JPS60234581A - Preparation of pullulanase - Google Patents

Abstract

PURPOSE:To mass produce pullulanase safely, by cultivating a specific transformed bacterium belonging to the genus Klebsiella, collecting its product. CONSTITUTION:(i) Escherichia coli (e.g., strain C-600) is transformed with a plasmid integrated with a bacterium [e.g., Klebsiella aerogenes strain W70 (FERM-P 7575)] belonging to the genus Klebsiella, capable of producing pullunalase, then, (ii) the prepared Escherichia coli a recombinant plasmid capable of producing pullulanase is taken out, treated with a restriction enzyme and miniaturized. Then, (iii) the a bacterium (host) belonging to the genus Klebsiella is transformed with the low-molecular plasmid, it is cultivated at about 5-10pH at about 15-40 deg.C for about 1-15 days, and pullulanase produced from the concentrated culture solution is collected.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing pullulanase in large amounts using Klabsiella bacteria obtained by genetic recombination technology.

(Field of Application) Pullulanase is an enzyme useful in the production of maltose and glucose in the field of starch processing because it cleaves α-1,6-glucoside bonds in starch.

Traditionally, Crepe Dura aerogenes (Crepe dura aerogenes) has been used as an industrial Prusin 4ase-producing bacterium in terms of enzyme productivity and safety.
-1 帥1e11aaatsu"μtes) has been used.

However, the amount of enzyme produced is small, and pullulanase is relatively expensive as an enzyme for starch processing, limiting its use.

The present invention makes it possible to supply pullulanase in large quantities and can greatly contribute to starch processing technology.

(Prior art) Regarding pullulanase production by genetic recombination technology,
Bacillus bacteria or Klebsiella neumo
There is an example (Japanese Unexamined Patent Application Publication No. 128796/1983) in which the DNA was cloned into E. coli as a host via λ phage, plasmid pACYC184, etc. using .niaL) as a DNA donor.

However, it is said that there are nuclease barriers, replication mechanism barriers, transcription barriers, translation barriers, protease barriers, etc. regarding the expression and propagation of foreign genetic material within heterologous bacteria, and it is said that there are barriers such as nuclease barriers, replication mechanism barriers, translation barriers, protease barriers, etc. It is difficult to obtain.

(Means for Solving the Solution) As a result of intensive research to overcome each of the above-mentioned obstacles, the present inventors obtained DNA from Crepesciella aerogenes, which is currently used in the industrial production of pullulanase and whose safety has been confirmed. We discovered that pullulanase can be produced safely and in large quantities by using it as a fungus and a host bacterium.
This completes the present invention.

That is, using a Crepe Dura bacterium as a DNA donor, D
When NA is cloned using a Crepsiella bacterium as a host, the DNA carrying the genetic information for pullulanase production is cloned.
can be amplified to produce a significantly larger amount of pullulanase than the production level of the DNA donor bacterium.

(Structure of the Invention) The production of the pullulanase high-producing bacterial strain according to the present invention is as follows.
This is carried out by the step (2).

(2) Total DNA is extracted from the bacteria that is the DNA donor, and then fragmented with restriction enzymes.

(2) Fragment the hector DNA with a restriction enzyme, and incorporate the DNA fragment obtained in (2) into the cleavage site.

(2) Transform E. coli with the recombinant plasmid, and select E. coli that has the ability to produce pullulanase.

(2) A recombinant plasmid is extracted from Escherichia coli capable of producing pullulanase and is reduced in size by treatment with restriction enzymes.

■ Transform the small-molecule miniaturized plasmid into a host strain of Crepsiella bacteria, and screen for host bacteria that exhibit high productivity.

The DNA donor having pullulanase production information used in the present invention is a Crepe Jura bacterium capable of producing pullulanase, preferably Crepe Jura aerogenes, and more preferably Crepe Jura aerogenes strain W70 (Feikoken Deposit No. No. 7575) is available. Total DNA was extracted using a known method (J, Marmur
+J, Mol, Biol, +3208 (1961). The obtained total DNA is partially digested with restriction enzymes, and then recombined with a plasmid digested with the same restriction enzymes. Restriction enzymes used here include BamHI, Sal
I + Hinder, Sma I, EcoRI
, CIa I, and the like. Furthermore, known plasmids such as pBR322 and its derivatives can be used.

The recombinant plasmid is then transformed into E. coli strain C-600 by a conventional method, and E. coli strains capable of producing pullulanase are selected from among the transformed strains.

This selective isolation of E. coli was carried out as follows.

For example, when BamHI is used as the restriction enzyme, an ampicillin-resistant colony growing in an anhisillin-containing Petri dish medium is replicated on a pullulanase detection medium, and after culturing at 37°C for 2 days, ethanol is poured into the Petri dish, and 2-
Colonies forming a halo were isolated after 4 hours.

Next, a recombinant plasmid carrying pullulanase-producing genetic information is extracted from an Escherichia coli strain capable of producing pullulanase, and further partially digested with restriction enzymes to obtain a miniaturized plasmid. This miniaturized plasmid was transformed into a host bacterium of the genus Crepesciella using the calcium method, and a high-producing strain was selected. As the host bacterium, Crepesciella aerogenes strain having pullulanase-producing ability, preferably Crepesciella aerogenes, more preferably Crepesciella aerogenes strain W70 (Feikoken Bacteria Deposit No. 7575) can be used.

For the culture, known methods such as ordinary aerated liquid culture can be applied, and the medium contains a carbon source, a nitrogen source, an inorganic salt, and, if necessary, a trace nutrient source that can be assimilated by the microorganism. In particular, if pullulan and maltose are added as a carbon source, and glutamic acid is added as a nitrogen source, the amount of enzyme production will be further improved. Culture pH
The cells are cultured for 5 to 10 days at a temperature of 15 to 40°C for 1 to 15 days.

To isolate pullulanase from the culture obtained in this way, the extracellular pullulanase and bacterial cells are simultaneously concentrated from the culture using an ultrafiltration device equipped with a hollow fiber cartridge, and the concentrated liquid is used for the purpose. It may be subjected to post-processing as appropriate. If you want to obtain a purified pullulanase sample, this concentrated solution is subjected to bacterial cell disruption treatment by a conventional method, and the supernatant obtained by centrifugation is subjected to a conventional protein purification method (salting out, adsorption chromatography, gel filtration, etc.). ) to purify. In addition, to use in the form of bacterial cells, collect the bacteria from the concentrate by centrifugation.

(Effects) The strain with high pullulanase production obtained by the present invention is cultured in large quantities by a conventional culture method, and pullulanase is isolated from the culture. In the brother-sister culture that does not contain the recombinant plasmid, pullulanase is present in the culture supernatant and in the bacterial cells to approximately the same extent, but in the strain that contains the recombinant plasmid, pullulanase is unevenly distributed within the bacterial cells. In addition, the obtained clone strain has enzyme levels higher than that of fluorine (32-110
(fold), it is easy to separate and purify the enzyme. Furthermore, since high bacterial cell activity is maintained over a long period of time, it can be used in the form of bacterial cells.Therefore, it can be used as an immobilized bacterial cell for continuous reactions in the starch processing field, and has great industrial value. .

(Example % in the example is weight %.)+IJ K
, a is 1. Isolation of total DNA from W2O overnight in LB medium (1% polypeptone, 0.5% yeast extract, 0.5%
After centrifuging the LJy Liaojiao Danbang-W2O strain cultured in 500 mL of sodium chloride, pH 7.2), total DNA was isolated using a partially modified version of the Murmur method. In other words, wet bacterial cells 3
g to 20ml of 5aline-EDTA (0,15M
NaCl, 0, IM E, DTA-Nat, pH 8
, 0) and centrifuged to collect bacteria.

This was resuspended in 24ml of 5aline EDTA,
Lylithium solution (10n+g/ml 5aline-E
1 ml of DTA) was added and shaken at 32°C for 30 minutes to lyse the bacteria. After adding 1 ml of 25% SDS solution to this and thoroughly mixing, 6.75 ml of 5M sodium perchlorate solution was added and mixed evenly. Furthermore, 33.8ml
of chloroform-isoamyl alcohol (24:1) was added, and the mixture was shaken gently for 30 minutes to remove protein. 400
An upper layer was obtained by centrifugation at 0 rpm for 15 minutes, and the precipitated DNA was wrapped around a glass rod while gradually adding twice the amount of 99% ethanol.

This was mixed with 10ml of DNA buffer 1 (Tris-11C
I 50mM, EDTA-Nat 10mM, pH 8
, 0) and 0.2 ml of RNAase solution (2 m
g/ml of water) was added and shaken at 37°C for 20 minutes. To this was added an equal amount of chloroform-isoamyl alcohol, and after shaking for 20 minutes, an aqueous layer was obtained by centrifugation. To this aqueous layer was added 1n+1 3M sodium acetate solution, and twice the amount of ethanol was added, and the DNA was wound around a glass rod. After dissolving this in 9 ml of DNA buffer, 1 ml of sodium acetate solution was added, and the DNA was wound around a glass rod while dropping 5.4 ml of isopropanol. After soaking this in 75% ethanol overnight, 2 ml of DNA buffer solution (Tris-IC 110 mM, EDT^-Na
c 1mM, pH 7.5) and stored at 4°C.

(2) Restriction enzyme treatment of all DNA of L 5W70 (
10μj2 (20Mg D
NA/ Itρ) to 11.1ml l Jll(]i
%i/Port (N++CI 500mM, ■BSOa100
mM, dithiothrei 1--10+nM, Tris-H
CI 100mM.

pH7,4) 2 /Z I! , water 3 /Z e and B
10 U of am1l I was added, and after decomposition at 37°C for 40 minutes, the mixture was incubated at 65°C for 10 minutes to inactivate the restriction enzyme.

This was centrifuged using 10-40% sucrose density gradient centrifugation (32,000
5 to 20 Kb of DNA fractionated by rpm, 22 hours)
The fragment is iM. After ethanol precipitation, these fractions were
After washing with % ethanol and dissolving in 50 μl of DNA mild solution H, the concentration was measured using OD2[J] and stored at 4°C.

(3) Separation and purification B of Hector plasmid pBR322
irnboim+H, C, and Doly, J, (
Nucleic Ac1ds Research, 7.
1513 (1979)) was modified.

Using the E.Co11 C600 strain carrying pBR322, 800 ml was cultured overnight at 37°C, and the cells were collected by centrifugation.
After washing with 10 mM phosphate buffer, the cells were placed in two 50 ml centrifuge tubes and centrifuged to collect bacteria. 10ml of solution in each■
(Glucose 50mM, Tris-IC 125mM,
EDTA Nat (10mM, pH 8.0) was added and suspended, then 50mg of lylithium was added, and after mixing, the mixture was allowed to stand at room temperature for 5 minutes. Add 20ml of fg liquid U (NaOHO
, 2N, SO 51%) and kept on ice for 10 minutes. Then 15 ml of potassium acetate solution (5M, p
H4, 8,0°C) was added thereto, and after thorough mixing, the mixture was placed on ice again. After 10 minutes, the mixture was centrifuged at 15,000 rpm and 4°C for 20 minutes to obtain a supernatant. Add 0.6 volumes of isopropanol to this, mix and leave to stand at room temperature for 15 minutes.
The precipitate was collected by centrifugation in Orpm for 30 minutes.

After dissolving this in 4 ml of DNA buffer, the volume was increased to 6 ml, and 6.3 g of cesium chloride and 0.6 ml of bromide bromide (1 g/ml) were added, using a vertical rotor at 39.00 Orpm, 16 Centrifuged for hours. The DNA showed two bands by this density gradient centrifugation, but the lower plasmid band was extracted, and after decolorizing with isopropanol saturated with 5M saline, it was diluted with 2 times the amount of DNA buffer solution and 6 times the amount. Add ethanol to precipitate the plasmid,
It was collected by centrifugation at 2.00 rpm for 15 minutes. After dissolving this in 50 μl of DNA buffer, the concentration was measured from the 0Dan value and stored at 4°C.

(4) Plasmid 2μm2 obtained in (3) by cutting the plasmid and creating a recombinant plasmid, BamHI
Board ih/&2p (!, water 16,1711, B
After mixing 5 U of am11 I and reacting at 37° C. for 60 minutes, 40 μl of slow induction liquid (saturated phenol) was added to stop the reaction. Separate the aqueous layer by centrifugation, remove residual phenol by ether extraction, ethanol precipitation,
After washing, it was dried under reduced pressure. Add to this the DNA obtained in (2)
Total volume of fragment solution, water and Iigation buffer (66
0mM Tris-HCI, pH 7, 6, 66mM magnesium chloride, 100mM dithiothreitol, 10m
M ATP) was added to bring the total reaction solution to 20 ml, and 0
．． 5 U of T4 ligase was added and reacted at 16°C for 16 hours. At this time, the molar ratio of plasmid and DNA is approximately 1=
I set it to 2.

Transformation to f51 E, Co11 C600 and isolation of plasmid producing strain ゛Add 0.4 ml of E, Co11 C600 (sorry) that was cultured overnight in LB medium to 40 ml of LB medium,
The cells were cultured at 37°C until reaching 00 klett units. After cooling this in ice water, 4000 rpm,
After collecting bacteria by centrifugation at 2℃ for 10 minutes, cold calcium buffer? & (
50111M Calcium Chloride, 10+IIMTris-
HC1, pH 7, 2) Suspended in 20ml and diluted on ice water for 30 minutes.
It was held for 0 minutes. After collecting the cells by centrifugation again, they were resuspended in cold 2I111 calcium buffer. After keeping it in ice water for 60 minutes, add 20 μl of the gauze reaction solution obtained in (4) to this 0.1 mM, keep it in ice water for 20 minutes, and then incubate at 40°C.
A heat pulse was applied for 2 minutes. Add 1 ml of LB medium and
The mixture was kept at 30° C. for 30 minutes, divided into 0.1 ml portions, spread on LB-ampicillin (100 μg/ml) agar plates, and cultured overnight at 37° C. The transformed strain exhibiting ampicillin resistance was replicated onto a selective medium (synthetic medium containing 1% pullulan and 0.5% maltose), cultured at 37°C for 2 days, and then ethanol was injected onto the plate. As a result, we obtained a strain that formed a halo around the colony. The plasmid carried by this strain was named ppH17 (17Kb).

The composition of the synthetic medium is as follows.

Potassium phosphate hasohar 50mM, Ph7.2M
g CI 2 ・6H200,01%NaC1O,00
1% Fe C13・6H200,001%MnCl
t 4 H2O0,001% (NH4) 2304
0.2% Agar 1.5% (6) Preparation of p5P19 and pMP1 p pH
No. 17 had five BamHI fragments in addition to pBR322. After separating and purifying this (BtBr-CsC1 density gradient centrifugation), it was partially decomposed with BamHI and Iigatior+
L, plasmid pPB174 with two fragments deleted (
14.7 Kb) was obtained. Separated and purified p pH1741
μl1 (4μg/, water 17.u#, Smal buffer (100mM Tris-11C1, pH8,0
, 70mM MgC1z, 200mM KCI, 70
2 μl of mM 2-mercaptoethanol) and Sma
10 U of I was mixed, reacted at 37°C for 60 minutes, partially decomposed, treated with phenol, treated with ether, washed with ethanol, and treated with E and Co in the same manner as in (5).
11 After transformation into C600, 2 from p f'B174
~4Kb small plasmid pSP19 (12,5Kb
) was obtained. This was mixed with EcoRI (EcoRI buffer:
LM Tris 1 (C1p+17.5.70mM magnesium chloride, 500mM NaCl, 70mM 2-mercaptoethanol, 0.1% bovine serum albumin) and Xhol (Xhol buffer: 100mM Tris
Mung Bean Nucleas
e (slow induction liquid 20.3 sodium acetate slow induction liquid, P
H4,6, IM NaC1,10IIIM zinc chloride) 0
．． After treatment with 5 U at 37°C for 15 minutes to obtain plant ends, phenol treatment, ether treatment, ethanol precipitation washing, 1igatior + Lz, transformation and Ec
A plasmid pMPI with a 2.2 Kb deletion of oRI-Xho I was obtained. - (7) Transformation of atkawa μbis W7L\ was carried out in the same manner as in the case of E and Co11 for transformation into L Kenchogin 5-W2O. However, when selecting convertible stocks, ampicillin is 6
00μg/m14 LB agar plate, 37℃,
Colonies formed in 12 hours were isolated.

(8) Production of pullulanase using acμ4μ+es W2O The transformed strain obtained under the conditions of (7) was grown in a synthetic medium (phosphoric acid 1
Compatibility: 50mM, sodium glucinate 0.
5%, MgCl26 +120 0.01%, NaC
1, MnCIz 4H20, FeCl2 6H20, each 0.001% (NH4)2SOto, 2%,
In a test tube containing 5 ml of carbon source (0.5%), heat at 28°C for 30 minutes.
The supernatant obtained by culturing for 1 day and centrifuging at 5000 rpm for 15 minutes was measured for pullulanase activity as extracellular activity. In addition, the bacterial cells were diluted with 10mM phosphoric acid (sealing solution (pH
7,2) Pullulanase activity was measured as the intracellular activity of the extract obtained by suspending the suspension in 5 ml and subjecting it to ultrasonication. These results are shown in Table 1.

The enzyme activity was measured using 1 ml of enzyme solution and 1 ml of 1%
A mixture of pullulan solution (0.1 M acetate buffer, pH 5.5) was reacted at 40°C, and the aldehyde groups generated in 15 minutes were quantified by the Somogyi-Nerlan method.
The production of μmole of aldehyde groups was defined as IU.

Furthermore, isolation of small-scale plasmids used for analysis of transformed strains is performed using Birngo in much the same way as large-scale isolation.
A modification of the method of im and Doly was used.

That is, the bacterial cells obtained by centrifuging 0.2 ml of the culture solution (
Alternatively, 1 platinum loop of colony) was suspended in 100 μl of solution (2) containing 4 mg/ml lylithium, and after being left at room temperature for 5 minutes, 200 μl of solution (2) was added, mixed, and placed on ice for 5 minutes. 150μβ solution ■ (sodium acetate 5M,
After adding pH 4,8) and leaving it on ice for 5 minutes,
Centrifugation was performed at 2.00 rpm for 5 minutes. 500μp for this
After adding and mixing chloroform-isoamyl alcohol,
The upper layer was separated by centrifugation at 12.00 rpm for 5 minutes. Add 800μβ of 99% ethanol to this, and add DNA
was precipitated, collected by centrifugation, and washed twice with 70% ethanol. A 10-fold concentrated buffer solution, water, and a restriction enzyme were added to this pellet to cause a reaction, and then used for analysis.

Table 1

Claims (3)

[Claims] (1) A method for producing pullulanase, which involves culturing a Klebsiella bacterium containing a plasmid incorporating DNA carrying pullulanase-producing genetic information derived from a Klebsiella bacterium to obtain pullulanase. (2) Bacteria of the genus Klebsiella are known as Klebsiel aerogenes.
1. The method for producing pullulanase according to claim 1. (3) Klebsiella aerogenes (Klebsi
3. The method for producing pullulanase according to claim 2, wherein the strain is Crepe Jura aerogenes (U-11ha'an' and leaves) wio strain.