JPH0440896A - New recombinant dna and production of sucrose-phosphorylase - Google Patents

Abstract

PURPOSE:To produce recombinant DNA sucrose.phosphorylase in high yield, by inserting a DNA containing sucrose.phosphorylase gene to a vector DNA. CONSTITUTION:A bacterium containing recombinant DNA, capable of producing sucrose.phosphorylase, is cultured in a medium and sucrose.phosphorylase is collected from the culture mixture. The recombinant DNA, for example, is obtained by collecting culture cells by H. C. Birnboim method, etc., subjecting to lysozyme baceteriolysis, treating with an alkali, neutralizing and precipitating with ethanol. In order to separate and purify sucrose.phosphorylase after the culture and collection of sucrose.phosphorylase from the culture mixture, the cells are collected from the culture mixture, subjected to bacteriolysis, treated with protamine, nucleic acid is removed and the resulting substance is subjected to ammonium sulfate fractionation, gel filtration and ion exchange chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] Sucrose phosphorylase is extremely useful, such as being used for the determination of inorganic phosphorus.

Conventionally, sucrose phosphorylase has been produced by, for example, culturing bacteria of the genus Leuconostoc or Pseudomonas, and separating and purifying sucrose phosphorylase from the bacterial cells [Adv. Adv.

AppL, Microbiol, Volume 32, No. 16
3-201 (1987)], and it has recently been confirmed that bacteria of the genus Streptococcus, a type of cariogenic bacteria, produce sucrose phosphorylase [Infection and Immunity (1987)].
ion and immunity), Vol. 56, pp. 2763-2765 (1988)].

[Problem to be solved by the invention]

However, when producing sucrose phosphorylase using these bacteria, there were problems such as extremely low yields of the enzyme.

As a result of intensive research to solve the above problems, the present inventors prepared DNA containing the sucrose phosphorylase gene, inserted it into vector DNA, and transformed it with the recombinant DNA. The present inventors have discovered that sucrose phosphorylase can be produced in high yield by using microorganisms that have been used to produce sucrose phosphorylase, and have completed the present invention.

[Means to solve the problem]

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

(In the formula, E is EcoRI, H is HindI[[, P is PstlST and EcoT141]) Furthermore, the present invention includes the recombinant DNA described in 1 above,
A method for producing sucrose phosphorylase, which comprises culturing a microorganism capable of producing sucrose phosphorylase in a medium, and collecting sucrose phosphorylase from the culture.

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

DN containing the sucrose phosphorylase gene
Any donor may be used as the donor for A, for example,
Lactobacillus leucotosdoc mesenteroides (Leuc)
onostoc mesenteroides) AT
CC12291 etc., and this microorganism can be
Methods in Enzymology
zymology) vol. 1, pages 227 to 228 (1955) to obtain cultured bacterial cells.

From this bacterial cell, for example, Current Protocols in Molecular Biology (CurrentPro
tocols in Molecular Biolo
gy) unit 2.4.3, (John Will
Harvest the culture and treat with brotinase in the presence of SDS, followed by treatment with hexadecylmethylammonium bromide and chloroform, followed by ethanol precipitation to obtain chromosomal DNA. I can do it.

Next, a restriction enzyme that produces overhanging ends, such as EcoRI (manufactured by Boehringer Mannheim Yamanouchi), is added to this chromosomal DNA at a temperature of 30'C or higher, preferably at 37°C.
Digest for 1-2 hours at °C to obtain a mixture of chromosomal DNA fragments. From this chromosomal DNA fragment mixture,
DNA inferred from the N-terminal amino acid sequence of purified protein
Using a DNA probe based on the A mixed sequence, colony hyfridization method [Molecular Cloning, Vol. 3]
72-328. :7-Led Spring Harbor
Laboratory (Cold Spring Harbor
Laboratory) (1982)], the desired sucrose phosphorylase gene can be obtained.

On the other hand, vector DN that can be used in the present invention
A may be anything, including plasmid vector DNA, bacteriophage DNA, etc., and specifically, for example, plasmid pBR322DNA.
(manufactured by Takara Shuzo Co., Ltd.) etc. are preferred.

a restriction enzyme that generates a protruding end in the vector DNA;
For example, EcoR (manufactured by Boehringer Mannheim Yamanouchi Co., Ltd.) is digested at a temperature of 30° C. or higher, preferably 37° C., for 1 hour or longer, preferably 1 to 3 hours to obtain cleaved vector DNA.

Next, the lactic acid bacterium Leuco-n obtained as described above
ostoc mesenteroides ATCC1
A mixture of DNA fragments derived from 2291 and containing the gene encoding sucrose phosphorylase and the cut vector DNA is added with, for example, E. coli DNA ligase (manufactured by Niney England Bio-Lapse) or T4 DNA ligase. (manufactured by Boehringer Mannheim Yamanouchi Co., Ltd.), preferably T4 DNA ligase, at a temperature of 4 to 37°C, preferably 4 to 16°C, for 1 hour or more, preferably 6 to 24 hours.
get an A.

This recombinant DNA is used to inoculate a suitable host microorganism, such as E. coli -12, preferably E. coli JM101 (AT).
CC33876), Escherichia coli HB 101 (ATCC3
3694), Escherichia coli D H1 (ATCC 33849), etc. to obtain the respective strains. This transformation may be carried out by a conventional method, for example, by DM Morrison (
Methods in Enzymology (Methods in Enzymology)
ymology), Vol. 68, pp. 321-326 (
(1979)].

Next, by screening the microorganisms subjected to the transformation for a strain having the ability to produce sucrose phosphorylase, a recombinant DNA containing a DNA containing a gene encoding sucrose phosphorylase is inserted into the vector DNA, A microorganism capable of producing sucrose phosphorylase can be obtained.

In order to obtain a novel recombinant DNA purified from the microorganism thus obtained, for example, H.C.
The method of Birnboim (H, C, Birnboim) [
Nucleic Acid Research (NucleicA)
cids Re5erch), Volume 7, No. 1513~
1523 (1979) etc., cultured bacteria were collected,
It can be obtained by lysis of lysozyme, followed by alkali treatment, neutralization, and ethanol precipitation. Using a microorganism, especially a strain belonging to the genus Escherichia, which contains a recombinant DNA obtained by inserting the DNA containing the gene encoding sucrose phosphorylase obtained as described above into a vector DNA and has the ability to produce sucrose phosphorylase. To produce sucrose phosphorylase, a culture is obtained by culturing as described below.

That is, to culture the above-mentioned microorganism, although it may be cultured by a normal solid culture method, it is preferable to adopt a liquid culture method if possible.

The medium for culturing the above-mentioned microorganisms may be one or more nitrogen sources such as yeast extract, peptone, meat extract, corn sweep liquor, or exudate of soybean or wheat malt, for example, monopotassium phosphate, potassium phosphate, etc. One or more inorganic salts such as dipotassium, magnesium sulfate, sodium chloride, magnesium chloride, ferric chloride, ferric sulfate, or manganese sulfate are added, and if necessary, carbohydrate raw materials, vitamins, etc. are added as appropriate. is used.

In addition, it is appropriate to adjust the initial pH of the culture medium to 7-9. In addition, the culture is carried out at 30-42°C, preferably 37°C.
It is preferable to carry out the cultivation by submerged culture with aeration and stirring, shaking culture, or static culture for 4 to 24 hours, preferably 6 to 24 hours, before and after the cultivation.

After completion of the culture, sucrose phosphorylase is collected from the culture and separated and purified using Methods in Enzymology.

1ogy) Volume 1, pp. 225-229 (1955)
Purification can be achieved by the method described above, that is, the culture is harvested and lysed, treated with protamine to remove nucleic acids, fractionated with ammonium sulfate, and then subjected to gel filtration and ion exchange chromatography.

The physicochemical properties of purified sucrose phosphorylase obtained by the above purification method are as follows: [Adv, Appl, Microbiol]
ol), Vol. 32, pp. 163-201 (1987)
] The physicochemical properties of sucrose phosphorylase are exactly the same as those described.

〔Effect of the invention〕

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, it is possible to obtain the enzyme in high yield. The invention is extremely useful industrially.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

〔Example〕

(1) Preparation of chromosomal DNA Lactic acid bacterium Leuconostoc mesenteroides ATCC1
2291 as a medium [1% tryptone (manufactured by Difco).

1% yeast extract (manufactured by Difco), 0,
5%! HPO, 1% sucrose, 0.00
1% thiamine/hydrochloric acid, 0.04% MgSO4/7H20
,0,001%Fe50. -7HzO, 0.02%Mn
SO4' 4HzO, 0,005% ascorbic acid]
11 and statically cultured for 2 days at a temperature of 30°C to obtain a culture. This culture was incubated at 5.00 Or, p, m,
After centrifuging for 15 minutes at
Protocols in Molecular Biology (
Current Protocols in Mole
cularBiology) unit2.4.3 (J
ohn Willey & 5ons, inc.

100μ of chromosomal DNA by the method described in (1987)
It was obtained by extracting g.

Next, 0 μg of this chromosomal DNA and restriction enzyme Ec
oRI (manufactured by Boehringer Mannheim Yamanouchi)'l
Q"/t, 50mM) Lis-HCl buffer (100mM)
(containing 0mM NaCl, 10mM MgClz, and 1mM dithiothreitol, pH 7.4), and reacted at a temperature of 37°C for 1 hour. After the reaction, the reaction solution is extracted by a conventional method.
After phenol extraction and ethanol precipitation, Ec
8μ chromosomal DNA fragment of lactic acid bacterium Leuconostoc mesenteroides ATCC12291 strain digested with oRI
I got g.

(2) Preparation of lactic acid bacteria chromosomal DNA library using plasmid vector pBR322 Plasmid vector pBR322 (manufactured by Zenshuzo Co., Ltd.) 10μ
g and 20 units of restriction enzyme EcoR1 (Boehringer Manno Im Yamanouchi Co., Ltd.) were added to 50mM Tris-HCl buffer (100mM NaCl, 10mM Mg5Ot).
, containing 1 mM dithiothreitol, pH 7.4) and reacted at a temperature of 37°C for 2 hours to obtain a digestive fluid, which was subjected to phenol extraction and ethanol precipitation in a conventional manner. In order to prevent the recombination of DNA fragments, molecular cloning (Molecular Cloning)
Bacterial alkaline phosphatase (Bacterial alkaline phosphatase) was prepared by the method described on pages 133-134 (1982), Cold Spring Noah-Barr Laboratory).
The DNA fragments were dephosphorylated by treatment with Alkaline Phosphatase, followed by phenol extraction by a conventional method, followed by ethanol precipitation, resulting in Ec
Plasmid vector pBR322D digested with oRl
8 μg of NA was obtained.

Next, 222 μg of this EcoRI-digested plasmid vector pBR3, P. aeruginosa obtained in the above item (1)
4 μg of the chromosomal DNA fragment of lactic acid bacterium Leuconostoc mesenteroides strain ATCC 12291 digested in 1 and 2 units of T4 DNA ligase (manufactured by Boehringer Mannheim Yamanouchi) were added to 66 mM MgCl2.10.
The DNA was added to a 66mM Lis-HCl buffer (pH 7.5) containing mM dithiothreitol and 10mM ATD, and reacted at a temperature of 16°C for 16 hours to perform a DNA ligation reaction.

Using this reaction solution, the Journal of Bacteriology
), Vol. 119, pp. 1072-1074 (1974), E. coli D HI (ATC
C33849) was transformed into LB-anp medium [Bactodrypton 1% (w/v), yeast extract 0.5% (w/v)].
/v), NaC1O, 5% (W/V), agar 1.2
% (w/v), and ampicillin 50 (μg/mj))
Approximately 6,000 colonies were obtained growing on the plate.
I used this as a library.

(3) Sucrose phosphorylase (hereinafter referred to as SPL)
It is abbreviated as ) Recombinant plasmid p containing the N-terminal side of the gene
Preparation of sPLO2D NA Lactic acid bacterium Leuconostoc
The method for purifying sucrose phosphorylase of Mesenteroides ATCC 12291 strain is described in Methods in Enzymolo.
gy), Vol. 1, pp. 225-228 (1955). By this method, the electrophoretically purified homogeneous protein was analyzed using a gas phase protein sequencer (manufactured by Applied Biosystems, model 37).
The N-terminal amino acid sequence of 30 residues was determined. Me in this
There is an amino acid sequence t-Glu-11e-Gln-Asn~Lys, and this amino acid AA is
SP using 4 types of mixed oligo DNA as probes.
Screening for the L gene was performed.

These 24 mixed oligonucleotides of 17 bases were synthesized using a DNA synthesizer (manufactured by Beckman).
The 5' end of this 2Otg oligonucleotide was subjected to molecular cloning (Molecular cloning) using (22P) ATP (manufactured by Amajam).
r C1゜ning), pp. 122-126, Cold Spring Harbor Laboratory (Cold
Spring Harbor Laboratory (
(1987).

Using the oligonucleotide corresponding to the DNA sequence corresponding to the 32P-labeled SPL protein N-terminal amino acid sequence prepared by the above method as a probe, lactic acid bacterium Leuconostoc. Mesenteroides ATCC1
2291 strain chromosomal DNA library was analyzed by colony hybridization method [Molecular Cloning (
Molecular Cloning), No. 312~
328L Cold Spring Harbor Laboratory
(1982)], and colonies containing the N-terminus of the SPL gene were obtained. Molecular cloning was performed from this colony.
Cold Spring Harbor Laboratory, pp. 86-94.
Harbor Laboratory (1982)
] According to the method described, the recombinant plasmid was
μg/l was obtained, and this recombinant plasmid DNA was transformed into pSP.
It was named LO2.

After 1% agarose gel electrophoresis of this 5au3AI digest of psPLO2, it was transferred to a nitrocellulose filter, and a 24-link synthesized oligonucleotide DNA corresponding to the N-terminal amino acid sequence was used as a probe to conduct current protocols.・Molecular Biology (CP in MB) (John Wil
Southern blot hybridization was performed according to the method described by Ley & 5ons).

As a result, Hind I[[-5au3AI 250b
It was found that the N-terminus of the SPL structural gene was present in 1). So this 250bp Hind m-3au
The nucleotide sequence of the 3AI fragment was performed using the Sequence Kit (manufactured by Toyobo Co., Ltd.), and the D
The existence of the NA base sequence was confirmed.

(4) Preparation of recombinant plasmid pSPL09 containing the C-terminal side of the sucrose phosphorylase gene Recombinant plasmid pSPL obtained in item (3)
Southern blot hybridization and base sequence analysis revealed that the 02 DNA is approximately 1 part of the N-terminal side of the SPL gene.
．． It has a DNA insert containing 1 kb.

The SPL gene has a molecular weight of approximately 1.6 calculated from 55,000.
kb, and the remaining approximately 0.5 kb is required.

Using the C-terminal 5au3AI-EcoRI 350bp fragment of the SPL structural gene of psPLO2DNA as a probe, colony hybridization was performed again [Molecular Cloning].
Cloning) pp. 312-328, Cold Spring Harbor Laboratory (C, S, H, L
) (1982)], and five positive clones were obtained.

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

Plasmid vector DNA pUcl19 (manufactured by Takara Shuzo Co., Ltd.) 5 μg and restriction enzyme BamHI (manufactured by Takara Shuzo Co., Ltd.) 10
Units were added to 20mM Tris-HCl buffer (100mM
KCl, 10mM MgC12, 1mM dithiothreitol, pH 8,5) +: mixed and reacted for 2 hours at a temperature of 37°C to obtain a digestive fluid, which was subjected to phenol extraction and ethanol precipitation in a conventional manner. To prevent the recombination of digested DNA fragments, molecular cloning (Molecular
Cloning), pp. 133-134, Cold Spring Harbor Laboratory (1982).
Bacterial Alkalinephospha
tase) treatment to dephosphorylate the DNA fragment,
The mixture was subjected to phenol extraction using a conventional method and further subjected to ethanol precipitation to obtain 3 μg of plasmid vector ptlc119DNA digested with BamHI.

Next, the lactic acid bacterium Leuconostoc mesenteroides ATCC12291 chromosomal DNA obtained in (11)
Pg and restriction enzyme 5au3AI (manufactured by Takara Shuzo Co., Ltd.) 20 units in 50mM) Lis-hydrochloric acid buffer (100mM N
aC1, 10mM MgCl2, 1mM dithiothreitol, pH 7.5) and reacted for 2 hours at 37°C, followed by phenol extraction and ethanol precipitation using a conventional method to obtain lactic acid bacteria Leuconostoc. 7 μg of chromosomal DNA of Mesenteroides ATCC12291 strain was obtained.

Next, 3 μg of plasmid vector pUc119D N A digested with BamHr and lactic acid bacterium Leuconostoc mesenteroides ATCC digested with Bi3AI were added.
12291 strain chromosomal DNA 5 μg and 2 units of T
4 DNA ligase (manufactured by Boehringer Manno\Im Yamanouchi Co., Ltd.) was added to 66mM containing 66mM MgClt, 10mM dithiothreitol, and 10mM ATP).
Lith-hydrochloric acid buffer (pH 7,5) and temperature 16°
The mixture was reacted at C for 2 hours to perform a DNA ligation reaction.

Using this reaction solution, E. coli JMIOI ATCC33876 was transformed in the same manner as in item (2), and about 10,000 colonies were extracted from this library.
The most C-terminal 5a of the SPL structural gene of sPLO2 DNA
u3A I-Ec.

Colony hybridization was performed again using the RI 350 bp fragment as a probe [Molecular Cloning Part 3].
12-328, Cold Spring Harbor Laboratory (C, S, H, L, X 1982)], and five positive clones were obtained.

From this colony, molecular cloning (Mol
ecular Cloning), pp. 86-94,
Cold Spring Harbor Laboratory (19
700 μg of the recombinant plasmid was obtained by IL culture according to the method described in 1982), and this recombinant plasmid DNA was named psPLO9.

This recombinant plasmid psPLO9 is psPLO
An insert fragment 2.2c overlapping the 5au3A I-EcoRI fragment of about 350 bp on the terminal side of 2c and containing about 850 bp on the C-terminal side of the SPL gene. This is a recombinant plasmid of Okb.

(5) Recombinant plasmid psPL10D N A containing the entire region of the sucrose phosphorylase gene
Recombinant plasmid DNAps obtained in production item (3)
20 μg of PLO2D NA, 30 units each of restriction enzyme cells RI and Sato I (both manufactured by Takara Shuzo Co., Ltd.) at 50 mM)
Lis-HCl buffer (100mM NaCl, 10mM
MgC1z, 1mM dithiothreitol, pH 7.5
) and reacted for 3 hours at a temperature of 37°C.
oning), pp. 156-161, Cold Spring Harbor Laboratory (1982 IE)), and a 0.7% (W/V) agarose gel electrophoresis treatment was performed on the DNA fragment containing approximately 4.9 kb DNA fragment. Cut out the gel part from the gel, put it in a dialysis tube, and
E buffer (10mM) Lis-HCl, 1mM EDT
After adding A, pH 7.5), the dialysis tube was sealed, and the DNA was eluted from the gel into the buffer solution by electrophoresis. This solution was subjected to phenol extraction and ethanol precipitation, and EcoRI-PstI4. Approximately 8 μg of Okb fragment was obtained.

Next, the recombinant DNApSP obtained in item (4)
20μg of O9 DNA, restriction enzyme cell R1 and wholesaler (both manufactured by Takara Shuzo Co., Ltd.) 30 units each at 50mM) 'J-HCl (100mM NaC1, lomM MgC1z)
, 1mM dithiothreitol, pH 7.5) and reacted for 3 hours at 37°C, followed by agarose gel electrophoresis, extraction and purification of DNA fragments in a dialysis tube as described above, and EcoRI-Pstl 1 .7kb
Approximately 5 μg of fragment was obtained.

EcoRI-PstI obtained as described above 4. Ok
5 μg of b fragment, 5 μg of 1.7 kb fragment, and 2 units of T4 DNA ligase (manufactured by Boehringer Mannheim Yamanouchi) in 66 mM containing 66 mM MgClt, 10 mM dithiothreitol, and 10 mM ATP.
Added to Tris-HCl buffer (pH 7.5) and heated to temperature 1.
The reaction was carried out at 6°C for 17 hours to perform a DNA ligation reaction.

Using this reaction solution, E. coli DHI ATCC (33849) was transformed in the same manner as in item (2) to obtain the colony, and the recombinant plasmid DNA contained in this strain was transformed.
A was named pspt, to. Escherichia coli (E. coli) DHI (1) SPLIO obtained in this way
) is the first microbiological research institute at the Institute of Microbial Technology, Agency of Industrial Science and Technology.
No. 1421 (FERMP-11421).

In addition, E. coli E and coli DHI (psPLlO) were subjected to molecular cloning (Molecular C
Cold Spring Harbor Laboratory, pp. 86-94.
g Harbor Laboratory) (1982
430 μg of recombinant plasmid psPL10 was obtained from 11 cultures according to the method described in 2010). This recombinant plasmid pspt, to EcoR of pBR322
I-Pst I 2220kd and Leuconosf
oc mesentenoides chromosomal DNA 180
0kd are concatenated. The restriction enzyme map of the recombinant plasmid psPL10 is shown in FIG.

E. coli DHI (psOLIo)
TY medium containing 1mM rPTG [tryptone 1%
(w/v), yeast extract 0.5% (w/v) and NaC
1 (w/v)) After culturing with shaking in 10rrLl at a temperature of 37°C for 8 hours, the bacteria were collected by centrifugation, and the crude enzyme solution obtained by sonication had a sucrose phosphorylase enzyme activity of 0. It was .64tJ/rILl.

For comparison, plasmid pBR322D N A
E. coli DHI (ATCC338) (manufactured by Zenshuzo Co., Ltd.)
49), sucrose phosphorylase activity was measured in the same manner as above, and no enzyme activity was detected.

[Brief explanation of drawings]

Figure 1 shows the recombinant plasmid psPL10D N
FIG. 3 is a diagram showing a restriction enzyme cleavage map of A. Diagram

Claims (1)

[Claims] 1. Has the following restriction enzyme map and has a molecular weight of 1800k
Sucrose phosphorylase gene DN with d
A novel recombinant DNA in which A is inserted into vector DNA
. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, E is ¥Eco¥R I, H is ¥Hin¥dIII,
2. A microorganism containing the recombinant DNA according to claim 1 and having the ability to produce sucrose phosphorylase is cultured in a medium, and sucrose is extracted from the culture. - A method for producing sucrose phosphorylase, which comprises collecting phosphorylase.