JP2904436B2 - Method for producing novel recombinant DNA and sucrose phosphorylase - Google Patents

Description

The present invention relates to a method for producing a novel recombinant DNA and sucrose phosphorylase.

[Conventional technology]

Sucrose phosphorylase
ylase (EC 2.4.1.7)] is an enzyme that catalyzes a reaction represented by the following reaction formula.

Sucrose phosphorylase is extremely useful, for example, used for the determination of inorganic phosphorus.

Conventionally, sucrose phosphorylase, for example,
Culture bacteria of the genus Leuconostoc or Pseudomonas and isolate sucrose phosphorylase from the cells,
It is manufactured by refining [Adva Apple
Microviol (Adv. Appl. Microbiol.) Vol. 32, No. 1
63-201 (1987)], and in recent years, it has been confirmed that a bacterium belonging to the genus Streptococcus, a kind of dental caries, produces sucrose phosphorylase [Infection and Immuity (Infection and Immunity)].
nity), 56, 2763-2765 (1988)].

[Problems to be solved by the invention]

However, when sucrose phosphorylase is produced using these bacteria, there have been problems such as the extremely low yield of the enzyme.

Thus, the present inventors have conducted intensive studies to solve the above problems, and as a result, prepared a DNA containing a sucrose phosphorylase gene, inserted this into a vector DNA, and transformed with the recombinant DNA. The present inventors have found that sucrose phosphorylase can be produced at a high yield by using the above microorganism, and have completed the present invention.

[Means for solving the problem]

That is, the present invention resides in a novel recombinant DNA having the following restriction map and a sucrose phosphorylase gene DNA having a molecular weight of 1800 kd inserted into a vector DNA.

(Where E is EcoR I, H is Hind III, P is Pst I, T is E
The present invention further includes the recombinant DNA according to the above 1,
A method for producing sucrose phosphorylase, comprising culturing a microorganism capable of producing sucrose phosphorylase in a medium and collecting sucrose phosphorylase from the culture.

First, preparation of a DNA containing the sucrose phosphorylase gene will be described.

DN containing sucrose phosphorylase gene
Any donor may be used as A, for example,
Lactic acid bacteria Leukotostock mesenteroides (Leuconos)
toc mesenteroides) ATCC 12291 and the like. These microorganisms are used in Methods in Enzymology (Methods in Enzymology).
Enzymology), Vol. 1, pages 227 to 228 (1955), and cultured to obtain cultured cells.

From these cells, for example, Current Protocols in Molecular Biology (Current Protocols
in Molecular Biology) unit 2.4.3, (John Willey &
Sons. Inc. 1987).
Chromosomal DNA can be obtained by treating with proteinase K in the presence of SDS, followed by treatment with hexadecylmethylammonium bromide and chloroform, followed by ethanol precipitation.

Next, a restriction enzyme which generates a protruding end, for example, EcoRI (manufactured by Boehringer Mannheim Yamanouchi) is added to the chromosomal DNA at a temperature of 30 ° C. or higher, preferably at 37 ° C.
Digestion is allowed to act for a time to obtain a chromosomal DNA fragment mixture.
From this chromosomal DNA fragment mixture, a colony hybridization method [Molecular Cloning (Molecular Cloning)] was performed using a DNA probe based on the DNA mixed sequence deduced from the N-terminal amino acid sequence of the purified protein.
oning), 372-328, Cold Spring Harbor Laboratory (1
982)] to obtain the desired sucrose phosphorylase gene.

On the other hand, a vector DN that can be used in the present invention
A may be anything, for example, plasmid vector DNA, bacteriophage DNA, etc., and specifically, for example, plasmid pBR322 DNA (manufactured by Takara Shuzo) is preferred.

A restriction enzyme that generates a protruding end in the vector DNA,
For example, EcoRI (manufactured by Boehringer Mannheim Yamanouchi) is digested by allowing it to act at a temperature of 30 ° C. or more, preferably 37 ° C. for 1 hour or more, preferably 1 to 3 hours to obtain a digested vector DNA.

Then, the lactic acid bacteria Leuconostoc obtained as described above
A mixture of a DNA fragment mixture containing a gene encoding sucrose phosphorylase derived from mesenteroides ATCC 12291 and a cleaved vector DNA, for example, Escherichia coli DNA ligase (New England Biolabs) or T4 DNA Ligase (Boehringer Mannheim Yamanouchi), preferably T4 DNA ligase at a temperature of 4 to 37 ° C, preferably 4 to 16 ° C for 1 hour or more,
Preferably, it is allowed to act for 6 to 24 hours to obtain a recombinant DNA.

Using this recombinant DNA, a suitable host microorganism such as E. coli K-12, preferably E. coli JM101 (ATCC 3387
6), E. coli HB101 (ATCC 33694), E. coli DH1 (ATCC 33694)
849) to obtain the respective strains. This transformation may be performed by a conventional method, for example, the method of DM Morrison (Methods in Enzymology, Vol. 68, No. 321).
326 326 (1979)].

Next, by screening a strain having sucrose phosphorylase-producing ability from the microorganisms subjected to the transformation, a recombinant DNA obtained by inserting a DNA containing a gene encoding sucrose phosphorylase into a vector DNA, It is possible to obtain a microorganism having the ability to produce sucrose phosphorylase.

In order to obtain a novel recombinant DNA purified from the microorganism thus obtained, for example, HCC
Cultured cells are collected by a method such as HC Birnboim (Nucleic Acids Research, Vol. 7, pp. 1513-1523 (1979)), lysozyme lysed, and alkali-treated. A sucrose phosphorylase is produced by inserting a DNA containing the gene encoding sucrose phosphorylase obtained as described above into a vector DNA and then transforming the resulting DNA into a sucrose phosphorylase. In order to produce sucrose phosphorylase using a microorganism having an ability, particularly a strain belonging to the genus Escherichia, culture is performed as described below to obtain a culture.

That is, in order to culture the microorganism, the microorganism may be cultured by a usual solid culture method, but it is preferable to culture by using a liquid culture method as much as possible.

The medium for culturing the microorganism may be, for example, yeast extract, peptone, meat extract, corn sweeper or one or more nitrogen sources such as leachate of soybean or wheat koji, for example, potassium monophosphate, phosphate At least one kind of inorganic salts such as ferric potassium, magnesium sulfate, sodium chloride, magnesium chloride, ferric chloride, ferric sulfate or manganese sulfate was added, and if necessary, saccharide raw materials, vitamins, etc. were appropriately added. Things are used.

It is appropriate that the initial pH of the medium is adjusted to 7 to 9. Cultivation is carried out at 30-42 ° C, preferably at about 37 ° C,
It is preferably carried out for 24 hours, preferably for 6 to 24 hours, by aeration and stirring deep culture, shaking culture, and stationary culture.

After completion of the cultivation, sucrose phosphorylase is collected from the culture and separated and purified in accordance with Methods in Enzymology (Vol. 1, 2).
25-229 (1955)], that is, cultures are collected and lysed, treated with protamine to remove nucleic acids, fractionated with ammonium sulfate, purified by gel filtration and ion exchange chromatogram treatment. It is possible.

The physicochemical properties of the purified sucrose phosphorylase obtained by the above-mentioned purification means are described in [Adv. Appl. Microbiol, Vol. 32, Vol.
3 to 201 (1987)], which are exactly the same as the physicochemical properties of sucrose phosphorylase.

〔The invention's effect〕

The ability of the microorganism transformed with the novel recombinant DNA of the present invention to produce sucrose phosphorylase is extremely high, and by culturing the microorganism in a medium, the enzyme can be obtained in high yield. The invention is industrially very useful.

 Hereinafter, the present invention will be described in more detail with reference to examples.

〔Example〕

(1) Preparation of chromosomal DNA Lactic acid bacteria Leuconostoc mesenteroides ATCC 122
91 was added to the medium [1% tryptone (Difco), 1% yeast extract (Difco), 0.5% K ₂ HPO ₄ , 1
% Sucrose, 0.001% thiamine / hydrochloric acid, 0.04% MgSO _{4
· 7H 2 O, 0.001% FeSO} 4 · 7H 2 O, 0.02% MnSO 4 · 4H 2 O, 0.05%
Ascorbic acid] 1 and cultivation was carried out at 30 ° C. for 2 days. A culture was obtained.
After centrifugation for 5 minutes, the cells were subjected to Current Protocols in Molecular Biology (Curr).
ent Protocols in Molecular Biolgy) unit 2.4.3 (Joh
n Willey & Sons, Inc. 1987) by extracting 100 μg of chromosomal DNA.

Next, 10 μg of this chromosomal DNA and the restriction enzyme EcoR I
20 units (manufactured by Boehringer Mannheim Yamanouchi) in 50 mM Tris-HCl buffer (100 mM NaCl, 10 mM MgCl ₂ ,
(1 mM dithiothreitol, pH 7.4) and reacted at 37 ° C. for 1 hour. The reaction-terminated liquid is prepared in the usual manner.
Phenol extraction treatment, ethanol precipitation treatment, EcoR
8 μg of a chromosomal DNA fragment of the lactic acid bacterium Leuconostoc mesenteroides ATCC 12291 strain digested with I was obtained.

(2) Preparation of lactic acid bacteria chromosomal DNA library using plasmid vector pBR322 10 μg of plasmid vector pBR322 (manufactured by Takara Shuzo) and 20 units of restriction enzyme EcoR I (manufactured by Boehringer Mannheim Yamanouchi) were mixed with 50 mM Tris-HCl buffer (100 mM NaC).
l, 10 mM MgSO ₄ , containing 1 mM dithiothreitol, pH 7.4), and reacted at 37 ° C. for 2 hours to obtain a digested solution. The solution was subjected to phenol extraction and ethanol precipitation by a conventional method. To prevent recombination of the EcoRI digested DNA fragments, bacterial cloning was performed by the method described in Molecular Cloning (Molecular Cloning, pp. 133-134 (1982) Cold Spring Harbor Laboratory). Le alkaline phosphatase (Bact
dephosphorylation of DNA fragments by erial Alkaline Phosphatase)
After further ethanol precipitation, 8 μg of the plasmid vector pBR322 DNA digested with EcoRI was obtained.

Next, the plasmid vector digested with EcoRI
2 μg of pBR322, lactic acid bacteria Leuconostoc mesenteroides ATCC digested with EcoRI obtained in the above item (1)
4 μg of chromosomal DNA fragment of 12291 strain and 2 units of T4 DNA
Ligase (Boehringer Mannheim Yamanouchi)
Add to 66 mM Tris-HCl buffer (pH 7.5) containing 66 mM MgCl ₂ , 10 mM dithiothreitol and 10 mM ATD,
The reaction was carried out at a temperature of 16 ° C. for 16 hours to perform a ligation reaction of DNA.

Using this reaction solution, Journal of Bacteriology, Vol. 119, No. 1072-
By the transformation method described on page 1074 (1974),
(ATCC 33849), and LB-anp medium [Bactotryptone 1% (w / v), yeast extract 0.5% (w / v), NaCl 0.
5% (w / v), agar 1.2% (w / v), and ampicillin 50
(Μg / ml)] about 6,000 colonies growing on the plate
And used it as a library.

(3) Sucrose phosphorylase (hereinafter SPL)
Abbreviated. ) Recombinant plasmid pS containing gene N-terminal
Preparation of PL02 DNA Lactic acid bacteria Leuconostoc mesenteroides ATCC 122
The purification method of sucrose phosphorylase of 91 strains is described in Methods in Enzymolog (Methods in Enzymolog).
y), Volume 1, pp. 225-228 (1955). According to this method, 30 residues of the N-terminal amino acid sequence of a protein purified uniformly by electrophoresis were determined using a gas-phase protein sequencer (Applied Bio Systems, Inc., type 37). In this, Met-Glu-Ile-Gln-
There is an amino acid sequence Asn-Lys, the DNA base sequence corresponding to this amino acid sequence is The SPL gene was screened using these 24 types of mixed oligo DNAs as probes.

This mix of 24 mix oligonucleotides of 17 bases
Were synthesized using a DNA synthesizer to [Bachman (Beckman) Co.], the 5 'end of this 20ng of oligonucleotides using ^[32 P] ATP (Amersham), Molecular Cloning (Molecular Cloning), No. 122-126
Page, Cold Spring Harbor Laboratory (1987).

A lactic acid bacterium leuconostoc stock using the plasmid vector pBR322 prepared in item (2) as a probe, using an oligonucleotide corresponding to a DNA sequence corresponding to the N-terminal amino acid sequence of SPL protein labeled with ³² P prepared by the above method as a probe.・ Mesenteroides ATCC 12291 strain chromosomal DNA
The library was cloned using a colony hybridization method [Molecular Clonin
g), pp. 312-328, Cold Spring Harbor Laboratory (1)
982)] to obtain colonies having the SPL gene N-terminal. Molecular Cloning from the colonies, pages 86-94, Cold Spring Harbor Laboratory [Cold Spring Harbor]
Laboratory (1982)], a recombinant plasmid was obtained in an amount of 350 µg / mouse, and this recombinant plasmid DNA was named pSPL02.

After 1% agarose gel electrophoresis of the digested product of Sau3A I of pSPL02, the product was transferred to a nitrocellulose filter, and the 24-fold mix-synthesized oligonucleotide DNA corresponding to the N-terminal amino acid sequence was used as a probe. -Southern blot hybridization was performed according to the method described in Molecular Biology (CP in MB) (John Willey & Sons). As a result, it was found that the N-terminus of the SPL structural gene was present in HindIII-Sau3AI 250 bp. So this
The nucleotide sequence of the 250 bp Hind III-Sau3A I fragment was determined using a Sequence Kit (manufactured by Toyobo), and it was confirmed that a DNA nucleotide sequence corresponding to the N-terminal 10 amino acid sequence was present.

(4) Preparation of the recombinant plasmid pSPL09 containing the sucrose phosphorylase gene C-terminal side The recombinant plasmid pSPL02 obtained in the preceding item (3)
DNA contains about 1.1 kb N-terminal side of SPL gene from Southern blot hybridization and nucleotide sequence analysis
Has a DNA insert. The SPL gene is about 1.6 kb in terms of molecular weight of 55,000, and the remaining about 0.5 kb is required.

Sau3A I-Eco, the most C terminal of the SPL structural gene of pSPL02DNA
Using the RI 350 bp fragment as a probe,
Hybridization was performed as described in Molecular Cloning, pp. 312-328, Cold Spring Harbor Laboratory (CSHL) (1982).
Year)], and 5 positive clones were obtained.

 First, a method for manufacturing a bank will be described below.

Plasmid vector DNA pUC119 (Takara Shuzo) 5 μg
And 10 units of restriction enzyme BamHI (Takara Shuzo) were mixed with 20 mM Tris-HCl buffer (100 mM KCl, 10 mM MgCl ₂ , 1 mM dithiothreitol, pH 8.5) and reacted at 37 ° C. for 2 hours to digest. A solution was obtained, and the solution was subjected to phenol extraction and ethanol precipitation by a conventional method, and then digested with this BamHI.
Molecular cloning (Molecular Cloning), 133-13, to prevent DNA fragments from recombining.
Page 4 Cold Spring Harbor Laboratory (1
982), the DNA fragment was dephosphorylated by treatment with bacterial alkaline phosphatase (Bacterial Alkaline phosphatase), subjected to phenol extraction by a conventional method, further ethanol-precipitated, and digested with BamHI. 3 μg of the plasmid vector pUC119 DNA was obtained.

Next, 10 μg of the lactic acid bacteria Leuconostoc mesenteroides ATCC 12291 chromosomal DNA obtained in (1) and 20 units of the restriction enzyme Sau3A I (Takara Shuzo) were added to 50 mM Tris-HCl buffer (100 mM NaCl, 10 mM MgCl ₂ , 1 mM dithiothreyl). Tol, pH 7.5), and reacted at 37 ° C for 2 hours, and then subjected to phenol extraction and ethanol precipitation, followed by digestion with Sau3A I, by a conventional method.
7 μg of chromosomal DNA of Mesenteroides ATCC 12291 strain was obtained.

Then, the plasmid vector pU digested with BamHI
3 μg of C119 DNA, 5 μg of chromosomal DNA of Leuconostoc mesenteroides ATCC 12291 strain digested with Sau3A I, and 66 μM of 2 Units T4 DNA ligase (Boehringer Mannheim, Yamanouchi) containing 66 mM MgCl ₂ , 10 mM dithiothreitol and 10 mM ATP Tris-HCl buffer (pH
7.5) and reacted at a temperature of 16 ° C. for 2 hours to carry out a ligation reaction of DNA.

Using this reaction solution, Escherichia coli JM101 ATCC 33876 was transformed in the same manner as in item (2), and colonies were grown to about 10,000
Using a 350 bp fragment of Sau3A I-EcoR I on the most C-terminal side of the SPL structural gene of pSPL02 DNA as a probe, colony hybridization was performed again from this library [Molecular Cloning
g) Pages 312-328, Cold Spring Harbor
Laboratory (CSHL) (1982)] to obtain 5 positive clones.

From the colony, molecular cloning (Mole
According to the method described in Cold Spring Harbor Laboratory (1982), Cold Spring Harbor Laboratory (1982), 700 μg of a recombinant plasmid was obtained from one culture, and this recombinant plasmid DNA was designated as pSPL09.

This recombinant plasmid pSPL09 has about 3
This is a recombinant plasmid of about 2.0 kb of insert fragment which overlaps a 50 bp Sau3A I-EcoRI fragment and contains about 850 bp of the C-terminal of the SPL gene.

(5) Preparation of recombinant plasmid pSPL10 DNA containing the entire sucrose phosphorylase gene region The recombinant plasmid DNA pSPL0 obtained in item (3)
2 DNA 20 μg, restriction enzymes EcoR I and Pst I (both manufactured by Takara Shuzo Co., Ltd.) were each 30 Units in 50 mM Tris-HCl buffer (100 mM
NaCl, 10 mM MgCl ₂ , 1 mM dithiothreitol, pH 7.5), and reacted at 37 ° C. for 3 hours [Molecular.
Cloning (Molecular Cloning), pp. 156-161,
Cold Spring Harbor Laboratory (1982
Year)], electrophoresis was performed on a 0.7% (w / v) agarose gel according to the method described above. A gel portion containing a DNA fragment of about 4.9 kb was cut out from the gel, placed in a dialysis tube, and placed in 2 ml of a TE buffer solution (10 mM Tris. Hydrochloric acid, 1mM EDTA, pH7.5)
The dialysis tube was sealed and the DNA was eluted from the gel into the buffer by electrophoresis. This solution was subjected to phenol extraction and ethanol precipitation to obtain about 8 μg of a EcoRI-PstI 4.9 kb fragment.

Next, the recombinant DNApSP09 D obtained in item (4)
NA20μg, restriction enzymes EcoR I and Pst I (both from Takara Shuzo)
30 Units of 50mM Tris-HCl (100mM NaCl, 10mM)
M MgCl ₂ , 1 mM dithiothreitol, pH 7.5), and reacted at 37 ° C. for 3 hours. Then, as described above, agarose gel electrophoresis, DNA fragment extraction and purification in a dialysis tube were performed, and EcoR About 5 μg of I-PstI 1.7 kb fragment was obtained.

The EcoRI-PstI 4.9 kb fragment 5μ obtained as described above
g, 1.7 kb fragment 5 μg and 2 units of T4 DNA ligase (Boehringer Mannheim Yamanouchi) 66 mM MgCl ₂ , 1
It was added to 66 mM Tris-HCl buffer (pH 7.5) containing 0 mM dithiothreitol and 10 mM ATP, and reacted at a temperature of 16 ° C. for 17 hours to carry out a ligation reaction of DNA.

Using this reaction solution, Escherichia coli DH1 ATCC (33849) was transformed in the same manner as in item (2), and the colonies were obtained.
The recombinant plasmid DNA contained in this strain was transformed into pSPL10
It was named. Escherichia coli (E.coli) thus obtained
DH1 (pSPL10) has been deposited at the Research Institute of Microbial Industry, National Institute of Advanced Industrial Science and Technology as Microfabrication Research Article No. 3356 (FERMBP-3356).

In addition, E. coli DH1 (pSPL10) was
Molecular Cloning, pp. 86-94, Cold Spring Harbor Laboratory (Cold S)
pirng Harbor Laboratory) (1982).
μg was obtained. This recombinant plasmid psPL10
EcoR I-Pst I 2220kd and Leuconosfoc mesentenoides
Chromosomal DNA 1800kd is ligated. The restriction map of the recombinant plasmid pSPL10 is shown in FIG.

This E. coli DH1 (pSOL10) was added to a TY medium containing 1 mM IPTG [tryptone 1% (w / v), yeast extract 0.5
% (W / v) and NaCl (w / v)] for 10 hours with shaking at 37 ° C. for 8 hours, collecting cells by centrifugation, and sonicating a crude enzyme solution obtained by sonication. The phosphorylase enzyme activity was 0.64 U / ml.

For comparison, when Escherichia coli DH1 (ATCC 33849) having plasmid pBR322DNA (manufactured by Takara Shuzo Co., Ltd.) was used, no sucrose phosphorylase activity was measured in the same manner as above, and no enzyme activity was detected.

[Brief description of the drawings]

FIG. 1 is a diagram showing a restriction enzyme cleavage map of the recombinant plasmid pSPL10 DNA.

──────────────────────────────────────────────────続 き Continued on the front page (56) References Journal of Bacterology Vol. 153, no. 1 p. 211-221 (1983) Gene Vol. 47, p. 201-209 (1986) Inection And Immunity Vol. 56, No. 6, p. 1585-1588 (1988) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12N 15/00-15/90 C12N 9/10 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] The present invention has the following restriction enzyme map and a molecular weight of 18:
Sucrose phosphorylase gene DN with 00kd
Novel recombinant DNA with A inserted into vector DNA. (Where E is EcoR I, H is Hind III, P is Pst I, T is E
indicates coT14 I) 2. A sucrose phosphorylase comprising culturing a microorganism containing the recombinant DNA according to claim 1 and capable of producing sucrose phosphorylase in a medium, and collecting sucrose phosphorylase from the culture. Manufacturing method.