JPH0354551B2 - - Google Patents

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 impurities, making it difficult to separate and purify cyclodextrin glucanotransferase. The present inventors succeeded in cloning the cyclodextrin glucanotransferase structural gene of Bacillus macerans in Bacillus subtilis. It was possible to produce cyclodextrin glucanotransferase. The present invention is Bacillus subtilis transformed by introducing a plasmid into which DNA encoding cyclodextrin glucanotransferase derived from Bacillus macellans is introduced.

The mutant Bacillus subtilis of 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 to the outside of the bacterial body. Saito, Miura method (Saito, H. and Miura,
K., Biochem.Biophys.Acta, 72 , 619, (1964))
Prepare chromosomal DNA by This is a restriction enzyme
Partially digest with Sau3AI, and separate chromosomal DNA fragments of approximately 2 kb or more from the digested product using sucrose density gradient ultracentrifugation.

Separately, extract Phage λD69 DNA with the restriction enzyme BamHI.
, mix this with the previous chromosomal DNA fragment,
Furthermore, T4 DNA ligase is added to perform the ligation process. Using this treatment solution, the in vitro packaging method (Horn, B., Methods in
Enzymology, 68 , 299, (1979)) to form lambda phage particles, and add the lambda phage particles to a culture suspension of Escherichia coli SM32 to determine the ability to produce cyclodextrin glucanotransferase. Obtain specialized transducing phages harboring . From the obtained special transduction phage particles, λ phage containing the cyclodextrin glucanotransferase gene was detected.
Prepare DNA. This DNA is digested with the restriction enzyme Sau3AI
Partially digest plasmid pUB110 with restriction enzymes
Bind to the BamHI cleavage site using T4 ligase,
Obtain a hybrid plasmid mixture. This mixture was used for Bacillus subtilis heprotoplast transduction (Chang, S. and Cohen, SN,
Mol.Gen.Genet. 168 , 111, (1979)). Among the transformed strains, a strain having kanamycin resistance (10 μg/ml) and cyclodextrin glucanotransferase activity is selected.
This strain is cultured, and plasmids are extracted from the cultured cells.
pDS10 was obtained.

The physical map (restriction enzyme cleavage map) of plasmid pDS10 is shown in Figure 4. The white part is derived from vector pUB110, the black part is the chromosomal cut part, and the size of this cloned fragment is It is approximately 2.2kb. The transformant obtained here is an excellent mutant Bacillus subtilis that can stably and well secrete cyclodexrin glucanotransferase outside the bacterial cell. This bacterium is Bacillus subtilis NA64-pDS10 (FERM P-
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 for industrial production of cyclodextrin glucanotransferase.

Next, examples and test examples of the present invention will be shown.

Example Creation of mutant Bacillus subtilis Bacillus macerans strain IAM1243 was grown in bran medium (6% bran extract, 0.5% malt extract, 0.5% yeast extract, 0.5%
The method of Saito, Miura (Saito, H. and
Miura, K., Biochim. Biophys. Acta, 72 , 619,
(1964)) was used to isolate chromosomal DNA, dissolve it in Tris-HCl/EDTA buffer, and use restriction enzymes.
After adding Sau3AI (Takara Shuzo Co., Ltd. product) and partially decomposing the mixture at 37°C, chromosome fragments of approximately 2 kb or more were isolated and obtained from the decomposed product using sucrose density gradient ultracentrifugation.

The schematic cleavage map of the used phage vector λD69 (provided by the Institute of Medical Science, the University of Tokyo) is shown in Figure 1. This phage λD69 is 38.8 kb and can be cleaved at one site by the restriction enzyme BamHI. It is.

λD69 cut at one site with BamHI and the chromosomal fragment of approximately 2 kb or more obtained from Bacillus macerans strain IAM1243 were mixed and ligated by adding T4 DNA ligase (Weiss, B., Sablon, A.
J., Live, T.R., Fareed, GCandRichardson,
CC, J. Biol. Chem., 243 , 4543, (1968)). The treatment solution was added to an in vitro packaging kit (Takara Shuzo Co., Ltd.) to perform the in vitro packaging method (Horn, B., Methods in
Enzymology, 68 , 299, (1979)).
DNA was introduced into phage particles. The phage particles were added to a bacterial cell suspension of Escherichia coli SM32, and λ containing 1% soluble starch and 1.2% agar was added.
Medium (10 g of bactotryptone, 2.5 g of NaCl, 1 part of water)
The phages that did not undergo an iodine reaction were isolated as cyclodextrin-glucanotransferase-producing phages by culturing at 37°C and spraying an iodine solution onto the plaques that appeared.

The resulting cyclodextrin glucanotransferase producing phages were isolated and cultured with Escherichia coli SM32 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 new phages. This phage was named λCD.

The phage particles were dialyzed against a phage suspension buffer containing 50% formamide to obtain λCD phage DNA. Phage λCDDNA is 49.8kb
The size of the physical map (restriction enzyme cleavage map) is shown in Figure 2. Here, the white part is derived from vector λD69, and the black part is a chromosome fragment. All abbreviations are restriction enzymes.

This phage λCDDNA was labeled with ^32P , and Bacillus macerans strain IAM1243 chromosomal DNA, Escherichia coli strain SM32 chromosomal DNA and α
-EcoRI-digested products of NA64 chromosomal DNA of high amylase producing strain Bacillus subtilis and Southern hybridization (Southern, EM, J.Mol.Biol., 98 , 503,
(1975)), Esierhia coli,
It did not hybridize at all with Bacillus subtilis chromosomal DNA, but Bacillus macerans
It specifically hybridized with the chromosomal DNA of strain IAM1243 at two locations, and it was confirmed that the cloned gene was derived from Bacillus macerans strain IAM1243.

The obtained λCD phage DNA was dissolved in Tris-HCl/EDTA buffer, and the restriction enzyme Sau3AI was added.
(Takara Shuzo Co., Ltd. product) was added and partially decomposed at 37°C.
Chromosome fragments of approximately 2 kb or more are separated from the digested product using sucrose density gradient ultracentrifugation, obtained, and converted into plasmids.
Recloning was attempted using pUB110.

A schematic cleavage map of plasmid pUB110 used for recloning is shown in Figure 3.
pUB110 is 4.5 kb, has kanamycin resistance (Km ^r ), and can be cut at one site with the restriction enzyme BamHI.

pUB110 cut at one site with BamHI and the chromosome fragment of about 2 kb or more obtained from the λCD phage described above were mixed and ligated by adding T4 DNA ligase. A protoplast transformation method using Bacillus subtilis strain NA64 as a host using this treatment solution (Chang, S., Cohen, SN, Mol.gen.
Genet., 168 , 111, (1979)), and the plasmid was introduced.

The resulting transformed cells were mixed with selective media of 1% soluble starch, 100 μg/ml of kanamycin, and agar.
DM3 medium containing 0.8% (5% bactocasamino acid)
100ml, 1M sodium citrate (PH7.3) 500ml,
3.5% dipotassium hydrogen phosphate 50ml, 1.5% potassium dihydrogenphosphate 50ml, 10% yeast extract 50ml, 20% glucose 25ml, 1M magnesium chloride 20ml, 2%
(mixed with 5 ml of bovine serum albumin) and cultured at 37°C to obtain a kanamycin-resistant strain. This kanamycin-resistant strain was grown in LG medium containing 10 μg/ml of kanamycin and 1% soluble starch (10 g of bactotryptone,
5g of yeast extract, 5g of NaCl, 2g of glycose in 1
(dissolved in water) 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 is n-butanol/n-propanol/water (3/5/4).
After two upward developments at 60°C in a solvent system of 90
A paper chromatogram was immersed in a % acetone iodine solution to search for the coloration of cyclodextrin, and those that developed color were able to be isolated as cyclodextrin glucanotransferase-secreting strains.

The obtained cyclodextrin glucanotransferase producing strain was isolated and cultured at 37°C in LG medium containing 10 μg/ml of kanamycin.
The culture solution was centrifuged to collect bacteria, and after washing, the isolated bacteria were extracted using an alkaline extraction method (Birnboim, HCand
Doly, J., Nucleic Acids Res., 7 , 1513.
(1979)), the plasmid was isolated and searched, and a new plasmid was obtained. This plasmid was named plasmid pDS10.

Plasmid pDS10 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 100 ml of Bacillus subtilis NA64-pDS10 was dispensed into a 500 ml Sakaguchi flask using LG medium containing 10 μg/ml of kanamycin, and cultured with shaking at 37°C for 30 hours. . During this time, 1 ml of the culture was collected and the activity of cyclodextrin glucanotransferase in the medium was measured.

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

As is clear from Figure 5, the cyclodextrin glucanotransferase activity was
It reaches its maximum value in 12 hours.

[Brief explanation of drawings]

Figure 1 is the physical map of phage λD69 (restriction enzyme cleavage map), Figure 2 is the physical map of phage λCD, and Figure 3 is the plasmid pUB110.
Figure 4 shows the physical map of pDS10. FIG. 5 is a graph showing changes over time in cyclodextrin glucanotransferase activity and bacterial growth in test examples.

Claims (1)

[Claims] 1 Bacillus subtilis was transformed by introducing a plasmid into which DNA encoding cyclodextrin glucanotransferase derived from Bacillus macerans was introduced.