JP2651811B2 - Novel Escherichia coli having oligo-1,6-glucosidase gene and method for producing oligo-1,6-glucosidase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel Escherichia coli having an oligo-1,6-glucosidase gene and a method for producing oligo-1,6-glucosidase.

[0002]

2. Description of the Related Art Oligo-1,6-glucosidase is an enzyme that specifically cleaves the α-1,6-glucosidic bond of dextrin produced from starch or glycogen by isomaltose or α-amylase, and is generally used for Streptococcus sp. Mitis ( Biochem. J. , 105, 937 (197
6)) and Lactobacillus bifidus ( Biochem. J. , 82 ,
272 (1962)) and Bacillus thermoglucosidius ( Biochim. Biophys. Acta, 445, 386 (19
It has been reported that it is produced by thermophilic bacteria such as 76)). However, these strains are not used industrially because they are difficult to culture, have low productivity, have low enzyme substrate specificity, and have a high reverse reaction rate. When glucose is industrially produced, pullulanase or isoamylase, which is another branching enzyme of glucoamylase, is often used in combination. However, in order to further increase the decomposition rate, oligosaccharides containing α-1,6-glycosidic bonds, such as isomaltose, panose, and isomalttriose, produced by the reverse reaction of glucoamylase, are decomposed in the presence of high-concentration glucose. There must be. The present inventors have previously discovered and obtained an alkaliphilic bacterium that produces oligo-1,6-glucosidase that efficiently degrades isomaltose (Japanese Patent Application Laid-Open No. 60-192590).

[0003]

Further, the present inventors have disclosed oligo-1,6-glucosidase as disclosed in Japanese Patent Application Laid-Open No. 60-192.
The present inventors have conducted intensive studies on a method for producing the cell more efficiently than that disclosed in JP-A-590. As a result, the cells were successfully introduced into Escherichia coli via DNA carrying the genetic information of the alkalophilic Bacillus genus, and oligo-1,6-glucosidase was industrially advantageous from the cells obtained by culturing the Escherichia coli. And succeeded in completing the present invention.

[0004]

According to the present invention, a DNA fragment carrying the genetic information of oligo-1,6-glucosidase obtained from an alkalophilic Bacillus bacterium is transformed into an E. coli vector D.
E. coli transfected with recombinant DNA incorporated into NA,
And a method for producing oligo-1,6-glucosidase, comprising: collecting the cultured cells, culturing the Escherichia coli, and collecting oligo-1,6-glucosidase from the cells.

[0005]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail. First, in the present invention, the alkalophilic Bacillus sp. Strain F-2 (FERM P-7496) and F-5 strain (FE
RM P-7497), F-14 strain (FERM P-7498), G-1 (FERM
P-7499), G-4 (FERM P-7500) and R-18 (FERM P
-7501). DNA fragments carrying the genetic information of oligo-1,6-glucosidase (hereinafter referred to as chromosome D
NA) can be isolated and purified from an alkalophilic Bacillus bacterium according to a conventional method. For example, the strain can be prepared by culturing the above strain in a liquid medium for about 1 to 3 days under aeration and stirring, collecting the resulting culture by centrifugation or filtration, and then lysing it. As the lysis method, for example, treatment with a cell wall lysing enzyme such as lysozyme or β-glucanase, or ultrasonic treatment is used. Further, if necessary, other enzyme agents such as protease and a surfactant such as sodium lauryl sulfate can be used in combination, and further, a freeze-thawing treatment can be performed.

[0006] DNA from the lysate thus obtained
Separation and purification can be performed according to a conventional method by appropriately combining methods such as phenol extraction, protein removal treatment, protease treatment, ribonuclease treatment, alcohol precipitation, and centrifugation. This chromosome D
The incorporation of NA into the vector DNA can be performed by cutting the chromosomal DNA and the vector DNA to prepare a chromosomal DNA fragment and a vector DNA fragment, and then linking a mixture of both. The DNA can be cleaved by, for example, sonication, restriction enzyme treatment, or the like.However, in order to facilitate the binding of the obtained DNA fragment to the vector fragment, restriction enzymes, especially specific nucleotide sequences, are used. Works, eg EcoR I, Hind I
II, BamH I, Sal I, Xho I, Xma I, Mbo I, Xba I
Type II restriction enzymes such as SacI, PstI, etc. are suitable.

The vector DNA used here includes pBR322, pBR325, pUC18, pUC19, λ phage and the like. In particular, pBR322 is preferably used. DNA
A method for joining the fragment and the vector fragment is known in the art.
Any method may be used as long as it uses A ligase. For example, as the DNA ligase, a DNA ligase derived from T4ase (T4 DNA ligase) is suitably used. Introduction of the recombinant DNA obtained by the above method into E. coli was performed using Proc.
Nat. Acad. Sci. USA 69 , 2110 (1972).

[0008] The method of selecting a strain having a vector into which a DNA fragment carrying the genetic information of oligo-1,6-glucosidase is incorporated also depends on the type of restriction enzyme and vector DNA used in preparing the recombinant DNA. For example, when BcoRV is used as a restriction enzyme and pBR322 is used as a vector DNA, the procedure can be performed as follows. That is, E. coli K12 strain, for example,
A transformant using the HB101 strain as a host was cultured on tryptone, yeast extract, NaCl, ampicillin, p-nitrophenyl-α-D-glucopyranoside (p-NPG) agar medium (hereinafter referred to as LBP agar medium), A p-NPG-degraded strain in which the periphery of ampicillin-resistant colonies that appeared on the plate turned yellow was selected. The recombinant DNA is isolated from the recombinant DNA-containing strain obtained by the above method.

The method for producing oligo-1,6-glucosidase according to the present invention comprises the steps of culturing the novel recombinant Escherichia coli obtained as described above by a conventional method, collecting the bacteria,
This is performed by collecting the cells from the treated sample.
The culturing method is, for example, a method in which a liquid medium is maintained at around pH 7 (pH 6.0 to 8.0.
0), while maintaining the temperature around 37 ° C (30-45 ° C)
Culture may be performed under aerobic conditions such as aeration and stirring for about 1 to 3 days to produce and accumulate oligo-1,6-glucosidase. Next, according to a conventional method, the bacterial cells separated by filtration, centrifugation, etc., are treated with ultrasonic waves, a surfactant, a cell wall lysing enzyme, and the like, and then filtered, centrifuged, etc., to give an oligo-1,6-glucosidase solution. Collect. The enzyme solution thus obtained is purified, for example, by concentration under reduced pressure and membrane concentration, and further by appropriately combining salting out with ammonium sulfate, sodium sulfate and the like, and fractional precipitation with methanol, ethanol, acetone, and the like. Oligo-1,6-glucosidase of purity can be collected. The obtained enzyme can be advantageously used as an industrial enzyme preparation. One unit of the enzyme titer of oligo-1,6-glucosidase in the present invention is 3.6 units.
50 mM phosphate buffer (pH 6.8) containing mg of isomaltose
Add 0.05 ml of appropriately diluted enzyme solution to 0.45 ml and add 40 ° C
After heating for 10 minutes in a boiling bath, the reaction was stopped by heating for 3 minutes, and the concentration of glucose in the reaction-stopping solution was determined by the known glucose oxidase-peroxidase method (Biochemical Experiment Method 1, "Quantitative method for reducing sugars"). Quantitative analysis at p.135 Society Press) and 1 μmole / min.
Means the amount of an enzyme that hydrolyzes isomaltose.

Hereinafter, the present invention will be further described with reference to examples.

【Example】

Example 1 Isolation of oligo-1,6-glucosidase gene and pMC
1. Construction of Chromosome D of Alkalophilic Bacillus sp. Strain F-5
NA is Saito. The method of Miura (Saito, Miura) (Bioc
him. Biophys, Acta. 72 p. 619-629 (1963)). Restriction enzyme E is added to this chromosomal DNA (50 μg).
coR V (15 units) was added and reacted at 37 ° C. for 1 hour. The obtained fragment was subjected to agarose electrophoresis, and a large fragment of 2 Kb or more was recovered. Expression vector pBR322 (7μ
g) to which the restriction enzyme EcoRV (35 units) was added.
The mixture was reacted for a period of time to completely decompose, and further treated with alkaline phosphatase (E. coli C75 origin, 1 unit) at 55 ° C for 30 minutes. T4 DNA ligase (14 units) was added to the chromosomal DNA fragment (4 μg) and the vector (1.4 μg) obtained as described above, and the mixture was ligated by reacting at 16 ° C. overnight.

The resulting recombinant DNA was transformed into E. coli HB101 by the competent cell method by calcium ion treatment (described above). That is, the recombinant DN
A (1 μg), 50 μl of the above reaction mixture, 5 μl of 0.3 M calcium chloride solution and 100 μl of a competent cell suspension
The mixture was left on ice for 1 hour and heated at 42 ° C. for 70 seconds. The suspension was mixed with 3 ml of L-broth (1% tryptone, 0.5% yeast extract, 1% NaCl, 0.1%).
% Glycol) and cultured with shaking at 37 ° C. for 3 to 5 hours. Dilute the obtained culture solution appropriately and dilute 100 μl.
When l was spread on an LBP agar plate and cultured at 37 ° C. for 1 to 3 days, a strain in which the colony periphery was yellowed was obtained.
This strain was inoculated into L-broth containing 100 μg / ml of ampicillin and cultured overnight, and after collecting the cells, the cells were washed with physiological saline.
The suspension was suspended in 20 mM phosphate buffer (pH 6.8), sonicated and centrifuged, and the supernatant was subjected to oligo-1,6-glucosidase activity measurement (40 ° C., 10 minutes). Confirmed that it has been converted. Plasmids were transformed from this transformant by the rapid boiling method (Holmes,
DS and M. Quigley, Anal.Biochem. 114 p. 193 (1
981)) and cut with EcoR V to produce 9.1 Kb
Fragment was found to be inserted. This plasmid was named pMC1 (shown in FIG. 1).

Example 2 Construction of plasmid pMC2 In order to obtain a plasmid in which the direction of the inserted fragment of pMC1 was reversed, pMC1 (1 μg) was restricted to EcoR V (15 μg).
), And reacted at 37 ° C for 30 minutes to completely decompose. T4 DNA ligase (5 units) was added, followed by reaction at 16 ° C overnight and religation. Obtained recombinant DNA
(1 μg) was similarly transformed into E. coli HB101 by the competent cell method. From the ampicillin-resistant transformant thus obtained, a plasmid was prepared by a rapid boiling method, and then cut with EcoR1,
In addition to pMC1 showing two bands of 11.2 Kb and 2.3 Kb by agarose gel electrophoresis, a new plasmid showing two bands of 7.2 Kb and 6.3 Kb was obtained. From this, it was confirmed that the inserted fragment of pMC1 was inverted in the new plasmid as shown in FIG. This plasmid is called p
MC2 (shown in FIG. 1). As in Example 1, it was confirmed that oligo-1,6-glucosidase activity was present, and it was confirmed that Escherichia coli containing pMC2 was transformed.

Example 3 Construction of Plasmid pMC21 In order to enhance the stability of the plasmid, subcloning was performed to reduce the size of the plasmid. pMC2
(1 μg), add restriction enzyme Pvu 1I (12 units) and add 3
The reaction was carried out at 7 ° C. for 1 hour to completely decompose, and T4 DNA ligase (5 units) was added, followed by reaction at 16 ° C. overnight and religation. The obtained recombinant DNA (1 μg) was similarly transformed into E. coli HB101 by the competent cell method. It was confirmed that the obtained transformant had oligo-1,6-glucosidase activity similarly to pMC1 or pMC2. The prepared plasmid was used for pMC2
No. 1 (shown in FIG. 1). pMC21 is pMC2
Had a 3.1 Kb insert fragment with a part of the insert fragment (6 Kb) and a part derived from pBR322 being deleted.
FIG. 2 shows a detailed cleavage map of pMC21 by various restriction enzymes. Oligo-1,6-glucosidase genes are restriction enzymes SacII, SalI, HincII, AccI, XhoI, Hi
nd III, one Pst I recognition site each, restriction enzymes BamH I, B
It possessed two anII and SacI recognition sites, respectively.

Example 4 Identification of Gene Chromosome DN of Bacillus bacterium Alkalophilic Bacillus sp. Strain F-5
A, recombinant DNA pMC2 and E. coli HB1
01 Chromosomal DNA was cut with EcoRV according to Examples 1 and 2, and then subjected to agarose gel electrophoresis. After electrophoresis, the gel was stained with ethidium bromide and photographed. Southern method (J. Mol. Biol., 98 p. 503-517).
(1975)) and blotted (fixed) to a Durapore filter (Millipore). pMC2
After cutting with EcoRV, nick translation kit and DNA detection system (both from BRL)
Was used to prepare a biotin-labeled probe according to the company manual. Hybridization was performed by immobilizing the fixed filter with the probe. As a result, a band appeared at 9.1 Kb on the chromosomal DNA of the F-5 strain, and no corresponding band appeared on the chromosomal DNA of E. coli. From the above, it was shown that the DNA inserted into the plasmid pMC2 was certainly derived from the chromosomal DNA of the Bacillus bacterium strain F-5.

Example 5 Method for Producing Oligo-1,6-Glucosidase in E. coli Plasmid pMC21 prepared according to Example 1 was used in E. coli.
coli HB101. The obtained transformed strain E.
coli HB101 (pMC21) at 100 μg / ampicillin /
After inoculating 5 ml of L-broth medium containing ml, the cells were pre-cultured overnight. The pre-culture solution was prepared using L-containing 100 μg / ml ampicillin
After inoculating 1% in 50 ml of broth medium and culturing at 37 ° C. for 5 to 24 hours, the cells were collected, and the culture supernatant was used as an extracellular fraction. On the other hand, the cells were washed with a physiological saline, and a 20 mM phosphate buffer (pH 6.
After suspension in 8), the suspension was subjected to ultrasonic treatment, and centrifuged to remove the crushed cells, and the supernatant was used as the intracellular fraction. When the oligo-1,6-glucosidase activity of each fraction was measured, oligo-1,6-glucosidase activity was observed only in the intracellular fraction, and the activity was 4.9 u / ml for 24 hours in culture. The activity is bacillus bacterium alkalophilic
Bacillus sp. (Alkalophilic Bacillus sp.) F-
Since the activity of the five strains after culturing for 24 hours is 0.5 u / ml on average, their productivity is about 10 times per culture and about 13 times per amount of cultured cells. The resulting oligo-1,
When the properties of 6-glucosidase were examined, the optimum pH was 6.0-6.5 and the optimum temperature was 45 ° C. as in the case of oligo-1,6-glucosidase derived from Bacillus bacterium strain F-5 (FIG. 3). The pH and temperature dependencies of the enzyme activity are shown in Table 1), the isoelectric point was 4.2 ± 0.1, and the molecular weight was 60,000 ± 3,000 daltons.

[Brief description of the drawings]

FIG. 1. Plasmid pM containing oligo-1,6-glucosidase gene integrated in plasmid pBR322.
C1, the plasmid pMC containing the reverse insert
2, and the subcloned plasmid pMC21
3 is a cleavage map of various restriction enzymes showing the structure of the enzyme. Bold line in the figure indicates Bacillus bacterium Alkalophilic Bacillus
This indicates that the DNA is derived from sp (Alkalophilic Bacillus sp.) Strain F-5.

FIG. 2 is a detailed cutout map of plasmid pMC21 by various restriction enzymes. The numbers in the figure represent approximately the distance (Kb) between each cutting point.

FIG. 3 shows oligo-1,6-glucosidase (●) produced from Escherichia coli by genetic recombination of the present invention, and Bacillus strain Alkalophilic Bacillus sp.
2 shows the optimum pH and optimum temperature of oligo-1,6-glucosidase (（) produced from kalophilic Bacillus sp. strain F-5.

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number in the agency FI Technical indication (C12N 9/44 C12R 1:19) (C12N 15/09 C12R 1:07)

Claims (4)

(57) [Claims] 1. D which carries genetic information of oligo-1,6-glucosidase obtained from an alkalophilic Bacillus bacterium
Escherichia coli into which a recombinant DNA having an NA fragment incorporated into a vector DNA for Escherichia coli has been introduced. 2. The DNA fragment carrying the genetic information of the oligo-1,6-glucosidase is composed of restriction enzymes SacII and SalI.
2. The Escherichia coli according to claim 1, wherein the Escherichia coli possesses one recognition site for each of HincII, AccI, XhoI, HindIII, and PstI, and two recognition sites for each of the restriction enzymes BamHI, BanII, and SacI. 3. DNA carrying the genetic information of oligo-1,6-glucosidase obtained from an alkalophilic Bacillus genus
Recombinant D in which the fragment was incorporated into Escherichia coli vector DNA
After culturing E. coli into which NA has been introduced, the cultured cells are collected,
A method for producing oligo-1,6-glucosidase, comprising collecting oligo-1,6-glucosidase from the cells. 4. A DNA fragment that carries the genetic information of the oligo-1,6-glucosidase comprises restriction enzymes SacII and SalI.
4. The production method according to claim 3, wherein each of the HcII, HincII, AccI, XhoI, HindIII, and PstI recognition sites has one site, and the restriction enzymes BamHI, BanII, and SacI sites each have two sites. .