JPH0773495B2 - Heat and salt resistant glutaminase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of use] The present invention relates to a novel heat- and salt-resistant glutaminase.

[Conventional technology]

Glutaminase is an enzyme that plays an important role in the food industry, particularly when a protein is enzymatically decomposed to produce a seasoned food (see, for example, Japanese Patent Publication No. 49-48759).

[Problems to be solved by the invention]

The conventionally known glutaminase is the above-mentioned Japanese Patent Publication No. 49-4875.
There is no salt tolerance at all, including those described in No. 9, or it is relatively weak, so there is a problem in producing foods such as seasonings containing a large amount of salt using such glutaminase. was there.

When a protein is enzymatically decomposed, a raw material protein is finally decomposed into constituent amino acids through peptides by the action of various proteolytic enzymes. Glutamine produced in such a case is easily converted into pyroglutamic acid having a phenol odor and a bitter taste in a non-enzymatic manner in the absence of glutaminase. Also, the rate of conversion of glutamine to proglutamic acid increases as the temperature increases. It is known that coexistence of glutaminase in such a decomposition system can prevent glutamine from changing into pyroglutamic acid, and can be converted into glutamic acid in an amount commensurate with free glutamine.
Producing amino acid seasoned foods by enzymatically decomposing proteins at high temperature in the presence of salt is an extremely effective method as a means for producing good seasonings by increasing the rate of protein degradation and preventing contamination by various bacteria as much as possible. Is. However, in order to adopt such conditions, it is necessary that the glutaminase used has heat resistance and salt resistance.

[Means for solving problems]

In view of such a current situation, the present inventor has conducted extensive studies to provide a heat-resistant and salt-tolerant glutaminase that can be used for the production of foods containing high-concentration salt, and as a result, newly cultivated bacteria belonging to the genus Prera. It was found that a novel glutaminase excellent in heat resistance and salt resistance and free from food hygiene problems can be obtained from a product, and the present invention has been completed.

That is, the present invention provides a thermostable and salt-tolerant glutaminase produced by a bacterium belonging to the genus Brera and having the following physicochemical properties.

(A) Working This is an enzyme that hydrolyzes L-glutamine to produce L-glutamic acid and ammonia.

(B) Substrate specificity The Km value (Michaelis constant) for L-glutamine is 30.
It is 8.1 × 10 ^-4 M at pH 7.5 (phosphate buffer) at ℃.

(C) Optimum pH and constant pH constant The optimum pH is 5.0 to 9.0 based on L-glutamine,
The stable pH range is 5.-8.0.

(D) Thermostable The residual rate of glutaminase activity when stored at pH 7.5 for 30 minutes at each temperature is 90% or more at 50 ° C and 71% at 60 ° C.

(E) Salt Tolerance 10% w / when the enzyme activity in the absence of salt is 100%
v) Relative activity of about 80% or more at salt concentration, 20% (w /
v) About 70% or more relative activity at salt concentration (pH 7.5, 30
C).

(F) Molecular weight about 150,000 (gel permeation method) Hereinafter, the physicochemical properties of the heat and salt tolerant enzyme of the present invention will be described in detail.

(1) Operation This enzyme is an enzyme that hydrolyzes L-glutamine to produce L-glutamic acid and ammonia.

(2) Substrate specificity 1.0 ml of each substrate (0.25M) shown in Table 1 was added to 0.25 ml of 0.4M phosphate buffer (pH 7.5), and 1.25 ml of this enzyme was added to this.
The reaction was carried out at 60 ° C for 60 minutes. 0.75N perchloric acid solution 0.625ml
Was added to stop the reaction, and 0.75N sodium hydroxide 0.6
The reaction solution was neutralized by adding 25 ml. The blank value was obtained by adding 0.75N perchloric acid solution 0.625 ml to 1.25 ml of this enzyme, and adding 1.0 ml and 0.25 ml of each substrate and buffer solution (pH 7.5) under the same conditions as above. The treated product (with a reaction time of 0 hours) was used.

Next, the ammonia produced by the enzyme reaction was measured using an F-kittourea / ammonia quantifying kit (manufactured by Boehringer). The relative activity (%) was shown as a comparative value (%) when the amount of ammonia obtained by enzymatic reaction with L-glutamine as a substrate was 100% and other substances were used as substrates.

The Km value (Michaelis constant) of this enzyme for L-glutamine is 8.1 × 10 ⁻⁴ M at 30 ° C. and pH 7.5 (phosphate buffer solution).

(3) Optimum pH and stable pH range When L-glutamine is used as a substrate, the optimum pH of this enzyme is pH 5.0 to 9.0, as shown in FIG. In addition, in Figure 1
◯ indicates that McIlvein buffer (pH 4.5 to 8.0) and Δ-Δ indicates that phosphate buffer (pH 7.5 to 9.0) was used.

The stable pH range was examined by measuring the residual activity after leaving 6 ml of each buffer solution containing 0.25 unit of the enzyme at 30 ° C. for 24 hours. The results are shown in Fig. 2, and were stable at pH 5.0 to 8.0. Incidentally, in FIG. 2, the buffer is a Macilvain buffer (pH 4.5 to 8.0).

(4) Method for measuring glutaminase activity 0.4 M phosphate buffer solution was added to 4.0 ml of 250 mM L-glutamine solution.
Add 0 ml (pH 7.5) and 5.0 ml of this enzyme solution, react at 30 ° C for 60 minutes, and then heat in boiling water for 10 minutes to stop the reaction. Next, using a kit for measuring L-glutamic acid manufactured by Boehringer, 2.3 ml of 25 mM phosphate buffer solution (pH 8.6) containing 6.7 mM of 200 mM triethanolamine in 0.2 ml of the above reaction solution, 6.7.
mM NAD ⁺ solution 0.2 ml, 1.19 mM iodonitrotetrazolium chloride solution 0.2 ml, 0.1% (w / v) diaphorase solution
Add 0.05 ml and 0.05 ml glutamate dehydrogenase, react at room temperature, and measure the absorbance value at 492 nm with a spectrophotometer.

Furthermore, the amount of L-glutamic acid produced was determined from the calibration curve prepared in advance. The amount of enzyme that produces 1 micromol of L-glutamic acid per minute at 30 ° C. was defined as 1 unit.

(5) Optimum temperature range for action As shown in Fig. 3, the optimum temperature range for this enzyme is 55-65 ° C.
(Reaction time 60 minutes, pH 7.5).

(6) Thermostability 6 ml of enzyme solution (pH 7.5) containing 0.25 unit of this enzyme was left at each temperature for 30 minutes, and the residual enzyme activity was measured.
As shown in FIG. 4, 70% residual activity was exhibited at 60 ° C. and 95% residual activity was exhibited at 50 ° C.

(7) Molecular Weight The enzyme has a molecular weight of 0.2M sodium chloride by gel permeation method using Cefacrylic S-300 (manufactured by Pharmacia).
As a result of measurement with 0.1 M phosphate buffer (pH 7.0), it was about 150,000.

(8) Salt tolerance To 5.0 ml of enzyme solution containing 0.25 unit of this enzyme, salt was added so that the salt concentration in the reaction solution was 0 to 20% 250 mM L
-5.0 ml of 0.4 M phosphate buffer containing glutamine was added, and the enzyme activity (pH 7.5, 30 ° C, reaction time 60 minutes) was examined. As a result, as shown in Fig. 5, when the enzyme activity in the absence of salt was 100%, it was about 10% (w / v) in salt concentration.
The relative activity was 80% or higher, and about 70% or higher at 20% (w / v).

(9) Purification method The present enzyme can be purified by the purification method described below.

(10) disc electrophoresis Devuisu [BJDavis, Ann.New York Acad.Sei., 121 , 4
04 (1964)] pH 8.9 gel was run on a 2 mA / gel column at 5 ° C for 90 minutes (migration time of the separating gel), and then stained with amide black, which migrated to the anode side. One band was recognized.

(11) Isoelectric point Ampholin, Carrier, Ampholite (pH3.5
~ 10) was used to measure the isoelectric point of this enzyme.
It was 5 ± 0.2.

As described above, this enzyme is different from the conventional glutaminase in various enzymatic and physicochemical properties, and is particularly highly thermostable and salt resistant. Therefore, when the present enzyme is used in the presence of high-concentration salt, the salt hardly inhibits the enzyme activity, which is advantageous.

Next, a method for producing the heat-resistant and salt-resistant glutaminase according to the present invention will be described.

Examples of the bacterium belonging to the genus Brera used in the present invention include, for example, Brera oryzae JCM528.
One is. This bacterium is stored in RIKEN and is always easily available to any person.

Brerera oryzae JCM5281 is cultivated and heat-resistant from the culture.
The method for producing salt-tolerant glutaminase is carried out as follows.

First, as the culture method, a liquid aerobic culture method is usually advantageous, and examples thereof include a shake culture method using a flask and an aerobic culture method using a jar fermenter equipped with an aeration and stirring device, a tank fermenter, and the like. Culture temperature is 15 to 30 ℃,
The temperature is preferably 20 to 25 ° C., and the culture time is usually about 16 hours or longer.

The medium used is preferably a liquid medium, and various nutrient sources generally used for culturing microorganisms can be used as the nutrient source added to the medium. That is, as the carbon source, for example, glucose, maltose and other monosaccharides and oligosaccharides are used, and as the nitrogen source, for example, peptone, meat extract, yeast extract, corn steep liquor, casamino acid, defatted soybean extract, A hydrolyzate of wheat gluten, an ammonium salt, a nitrate or the like is used. In addition, salts such as magnesium, calcium, sodium and phosphate, trace nutrients and the like may be added.

A culture solution obtained by using such a culture method, or a culture cell obtained by solid-liquid separation of the culture solution by centrifugation, centrifugation or the like, and collecting the cells, a cell wall lysing enzyme-containing substance, for example, a cell wall lysing enzyme The glutaminase is solubilized by adding a culture broth originating from the genus Rhizopus or an enzyme solution obtained from the culture broth having productivity.

The temperature for solubilizing the heat- and salt-resistant glutaminase is 30 to 50 ° C, preferably 35 to 45 ° C, and the liquidity is pH 3.0.
~ 7.0, preferably pH 4.0 ~ 6.0, time is 2 ~ 72 hours, preferably about 16 ~ 48 hours, the concentration of cell wall lysing enzyme during action is 0.1% (w / v) or more, preferably 0.5 ~ 1.5%
(W / v).

As a means for collecting the enzyme from the obtained salt-tolerant glutaminase-containing liquid, for example, the enzyme-containing liquid is centrifuged by a conventional method to remove bacterial cells, and further dialyzed, if necessary, adsorbed on an ion exchange resin and eluted. The heat- and salt-resistant glutaminase according to the present invention can be isolated by a method, gel filtration, or the like.

〔The invention's effect〕

The heat- and salt-tolerant glutaminase according to the present invention has almost no inhibition of the enzyme activity by salt even in the presence of high-concentration salt, so if it is used for producing a food such as a seasoning containing high-concentration salt, the umami taste is improved. It is possible to efficiently obtain a food in which glutamic acid, which is a component, is remarkably enhanced, which is extremely significant in food production.

〔Example〕

 Examples of the present invention are shown below.

Example 1 Bullera Oryzae JCM5281 was inoculated in a 500 ml Sakaguchi flask in 100 ml of a liquid medium containing glucose 1%, yeast extract 0.3%, malt extract 0.3% and peptone 0.5% and adjusted to pH 5.5, It was cultured at 25 ° C for 48 hours. This seed culture was inoculated into the medium 3 having the same composition as described above in a 5-jar fermenter, the aeration rate was 1 / min, and the stirring speed was 300r.
After culturing at 25 ° C. for 48 hours under the condition of pm, the culture was centrifuged with a centrifuge to collect the cells.

Of the cells obtained, 150 g of 0.2 M acetate buffer (pH 5.0)
1 was added to disperse the bacteria well. Next, 8 g of Gluczyme 6000 (manufactured by Amano Pharmaceutical Co., Ltd.) as a cell wall lytic enzyme was added to this.
In addition, the mixture was stirred at 40 ° C. for 16 hours, then centrifuged and then centrifuged (10,000 rpm, 10 minutes) to obtain a supernatant.

Amberlite CG-50 buffered with 0.02M acetate buffer (pH 5.0) was added to the above supernatant, and the mixture was stirred for 1 hour to adsorb the enzyme, and 0.02M acetate buffer (pH 5.0) was added. Wash well with
Then, the active fraction was collected by elution with 7% (w / v) ammonium sulfate (pH 7.0).

Next, ammonium sulfate was added to this enzyme solution to 0.8 saturation, followed by centrifugation (10,000
At 10 rpm for 10 minutes), a precipitate was obtained.

The obtained precipitate was dissolved in 0.02 M phosphate buffer (pH 6.5) and dialyzed against 0.02 M phosphate buffer (pH 6.5) to obtain a crude enzyme solution.

DEAE-Toyopearl 650M column (manufactured by Toyo Soda Co., Ltd.) obtained by buffering the above crude enzyme solution with 0.02M phosphate buffer (pH 6.5)
The enzyme was adsorbed through the column, washed well with 0.02 M acetate buffer (pH 6.5), and then eluted with a concentration gradient of NaCl 0 to 0.5 mol to collect the active fraction. Next, this enzyme solution was concentrated with an ultrafiltration device (fractionation membrane 10,000, manufactured by Amicon). The concentrated enzyme solution was added with 0.1 M phosphate buffer solution containing 0.2 M sodium chloride (pp. 7.
The gel was applied to a column (5.0 × 80 cm) packed with Cefacrylic S-300 buffered in (0).

The obtained active fraction was concentrated with an ultrafiltration device to obtain 4.7 mg of a single enzyme preparation. As a result, the specific activity of this enzyme was 5.5 units.
It was / mg.

Example 2 Bullera Oryzae JCM5281 was inoculated in a 500 ml Sakaguchi flask into 100 ml of a liquid medium containing glucose 1%, yeast extract 0.3%, malt extract 0.3% and peptone 0.5% and adjusted to pH 5.5, It was cultured at 25 ° C for 48 hours. This seed culture was inoculated into the medium 3 having the same composition as described above in a 5-jar fermenter, the aeration rate was 1 / min, and the stirring speed was 300r.
After culturing at 25 ° C. for 48 hours under the condition of pm, the culture was centrifuged with a centrifuge to collect the cells.

Of the cells obtained, 150 g of 0.2 M acetate buffer (pH 5.0)
1 was added to disperse the bacteria well. Next, Gluczyme 6000 (manufactured by Amano Pharmaceutical Co., Ltd.) as a cell wall lysing enzyme was dissolved in 0.02M acetate buffer and filled with Sephadex G-50 (manufactured by Pharmacia) buffered with 0.02M acetate buffer (pH 5.0). The gel was applied to a column (580 cm), and the whole amount of the obtained fraction having cell wall lysing activity was added, and the mixture was stirred at 40 ° C for 16 hours and then centrifuged (10,000 rpm, 10 rpm).
To give a supernatant.

Amberlite CG-50 buffered with 0.02M acetate buffer (pH 5.0) was added to the above supernatant and stirred for 1 hour to adsorb the enzyme, and then 0.02M acetate buffer (pH 5.0) was added. The active fraction was collected by thorough washing, followed by elution with 7% (w / v) ammonium sulfate (pH 7.0). Next, ammonium sulfate was added to this enzyme solution so as to have a saturation of 0.8, and then centrifuged (10,000 rpm, 10 minutes) with a centrifuge to obtain a precipitate. The obtained precipitate was dissolved in 0.02 M phosphate buffer (pH 6.5) and dialyzed against 0.02 M phosphate buffer (pH 6.5) to obtain a crude enzyme solution.

The above crude enzyme solution was passed through DEAE-Toyopearl 650M (manufactured by Toyo Soda Co., Ltd.) buffered with 0.02 M phosphate buffer (pH 6.5) to adsorb the enzyme, and 0.02 M phosphate buffer (pH 6.5). ), Followed by elution with a concentration gradient of NaCl 0 to 0.5 M to collect active fractions. Next, this enzyme solution was concentrated with an ultrafiltration device (fractionation membrane 10,000, manufactured by Amicon). The concentrated enzyme solution was added with 0.1 M phosphate buffer (pH 7.
The gel was applied to a column (5 × 80 cm) packed with Cefacrylic S-300 buffered in (0).

The obtained active fraction was dialyzed against 0.02M acetate buffer (pH 5.0) and passed through DEAE-Toyopearl 650M (manufactured by Toyo Soda) buffered with 0.02M acetate buffer (pH 5.0) to adsorb the enzyme. And wash well with 0.02M acetate buffer (pH 5.0), followed by Na
The active fractions were collected by elution with a gradient of Cl0 to 0.1M. Next, this enzyme solution was concentrated with an ultrafiltration device (fractionation membrane 10,000, manufactured by Amicon). A column (5 x 80 cm) packed with Cefacryl S-300 in which the concentrated oxygen solution was buffered with 0.1 M phosphate buffer (pH 7.0) containing 0.2 M sodium chloride.
I ran through the gel. The obtained active fraction was concentrated with an ultrafiltration device to obtain 1.15 mg of a single enzyme preparation. As a result,
The specific activity of this enzyme was 22.5 units / mg.

[Brief description of drawings]

FIG. 1 is a drawing showing the optimum pH of the enzyme, FIG. 2 is a drawing showing a stable pH range, FIG. 3 is a drawing showing a suitable temperature range of action, FIG. 4 is a drawing showing thermostability, and FIG. The figure is a drawing showing salt resistance.

Front page continuation (72) Inventor Toshio Tanaka 10-52 Kamimae-cho, Nagahama-shi, Shiga Excellent No. 105 Kamimae (72) Inventor Fumio Hara 947-3, Masagoba, Kawagoe-shi, Saitama Prefecture (72) Hiroshi Nagi, Saitama Prefecture 2-23-16 Midorigaoka, Oi-cho, Iruma-gun

Claims (1)

[Claims] 1. A heat- and salt-resistant glutaminase having the following physicochemical properties, which is produced by a bacterium belonging to the genus Brera. (A) Action It is an enzyme that hydrolyzes L-glutamine to produce L-glutamic acid and ammonia. (B) Substrate specificity The Km value (Michaelis constant) for L-glutamine is 30.
It is 8.1 × 10 ^-4 M at pH 7.5 (phosphate buffer) at ℃. (C) Optimum pH and stable pH range The optimum pH is 5.0 to 9.0 when L-glutamine is used as a substrate, and the stable pH range is 5.0 to 8.0. (D) Thermostability The residual rate of glutaminase activity when stored at pH 7.5 for 30 minutes at each temperature is 90% or more at 50 ° C and 71% at 60 ° C. (E) Salt-tolerance 10% (w
/ v) Relative activity of about 80% or more at salt concentration, 20% (w /
v) About 70% or more relative activity at salt concentration (pH 7.5, 30
C). (F) Molecular weight about 150,000 (gel method)