JPS61132183A - Production of cyclodextrin glucanotransferease - Google Patents

Abstract

PURPOSE:To provide the titled substance free from contamination of impurities, by culturing mutant of Bacillus subtilis capable of producing cyclodextrin glucanotransferase, and separating the cyclodextrin glucanotransferase from the culture product. CONSTITUTION:A chromosome DNA is prepared by Bacillus macerans IAM1243 strain. The chromosome DNA is partially decomposed with the restriction enzyme Sau3AI, and the chromosome DNA fragment of about >=2 Kb is separated from the decomposition product. Separately, phase lambdaD69 DNA is incised with the restriction enzyme BamH1, mixed with the above chromosome DNA fragment, and linked by the addition of T4DNA ligase to obtain the plamid pDS10. A mutant Bacillus subtilis is prepared therefrom. The Bacillus subtilis is cultured, and the objective cyclodextrin glucanotransferase is separated from the culture product.

Description

DETAILED DESCRIPTION OF THE INVENTION Generally, bacteria of the genus Bacillus and Klebsiella have been used as cyclodextrin glucanotransferase producing bacteria. However, its production was small and unstable, and the culture medium contained many contaminants, making it difficult to separate and purify cyclodextrin glucanotransferase. The present inventors have discovered that Bacillus
We succeeded in cloning the structural gene for cyclodextrin glucanotransferase from Bacillus subtilis, and when we cultured this mutant strain, we were able to stably produce cyclodextrin glucanotransferase in a short period of time. be.

The present invention is a method for producing cyclodextrin glucanotransferase, which comprises culturing Bacillus subtilis that produces cyclodextrin glucanotransferase, and collecting cyclodextrin glucanotransferase from the culture.

Bacillus subtilis that produces the cyclodextrin glucanotransferase used in the present invention can be created as follows.

The cyclodextrin glucanotransferase gene is prepared as follows.

Bacillus macerans strain IAM1243 is known as a bacterium that secretes cyclodextrin glucanotransferase outside the bacterial body. 5aito from this bacterial body,
Miura method (Saito, H, and Miur
a.

In,,Biochem,Biophys,Acta,7
2,619. (1964)) to prepare chromosomal DNA. This was partially digested with restriction enzymes Sau3A, I, and the digested product was separated into approximately 2 to 5 pieces by sucrose density gradient ultracentrifugation.
Separate the above chromosomal DNA fragments.

Separately, phage λD69 DNA was digested with restriction enzyme Ba1llH.
After cutting with I, the previous chromosomal DNA fragment is mixed with this, and T4 DNA licase is added to perform ligation. Using this treatment solution, the in vitro packaged blowfish method (Ho
rn, B,, Methods in Enzymol
ogy, 68.299. (1979)), and the λ phage particles are added to a culture suspension of Escherichia coli 5M32 to obtain a special transducing phage having the ability to produce cyclodextrin glucanotransferase. Lambda phage DNA containing the cyclodextrin glucanotransferase gene is prepared from the obtained special transduction phage particles. This DNA is partially digested with restriction enzyme 5au3A I, and ligated to the restriction enzyme BamHI cut portion of plasmid pUB110 using 741J gauze to obtain a hybrid plasmid mixture. This mixture is used to transform Bacillus (1979). Among the transformed strains, a strain with kanamycin resistance (10 μg/mJ) and cyclodextrin glucanotransferase activity is selected. This strain was cultured, and plasmid pDs10 was obtained from the cultured cells.

The physical map (restriction enzyme cleavage map) of plasmid pDs10 is shown in Figure 4, where the white part is derived from vector pUB110, the black part is the chromosomal cut part, and the size of this cloned fragment. is about 2.2
Kb. The transformant obtained here is an excellent mutant Bacillus subtilis that secretes cyclodextrin glucanotransferase stably and well outside the bacterial cell. This bacterium is Bacillus subtilis NA64-pD
S 10 (FERMP-7959) and has been deposited with the Institute of Fine Technology.

This mutant Bacillus subtilis is completely new in its productivity of cyclodextrin glucanotransferase, and is extremely useful in the industrial production of cyclodextrin glucanotransferase.

In the present invention, any desired cyclodextrin obtained by culturing this mutant Bacillus subtilis in liquid may be used, but starch can be used as the carbon source.

Examples of nitrogen sources include liquefied starch, glucose, glycerin, molasses, and blackstrap molasses. Nitrogen sources include various protein digests, soy flour, meat extract, peptone, urea, nitrates, ammonium salts, yeast extract, and cornstarch liquor. Other nutrients such as biotin and trace metals are used as appropriate.

Cultivation is carried out at 37°C with aeration.

Since the cyclodextrin glucanotransferase in the medium shows the maximum accumulation amount after 12 to 20 hours of culture, the culture is terminated at this point and the culture solution is centrifuged to remove the bacterial cells.

Cyclodextrin glucanotransferase can be isolated from the obtained culture filtrate by general enzyme purification methods such as salting out, dialysis, ion exchange resin, and affinity chromatography.

Next, the present invention will be explained in more detail by Examples and Test Examples.
Yeast Extract, 0.5% Yeast Extract 0.5% Polypeptone.

The cells were cultured for 48 hours in 0.05% calcium chloride), collected by centrifugation, and washed.
.

Miura's method (Saito, H, and Mtu
ra, K., Biochgm.

Biophys, Acta, 72.619. (1
The chromosomal DNA was isolated by 964)), dissolved in Tris-HCl/EDTA buffer, and treated with the restriction enzyme 5au3A.
[(Takara Shuzo Co., Ltd. product) was added, and after partial decomposition at 37°C, chromosomal fragments of approximately 2 Kb or more were separated and obtained from the decomposed product by sucrose density gradient ultracentrifugation.

The schematic cleavage map of the phage vector λD69 (provided by the Institute of Medical Science, University of Tokyo) used is shown in Figure 1. This phage λD69 is 38.8 Kb and is
It is cut at one place.

λD69 cut at one site with Ban+HI and about 2 Kb obtained from the above Bacillus macerans IAM1243 strain
The above chromosome fragments were mixed and ligated by adding T4 DNA licase (Weiss+8.+5ablo
n, A, J,, Live, T, R,, Far
eed+G, C.

and Richardson, C.C., J.B.
iol, Chem, +243+4543, (19
68)). In vitro pathocaging of the treatment solution
kit (Takara Shuzo Co., Ltd.) to perform the in vitro backsaging method (Horn+ 8.+Methods i).
n Bnzymology+ 68+ 299+ (1
979)), the DNA was introduced into phage particles. These phage particles were added to a bacterial cell suspension of Escherichia coli 5M32, and λ medium (10 g of Batatotryptone, N a C1) containing 1% soluble starch and 1.2% agar was added.
2.5g dissolved in water 11) and cultured at 37°C,
By spraying an iodine solution onto the appearing plaques, we were able to isolate phages that did not undergo an iodine reaction as cyclodextrin glucanotransferase producing phages.

The resulting cyclodextrin glucanotransferase producing phage was isolated and cultured with Escherichia coli 5M32 in lambda medium at 37°C. The culture solution was centrifuged to remove host cells, polyethylene glycol was added to aggregate the phage particles, and the phage particles were collected by centrifugation to obtain a new phage. This phage was named λCD.

The phage particles were dialyzed against phage suspension buffer containing 50% formamide, and the λCD phage DNA
I got it. Phage λCD DNA is 49.8 Kb in size, and its physical map (restriction enzyme cleavage map) is
Show the diagram. Here, the white part is derived from vector λD69, and the black part is a chromosome fragment. All abbreviations are restriction enzymes.

This phage λCD DNA was labeled with 32P and converted into Bacillus macerans IAM1243 strain chromosomal DNA.

Escherichia coli 5M32 strain chromosomal DNA and α-
amylase high producing strain Bacillus subtilis
lus 5ubtilis) NA64 chromosomal DNA
Southern hybridization with each EcoRI-digested product (5Outhernl E, M-+, r,
Mo1. Biol, +98+503, (197
5)), it did not hybridize at all with the chromosomal DNA of Escherichia coli and Bacillus subtilis, but it specifically hybridized with the chromosomal DNA of Bacillus macerans strain IAM1243 at two locations, indicating that the clone The transformed gene is Bacillus macerans IA
It was confirmed that it was derived from the M1243 strain.

The λCD phage DNA obtained here was treated with Tris-HCl/E.
Dissolve in DTA buffer and add restriction enzyme Sau 3AI (
Takara Shuzo Co., Ltd. product) was added and partially decomposed at 37°C. A chromosomal fragment of approximately 2 Kb or more was isolated and obtained from the digested product by C1115 density gradient ultracentrifugation, and recloned using plasmid pUB110.

A schematic cleavage map of plasmid pUB110 used for recloning is shown in FIG.
is 4.5 Kb, has kanamycin resistance (K1), and is cleaved at one site by the restriction enzyme BamHI.

pUBllo cut at one site with Bam Hf and the above λ
Chromosome fragments of about 2 Kb or more obtained from CD phage were mixed and ligated by adding T4 DNA ligase.

Using this treatment solution, a protoplast transformation method using Bacillus subtilis strain NA64 as a host (Chang,
S,, Cohen+ S, N,, Mo1. Qe
n.

Genet, 168.111. (1979)), and the plasmid was introduced.

The resulting transformed bacterial cells were incubated with selective media of 1% soluble starch, 100 μg/ml of kanamycin, and 0 agar.
．． DM3 medium containing 8% (5% Batatocasamino acids 100
mC1M sodium citrate (pH 7,3) 500 m
l, 3.5% dipotassium hydrogen phosphate 50ml 1°1.
5% potassium dihydrogen phosphate 50mL 10% yeast extract 5
0mC 20% glucose 25ml 1. A kanamycin-resistant strain was obtained by adding 20 mL of LM magnesium chloride (mixed with 5 mJ of 2% bovine serum albumin) and culturing at 37°C. This kanamycin-resistant strain was treated with kanamycin at 10 μg/ml.
LG medium containing % soluble starch (10 g
, yeast extract 5g.

425g NaC, 2gt-11 glucose dissolved in water)
The cells were cultured at 37° C. for 24 hours, and the presence or absence of cyclodextrin in the culture solution was examined by paper chromatography and high performance liquid chromatography.

For paper chromatography, the culture solution was diluted with a solvent system of n-butanol/n-propanol/water (315/4) at 60%
After increasing the temperature twice, the paper chromatogram was immersed in a 90% acetone collagen solution to search for the coloration of cyclodextrin, and those that developed color could be isolated as cyclodextrin glucanotransferase-secreting strains. did it.

The resulting cyclodextrin glucanotransferase producing strain was isolated and treated with 10 μg of kanamycin.
The cells were cultured at 37°C in LG medium containing /mβ, the culture solution was centrifuged to collect the bacteria, and after washing, the isolated cells were subjected to alkaline extraction method (Birnboim, El, C, and Doly,
J., Nucleic Ac1ds Res, 1513. (1979)), the plasmid was isolated and searched for, and a novel plasmid was obtained. This plasmid was named plasmid pDs10.

Plasmid pDslo has a size of 6.7 Kb, and its physical map (restriction enzyme cleavage map) is shown in FIG.

Here, the white part is derived from vector pUB110, the black part is a chromosome fragment part, and each abbreviation is a restriction enzyme.

Test Example 1 Production of cyclodextrin glucanotransferase Bacillus subtilis NA64-p DSIO was dispensed into a 500 mJ Sakaro flask using LG medium containing 10 μg/ml of kanamycin, and shaken at 37°C for 30 hours. Cultured. During that time, 1 culture
The activity of cyclodextrin glucanotransferase in the culture medium was measured by collecting ml of the culture medium.

FIG. 5 shows changes over time in cyclodextrin glucanotransferase activity. Here A
1 shows bacterial growth, and B shows cyclodextrin glucanotransferase activity.

As is clear from FIG. 5, cyclodextrin glucanotransferase activity reaches its maximum value 12 hours after culture.

Composition of Example LG medium Bactodorypton 10g Yeast extract 5g NaCβ 5g Water 11 Kanamycin 10mg Bacillus subtilis NA64-p D S 10
(FERMP-7959) was inoculated into two 500 m1 slope flasks containing 100 mJ of the above medium, and
A seed culture solution was obtained by culturing with shaking overnight at C.

Seed culture solution 200mj+ was added to a jar fermenter containing medium 20j2 with the same composition as above, and incubated at 37°C for 1 hour.
Culture was carried out with aeration for 5 hours. The resulting culture solution was centrifuged to remove the bacterial cells and a culture filtrate 19N was obtained.

Add 200 g of corn starch to this culture filtrate,
The mixture was stirred overnight at 5°C to adsorb cyclodextrin glucanotransferase.

The starch was washed with 11 33% ethanol and filtered under reduced pressure through a glass filter to remove the ethanol solution.

Starch was suspended in 11 distilled water, and the enzyme was desorbed at 50°C for 15 minutes.

Filter the starch and add 0.05M containing 1mM calcium chloride.
Column chromatography was performed using a DEAE-cellulose column equilibrated with acetate buffer (pH 6,0). Elution was performed using the same buffer solution using a concentration gradient method from 0 to 0.5M sodium chloride. The active fractions were collected, dialyzed in a 1mM calcium chloride solution using a collodion bank, and concentrated to a fluorescent concentration of 3-4%.

A saturated ammonium sulfate solution containing 1 mM calcium chloride was added dropwise to this concentrated enzyme solution. Since the mixture became slightly cloudy at about 0.1 saturation, a small amount of 1mM calcium chloride solution 11 was added to make it transparent, and the mixture was allowed to stand at 3 to 5°C. After 4 weeks, the crystals were taken out onto a glass filter and washed with a 1mM calcium chloride solution, and then the crystals were collected, suspended in a 1mM calcium chloride solution, and frozen to obtain a purified enzyme.

[Brief explanation of the drawing]

FIG. 1 shows a physical map (restriction enzyme cleavage map) of phage λD69, FIG. 2 shows a physical map of phage λCD, FIG. 3 shows a physical map of plasmid pUB110, and FIG. 4 shows a physical map of pDslo. FIG. 5 is a graph showing changes over time in cyclodextrin glucanotransferase activity and bacterial growth in test examples. Bamf-H Figure 4 BgLπ Procedural amendment form button January to date, 1985

Claims (1)

[Claims] A method for producing cyclodextrin glucanotransferase, which comprises culturing Bacillus subtilis, which produces cyclodextrin glucanotransferase, and collecting cyclodextrin glucanotransferase from the culture.