducing the expression of the TG gene before the time when logarithmic growth slows down,
Production is low, or high T
Although G forms inclusion bodies and produces only a small amount of active TG, when TG gene expression is induced during the period when logarithmic growth slows down to the stationary phase, part of TGs Even if formed, active TGs accumulate in the cells in significant amounts.

The time when the logarithmic growth slows down or reaches the stationary phase depends on the type of Escherichia bacterium, vector, promoter and medium used, the culturing conditions, etc., but preliminary culturing is performed under the set conditions.
By measuring the number of viable cells or absorbance over time and plotting it on a graph to create a growth curve, it can be easily examined. In addition, a particularly preferred induction time can be determined by measuring the TG activity of the bacterial cell extract over time. Furthermore, the culture time after the induction is applied may be appropriately set in the same manner.

The produced TG derived from Bacillus bacterium is
A crude enzyme solution can be obtained by lysing or disrupting the cells of the genus Escherichia and removing the insoluble fraction from the lysate or lysate. Further, if necessary, T is obtained by a technique such as ordinary precipitation, filtration, and column chromatography.
G can be used after purification. In this case, TG
A purification method using an antibody against is also available.

[0033]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. The present invention is not limited to the description of the embodiments.

<1> Construction of a direct expression system for Bacillus subtilis-derived TG in Escherichia coli (1) Preparation of chromosomal DNA library 1 μg of chromosomal DNA of Bacillus subtilis AJ1307 strain
It was completely digested with Hin dIII. After recovering the DNA by ethanol precipitation, it was dissolved in 10 μl of 10: 1 TE. And 5μl of this, further B is digested with Hin dIII
PUC118 (Takara Shuzo) 1 dephosphorylated by AP
ng and a ligation reaction was performed using DNA Ligation Kit Ver.2 (Takara Shuzo). 100 μl of competent cells of Escherichia coli JM109 strain (Takara Shuzo) were mixed with 3 μl of a ligation reaction solution to transform Escherichia coli JM109 strain. This was applied to an appropriate solid medium to prepare a chromosomal DNA library.

(2) Preparation of Probe The probe includes Hindase of Bacillus subtilis chromosomal DNA containing the TG gene.
Plasmid (pBSTG) cloned dIII fragment into pUC118
75-11) (see Example 10 of JP-A-9-131180). The sequence encoding TG contained in pBSTG75-11 corresponds to base numbers 118 to 1042 in SEQ ID NO: 1 in the sequence listing. The Escherichia coli JM109 strain transformed with pBSTG75-11 (AJ1
3172) was issued to Life Research Institute on December 20, 1995,
This is the deposit number of RM BP-5346 and is deposited internationally under the Budapest Treaty.

Using pBSTG75-11 as a template, a PCR reaction was performed using Primer S2 (SEQ ID NO: 2) and Primer S3 (SEQ ID NO: 3). The PCR reaction was performed according to TaKaRa LA PCR Kit Ver.

10 ng of pBSTG75-11 as a template, Primer S2
Then, 100 μl of a reaction solution containing 20 pmol of each of Primer S3 and Primer S3 was prepared and reacted. In addition, Primer S2 is a primer complementary to 35 bases from the 118th to 152nd base sequence of SEQ ID NO: 1 of TG gene, and PrimerS3 is a primer complementary to 35 bases from 818th to 852nd base of SEQ ID NO: 1 of TG gene. It is. The PCR reaction was performed for 30 cycles under the following conditions.

94 ° C. for 30 seconds 55 ° C. for 30 seconds 72 ° C. for 1 minute

The DNA fragment amplified by the above reaction is 1%
Separation was performed by agarose gel (Seaplaque GTG, manufactured by FMC) electrophoresis. Cut out the desired band and use EasyPrep Sys
The DNA was purified using tem (Pharmacia) and PCR Products Prep Kit (Pharmacia). Finally
200 μl of a 4 ng / μl DNA solution was obtained.

This DNA fragment was labeled with ³² P and used as a probe. Probe labeling was performed using [α- ³² P] dCTP 3000 Ci / mmol (Amersham) and Random Primer DNA Labeling Kit Ver. 2 (Takara Shuzo) according to the instructions.

(3) Colony Hybridization The procedure for colony hybridization is Molecular
Cloning, 2nd edition, Cold Spring Harbor press (19
89) was performed according to the method described in.

The colony of the chromosomal DNA library was transferred to a nylon membrane filter (Hybond-N, manufactured by Amersham) and subjected to alkali denaturation, neutralization, and immobilization. Hybridization was performed using a Rapid-hyb buffer (Amersham). The filter was immersed in the buffer and prehybridized at 65 ° C. for 4 hours. Thereafter, the labeled probe prepared above was added, and hybridization was performed at 65 ° C. for 2 hours.
After this, the filter was washed at room temperature with 2 × SSC containing 0.1% SDS.
Washed for 0 minutes. 65 in 0.1 × SSC containing 0.1% SDS
Washing was performed twice at 15 ° C. for 15 minutes. As a result, five colonies hybridizing with the probe were confirmed.

(4) DNA sequence of TG gene One of the plasmids (pBST
G3-1), Molecular Cloning, 2nd edition,
It was prepared according to the method described in Cold Spring Harbor press (1989), and the nucleotide sequence of the DNA fragment inserted into pUC118 was determined. The sequencing reaction is performed using the Dye Terminator Cyc
This was performed using the le Sequencing Kit (ABI) according to the instructions. The electrophoresis was performed using DNA Sequencer 373 (manufactured by ABI). As a result, it was confirmed to have the nucleotide sequence shown in SEQ ID NO: 1 in the Sequence Listing.

(5) Construction of TG Direct Expression Plasmid From the plasmid pBSTG3-1 obtained as described above, a HindIII-BamHI fragment containing the TG gene derived from Bacillus subtilis was pHSG399.
Was subcloned into pHSG9TG (FIG. 1). p
HSG399 has lacZ 'expressed under the control of the lac promoter, and has a HindIII site and a BamHI site immediately below it.

The base sequence immediately before the N-terminal methionine codon in the B. subtilis-derived TG gene was synthesized using a synthetic oligonucleotide [NDE2] having the base sequence shown in SEQ ID NO: 4.
Two base substitutions were made by site-directed mutagenesis to introduce an NdeI site at the codon of the N-terminal methionine (FIG. 1, pH
SG9TG (Nde)). This made it possible to connect an arbitrary nucleotide sequence upstream of T (thymine) immediately before the N-terminal methionine codon by digesting the obtained plasmid with the restriction enzyme NdeI.

Next, a DNA fragment obtained by annealing synthetic oligonucleotides [SD1F and SD1R] having the nucleotide sequences shown in SEQ ID NOs: 5 and 6 was ligated to pHSG9TG (Nde) digested with HindIII and NdeI, and pHSG9TG ( Nde) HindII
By replacing the I-NdeI fragment with the above-mentioned synthetic DNA fragment, an expression system capable of directly expressing Bacillus subtilis-derived TG under the control of the lac promoter was constructed (FIG. 2, pHSD1TG). The synthetic DNA fragment (hereinafter, referred to as a 5 ′ flanking region) has a HindIII cleavage sequence and an NdeI cleavage sequence at both ends, and internally a part of the 3 ′ terminal of lacZ ′ and SD (Shine-Dal).
garno) sequence. In pHSD1TG, synthetic D
Part of the 3′-terminal side of lacZ ′ in the NA fragment was derived from pHSG399.
It is connected to the 5 'end of lacZ' following the lac promoter, and can express lacZ 'and TG genes under the control of the lac promoter. In addition, a termination codon in the same frame as lacZ 'and an SD sequence exist between the coding sequence of lacZ' and the TG gene, and TG is designed to be expressed alone, not as a fusion protein. .

<2> Direct Expression of Bacillus subtilis-derived TG in Escherichia coli From the pHSD1TG constructed above, a HindIII-BamHI fragment containing the Bacillus subtilis-derived TG gene (including the 5 ′ flanking region) was excised and excised. PUC1 digested with HindIII and BamHI
PUSD1TG was constructed by ligation to 9 (Figure 2).

Escherichia coli JM109 strain was transformed with pUSD1TG, and the resulting transformant was treated with 100 μg of ampicillin.
2 × YT medium (Bctotrypton 1.6%, Yest extract
(1.0%, NaCl 0.5%) at 37 ° C. overnight. 1 ml of this culture was inoculated into 100 ml of 2 × YT medium containing 0.2% casamino acid and 100 μg / ml of ampicillin and cultured at 37 ° C. with shaking. When the absorbance of the medium at a wavelength of 660 nm reached 0.4 to 0.5 (midlog phase), 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added to the medium, and the lac promoter was added. Expression was induced, and the cells were further cultured with shaking at 37 ° C. for 6 hours. Microscopic observation of the cells at this stage revealed that inclusion bodies had been formed in the cells. No inclusion body formation was observed in the strain retaining the vector (pUC19) alone.

After collecting the cells, the cells were washed with 0.85% NaCl,
bufer (20 mM Tris-HCl, 5 mM EDTA, 30 mM NaCl, pH 8)
And add 0.2mg / ml lysozyme and add on ice.
After a time treatment, it was sonicated. 10,000 × g of crushed liquid
Centrifugation at 0.5% for 10 minutes.
The extract was washed twice with a buffer, suspended in a 10 mM EDTA (pH 8) solution, and used as an inclusion body fraction.

When the obtained inclusion body was subjected to SDS-PAGE, its molecular weight was consistent with the molecular weight (about 28,000 to about 30,000) of TG derived from Bacillus subtilis disclosed in JP-A-9-131180. After dissolving the inclusion body with 8M urea, dilute the urea concentration to 2.5M, infiltrate the centrifuged supernatant into a PVDF membrane, wash with 20% methanol for 1 hour, and use Applied B
The N-terminal amino acid sequence of the protein that formed the inclusion body was determined using a protein sequencer manufactured by iosystems. As a result, 10 residues of the N-terminal amino acid of the protein forming the inclusion body coincided with the N-terminal amino acid sequence of TG derived from Bacillus subtilis disclosed in JP-A-9-131180. From the above, it was confirmed that TG derived from Bacillus subtilis was directly expressed in pUSD1TG.

<3> Increase in TG Activator Accumulation by Changing Expression Induction Timing The TG gene derived from Bacillus subtilis on pUSD1TG was replaced with pTrc99A (Pharma
Bacillus subtilis-derived TG is trc
A plasmid pTcSD1TG capable of expression under the control of a promoter was prepared (FIG. 3). That is, after digesting pUSD1TG with NheI, the cleaved ends are blunted, and the TG gene fragment obtained by digesting with BamHI (including the 5 ′ flanking region)
Was ligated to pTrc99A digested with BamHI and SmaI. pTrc
99A has a trc promoter, and SmaI
Site and a BamHI site.

Escherichia coli JM109 was transformed with pTcSD1TG, and the resulting transformant was transformed into ampicillin 100.
2 x YT medium containing μg / ml (Bctotrypton 1.6%, Yest extr
(act 1.0%, NaCl 0.5%) overnight at 37 ° C., 1 ml of the culture was inoculated into 100 ml of 2 × YT medium containing 0.2% casamino acid and 100 μg / ml ampicillin, and cultured at 37 ° C. with shaking. When the absorbance of the culture solution at a wavelength of 660 nm reaches 0.4 to 0.5 (mid-log phase), or the absorbance at a wavelength of 660 nm of 3.2 to 4.0
At the time the logarithmic growth slows, 1 mM IPTG
Was added to the medium to induce expression, followed by shaking culture at 37 ° C.

After collecting the cells, the cells were washed with 0.85% NaCl,
buffer (50mM TrisHCl, 10mM EDTA, 2mM DTT, pH7.5)
After adding 0.2 mg / ml lysozyme and 0.02 mg / ml DNase I, treating at 37 ° C. for 40 minutes, adding 15 mM MgSO ₄ and treating at 37 ° C. for another 15 minutes, and adding 50 mM NaHCO ₃ (pH 10). At 1M NaOH
After adjusting the pH to 10.2, the mixture was further treated at 37 ° C. for 30 minutes. This treated solution was centrifuged at 12,000 × g for 30 minutes, and the TG activity derived from Bacillus subtilis in the supernatant was measured according to the method described in Japanese Patent Application Laid-Open No. Hei 9-131180 to convert ¹⁴ C-labeled putrescine into dimethyl casein. It was measured as uptake activity. TG activity per WCW (Wet cellweight) and culture solution
FIG. 4 shows the TG activity per 0.1 ml.

As shown in FIG. 4, when the induction was applied after the logarithmic growth was slowed down, the wetness was about 4.5 times higher per culture solution than when the induction was applied during the middle logarithmic growth phase. Approximately 2.6 times higher TG activity per weight was obtained.

[0055]

According to the present invention, Bacillus subtilis-derived TG can be produced in an active form using a bacterium belonging to the genus Escherichia.

[0056]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1042 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Origin Organism: Bacillus subtilis Strain: AJ1307 Sequence Characteristic Characteristic symbol: CDS Location: 118..855 Method for determining the characteristic: Sequence CTGCTTAAAA AGTTTTAAAA TAAAAAATGG AAGAAGTTCT TTTTGGCAGT CTTCTGTCTT 60 TTTAGCTTTC ATTGCCCAAG CTCTTTGCAT ATCTTATATA AACAAGGGGG GCTA GAT CATGAT GATC ATTGA TGA TGA TGA TGA ATGA TGA TGA ATGA TGA TGA ATGA TGA TGA TGA ATGA TGA TGA TGA TGA AT GAT CATGATCA AAT 165 Met Ile Ile Val Ser Gly Gln Leu Leu Arg Pro Gln Asp Ile Glu Asn 1 5 10 15 TGG CAG ATT GAT CAA AAT CTG AAT CCG CTG TTA AAA GAG ATG ATT GAG 213 Trp Gln Ile Asp Gln Asn Leu Asn Pro Leu Leu Lys Glu Met Ile Glu 20 25 30 ACG CCT GTT CAG TTT GAT TAT CAT TCA ATT GCT GAA CTG ATG TTT GAG 261 Thr Pro Val Gln Phe Asp Tyr His Ser Ile Ala Glu Leu Met Phe Glu 35 40 45 CTT AAA CTG CGG ATG AAT ATT GTA GCA GCG GCA AAG ACG CTG CAC AAA 309 Leu Lys Leu Arg Met Asn Ile Val Ala Ala Ala Lys Thr Leu His Lys 50 55 60 AGC GGG GCG AAG TTT GCC ACT TTT TTA AAA ACA TAC GGG AAT ACA ACG 357 Ser Gly Ala Lys Phe Ala Thr Phe Leu Lys Thr Tyr Gly Asn Thr Thr 65 70 75 80 TAT TGG AGG GTT TCA CCG GAG GGC GCC TTG GAG CTG AAA TAC AGA ATG 405 Tyr Trp Arg Val Ser Pro Glu Gly Ala Leu Glu Leu Lys Tyr Arg Met 85 90 95 CCG CCT TCA AAA GCG ATT CGG GAC ATT GCA GAG AAC GGC CCG TTT TAT 453 Pro Pro Lys Ala Ile Arg Asp Ile Ala Glu Asn Gly Pro Phe Tyr 100 105 110 GCG TTT GAA TGC GCA ACC GCA ATC GTT ATC ATT TAT TAC TTG GCC TTA 501 Ala Phe Glu Cys Ala Thr Ala Ile Val Ile Ile Tyr Tyr Leu Ala Leu 115 120 125 ATC GAT ACA ATC GGT GAA GAT AAA TTC AAT GCC AGC TTT GAC AGA ATT 549 Ile Asp Thr Ile Gly Glu Asp Lys Phe Asn Ala Ser Phe Asp Arg Ile 130 135 140 ATT TTA TAT GAC TGG CAT TAT GAG AAA TTG CCG ATC TAT ACG GAA ACA 597 Ile Leu Tyr Asp Trp His Tyr Glu Lys Leu Pro Ile Tyr Thr Glu Thr 145 150 155 160 GGA CAC CAC TTT TTC CTT GGA GAT TGT TTG TAT TTT AAG AAT CCT GAA 645 Gly His His Phe Phe L eu Gly Asp Cys Leu Tyr Phe Lys Asn Pro Glu 165 170 175 TTT GAT CCG CAA AAG GCG CAA TGG AGA GGC GAA AAT GTG ATT TTA CTG 693 Phe Asp Pro Gln Lys Ala Gln Trp Arg Gly Glu Asn Val Ile Leu Leu 180 185 190 GGG GAA GAT AAA TAT TTT GCC CAT GGT CTT GGA ATC TTA AAC GGA AAG 741 Gly Glu Asp Lys Tyr Phe Ala His Gly Leu Gly Ile Leu Asn Gly Lys 195 200 205 CAA ATT ATA GAT AAG CTG AAT TCT TTT AGG AAA AAA GGA GCC TTA CAG 789 Gln Ile Ile Asp Lys Leu Asn Ser Phe Arg Lys Lys Gly Ala Leu Gln 210 215 220 TCA GCC TAC CTT CTG TCT CAG GCG ACC AGA CTG GAT GTT CCG TCT CTT 837 Ser Ala Tyr Leu Leu Ser Gln Ala Thr Arg Leu Asp Val Pro Ser Leu 225 230 235 240 TTC CGC ATC GTC CGC TAAAAAGCCC CATCGCCTAT TTTCGGGACG ATGGGGTTTC 892 Phe Arg Ile Val Arg 245 AAATGCCTTT CGTTTTCGAT AGAAGGGGGC TGTGCCGAAA TATTGGTTCG CAGCCCACTC 952 CATTTTTTCA AGGTCATTTC TTGTCACGAT TGGATCCTGG CTGCTCCATT TGATAAAGCG 1012 GACAAAATAG TAGCCTTTGA TAGGAACCAT 1042

SEQ ID NO: 2 Sequence length: 35 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Hypothetical: No sequence ATGATTATTG TATCAGGACA ATTGCTCCGT CCCCA 35

SEQ ID NO: 3 Sequence length: 35 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Hypothetical: No sequence GCGGACGATG CGGAAAAGAG ACGGAACATC CAGTC 35

SEQ ID NO: 4 Sequence length: 23 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Hypothetical: No sequence ATACAATAAT CATATGTAGC CCC 23

SEQ ID NO: 5 Sequence length: 39 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Hypothetical: No sequence AGCTTCGAAG CTAGCTAAAA CTTTAAGAAG GAGATATCA 39

SEQ ID NO: 6 Sequence length: 37 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Hypothetical: No sequence TATGATATCT CCTTCTTAAA GTTTTAGCTA GCTTCGA 37

[Brief description of the drawings]

FIG. 1 is a diagram showing the construction process of a plasmid pHSG9TG (Nde) containing a Bacillus subtilis-derived TG gene into which an NdeI site has been introduced.

FIG. 2 is a view showing the process of constructing plasmids pHSD1TG and pUSD1TG that directly express a TG gene derived from Bacillus subtilis.

FIG. 3 is a view showing a construction process of a plasmid pTcSD1TG which directly expresses a Bacillus subtilis TG gene by a trc promoter.

FIG. 4 is a view showing the TG activity of Escherichia coli JM109 (pTcSD1TG) in cells.

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Claims (2)

[Claims] 1. A method for producing a transglutaminase by culturing a bacterium belonging to the genus Escherichia carrying a DNA fragment containing a transglutaminase gene derived from a bacterium belonging to the genus Bacillus, and expressing the gene, wherein the logarithmic growth of the bacterium belonging to the genus Escherichia is reduced. A method comprising inducing the expression of the transglutaminase gene during a period from the time of blunting to the time when the growth of the bacterium reaches a stationary period. 2. The method according to claim 1, wherein transglutaminase is accumulated in bacterial cells belonging to the genus Escherichia.