JPS6181788A - Novel bacillus subtilis containing thermostable beta-galactosidase and production of thermostable beta-galactosidase - Google Patents

Abstract

PURPOSE:To product beta-galactosidase having improved thermostability industrially advantageously, by transducing DNA obtained by integrating a DNA fragment carrying specific genetic information into vector DNA for Bacillus subtilis into Bacillus subtilis, and cultivating the Bacillus subtilis. CONSTITUTION:Recombinant DNA obtained by integrating DNA fraction carry ing genetic information of thermostable beta-galactosidase collected from Bacillus stearothermophilus into a vector for Bacillus subtilis is transduced into Bacillus subtilis, which is cultivated. Then, thermostable beta-galactosidase accumulated in the culture material is collected. The integration of the chromosome DNA into vector DNA can be carried out by scissoring the chromosome DNA and the vector DNA with a restricted enzyme to prepare a chromosome DNA frag ment and a vector DNA fragment, and treating a mixture of them with DNA ligase. DNA ligase derived from phage T4 is preferably used as the DNA ligase.

Description

Detailed Description of the Invention (Objective of the Invention) Industrial Application Field The present invention relates to a novel Bacillus subtilis having a heat-stable β-galactosidase gene and a method for producing thermostable β-galactosidase (
= related.

β-galactosidase is an enzyme that hydrolyzes lactose into galactose and glucose, and is used in the production of low-lactose milk and extracts useful value from lactose in whey, which is produced in large quantities as a by-product during the production of f-su. It is widely used in food processing, such as when used to produce high galactose or glucose.

It is desirable that enzymes used in food processing be able to withstand high temperature use in order to prevent microbial contamination during processing.

Furthermore, this enzyme is also used as a drug for treating lactose intolerance, and even in this case, excellent heat resistance is preferred from the viewpoint of stability of the preparation.

In order to meet these demands, the present invention aims to establish an industrially advantageous method for producing β-galactosidase with excellent heat resistance.

PRIOR TECHNOLOGIES AND PROBLEMS It is known that thermophilic Bacillus bacteria produce heat-stable β-galactosidase, and that low-lactose milk can be obtained by immobilizing the microbial cells and processing milk. ■, ■, and ■ Document C: Described.

■ R, E, Goodman, et al; Ca
nadian Journal of Microbi
ology.

Dish, 817-825 (1976).

■ M, W, Griffiths, et al.
；Journal of the 5science
of Food and Agriculture, 2
9.753-761 (1978)-■ T, Kob
ayashi, et al ;Journal o
f Fermentation Technology
.

Parallel, 309-314 (1978).

However, these conventional methods have problems such as low enzyme productivity, low affinity for the enzyme's substrate (lactose), and insufficient heat resistance.

The present inventors have previously succeeded in introducing the thermostable β-galactonase gene of Pacillus stearothermophilus IAM 11001 into Escherichia coli (:: This genetically modified Escherichia coli [Escherichia coli 294-43 (pH02)]
Heat-resistant β-
Completed a method for producing galactosidase and applied for a patent. (
Patent Application No. 58-171077) c.

According to this method (=), it is possible to obtain β-galactosidase with very good heat resistance, and since the enzyme according to this method (=) can be highly purified by simple heat treatment, it is possible to simplify the purification process in industrial production. However, the drawback was that the production amount of the enzyme was rather low.

(Structure of the Invention) The present invention provides a DNA carrying the genetic information of thermostable β-galactosidase obtained from the thermophilic bacterium Pacillus stearothermophilus.
This is based on the successful introduction of A into Bacillus subtilis via a vector and the commercially advantageous acquisition of thermostable β-galactosidase from the culture obtained by culturing this Bacillus subtilis. It is.

Therefore, the present invention includes each of the following inventions.

(1) DN carrying the genetic information of thermostable β-galactosidase obtained from Bacillus stearothermophilus
A novel recombinant DNA in which the A fragment is integrated into the vector DNA for Bacillus subtilis.

(2) DN carrying the genetic information of thermostable β-galactosidase obtained from Bacillus stearothermophilus
Recombinant D in which fragment A is integrated into vector DNA for Bacillus subtilis
A new Bacillus subtilis with NA introduced.

(3) DN carrying the genetic information of thermostable β-galactosidase obtained from Pacillus stearothermophilus
Recombinant D in which fragment A is integrated into vector DNA for Bacillus subtilis
A method for producing heat-stable β-galactosidase, which is characterized by culturing Bacillus subtilis into which NA has been introduced and collecting heat-stable β-galactosidase accumulated in the culture. - DNA that carries the genetic information of galactosidase (hereinafter referred to as

Isolation and purification of chromosomal DNA (referred to as chromosomal DNA) from Pacillus stearothermophilus can be carried out according to conventional method C2. For example, Biochim, Biophys, Ac
It can be carried out by the phenol method described in Ta72, 619-629 (1963).

This chromosomal DNA can be integrated into vector DNA by cutting the chromosomal DNA and vector DNA with restriction enzymes to prepare chromosomal DNA fragments and vector DNA fragments, and then treating a mixture of the two with DNA ligase. Examples of vector DNA used here include 1) I) UBIIO, I) E194. pC
194, pBD9°p'rp 4, etc.

In addition, restriction enzymes include BamHl and Bgl 1ie.
Eco RI, Pst I, Mlu I, Sal
I, Xho I, etc.

Furthermore, as a DNA ligase, D
NA ligase is preferably used.

The recombinant DNA obtained by the above method is introduced into Bacillus subtilis using the protoplast transformation method (Molecularand
General Genetics, 168
．． 111-115 (1979),)
°“”kQ-T'fxb%, WdfA@I-N 5
The method for selecting a strain having a recombinant DNA (vector DNA incorporating a DNA fragment carrying genetic information for tosidase) is to prepare the recombinant DNA (; For example, using EcoRI as the restriction enzyme and p
When using UBllo, this can be done in a first-order manner. That is, the strain is 5-bromo-4-chloro-3
-indolyl-β-D-galactopyranoside (hereinafter Xg
The cells were cultured on DM3 agar medium containing (referred to as al) and kanamycin, and colonies that appeared blue were selected, and finally β
- Check the presence or absence of galactosidase activity.

Next, recombinant DNA is isolated from the recombinant DNA-containing bacterial strain obtained by the above method. Isolation of recombinant DNA can be carried out according to conventional method C2. For example, Nuclei
cAcids Research, 7. 1513-
1523 (1979). If the recombinant DNA obtained in this manner is introduced into Bacillus subtilis, Bacillus subtilis containing the recombinant DNA can be prepared. Bacillus subtilis containing recombinant DNA can be obtained as blue colonies appearing on a DM3 agar medium containing kanamycin and Xgal.

Naori M3 agar medium is prepared by separately sterilizing and mixing the following 8 solutions.

1) 4chi agar 200-2) I
M Sodium succinate (pH 7,3) 500d
3) 5% Casamino Acid 100-4) 1
0% yeast extract 50-3.5% dipotassium phosphate + 100d5) 1.5% phosphoric acid l
Potassium 6) 20 Tyglucose 25-7)
IMMget2 2
0d8) 2% Bovine Serum Albumin 5dThe thermostable β-galactosidase according to the present invention is produced by culturing the novel genetically modified Bacillus subtilis obtained as described above by a conventional method, collecting the bacteria, and then using a conventional method. This is done by crushing the bacterial cells and obtaining a fat-free extract. The heat-stable β-galactosidase thus obtained is purified by heat treatment and conventional protein purification methods such as ion exchange chromatography and gel filtration, but heat treatment is particularly effective.

This heat treatment purification method is different from conventional methods for obtaining heat-stable β-galactosidase from Pacillus stearochymophilus and Namus thermophilus, and is a new and effective method.

That is, since all proteins produced by thermophilic bacteria have good thermal stability, when the cell extract is heat-treated.

The entire protein is only gradually denatured, and only β-galactosidase is not purified.

On the other hand, the β'-galactosidase produced by the novel microorganism of the present invention, in which genes from a thermophilic bacterium are integrated into the thermophilic bacterium Bacillus subtilis, has a large difference in thermostability from the original Bacillus subtilis protein. Most of the proteins of Bacillus subtilis are denatured and precipitated by heat treatment at about 70° C. for about 15 to 30 minutes, but β-galactosidase is hardly denatured and is dissolved in the heat-treated solution.

By simply centrifuging this heat-treated solution, β-galactosidase with increased purity can be obtained as a supernatant.

This simple and efficient method for separating and purifying β-galactosidase using heat treatment is a new technology made possible for the first time by the genetic recombination technology of the present invention.

Next, examples will be shown to explain the present invention in detail. In addition,
In the example shown below, Escherichia coli 294-294-2, which carries a recombinant plasmid pH02 incorporating a DNA fragment carrying genetic information for heat-stable β-galactosidase of Pacillus stearothermophilus IAM11001, is used as a DNA donor. 43 (pH02), (Feikoken Bacterial Serial No. 7233) as vector DNA.
Blolo and Bacillus subtilis M as host Bacillus subtilis.
1111 (T, IMANAKA, et al.
JOURNAL OF EACTERI-OLOGY,
146. 1091-1097 (1981)-2] were used.

Example 1. Preparation and cleavage of plasmid DNA carrying genetic information for thermostable β-galactonase Escherichia coli 294-43 (pH 02) was transformed into M9
Medium (NazHPO45,8?/L, KH2PO4
3fμ, NaCl3? /l, NH2Cl 1
? /l, CaCl211F'μ, Mg5049
5m9/l, FeCl31.6m9/l, casamino acid 5? /l, glucose 4 f/l) 150
-The absorbance of the culture solution at 600 nm at 37℃ is 0.6-
After culturing until the concentration reached 1.0, 200 μm/− of chloramphenicol was added and the culture was continued overnight. After collecting and washing the bacterial cells, 25 mM T containing 2 my/- Norizozyme was added.
ris-HCt (pH 8,0), 50mM glucose, 10mM EDTA, 15ml: Suspended and left at 0°C for 30 minutes, then 0.2N NaOH
, 1% SDS (sodium lauryl sulfate) 30- was added to lyse the bacteria.

It was left at 0°C for 5 minutes. Then 3M sodium acetate (p
H4,8) 22.5- was added and left at 0°C for 1 hour.

A supernatant was obtained by centrifugation (8.00 rpm, 20 minutes).

After adding 2.5 times the amount of ethanol to the upper groove to precipitate the DNA, 5 times the amount of IQ mM Tris-HCt (
pH 7.5).

It was dissolved in 1 mM EDTA (hereinafter referred to as TE buffer). This DNA solution was subjected to ethidium bromide-sesicum chloride equilibrium density gradient centrifugation, and pH02 plasmid 5
00 μt was obtained. To separate plasmid DNA into vector DNA and Pacillus stearothermophilus chromosomal DNA that carries genetic information for thermostable β-galactosidase, 5 μt of DNA +: was added with 5 U of PstI + 20 mM Tris-HCt. (pH 7,5
), 10mMget2. 50mM(NH4)2
Cutting was carried out at 37°C for 3 hours in 50μ of a reaction solution of s04°0.1 m97at bovine serum albumin. 65℃,
Pst I was inactivated by heating for 10 minutes, the DNA was precipitated with ethanol, and then dissolved in 20 μt of TE buffer.

Example 2. EcoRI from the Pst I site at the end of the DNA fragment carrying genetic information for thermostable β-galactosidase.
In order to link the DNA fragment carrying the genetic information of thermostable β-galactosidase obtained in Example 1 to the vector DNA1) UBIIO, the terminal Pst I site was converted to E by the following method.
Converted to CORI site. Example 1 Te obtained Pst
I t77 cut DNA 5pf was treated with 20mM Tris-HC.
L (pH 8,0), 600mM NaCl, 1
2mM CaCl2.1mM BDTA reaction solution 25μ
Inside, Equine Nuclease BAL-31 (New
(manufactured by England Biolabs) 30 at 0.2U
℃ for 6 minutes. BAL from phenol-treated ζ2
-31 was inactivated and after ethanol precipitation.

Dissolved in 26μt TE buffer. Ec phosphorylated at the 5' end with T4-polynucleotide kinase.

R Nirinka (GGAATTCC) (manufactured by Takara Shuzo) 2
Add 5 pmol and 66 mM Tris-H
CL (pH 7,5), 10 mM kfgct2.

40μ of a reaction mixture of 10mM 9j Oslate-/I/, 1mM ATP with IU of T4-DNA ligase.

The reaction was carried out at 15°C for 16 hours. Heat at 65°C for 20 minutes.

T4-DNA! After inactivating J gauze, 14μυTE
The mixture was reacted with 50 U of EcoRI at 37° C. for 3 hours in a 6 μm reaction solution containing a buffer solution and 6 μt of IMNaCt.

After heating at 65° C. for 20 minutes to inactivate EcoRI, ethanol precipitation was performed and dissolved in 40 μt of TE buffer.

Example 3. Preparation and cleavage of vector DNA DNA of pUBIIO plasmid having kanamycin resistance was prepared as follows. Bacillus subtilis MIII, a known Bacillus subtilis that has pUBIIO as a plasmid
L medium (tryptone 1%, yeast extract 0.5%,
NaC10.5%, ';
pH 7, Q) 60% of the culture at 37°C in 500rnt.
Culture with shaking until the absorbance at 0 nm becomes 2 to 3, collect and wash the bacterial cells, and add 2 my/mt Norizozyme-containing 2.
5mM Tris-HCL (pH 8,0), 5Q
Suspend in mM glucose, 10mM EDTA50me, and leave at 37°C for 30 minutes.

Add 0.2M NaOH and 1% SDS 100- to lyse the bacteria.

It was left at 0°C for 5 minutes. Then 3M sodium acetate (p
H4,s) 75 ml was added and left at 0°C for 1 hour.

A supernatant was obtained by centrifugation (8.00 rpm, 20 minutes).

2.5 times the volume of ethanol was added to the supernatant to precipitate the DNA, which was then dissolved in 5 volumes of TE buffer. This DNA solution was subjected to ethidium bromide/sencum chloride equilibrium density gradient centrifugation to obtain pUBIIO plasmid DNA 5.
0μ2 was obtained. To cut the vector DNA, p
Add 5U of Eco RI to 1μ2 of UBIIO,
lQmMTris-HCz (pH 7,5), 10
0mM NaCL, 10mM Mg cz2
The reaction was carried out at 37° C. for 2 hours in 75μ of the reaction solution. 6
Heat at 5°C for 10 minutes.

After the DNA was precipitated with ethanol, it was dissolved in 10 μt of TE buffer.

Example 4. Insertion of a DNA fragment carrying genetic information for thermostable β-galactosidase into vector DNA. 5 μt of the Eco RI fragment of the DNA obtained in Example 2 and 1 μm of the Eco RI fragment of the vector DNA obtained in Example 3 were mixed at 66 mM. Tris-HCl (pf
47.5), IQmMMgCt2゜10mM dithiothreate-/L/, 1mMATP (7) 50μ of the reaction solution was reacted with 0.2U of T4-DNA ligase at 4°C for 16 hours. 'I'4-DNA ligase was inactivated by heating at 65°C for 10 minutes, the DNA was precipitated with ethanol, and then dissolved in 100 μt of TE buffer.
This was used as a DNA solution.

Example 5 Transformation of Bacillus subtilis with recombinant plasmid and selection isolation of Bacillus subtilis having ability to produce thermostable β-galactosidase Bacillus subtilis MI 111 was transformed into Panassay
broth (meat extract 0.15%, yeast extract 0.
15%, bebuto 10.5%, g/l/=r-so, 1%, NaCLO, 3%, dipotassium phosphate 0.37%, monopotassium phosphate 0.13%, pH
7,0) Culture with shaking at 37°C in 20 mt until the absorbance at +570 nm becomes 0.8-1.0, and collect the bacteria.

S M M P solution containing 24 lysozymes (2x concentration 8MM solution and 4x concentration Penassay bro
2.5 mt +:, l
Prepare protoplasts by stirring gently at 37°C for 2 hours.

Centrifuge the protoplasts (4,00 Orpm, 15
After washing with 8 MMP solution, the cells were centrifuged again.

Suspend in 2rnt S M M P solution.

Note that the 8MM solution contains 0.5M i/q sugar, 20mM maleic acid (pH 6,5), 20mM Mgcz
It is a mixed liquid consisting of 2.

3 μt of the DNA solution obtained in Example 4 and 8 MM at twice the concentration
For a mixture of 30 μt of solution, add 0.5 d of this protoplast suspension, and 1.5 d of 40 polyethylene glycol solution (40 μt of 1 polyethylene glycol 6000 in 100 d, 50 d of SN 1M solution of 2 times concentration). -) was added and left for 2 minutes, 57! of SMMP solution was added, and the protoplasts were collected by centrifugation. Protoplasts were suspended in 14 5A4i'LP solution, cultured with shaking at 30°C for 1.5 hours, and then plated on a DM3 regeneration agar medium containing kanamycin (1/*) and Xgol. 3
When cultured at 7°C for 2 days, colonies of Bacillus subtilis capable of producing β-galactosidase exhibit a blue color.

The novel Bacillus subtilis thus obtained was named Pacillus subtilis MI 111 (pH 05) and deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (-).The deposit number is FEIB Deposit No. 7831.

In addition, this new Bacillus subtilis, Bacillus subtilis MI 1
The mycological properties of No. 11 (closed G5) are almost the same as those of ordinary Bacillus subtilis, except that it exhibits kanamycin resistance and heat-stable β-galactosidase productivity.

Example 6 Production and heat resistance test of β-galactosidase Bacillus subtilis MI 111 (pH 05) was grown in LL medium containing kanamycin 5 μf/me (tryptone 1%, yeast extract 0.5%, NaCtO, 5%)
, lactose 0.2%.

The cells were cultured with shaking at 37°C for 16 hours in a 150°C (pH 7.0).

After collecting bacteria, add Z buffer (0.1M phosphate buffer (medium 17.0
').

10mM KCl, 1mM MgSO4,50
Suspend in mM 2-mercaptoethanol) 3-.

After ultrasonication, centrifugation (15,00 Orpm, 15
The supernatant obtained was used as a cell extract.

β- of this cell extract before and after heat treatment at 70°C for 30 minutes
Galactosidase activity was measured as follows using 0-nitrophenyl-β-D-galactopyranoside (hereinafter referred to as 0NPG) as a substrate.

2 buffer solution 2 containing 0.81n97me NOON PG
ml of the enzyme solution was mixed and left at 65° C. for a certain period of time, then 1 ml of IM Na2CO was added and cooled with water, and the amount of O-nitrophenol produced in one reaction was measured by absorbance at 420 nm. The amount of enzyme that released 1 μmol of O-nitrophenol per minute was defined as IU.

For comparison, Escherichia Ko! I 294-43 (p.
H02) with Tetracyclear (5μSF7'++4!
), and cultured in the same manner as above (= cell extract obtained by cell extraction and Pacillus stearothermophilus I
A similar C test was conducted using a cell extract obtained by culturing AMI100I in LL medium at 55°C.

The results are shown in Table 1.

Table 1 As is clear from Table 1, the β-galactosidase of the present invention and Comparative 1 had an activity residual rate of 90% and 81% after heat treatment at 70°C for 30 minutes, while that of Comparative 2 was 33%. In comparison, it showed a significantly higher value, and its heat resistance was extremely high.

Looking at the purification effect of each enzyme by heat treatment, the specific activities of the enzymes of the present invention and Comparison 1 were improved by 3.2 and 4.5 times, whereas in the case of Comparison 3, the specific activities were the opposite (20.8 times). decreased.

Productivity of thermostable enzymes of each microorganism [activity after heating (U/d
)] according to the present invention (: according to the comparison 1, about 29
The yield was improved by 2 times or 20 times that of Comparative 2.

In order to investigate in more detail the purification effect of this enzyme by heat treatment C: cell extraction was carried out for Pacillus subtilis MI 111 (pH 05), a symptomatic organism of the present invention, and its host microorganism, Pacillus subtilis MI 111 (PUB 110). 5DS-polyacrylamide gel electrophoresis (U, K, Laemmli et al.

Nature 227.680-685 (1970)
,) was performed.

In this test, purified β-galactosidase was used as a standard product, and RNA-polymerase (165,000, 155,000°, 39,000°) was used as a molecular weight marker protein.
), bovine serum albumin (68000'), )
A mixture of Rippun inhibitors (21500) was used. After electrophoresis, the results of staining with 0.0:1 Damassie Brilliant Pur-R250 are shown in FIG.

In FIG. 1, lanes 1 to 6 are as follows.

Lane Sample 1 Molecular weight marker 2 Purified β-galactosidase 3 Cell extract of a non-inventive organism 4 Solution 5 obtained by heat-treating the same as above at 70°C for 15 minutes 5 Cell extract of host microorganism 6 Solution 1 obtained by heat-treating the same as above for 700.15 minutes As is clear from the figure, a cell extract of a symptomatic organism of the present invention (lane 3)
and the host microorganism cell extract (lane 5) contain many identical components, except that the former contains β-galactosidase and the latter does not.

Components other than these many types of β-galactosidase are
Samples heat-treated for 700.15 minutes (lanes 4 and 6)
) has almost completely disappeared.

In addition, as a result of measuring the half-life of the activity of the heat-stable β-galactosidase purified product according to the present invention in another test, it was found that the half-life at 60C was 150 hours, 7 minutes in the above document (1) and 450 hours in the document (2). It was found to be significantly longer than 1 minute.

Example 7. Analysis of recombinant DNA retained by Bacillus subtilis The transformed strain obtained in Example 5 was treated with kanamycin (5 μC).
The plasmid DNA 50 was cultured at 370 °C in L medium 500 °C containing 50 °C as in Example 3 (2) to obtain plasmid DNA 50 μt. Using this plasmid DNA, Pacillus subtilis MIII was cultured in the same manner as in Example 5. All the transformed strains obtained were kanamycin resistant,
It had the ability to produce β-galactosidase.

This indicates that the 5 DNA fragment carrying the genetic information of β-galactosidase is incorporated into the plasmid DNA.

In addition, this plasmid DNA was cut with the restriction enzyme EcoRI in the same manner as in Example 2, and the size of the DNA fragment carrying the genetic information of thermostable β-galactosidase was measured by 1% agarose gel electrophoresis.2. It was 9 kilobase pairs (kb).

The structural diagram (restriction enzyme map) of this plasmid DNA (PH05) is shown in FIG.

For comparison, C2 in the same figure shows plasmid DNA (pH 02) prepared according to the prior invention of Japanese Patent Application No. 171077/1982.
A structural diagram is also shown.

[Brief explanation of drawings]

FIG. 1 shows the results of 8DS-polyacrylamide gel electrophoresis, and the samples in each lane are as follows. Lane Sample 1 Molecular weight marker 2 Purified β-galactosidase 3 Cell extract of a non-inventive organism 4 Solution obtained by heat-treating the same above at 70C for 15 minutes 5 Cell extract of host microorganism 6 Solution obtained by heat-treating the same above for 700.15 minutes Figure 2 is the novel recombinant plasmid DNA of the present invention (pi (G5)) and the plasmid DNA of the invention of Japanese Patent Application No. 171077/1982.
(pH02) shows a restriction enzyme map.

Claims (3)

[Claims] (1) A novel recombinant D in which a DNA fragment carrying the genetic information of thermostable β-galactosidase obtained from Bacillus stearothermophilus is incorporated into the vector DNA for Bacillus subtilis
N.A. (2) Recombinant DNA in which a DNA fragment carrying the genetic information of thermostable β-galactosidase obtained from Bacillus stearothermophilus is integrated into vector DNA for Bacillus subtilis
A new Bacillus subtilis that has been introduced. (3) Recombinant DNA in which a DNA fragment carrying the genetic information of thermostable β-galactosidase obtained from Bacillus stearothermophilus is integrated into vector DNA for Bacillus subtilis
1. A method for producing heat-stable β-galactosidase, which comprises culturing Bacillus subtilis into which B. subtilis has been introduced, and collecting heat-stable β-galactosidase accumulated in the culture.