JPH0789912B2 - Method for producing cyclodextrin glucanotransferase - Google Patents

Description

Detailed Description of the Invention Generally, as a cyclodextrin glucanotransferase-producing bacterium, bacteria of the genus Bacillus or Klebsiella have been used. However, its production is insignificant and unstable, and many contaminants are contained in the medium, and cyclodextrin
It was difficult to separate and purify glucanotransferase. For example, the amount of cyclodextrin glucanotransferase produced in the case of the 7th Annual Meeting of the Molecular Biology Society of Japan Program / Abstracts Collection (SHO 59-11-16) is 0.8 U / 1 ml.
It is a culture solution. The present inventors succeeded in cloning the Bacillus macerans cyclodextrin glucanotransferase structural gene in Escherichia coli, and when the mutant was cultured, it was stably stable. It was possible to produce cyclodextrin glucanotransferase in high yield over time. The present invention is
Cyclodextrin glucanotransferase producing Escherichia coli (C600-pEDS1 (FERM BP-
774) is cultured, and cyclodextrin glucanotransferase is collected from the culture, which is a method for producing cyclodextrin glucanotransferase.

Escherichia coli (C600-pE) producing cyclodextrin glucanotransferase used in the present invention
DS1 (FERM BP-774) can be created as follows.

The cyclodextrin glucanotransferase gene is prepared as follows.

Bacillus macerans IAM1243 strain is known as a bacterium that secretes cyclodextrin glucanotransferase to the outside of the cells. Saito, Miura method (Sai
to.H. and Miura, K..Biochim.Biophys.Acta, 72 , 619, (19
64)) to prepare chromosomal DNA. The restriction enzyme Sau
Partially decomposed with 3AI, and a chromosomal DNA fragment of about 2 kb or more is separated from the decomposed product by sucrose density gradient ultracentrifugation.

Separately, the phage λL47 DNA is cut with a restriction enzyme Bam HI, the chromosomal DNA fragment is mixed with the restriction enzyme, T4 DNA ligase is further added, and ligation is performed. Using this treatment solution
Vitro Packaging Method (Horn, B., Methods in Enzym
ology, 68 , 299, (1979)) forms λ phage particles, and these λ phage particles are added to a culture suspension of Escherichia coli WL95 to produce a special trait possessing cyclodextrin glucanotransferase-producing ability. Obtain the introduced phage. Λ phage DNA containing the cyclodextrin glucanotransferase gene is prepared from the obtained specially transduced phage particles. This DNA is partially digested with the restriction enzyme Sau 3AI and ligated to the restriction enzyme Bam HI digested site of plasmid pBR322 using T4 ligase to obtain a hybrid plasmid mixture. Using this mixture, the Escherichia coli Hederberg, Cohen method (Lederber
g, EMand Cohen, SN.J.Bacteriol., 119 , 1072, (197
4)). A strain having ampicillin resistance (50 μg / ml) and cyclodextrin glucanotransferase activity is selected from the transformants. This strain was cultured, and the plasmid pEDS1 was obtained from the cultured cells.

The physical map (restriction enzyme digestion map) of the plasmid pEDS1 is as shown in Fig. 4. The open part is derived from the vector pBR322, the black part is the chromosome fragment part, and the size of this cloned fragment. Is about 4.4 kb. The transformant obtained here is an excellent mutant Escherichia coli that stably and efficiently produces cyclodextrin glucanotransferase. This bacterium has been deposited with the Institute of Mechanical Engineering as Escherichia coli C600-pEDS1 (FERM BP-774).

This mutant Escherichia coli is cyclodextrin
It is completely novel in productivity of glucanotransferase and is extremely useful for industrial production of cyclodextrin glucanotransferase.

In the present invention, the mutant Escherichia coli is liquid-cultured. The medium may be any as long as it can be used for culturing Escherichia coli, but the carbon source includes starch, liquefied starch, glucose, glycerin, molasses, molasses, etc., and various proteolytic products as nitrogen source. , Soybean flour, meat extract, peptone, urea, nitrate, ammonium salt, yeast extract, corn steep liquor, and other nutrients such as biotin and trace metals are used as appropriate.

The culture is carried out at 37 ° C by aeration stirring. Since cyclodextrin glucanotransferase shows the maximum accumulation amount after 8 to 10 hours of culture, the culture solution is centrifuged to obtain bacterial cells and ultrasonically disrupt the cells.

The disrupted bacterial cells are removed by centrifugation, and cyclodextrin glucanotransferase can be isolated from the resulting supernatant by a general enzyme purification method such as salting out, dialysis, ion exchange resin, affinity chromatography treatment.

Next, the present invention will be described in detail with reference to Examples and Test Examples.

Example of creation Creation of mutant Escherichia coli Bacillus macerans IAM1243 strain is cultured for 48 hours in a fusuma medium (6% fusuma extract, 0.5% malto extract, 0.5% yeast extract, 0.5% polypeptone, 0.05% calcium chloride), The cells were collected by centrifugation, washed, and the cells obtained were subjected to the method of Saito, Miura (Saito, H. and
Miura, K., Biochim. Biophys.Acta, 72 , 619, (1964)) isolates chromosomal DNA, dissolves it in Tris-HCl / EDTA buffer, and adds restriction enzyme Sau 3AI at 37 ℃. After partially decomposing, the degradate is approximately sucrose concentration gradient ultracentrifugation method
A chromosome fragment of 2 kb or more was separated and obtained.

The schematic map of DNA cleavage of the used phage vector λL47 is the first
As shown in the figure, this phage λL47 DNA is 40.6 kb.
It is cleaved at two sites by the restriction enzyme Bam HI.

A λL47 DNA cleaved with Bam HI and a chromosome fragment of about 2 kb or more obtained from the Bacillus macerans IAM1243 strain were mixed, and ligated by adding T4 DNA ligase (Weiss, B.,
Sablon, AJ, Live, TR, Fareed, GC and Richardson, C.
C., J. Biol. Chem., 243 , 4543, (1968)). In-vitro packaging kit (product of Takara Shuzo)
In vitro packaging method (Horn,
B., Methods in Enzymology, 68 , 299, (1979)) to introduce the DNA into phage particles. The phage particles were added to a bacterial cell suspension of Escherichia coli WL95, and added to a λ medium containing 1% soluble starch and 1.2% agar (10 g of bactotryptone and 2.5 g of NaCl dissolved in 1 l of water). After culturing at 37 ℃, spray the emerging plaque with iodine solution to remove phages that did not react with iodine by cyclodextrin.
It could be isolated as a glucanotransferase-producing phage.

The obtained cyclodextrin glucanotransferase-producing phage was isolated and cultured with λ medium at 37 ° C. together with Escherichia coli WL66. The culture solution was centrifuged to remove the host cells, polyethylene glycol was added to aggregate the phage particles, and the phage particles were collected by centrifugation to obtain new phages. This phage was named λCD2.

The phage particles were dialyzed against a phage suspension buffer containing 50% formamide to obtain λCD2 phage DNA. The phage λCD2 DNA has a size of 45.0 kb, and its physical map (restriction enzyme cleavage map) is shown in FIG. Here, the white parts are derived from the vector λL47, and the black parts are the chromosome fragment parts. All abbreviations are restriction enzymes.

This phage λCD2 DNA was labeled with ³² P, and Bacillus macerans IAM1243 strain chromosomal DNA, Escherichia coli WL95
Strain chromosomal DNA and α-amylase high-producing strain Bacillus
When substituting Eco RI degradation products of Bacillus subtilis NA64 chromosomal DNA with Southern hybridization (Sauthern, EM, J. Mol. Biol., 98 , 503, (1975)), Escherichia coli and Bacillus It did not hybridize with the subtilis chromosomal DNA at all, but specifically hybridized with the chromosomal DNA of the Bacillus macerans IAM1243 strain at two sites, and the cloned gene was derived from the Bacillus macerans IAM1243 strain. I was confirmed.

The λCD2 phage DNA obtained here was dissolved in Tris-HCl / EDTA buffer, the restriction enzyme Sau 3AI was added, and partial digestion was carried out at 37 ° C. Approximately 2 kb from the degradation product by sucrose density gradient ultracentrifugation
The above chromosomal fragments were separated and obtained, and recloned using the plasmid pBR322.

A schematic cleavage map of the plasmid pBR322 used for recloning is shown in Fig. 3. This plasmid pBR322 is 4.4 kb, has ampicillin resistance (Ap ^r ), and is cleaved at one site by the restriction enzyme Bam HI. .

PBR322 cut at one site with Bam HI was mixed with a chromosome fragment of about 2 kb or more obtained from the above λCD2 phage, and T4 DNA ligase was added for ligation. Using this treatment solution, using Escherichia coli C600 strain as a host, Lederberg, Cohe
n method (Lederberg, EM and Cohen, SN, J. Bacteriol., 11
9 , 1072, (1974)).

The obtained transformant-treated cells were used as a selective medium in an L medium containing 1% of soluble starch, 50 μg / ml of ampicillin and 1.6% of agar (10 g of bactotryptone, 5 g of yeast extract, 5 g of NaCl dissolved in 1 l of water). The cells were cultured at 37 ° C to obtain an ampicillin resistant strain. This ampicillin resistant strain was treated with ampicillin 50 μg / ml,
After culturing in L medium containing soluble starch at 37 ° C. for 24 hours, the presence or absence of cyclodextrin in the culture was examined by paper chromatography and high performance liquid chromatography.

Paper chromatography was performed using n-butanol
After developing twice at 60 ℃ in a solvent system of / n-propanol / water (3/5/4), dip the paper chromatogram in a 90% acetone iodine solution to search for cyclodextrin coloration. , The colored one is cyclodextrin
It could be isolated as a glucanotransferase-secreting strain.

The resulting cyclodextrin glucanotransferase-producing strain was isolated, and it was treated with ampicillin 50 μg / ml.
Cultivated at 37 ℃ in L medium containing, and collect the cells by centrifuging the culture broth, wash, and then perform alkaline extraction from the separated cells (Birnbo
im, HCand Doly, J., Nucleic Asids Res., 7 , 1513, (19
79)), the plasmid was separated and searched to obtain a new plasmid. This plasmid was designated as plasmid pEDS1.

The plasmid pEDS1 has a size of 8.8 kb, and its physical map (restriction enzyme digestion map) is shown in FIG. Here, the white parts are derived from the vector pBR322, the black parts are the chromosome fragment parts, and all abbreviations are restriction enzymes.

Test Example 1 Production of cyclodextrin glucanotransferase L medium containing 50 μg / ml of ampicillin was poured into a 500 ml Sakaguchi flask in an amount of 100 ml to prepare Escherichia coli C600-pED.
S1 (FERM BP-774) was shake-cultured at 37 ° C. for 20 hours. During that period, 1 ml of the culture was collected to measure the activity of the produced cyclodextrin glucanotransferase.

FIG. 5 shows the time course of the cyclodextrin glucanotransferase activity. Where A
Indicates cyclodextrin glucanotransferase activity, and B indicates growth of the fungus.

As is clear from FIG. 5, the cyclodextrin glucanotransferase activity reaches the maximum value 8 hours after the culture.

Example L Composition of medium Bactotryptone 10 g Yeast extract 5 g NaCl 5 g Water 1 l Ampicillin 50 mg Escherichia coli C600-pEDS1 (FERM BP-774) was inoculated into two 500 ml Sakaguchi flasks containing 100 ml of the above medium, and 37 ° C. After culturing with shaking overnight, a seed culture solution was obtained. 200 ml of the seed culture was added to a jar fermenter containing 20 l of a medium having the same composition as described above, and the cells were subjected to aeration and stirring culture at 37 ° C for 10 hours. The obtained culture broth was centrifuged to obtain bacterial cells, which were suspended in 5 l of 10 mM Tris-HCl buffer (pH 7.5) containing 2 mM calcium acetate, and the bacterial cells were disrupted by an ultrasonic disruptor.

The disrupted cells were removed by centrifugation, 50 g of corn starch was added to the supernatant, and the mixture was stirred overnight at 5 ° C to adsorb cyclodextrin glucanotransferase. The starch was washed with 250 ml of 33% ethanol and filtered through a glass filter under reduced pressure to remove the ethanol solution.

The starch was suspended in 250 ml of distilled water and stirred at 50 ° C for 15 minutes to desorb the enzyme.

The starch was filtered off and column chromatography was performed using a DEAE-cellulose column equilibrated with a 0.05 M acetate buffer (pH 6.0) containing 1 mM calcium chloride. Elution was performed using the same buffer solution by a concentration gradient method of 0 to 0.5 M sodium chloride. The active fractions were collected, dialyzed against a collodion bag in a 1 mM calcium chloride solution, and concentrated to a protein concentration of 3 to 4%.

A saturated ammonium sulfate solution containing 1 mM calcium chloride was added dropwise to the concentrated enzyme solution. It became slightly cloudy at about 0.1 saturation, so add a small amount of 1mM calcium chloride solution to make it clear and make it 3-5.
It was left at ℃. After 4 weeks, the crystals were taken out on a glass filter, washed with a 1 mM calcium chloride solution, and then collected, suspended in a 1 mM calcium chloride solution and frozen,
It was a purified enzyme.

[Brief description of drawings]

FIG. 1 shows the physical map of phage λL47 (restriction enzyme cleavage map), FIG. 2 shows the physical map of phage λCD2, FIG. 3 shows the physical map of plasmid pBR322, and FIG. 4 shows the physical map of pEDS1. FIG. 5 is a graph showing changes in cyclodextrin glucanotransferase activity with time and growth of bacteria in Test Examples.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Shoichi Kobayashi 1-1410, Takemura, Sakuramura, Shinji-gun, Ibaraki Prefecture, No. 403, No. 401 (72) Inventor, Keiji Kainuma, 3121, 611 Namiki, Sakura-mura, Shinji-gun, Ibaraki (72) Inventor Hitoshi Hashimoto 4 31-10 Imaizumidai, Kamakura City, Kanagawa Prefecture (72) Inventor Takashi Okemoto 1-4-6, Uenomiya, Tsurumi Ward, Yokohama City, Kanagawa Prefecture, 1st dormitory of salt sugar (56) Reference JP 61- 135589 (JP, A) 7th Annual Meeting of the Molecular Biology Society of Japan Program / Abstracts (SHO59-11-16) B-78

Claims (1)

[Claims] 1. Escherichia coli C600-pEDS1 which produces cyclodextrin glucanotransferase.
(FERM BP-774) is cultured, and cyclodextrin glucanotransferase is collected from the culture, a method for producing cyclodextrin glucanotransferase.