JPH0614774A - Novel heat-resistant beta-galactosidase and its production - Google Patents

Abstract

PURPOSE:To find out the novel heat-resistant beta-galactosidase suitable for the production of low lactose milk and for the decomposition of lactose in whey, and to establish the method for producing the same. CONSTITUTION:The novel heat-resistant beta-galactosidase is contained in a culture product obtained by culturing Bacillus subtilis holding a recombined plasmid containing the heat-resistant beta-galactosidase gene of Bacillus stearothermophilus, and the method for producing the beta-galactosidase. The novel heat-resistant beta-galactosidase exhibits excellent heat resistance in comparison with a heat- resistant beta-galactosidase originated from known Bacillus.stearothermophilus, and also exhibits excellent substrate affinity in comparison with commercially available enzymes.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel thermostable β-galactosidase and a method for producing the same. β-Galactosidase is an enzyme that hydrolyzes lactose into galactose and glucose, and is used for the production of low-lactose milk, or produces galactose or glucose from lactose in whey, which is produced in large quantities as a by-product during cheese production. It is widely used in food processing such as used to

The enzyme used for food processing is preferably one that can withstand high temperature use from the viewpoint of preventing microbial contamination during processing. This enzyme is also used as a drug for treating lactose intolerance, and even in this case, it is preferable that the enzyme has excellent heat resistance from the viewpoint of stability of the preparation. In order to meet these demands, the present invention aims at establishing a method for industrially producing β-galactosidase having excellent heat resistance.

[0003]

2. Description of the Related Art Thermophilic Bacillus (Baci)
It is described in, for example, the following documents that bacterium belonging to the genus llus) produces thermostable β-galactosidase, and that microbial cells are immobilized and milk treatment is performed to obtain low-lactose milk. AR Goodman et al .; Canadian Journal of Microbiology, Vol. 22, 817-8.
Page 25 (1976) [R. E. Goodman, et
al; Canadian Journal of M
microbiology, 22 , 817-825 (19
76)] M W. Griffith, et al .; Journal of the Science of Hood and Agriculture, 29, pp. 753-761 (1978).
Year) [M. W. Griffiths, et al; Jo
urinal of the Science of Fo
od and Agricultural, 29, 753
-761 (1978)] and JP-A-54-16388.
No. 9, Gazette T. Kobayashi, et al .; Journal of Fermentation Technology, Volume 56, 309-3
P. 14 (1978) [T. Kobayashi, et
al; Journal of Fermentati
on Technology, 56 , 309-314.
(1978)] However, in these conventional methods, the productivity of the enzyme is low, and the affinity of the enzyme itself for the substrate (lactose) is small,
There were problems such as insufficient heat resistance.

The inventors of the present invention have previously used the gene recombination technique to utilize Bacillus stearothermophilus IAM.
The thermostable β-galactosidase gene of 11001 was successfully introduced into Escherichia coli via a vector, and this recombinant Escherichia coli (Escherichia coli 294-43 (p
HG2), and a method for producing thermostable β-galactosidase by culturing Microorganism Research Institute No. 7233], and filed a patent application. (Japanese Patent Application No. 58-171077).

According to this method, β-galactosidase having extremely excellent thermostability can be obtained, and since the enzyme by this method is highly purified by a simple heat treatment,
Although it has made it possible to simplify the purification process in industrial production, it has the drawback that the enzyme production is rather low.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention provides a thermostable β-galactosidase from a culture obtained by culturing a novel Bacillus subtilis in which a thermostable β-galactosidase gene of Bacillus stearothermophilus IAM11001 is introduced via a vector. It was based on the fact that the obtained β-galactosidase was a novel enzyme having both excellent thermostability and high substrate affinity.

Therefore, the present invention includes the following respective inventions. (1) Novel thermostable β-galactosidase having the following physicochemical properties 1) Action and substrate specificity A substrate having a β-D-galactoside bond is hydrolyzed to release D-galactose.

2) Optimum pH and stable pH range Optimum pH: 5.5 Stable pH range: 5.5 to 9.0 3) Optimum temperature range of action Optimum temperature of activity: about 70 ° C 4) Stable temperature range Temperature The half-lives of β-galactosidase activity at 55 ° C, 60 ° C and 65 ° C are about 620 hours, 150 hours and 55 hours, respectively.

5) Effect of metal ion When adding a concentration of 1 mM, Mg ²⁺ , Ca ²⁺ , Cu ²⁺ , Fe
²⁺ , Co ²⁺ and Li ⁺ do not inhibit the activity of this enzyme,
Ag ⁺ inhibits about 80% or more, and Hg ²⁺ inhibits about 90% or more. 6) Molecular weight: about 67,000 (by SDS-polyacrylamide gel electrophoresis) 7) Michaelis constant (Km) for lactose
2.4 mM (2) Heat resistance β- of Bacillus stearothermophilus
Bacillus subtilis MI carrying a novel recombinant plasmid pHG5 in which a DNA fragment containing a galactosidase gene is inserted into a vector plasmid pUB110.
111 (pHG5) was cultured in a medium containing an appropriate nutrient source,
A method for producing a thermostable β-galactosidase, comprising collecting the thermostable β-galactosidase according to claim 1.

In carrying out the present invention, the microorganism used is a recombinant plasmid pHG5 containing the thermostable β-galactosidase gene of Bacillus stearothermophilus.
Bacillus subtilis MI 111 (pHG5)
This microorganism has been deposited at the Institute of Microbial Engineering, Institute of Industrial Science and Technology as Micro Engineering Research Article No. 911 (FERM BP-911).

The β-galactosidase of the present invention is produced by inoculating Bacillus subtilis MI 111 (pHG5) in a nutrient-containing medium and aerobically culturing. During the culture, for example, glucose as a carbon source,
Lactose, starch, dextrin, glycerin, molasses and other assimilable organic carbon compounds are used, while nitrogen sources include, for example, soybean flour, cottonseed flour, casein, peptone, yeast extract, meat extract, amino acids, ammonium salts. Organic nitrogen compounds such as and inorganic nitrogen compounds can be used. If necessary, sodium salt, potassium salt,
Inorganic salts such as calcium salts, magnesium salts and phosphates may be used alone or in appropriate combination.

As the culturing method, a method generally used for enzyme production is adopted, but in the liquid culturing method, deep aeration stirring culturing is preferable in order to keep it aerobic, and in the laboratory, it is usually a flask. Shaking culture of is suitable. The culture temperature can be usually 20 to 45 ° C, but it is desirable to maintain it at 35 to 42 ° C. PH of culture is 6 ~
It is possible to be around 8, but it is desirable to keep around 7. The number of culture days can be 1 to 6 days.

In order to collect the enzyme of the present invention accumulated in the culture, a usual method for separating and purifying the enzyme can be used.
That is, since the enzyme of the present invention is contained in cultured cells, the cells are first obtained by vacuum filtration or centrifugation of the culture. The cells are suspended in an appropriate buffer such as phosphate buffer, disrupted by ultrasonic disruption method or French press method, and then centrifuged to collect the supernatant (cell extract). Purification of the present enzyme contained in this cell extract is carried out by heat treatment and usual protein purification methods such as ion exchange chromatography and gel filtration. The heat treatment is particularly effective. This purification method by heat treatment is a novel and effective method, unlike the conventional method for obtaining thermostable β-galactosidase from Bacillus stearothermophilus or Thermus thermophilus.

That is, since all the proteins produced by thermophiles have good thermostability, when the cell extract is heat-treated, all the proteins are gradually denatured, and in particular, only β-galactosidase is purified. Absent. On the other hand, the novel microorganism of the present invention in which the thermophilic bacterium gene is incorporated into Bacillus subtilis, which is a room temperature bacterium, is produced.

The enzyme of the present invention has a large difference from the original Bacillus subtilis protein in terms of heat stability. Most of the Bacillus subtilis protein is denatured and precipitated by heat treatment at about 70 ° C. for about 15 to 30 minutes. However, the enzyme of the present invention is hardly denatured and is dissolved in the heat treatment solution. By simply centrifuging the heat-treated solution, the enzyme of the present invention having an increased purity can be obtained in the supernatant.

The simple and efficient method of separation and purification by this heat treatment is a novel technique made possible for the first time by the present invention. Next, examples will be shown in order to explain the present invention in detail.

[0017]

【Example】

Example 1 Production of β-galactosidase and heat resistance test Bacillus subtilis MI 111 (pHG5) LL medium containing 5 μg / ml of kanamycin (tryptone 1%, yeast extract 0.5%, NaCl 0.5%, lactose 0. Two
%, PH 7.0), shake culture at 37 ° C. for 16 hours in 150 ml, and after collecting the bacteria, Z buffer (0.1 M phosphate buffer (pH 7.0), 10 mM KCl, 1 mM MgSO 4).
₄ , 50 mM 2-mercaptoethanol) 3 ml. After ultrasonic treatment, centrifuge (15,000 rp
m, 15 minutes) and the resulting supernatant was used as a cell extract.

The β-galactosidase activity of this cell extract before and after heat treatment at 70 ° C. for 30 minutes was used as a substrate for O-.
Nitrophenyl-β-D-galactopyranoside (hereinafter O
(Hereinafter referred to as "NPG").
2 ml of Z buffer solution containing 0.8 mg / ml of ONPG and 0.4 ml of enzyme solution were mixed and allowed to stand at 65 ° C. for a certain time, and then 1
1 ml of M Na ₂ CO ₃ was added and ice-cooled, and the amount of O-nitrophenol generated by the reaction was measured by the absorbance at 420 nm. The amount of enzyme that liberates 1 μmol of O-nitrophenol in 1 minute was set to 1U.

For comparison, Escherichia coli 294-
43 (pHG2) with tetracycline (5 μg / ml)
Cell extract and Bacillus stearothermophilus IAM1 obtained by culturing in LL medium containing
The cell extract obtained by culturing 1001 in LL medium at 55 ° C. was tested in the same manner. The results are shown in Table 1.

[0020]

[Table 1] As is clear from Table 1, the β-galactosidases of the present invention and Comparative Example 1 have 90% and 81% of residual activity after heat treatment at 70 ° C. for 30 minutes, which is 33% of Comparative Example 2. The value was extremely high, and the heat resistance was very excellent. Looking at the purification effect of each enzyme by heat treatment,
The enzyme of the present invention and the enzyme of Comparative Example 1 improved the specific activity by 3.2 times and 4.5 times, respectively, whereas in the case of Comparative Example 2, conversely, the specific activity was reduced to 0.
It has decreased eight times.

Regarding the productivity of the thermostable enzyme of each microorganism [activity after heating (U / ml)], according to the present invention, the yield was improved by about 29 times as compared with Comparative 1 or 20 times as much as Comparative 2. .
In order to investigate the purification effect of this enzyme by heat treatment in more detail, the microorganism of the present invention, Bacillus subtilis M
I 111 (pHG5) and its host microorganism, Bacillus subtilis MI 111 (pUB110), were subjected to SDS-polyacrylamide gel electrophoresis using a cell extract and a solution obtained by heat treating each of them for 15 minutes at 70 ° C.
K. Renmley; Nature, Volume 227, 680-6
Page 85 (1970) [U. K. Laemmli; Na
true 227 , 680-685 (1970)].

In this test, the purified β-galactosidase obtained in Example 2 described below was used as a standard, and RNA-polymerase (16) was used as a molecular weight marker protein.
5000, 155,000, 39000), bovine serum albumin (68000), trypsin inhibitor (215
00) was used. The results of staining with 0.02% Coomassie Brilliant Blue R250 after electrophoresis are shown in FIG.

In FIG. 1, lanes 1 to 6 are as follows, respectively. Lane specimen 1 molecular weight marker 2 purified β- galactosidase three invention cell extract 4 supra to 70 ° C. microbial cell extract 6 Same as above the liquid 5 host microorganism heat-treated 15 minutes 70 ° C., the liquid view was heat treated 15 minutes 1 As is clear from the above, the components of the cell extract of the microorganism of the present invention (lane 3) and the cell extract of the host microorganism (lane 5) are the same except that the former contains the enzyme of the present invention and the latter does not contain it. , Containing almost the same variety of ingredients.

The various components other than the enzyme of the present invention almost completely disappeared from the samples (lane 4 and lane 6) heat-treated at 70 ° C. for 15 minutes. Example 2 Purification and physicochemical properties of β-galactosidase Bacillus subtilis MI 1 obtained by culturing in the same manner as in Example 1 (medium solution amount: 4.5 liters) and centrifuging
650 ml of acetone was added to 72 g of wet bacterial cells of 11 (pHG5), and the mixture was stirred at room temperature for 1 hour and then filtered to obtain dried bacterial cells. Dried bacterial cells in 0.1M phosphate buffer (pH 7.0)
The suspension was suspended in 1.3 liters and shake-extracted at 4 ° C. overnight and at 30 ° C. for 21 hours, followed by centrifugation (10000).
The resulting supernatant was used as a cell extract. The cell extract (total activity 52,000 U) was heat-treated at 60 ° C. for 1 hour and then centrifuged (15,000 rpm, 20 minutes) to obtain a supernatant. Heat treated supernatant (50,000 U) 3 times with water
A 0.05M phosphate buffer solution (pH 7.
0) equilibrated with DEAE-Sephadex A-50
1 liter was added and the enzyme was adsorbed by the batch method. 0.
After washing the resin with 05 M phosphate buffer (pH 7.0), the enzyme was eluted twice with 1 liter of 0.05 M phosphate buffer (pH 7.0) containing 0.5 M NaCl. Ammonium sulfate was added to the eluate (39,000 U) to 70% saturation to precipitate the enzyme. Centrifuge the precipitate (1200
(0 rpm, 15 minutes), collected, dissolved in 15 ml of 0.02 M phosphate buffer (pH 7.0), heat treated at 70 ° C. for 15 minutes, and then centrifuged (36000 rpm, 1 hour).
The supernatant was obtained. Heat-treated supernatant (33,600 U) was added to 0.
The column (2.5 × 95 cm) of Toyopearl HW-55 equilibrated with a 05 M phosphate buffer (pH 7.0) was loaded onto the column, and each 4 ml was collected. Active fraction (fraction number 47-5
(8,10,200 U) were collected and loaded onto a DEAE-Sephadex A-50 column (4 × 40 cm) equilibrated with 0.05 M phosphate buffer. Elution was performed by changing the NaCl concentration linearly from 0 to 0.5 M, with a total volume of 2.5 liters and a flow rate of 100 ml / hour, and 15 ml each was collected. The active fractions (fraction numbers 123 to 135) were collected and subjected to ultrafiltration (fraction molecular weight 50,000, Toyo Roshi Paper UK50).
The membrane was concentrated to 10 ml to obtain a purified β-galactosidase standard (7,400 U, specific activity 170 U / mg protein).

Next, the physicochemical properties and test method of the enzyme of the present invention will be described in detail. (1) Action and Substrate Specificity A substrate having a β-D-galactoside bond is hydrolyzed to release D-galactose. A typical substrate is lactose or 4-O-β-D-galactopyranosyl-D.
-Glucopyranose, ONPG and P-nitrophenyl β-D-galactopyranoside. (2) Optimum pH and stable pH range Optimum pH 5.5 (see FIG. 2) Stable pH range 5.5 to 9.0 The optimum pH is determined from the measured values of the enzyme activity in the McKeelvine buffer solutions of various pHs. The stable pH range was determined from the measured value of the residual activity of the enzyme treated at 60 ° C. for 90 minutes in a McKeelvine buffer solution of various pH.

Each measured value was expressed as a ratio (relative activity rate and residual activity rate) to the maximum value. However,
In the measurement at a pH of 7.5 or higher, glycine-sodium hydroxide buffer solution was used instead of McKeelvine buffer solution. (3) Optimum temperature range for action Optimum temperature for activity Approx. 70 ° C. (see FIG. 3) This value was determined from the enzyme activity values measured in various pH values of McKeelvine buffer at pH 5.5. (4) Stable temperature range The stability with respect to temperature is as shown in Table 2.
It has a half-life at 60 ° C. of 150 hours and shows very good thermal stability.

This value was determined from the half-life of the enzyme activity measured at various temperatures in a McKeelvine buffer solution of pH 7.0.

[0028]

[Table 2] (5) Effect of metal ion As shown in Table 3, it is particularly inhibited by silver ion and mercury ion.

[0029]

[Table 3] The results shown in Table 3 were obtained by measuring the activity of this enzyme when various reagents were added to the reaction system of ONPG substrate at a predetermined concentration, and the values were shown as a percentage (relative activity rate) with respect to the value without addition. . (6) Molecular weight: about 67,000 The molecular weight (MW) was determined by the above-mentioned SDS-polyacrylamide gel electrophoresis method. (See FIG. 1) The molecular weight measured by the gel filtration method using a TSKG3000SW column manufactured by Toyo Soda Co., Ltd. was 24-2.
A value of 50,000 was shown, but this value seems to be an effect of enzyme association. (7) Michaelis constant for lactose The Michaelis constant (Km) for a lactose substrate is defined as Li.
newerer-Burk plot (edited by Nankodo, "Basic Experimental Methods for Proteins and Enzymes", edited by Takeichi Horio, Nihei Yamashita, 3
Page 87, 1981), the Km was 2.4 mM.

This value was measured simultaneously with the commercially available enzymes L and O.
And G have Km values of 43.1, 39.4 and 2 respectively.
Although it was 6.6, it was remarkably low, suggesting that the substrate affinity of this enzyme for lactose is remarkably large. Comparative Example
When compared with stearothermophilus β-galactosidase, as shown in Table 4, it was confirmed that the present enzyme is a novel enzyme which has not been heretofore known and has excellent heat resistance.

[0031]

[Table 4]

[Brief description of drawings]

FIG. 1 shows the results of SDS-polyacrylamide gel electrophoresis, and the samples in each lane are as follows. Lane specimen 1 molecular weight marker 2 purified β- galactosidase three invention cell extract 4 supra to 70 ° C. microbial, 70 ° C. The cell extract 6 Same as above the liquid 5 host microorganism heat-treated 15 minutes, a solution obtained by heat treatment 15 min

FIG. 2 shows the effect of pH on the activity of this enzyme.

FIG. 3 shows the effect of temperature on the stability of this enzyme.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] June 7, 1993

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] Brief description of the drawing

[Correction method] Delete

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12R 1: 125) (C12N 1/21 C12R 1: 125) (C12N 15/56 C12R 1:07)

Claims (2)

[Claims] 1. A novel thermostable β-galactosidase having the following physicochemical properties 1) Action and substrate specificity A substrate having a β-D-galactoside bond is hydrolyzed to release D-galactose. 2) Optimum pH and stable pH range Optimum pH: 5.5 Stable pH range: 5.5 to 9.0 3) Optimum temperature range of action Optimal temperature of activity: Approx. 70 ° C 4) Stable temperature range 55 ° C, The half-life of β-galactosidase activity at 60 ° C and 65 ° C is about 620 hours, 150 hours, and 55 hours, respectively. 5) Effect of metal ions When adding a concentration of 1 mM, Mg ²⁺ , Ca ²⁺ , Cu ²⁺ , Fe
²⁺ , Co ²⁺ and Li ⁺ do not inhibit the activity of this enzyme,
Ag ⁺ inhibits about 80% or more, and Hg ²⁺ inhibits about 90% or more. 6) Molecular weight: about 67,000 (by SDS-polyacrylamide gel electrophoresis) 7) Michaelis constant (Km) for lactose
2.4 mM 2. Bacillus subtilis Bacillus subtilis MI 111 (pHG5) carrying a novel recombinant plasmid pHG5 in which a DNA fragment containing the thermostable β-galactosidase gene of Bacillus stearothermophilus is incorporated into vector plasmid pUB110. A method for producing heat-resistant β-galactosidase, which comprises culturing in a medium containing an appropriate nutrient source and collecting the heat-resistant β-galactosidase according to claim 1.