JPS6062984A - Novel coliform bacilli having heat-resistant beta-galactosidase gene and production of heat-resistant beta-galactosidase - Google Patents

Abstract

PURPOSE:To produce heat-resistant beta-galactosidase in bacterial cells, by cultivating novel coliform bacilli having a heat-resistant galactosidase gene. CONSTITUTION:A recombinant DNA having DNA carrying genetic information about heat-resistant beta-galactosidase obtained from Bacillus thermophilus IAM 11001 is introduced into a vector plasmid for coliform bacilli to give Escherichia coil 294-43(pHG1) [FERM-P No.7232] and Escherichia coli 294-43(pHG2) [FERM- P No.7233]. The resultant bacteria are then cultivated by the conventional method and collected. The resultant bacterial cells are crushed and extracted to give the aimed heat-resistant beta-galactosidase.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel Escherichia coli having a thermostable β-galactosidase gene and a method for producing thermostable β-galactosidase.

β-galactosidase is an enzyme that has the ability to hydrolyze lactose into galactose and glucose. It is used in the production of low-lactose milk, and is utilized from lactose in whey, which is produced in large quantities as a byproduct during cheese production. It is widely used in food processing, such as for producing high-value galactose or glucose. As enzymes used in food processing, thermostable enzymes that can withstand high temperature use are desirable from the viewpoint of preventing microbial contamination during processing. As a result of intensive research into a method for producing thermostable β-galactosidase, the present inventors discovered that the DNA carrying the genetic information for thermostable β-galactosidase obtained from the thermophilic bacterium Pacilus stearothermophilus was transferred via a vector. and succeeded in introducing it into E. coli.

The present inventors succeeded in industrially advantageously obtaining heat-stable β-galactosidase from the bacterial cells obtained by culturing this Escherichia coli, thereby completing the present invention.

The present invention will be explained in detail below.

1) N that carries the genetic information of heat-resistant β-galactosidase
A (hereinafter referred to as chromosome 1) NA) can be produced by a single RIM from Bacillus stearothermophilus cells according to a conventional method. For example, Biocbim, B
iopbys, Acta, 72.619-629
(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. Vector used here 1) N
A and its derivatives (e.g. pHR322, I) AC
YC177, etc.) and λ phage, among others, p
HR322°pACYC177 is preferably used. In addition, restriction enzymes include BamH1, BgLIJ, Eco
Rl, Pst l, Mlu l.

Examples include SaL l and Xbo 1. moreover]
) N A ligase is D derived from T4 phage.
NA ligase is preferably used.

The recombinant DNA obtained by the above method was introduced into E. coli according to Proc. Nat. Acad. Sci. U.S.
A, 69°2110-2114 (1972). Recombinant DNA (i.e., vector DNA incorporating a DNA fragment carrying genetic information for thermostable β-galactosidase)
) The method for selecting a strain having
pAcYc177 as vector DNA
If you use .

This can be done as follows. That is, the bacterial strain was transferred to tryptone yeast extract medium (hereinafter referred to as TY
The ampicillin-resistant and kanamycin-sensitive strains are first selected. This strain was then treated with 5-bromo-4-chloro-3-indolyl-β-D.
- Cultivate on a TYE agar medium containing galactopyranoside (hereinafter referred to as Xgat) and ampicillin, select blue colonies, and finally confirm the presence or absence of β-galactosidase activity.

Next, recombinant 1) NA is isolated from the recombinant DNA-containing bacterial strain obtained by the method described in Section 2. Recombination break 1) N
Isolation of A can be carried out according to conventional methods. For example, Nu
cleic Ac1ds Re5earch, vol.
-7, pl) 1513-J523 (1979). By introducing the thus obtained recombinant DNA into E. coli, E. coli containing the recombinant DNA can be prepared. E. coli containing recombinant DNA can be obtained as blue colonies appearing on a TYE agar medium containing ampicillin and Xgal.

The following two representative E. coli strains containing the recombinant DNA thus obtained have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

Escherichia coli 9294-43 (pH 01) [Hui Industrial Research Institute Bacteria No. 7232] Escherichia coli 294-43 (plIG 2)
[Feikoken Bibori No. 7233] The heat-stable β-galactosidase according to the present invention is produced by culturing the novel genetically modified Escherichia coli obtained as described above by a conventional method, harvesting the bacteria, and then using a conventional method. This is done by disrupting the bacterial cells and obtaining a cell-free extract. The thermostable β-galactosidase thus obtained is purified by heat treatment and conventional protein purification methods 1, such as ion exchange chromatography, gel filtration, etc.
Heat treatment is particularly effective. This purification method by heat treatment is a new and effective method, unlike the conventional method for obtaining heat-stable β-galactosidase from Pacillus stearothermophilus or Thermus thermophilus.

That is, since all proteins produced by thermophilic bacteria have good thermostability, heat treatment of a cell extract only gradually denatures all the proteins, and in particular, 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 incorporated into the thermophilic Escherichia coli, has a large difference in thermostability from the original Escherichia coli protein, with approximately 70% Most of the E. coli proteins are denatured and precipitated by heat treatment for about 30 minutes at °C, 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.

Examples of the present invention are listed below.

Example 1 This example uses Bacillus stearothermophilus IAMI100I as a DNA donor, pACYC177 as a vector DNA, Xhol as a restriction enzyme, and Escherichia coli 294 (ATC) as a host E. coli.
This is an example in which a β-galactosidase-deficient strain (hereinafter referred to as ``Escherichia coli 294-43 (rk-mk)'') obtained by nitrosoguanidine mutation treatment of C31446) was used.

(1) Preparation and cutting of chromosomal DNA carrying the genetic information of thermostable β-galactosidase Bacillus stearothermophilus JAM11001 was grown in YE medium containing 0.2% lactose (1% tryptone).
, yeast extract 0.5%, NaCzO, 5%, p! 17
．． 0) 4 was cultured with shaking at 60°C for 6 hours. After collecting and washing the bacterial cells, 20 mM'Tris-HCt containing 200-1 m9/lnε lysozyme (pH 7,5),
5 mM (suspended in DTA buffer and left at 15°C for 15 minutes. Add 20% SDS solution to lyse the bacterium, add 205 m/m of phenol (205 mM/ml immediately before use)
Add 0mM Tris-HCt (saturated with plI 8.0), stir gently, and centrifuge. (7,OOQ rpm.

10 minutes) Take the aqueous layer (upper layer) and repeat the phenol extraction several times.Add 2 times the volume of 95-thiethanol to the resulting aqueous layer, and collect the resulting white precipitate by scattering it around a glass rod. This DNA was dissolved in 100+j of 0.1×SSC solution, 200×e of ethanol was added, and ethanol precipitation was performed again. Collect the resulting precipitate in the same manner as in 1.
-0. After dissolving in lX5SC solution, 5m7! 10X
Add 8SC. (So that the final concentration is 1xS8C)
The resulting I) NA solution '50me was subjected to ethidium bromide-cesium chloride equilibrium density gradient centrifugation.

Chromosome 1) N A of 750 μV was obtained. chromosomal dna
to cut 10 pff of I) NA
u (7) Add Xho 1, 10mM Tris-14
(1 (IA17.4), 50mM NaC110mM
The reaction was carried out in 50 pt of MgCl2 reaction solution at 37°C for 3 hours, and then heated at 65°C for 10 minutes.

) (ho I was inactivated and the DNA was precipitated with ethanol, then 251Lt of 10mM Tris-HCt (pT(
7,5) Dissolve in 1mM EDTA (hereinafter referred to as TE buffer).

(2) Preparation and cleavage of vector DNA pAc with ampicillin and kanamycin resistance
The DNA of Yc 177 plasmid was prepared as follows.

Escherichia coli C600 (rk-/mk-) carrying pAcYc177 as a plasmid [A, C, Y,
Chang.

et al., J. Hacteriol, 134
1141-1156 (H)78)-] was added to M9 medium (N
a2HPO45.8W/l, KH2PO43f/l.

NaCt5jμ, NH4Cl 1 f/l, Ca
cz211m9/L, Mg 80495m9/L,
17'e C/-31,6m9/l, casamino acids 57
μ, glucose 4 f/L) 3 in 150 me
The absorbance of 5QQnrn of the culture solution at 7°C is 0.6 to 1.0.
After culturing until

Chloramphenicol was added for 200 μs and the culture was continued overnight. After collecting bacteria, 2 mq/m1 (D lyso f
-25mM Tris-HCt (pl(8,0)
.

Suspend in 15mg of 50mM GTA and 10mM EDTA.

After standing at 0℃ for 30 minutes, 0.2NNa011.1%5D
S (butorium lauryl sulfate) 30- was added to lyse the bacteria, and the mixture was left at 0°C for 5 minutes. Then 3M sodium acetate (
After adding 22.5 ml (pH 4.8) and leaving it at 0°C for 1 hour, centrifugation (8.00 rpm, 20 minutes) was performed to obtain a supernatant. After adding 2.5 times the volume of ethanol to the supernatant to precipitate the DNA, 5 mg of 10 mM Tris-HCt (p
) 17.5). This DNA solution was subjected to ethidium bromide (J mito-cesium chloride equilibrium density gradient centrifugation,
l] AcYc177 plasmid 1) 100 μf of NA was obtained. Vector 1) pACYC17 to cut N-A
71/7 ri JL, 511's Xbox l, force 11er
10mM Tris-Ilct (pH 7,4), 50
The reaction was carried out at 37°C for 2 hours in 20μ of the reaction mixture of mMNaCt and 10mMMg (22).

Heat at 65° C. for 10 minutes to precipitate 1) NA with ethanol, and then dissolve in 20 pL of 'll'E buffer.

(3) Insertion of chromosomal DNA fragment into vector DNA Mix the chromosome 1) NAIOμ2 obtained in (1) with 0.57j of the vector DNA obtained in (2).
is -He (pH 7,5), 10rnMMgC/-
z, 10mM dithiothreate-#, 1mM AT
After reacting at 4°C for 16 hours with 0.20 T4-DNA ligase in 50pt of P reaction solution, the reaction was carried out at 65°C for 10 hours.
The mixture was heated for a minute to form a DNA solution.

(4) Transformation of Escherichia coli with recombinant plasmids Escherichia coli 294-43 was grown in L medium (1 g of tryptone, 0.5% tNa (Jo, 51-glucose, 0.1 g, p))7. 0) 507! 3rd year middle school
At 7°C, the absorbance of the culture solution at 5QQ nm is 0.6 to 1.0.
Culture by shaking until the bacteria are collected. 12.5mg of 0
．． After washing with 1 M MgCl2, 12.5 ml 0J
J) Suspended in 0.1 M Ca C12 at 0°C.

Leave for 20 minutes. After centrifugation, 2. ! 15% of 5ml
Suspend in glycerin SO, I M Ca Ct.

Add 10 μt of the DNA solution obtained in (3) to 0.2 μm of this suspension.
Add.

After being left at 0°C for 1 hour, it was treated at 42°C for 3 minutes.

This suspension was diluted 10 times with L medium and kept cool at 37°C for 30 to 90 minutes, then ampicillin (50pf 7th season)
It was spread on a TYB agar medium containing. E. coli that has become ampicillin resistant is obtained by culturing at 37° C. overnight.

(5) Selective isolation of Escherichia coli capable of producing thermostable β-galactosidase Among the ampicillin-resistant Escherichia coli obtained as described above, those that cannot grow on TYE agar medium containing kanamycin (5 μf/me) were selected.

Xgal (4o μf/mg) and Ambisiliy (5
TYE agar medium containing 0 pt/me) and incubated at 37°C.
Cultivate with

Colonies of E. coli that have the ability to produce β-galactosidase exhibit a blue color. The new Escherichia coli thus obtained was named Escherichia co9294-43 (psIGl).
Deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology. [Microtechnical Research Institute No. 7232: 1M5cliericha co
li 294-43 (pH 01) -) (6) Analysis of recombinant DNA retained by E. coli The transformed strain obtained in (5) was placed in M9 medium 15 containing ampicillin (50μ).
The cells were cultured in 0 mg at 37°C, and 100 μ2 of plasmid DNA was obtained in the same manner as in (2). This plasmid D
Using N.A.

When Escherichia coli 294-43 was transformed in the same manner as in (4), all of the resulting transformants were ampicillin resistant and had the ability to produce β-galactosidase. This indicates that a DNA fragment carrying genetic information for β-galactosidase has been incorporated into the plasmid DNA.

In addition, this plasmid DNA was cut with the restriction enzyme Xhol in the same manner as in (2), and the size of the DNA fragment carrying the genetic information of thermostable β-galactosidase was measured using 1% agarose gel electrophoresis. Mega Dalton (
Md).

(7) Thermostable β-galactosidase produced by Escherichia coli Escherichia coli 294-43 (pmlGl) was cultured with shaking in L medium 150- at 37°C for 6 hours.

After collecting bacteria, add Z buffer (0.1M phosphate buffer (pIt7.
o).

10mM KCt, 1mM Mg5O+, 50mM 2-
Suspend in mercaptoethanol]3-. A cell-free extract was prepared by ultrasonication.

β of this cell-free extract before and after heat treatment at 70°C for 30 minutes
- Galactosidase activity was measured as follows using O-nitrophenyl-β-D-galactopyranoside (hereinafter referred to as 0NPG) as a substrate. Mix Z buffer 2 containing 0.8 mν of 0NPG and 0.4 ml of enzyme solution.

After standing at 65℃ for a certain period of time, I MNa2 co31 tn
ε was added and cooled with water, and the amount of salt nitrophenol produced in one reaction was measured by absorbance at 420 nm.

The amount of enzyme that releases 1 μmol of orthonitrophenol per minute was set to 10.

As a control, the wild strain Escherichia coli 294 (
A'l'CC31446) containing 0.2% lactose
A cell-free extract obtained by culturing on an agar medium in the same manner as above was used.

The results are shown in the f81 table.

Table 1 shows that even after heat treatment at 70°C for 30 minutes, 80% of activity remained, and the specific activity increased by 4.5 times to I/X.

Example 2 This example uses plasmid pIIGl as a DNA donor, PGl as a restriction enzyme, and pIIGl as a vector DNA.
BR322 and Escherichia coli 2 as host E. coli.
This is an example using 94-43.

(1) Cleavage of plasmid pl (Gl) Plasmid DNA obtained in Example 1 (6) 1μf (2 pairs, 5u of psLl was added, 2QmMTris-1
1Ct (pH 7,5), 10mM Mg (E4z, 5
6mM (NH4) 2804.0.1mV - 20μ of bovine serum albumin reaction solution was reacted at 37℃ for 3 hours, heated at 65℃ for 10 minutes, and after ethanol precipitation, 20μ
Dissolve in μt Tll buffer.

(2) Vector 1) Preparation and cleavage of NA pBI<
The DNA of the 322 plasmid was transferred to Escherichia-7C600 (rlc-m), which has pBR322 as a plasmid.
k-) according to the method described in Example 1 (2). As a result, 1111 g of pBR322 plasmid DNA
I got it. pBR322i to cut the vector DNA
Add pf + 2 pair + 5u no Pst1, 20m
A4'l"ris-) ICt(p+17.5), 1
0mMMgC/-2゜50mM(NH4)2S04.0
．． 1my7,1 bovine serum albumin reaction solution (20μ) was reacted at 37°C for 3 hours, heated at 65°C for 10 minutes, and precipitated with ethanol.

Dissolve in 20μt of 'I'E buffer.

(3) Binding of DNA fragments Mix 0.5μ2 of the DNA obtained in (1) with 9.5 pf of the vector DNA obtained in (2), and add 66mM Tris.
-Hct (pH 7,5).

10fnMMg (J2. 10mM9 Thiothret-
/l/, 0.2 in 5QμA of 1mM ATP reaction solution.
After reacting with T4'-DNA ligase at 4°C for 16 hours, the temperature was increased to 65°C.

The mixture was heated for 10 minutes to prepare a DNA solution.

(4) Transformation of E. coli with recombinant plasmid In the same manner as in (4) of Example 1, the DNA solution prepared as above was introduced into Escherichia coli 294-43, and tetracycline (5μ2/m7) was introduced. and Xgal (
Colonies exhibiting blue color were selected on TYE agar medium containing 40 pf/me). The new Escherichia coli thus obtained was transformed into Escherichia coli 294-43 (p)lG2.
) and deposited it with the Institute of Microbial Technology, Agency of Industrial Science and Technology. [Microtechnology Research Institute No. 7233: hscheric
hia coli 294-43 (pH 02), ) (5) Recombinant hia coli retained by E. coli ]) Analysis of NA (
The transformed strain obtained in 4) was treated with tetracycline (5 μf
/ld) at 37°C in M9 medium 150-
In the same manner as in (2), 300 μf of plasmid JNA was obtained. This plasmid DNA was cut with the restriction enzyme PsLl in the same manner as in (2), and the size of the DNA fragment carrying the heat-stable β-galactosidase genetic information was measured by 1-layer agarose gel electrophoresis, and it was found to be 2 Md. .

(6) β-galactosidase produced by Escherichia coli The β-galactosidase activity of a cell-free extract prepared from Escherichia coli 294-43 (pH 02) in the same manner as in Example (7) was 12.1 u. /me
(0.53 u/my protein) at 70°C.

:The activity after heat treatment for 30 minutes was g, 7 u/mA (2
, 4''/'9 protein).

Furthermore, the mycological properties of the new Escherichia coli bacteria Escherichia coli 294-43 (p)Io 1 ) and Escherichia coli 294-43 (pl (02)) created in Examples 1 and 2 are ampicillin resistant and heat resistant. Its properties are almost the same as those of ordinary E. coli, except that it exhibits β-galactosidase productivity.

N1 person for patent Wakamoto Pharmaceutical Co., Ltd.

Claims (3)

[Claims] (1) DNA carrying genetic information for thermostable β-galactosidase obtained by Bacillus stearothermophilus
New recombinant DNA0 in which the fragment was integrated into E. coli vector DNA (2) DNA carrying the genetic information of thermostable β-galactosidase obtained from Bacillus stearothermophilus
Recombinant DNA in which the fragment was integrated into E. coli vector DNA
A new E. coli strain containing A. (3) DNA carrying the genetic information of thermostable β-galactosidase obtained from Pacillus stearothermophilus
Recombinant DNA in which the fragment was integrated into E. coli vector DNA
1. A method for producing heat-stable β-galactosidase, which comprises culturing E. coli into which A has been introduced, and collecting heat-stable β-galactosidase accumulated in the cultured cells.