JPS60141288A - Production of heat-resistant beta-galactosidase - Google Patents

Abstract

PURPOSE:To provide the titled enzyme having further improved heat-resistance, by culturing a beta-galactosidase-producing strain belonging to Aspergillus oryzae in a medium having suppressed alkaline protease activity. CONSTITUTION:The objective enzyme can be prepared by culturing a beta-galactosidase-producing-strain belonging to Aspergillus oryzae [preferably Aspergillus oryzae L-83 (FERM-P No. 7379), etc.] in a medium having an alkaline protease activity suppressed e.g. by the addition of an alkaline protease inhibitor.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing thermostable β-galactosidase using a microorganism belonging to Aspergillus oryzae.

β-galactosidase is an enzyme that hydrolyzes lactose into galactose and glucose, and is widely used as an enzyme that decomposes lactose in dairy products or as a medicine to treat diarrhea caused by lactose intolerance.

In particular, when processing milk in the food industry, it is preferable to process it at high temperatures to avoid bacterial contamination, and there is a strong need for inexpensive heat-stable β-galactosidase. Conventional commercially available β-galactosidase produced by Aspergillus oryzae is known to be almost completely inactivated by heat treatment at 60°C for 30 minutes, while others show 40-50% residual activity. cannot be said to be sufficient in terms of heat resistance.

The present inventors investigated various culture conditions for Aspergillus oryzae in order to create β-galactosidase with better heat resistance.

First of all, the present inventors varied the medium composition for culturing Aspergillus oryzae and measured the heat resistance of β-galactosidase produced therein.
As a result of measuring the alkaline protease activity, it was found that there was a very close correlation between the two, and that the lower the alkaline protease activity of the culture, the more excellent the heat resistance of β-galactosidase was.

Next, the present inventors used a medium in which alkaline protease activity was suppressed using two different methods.

The present invention was completed by culturing Aspergillus oryzae and confirming that the β-galactosidase produced therein has excellent heat resistance.

That is, the present invention provides ``β-
The present invention relates to a method for producing heat-stable β-galactosidase, which comprises culturing galactosidase-producing bacteria in a medium in which alkaline protease activity is suppressed.

β belonging to Aspergillus oryzae used in the present invention
- Galactosidase-producing bacteria are not particularly limited, but in order to obtain enzymes with particularly excellent heat resistance, it is of course better to use microorganisms that themselves have a high ability to produce heat-resistant β-galactosidase. It's advantageous. Conventionally, a mutant strain of Aspergillus 9 oryzae Y-22, YU-22B, has been reported as a thermostable β-galactosidase producing bacterium. [Journal of Fermentation
Technology, Vol. 58, pp. 115-122 (1980)] The present inventors have developed a wild strain that produces β-galactosidase, which exhibits superior heat resistance comparable to YU-22B, at the Sagami-Oi Plant of Wakamoto Pharmaceutical Co., Ltd. It was isolated from soil on the premises and confirmed to be a strain belonging to Aspergillus oryzae.

It was named Aspergillus oryzae L-83 and deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology.

The deposit number is Microtechnical Research Institute Deposit No. 737T.

Furthermore, the present inventors created a mutant strain by irradiating the L-83 strain with ultraviolet rays, isolated a strain with low alkaline protease production performance, cultured it pure, and used this strain as "Aspergillus oryzae U-8". This was also deposited with the Microbial Technology Research Institute.

The deposit number is FEK deposit number 737g.

Next, the mycological properties of Aspergillus oryzae L-83 and U-8 will be explained.

[l] Mycological properties of Aspergillus oryse L-83 strain Strain used: Aspergillus oryse L83 One platinum loop of conidia obtained by culturing the above strain on malt extract agar medium was inoculated into the specified medium. did.

Media used: malt extract agar, potato/glucose agar,
Three types of Czapek agar medium with a diameter of 9zw and a thickness of 1.5
It was used by dispensing 25 pieces into crn plastic petri dishes.

■, Morphological properties The results observed after culturing at 30°C for 87 hours are described.

(1) Malt extract agar medium: Growth is vigorous. Abundant velvety 1-2 myn white aerial mycelia with pale yellow-brown conidia on the entire surface are attached.

No soluble dyes are observed.

(2) Potato/glucose agar medium: Growth is vigorous.

The growth condition is similar to that of malt extract agar culture, and one or two aerial mycelia grow densely over the entire surface of the bacterial flora, forming a velvet-like shape, and the center is swollen by 2 to 3 meters. Conidia are greenish-brown in color.

The underside of the bacterial flora is smooth and white. No production of soluble pigment is observed.

(3) Zapek agar medium: Growth is good, but inferior to malt extract agar culture. The conidia are yellow, colored in the center of the flora, and the outer edge is slightly raised with white short (0.5 to 1 hectare) dense aerial hyphae. Aerial hyphae with conidia are 1-2 cm long.

The back surface is smooth and pale yellow, but no soluble pigment is observed.

■9 minutes The source of this bacterial strain was the premises of Wakamoto Pharmaceutical Sagami Oi Factory (
It was isolated from soil collected from 378 Kanate, Oimachi, Ashigarakami-gun, Kanagawa Prefecture.

(2) Microscopic observation The results of observations on bacteria cultured on malt extract agar plates (30°C, 87 hours) are described.

The tip of the conidiophore is a flask-shaped apical sac with a width of 25 to 50 μm, and the apical sac consists of numerous traculi-shaped probes (length 10
~20 μm×5 to 8 μm in diameter) is settled (1 stage), and conidia of 6 to 8 μm in size are produced from the tip. The surface of conidia is smooth. The surface of the conidiophore (approximately 300 to 1000 μm in length x 10 to 20 μm in width) is rough, especially in the area near the nuchal sac.

Angiosperms are not formed.・ ■Physiological properties (1) Optimal growth conditions: The optimal pH for growth is 4-11, and the optimal temperature is 25-11.
The temperature is 35°C and it is aerobic.

(2) Conditions under which it can grow: It can grow in a wide pn range of pH 3 to 12 and at a temperature of 15 to 40°C.

Based on the results of the observation of the mycological properties mentioned above, Zazyenus Aspergillus 357~
404 pages (1965) and Food Hygiene Journal, Vol. 13, 1-1
A comparative study was carried out according to the classification method described on page 1 (1972). As a result, there were 9 strains.

It was identified as Aspergillus oryzae because it had properties consistent with Aspergillus oryzae.

[■] Mycological properties of Aspergillus oryzae U-8 Strain used: Aspergillus oryzae U-8 One platinum loop of conidia obtained by culturing the above strain on malt extract agar medium in a specified medium was inoculated.

Medium used: The same medium as for Aspergillus oryzae L-83 strain was used.

■, Morphological properties The results observed after culturing at 30°C for 87 hours are described.

(1) Malt extract agar medium: Growth is good. It grows in abundance with 1 to 2 velvety white aerial mycelia covered with white conidia. No soluble dyes are observed.

(2) Potato/glucose agar medium: Growth is good.

The growth state is similar to that of malt extract agar culture, and aerial mycelium of 1 to 2 mm grows densely over the entire surface of the bacterial flora, forming a velvet-like shape, and the center is swollen by 2 to 3 meters. Conidia are pale yellow.

The underside of the bacterial flora is smooth and white. No production of soluble pigment is observed.

(3) Zapek agar culture medium: Although growth is somewhat good, it is inferior to malt extract agar culture. Conidia are white.

The characteristic of huge settlements is cavities) (CapiLat
e). Aerial hyphae are extremely short (01-0.5
mm).

The back surface is smooth and pale yellow, but no soluble pigment is observed.

11. Isolation The conidia of Aspergillus oryzae L-83 were isolated from a strain obtained by mutating them by irradiating them with ultraviolet light.

■・Microscopic observation The results of observations on bacteria cultured on malt extract agar plate 1 (30°C, 87 hours) will be described.

The tip of the conidiophore is flask-shaped and has an apical sac with a width of 10 to 15 μm.
0μm×width 5-8μm) in a relatively small number (1 stage),
From its tip, 6 to 5 μm of conidia are produced. A deformed type of apical capsule is also seen. The surface of conidia is smooth. Conidiophore (
The surface (around 150 to 500 μm in length x 7 to 10 μm in width) is somewhat rough. Angiosperms are not formed.

■Physiological properties (1) Optimal growth conditions: The optimal pH for growth is 4-11, and the optimal temperature is 25-11.
The temperature is 35°C and it is aerobic.

(2) Conditions for growth In one wide range of pH 3 to 12, the temperature is 15 to 401? .

It can be grown in

The present invention relates to a method for producing thermostable β-galactosidase, which is characterized by culturing these β-galactosidase-producing bacteria belonging to Aspergillus oryzae in a medium in which alkaline protease activity is suppressed. As a method for suppressing alkaline protease activity in the medium, for example, the following method is effective.

■ Add alkaline protease inhibitor to the medium.

■ Add an acidic buffer and a nitrogen source substance to the culture medium.

■ Use alkaline protease low titer mutants.

The above methods are effective even when used alone.

It is more effective to employ them in combination.

As alkaline protease inhibitors, known inhibitors such as potato extract and diisopropylhonufofloridate can be used as appropriate.

Since an acidic buffer and a nitrogen source substance are used together, the most convenient substances are substances that have both components, such as monoammonium phosphate and diammonium citrate. These are usually 40 cm or more per 12 wrinkles, preferably 50 to 150
It is preferable to add F#.

Of course, the acidic side buffer and the nitrogen source substance can be used in appropriate combinations of independent components.

Examples of acidic buffers include phosphates.

Weak inorganic and organic acid salts such as citrate, borate, acetate, etc. can be used.

Inorganic and organic nitrogen compounds can be used as nitrogen source substances. Representative examples include peptone, meat extract, and amino acids.

Examples include urea, ammonium nitrate, sodium nitrate, ammonium chloride, ammonium sulfate, and the like. In the case of urea, it is sufficient to add these in an amount of about 137n9 per branch If, but in other cases, it is generally about 50 to 15 (about 19 is appropriate).

Next, the effects of the present invention will be explained using test examples.

Test example 1. Heat resistance of β-galactosidase [Second and number 1]
Effect of alkaline protease activity on medium (a) Test method Add the components shown in Table 1 to a sterile medium containing 91.5 f and 2.5 m of water, and inoculate conidia of Asbergilnu oryzae L-83. and cultured at 30°C for 50 hours.

Water 15- was added to the culture, and the enzyme was extracted by shaking vigorously at 30°C for 1 hour to obtain liquid P. As for the residue.

Matsukirbain buffer (pH 4,5) 5- was added to make the mixture sufficiently homogeneous, and then centrifuged at 15,000 rpm for 10 minutes to obtain a supernatant. This was combined with solution F and used as a sample for measuring enzyme activity.

β-galactosidase activity and alkaline protease activity were measured for each of the extracts obtained from cultures in media of various compositions as described above, and the extracts were further incubated at pH 4 and 5 at 60°C.

The residual activity of β-galactosidase after heating for 30 minutes was measured, and the residual activity rate was calculated.

The thermostability of β-galactosidase was evaluated.

Table 1 The results of this test are as shown in Table 2 below. As is clear from the results shown in the table.

The heat resistance of β-galactosidase was better when the alkaline protease activity of the medium was lower.

Table 2 Test Example 2. Effect of addition of alkaline protease inhibitor on the heat resistance of β-galactosidase (a) Test method @1. st, -y-y stay liquor 75m9. water 2
．． Conidia of Aspergillus oryzem-83 strain were inoculated into a sterilized solid medium containing 5- and cultured at 30°C for 20 hours.
50 μt of potato extract solution with t2/− concentration.

Cultures were prepared by adding 100 μt, 250 μt, and 500 μt, and the same amount of water was added as a control for each.

After culturing each culture at 30° C. for 30 hours, the heat resistance of β-galactosidase and alkaline protease activity were measured in the same manner as in Test Example 1.

(b) Test results The results of this test are shown in Table 3.

Table 3 As is clear from the results in Table 3, adding potato extract to the medium to inhibit the alkaline protease activity of the medium significantly improved the heat resistance of β-galactosidase produced therein. .

Next, we used Aspergillus oryzae strain 83, a mutant strain with low alkaline protease activity obtained by irradiating this strain with ultraviolet rays, and Aspergillus oryzae U-8 strain. 9. We will compare the enzymes produced when culturing according to the method of the present invention and explain that the method of the present invention is effective regardless of the type of microorganism.

Test Example 3 A solid medium (normal medium) containing 152゜Shinsui 25゜ and a medium containing 115㎜ of 1 ammonium phosphate (
The conidia of Aspergillus oryzae strain 83 and Aspergillus oryzae strain U-8 were each inoculated into the medium of the present invention (30 tl).

The cells were cultured statically for 60 hours.

The enzyme was extracted from the culture in the same manner as in Test Example 1, and the heat resistance of β-galactosidase and the alkaline protease value were measured.

The results of this test are shown in Table 4.

Table 4 Example 1 45 tons of mosquitoes, 7.5 mg of water, (NH4)H2PO4,0
, 59 (11.1% for wrinkles) was inoculated with conidia of Aspergillus oryzem-83 strain,
Static culture was performed at 30°C for 60 hours.

Cultures were treated with 60-Mazukirpine buffer (pH 4,5).
) was added and shaken for 2 hours, and then left overnight at 4°C.

The crude enzyme was precipitated from this supernatant by treatment with 50 to 95% ammonium sulfate, and this was then added again to Matskilpine buffer (pH 4).
, 5), and the enzyme titer and protein of the suspension were determined.

As a result, the amount of β-galactosidase produced per gram of wrinkles was 38.0 units, the amount of crude protein produced was 27.6 gin, and the specific activity was 1438 U/n9 protein.

This β-galactosidase is p)! 4.5.60℃
.

It showed a residual titer of 68.9% after 30 minutes of heat treatment, and had significantly excellent heat resistance.

For comparison, culture was performed in the same manner as above except that (NH,)H2p04 was used at 0.03F (0.67% for mosquitoes).

A similar test was conducted. As a result, the specific activity of β-galactosidase (1,19 U/m9 protein) and the production amount per wrinkle 12 (36,9 units) showed similar values, but the heat resistance (31,3%) was about 100%. showed the value of

The above test examples and examples are for explaining the present invention.1 The present invention provides that when a microorganism belonging to Aspergillus oryzae is cultured to produce β-galactosidase, if the alkaline protease activity of the medium is suppressed, This invention relates to a basic invention in which the heat resistance of β-galactosidase produced therein is significantly increased.

Specific methods for suppressing alkaline protease activity in the culture medium are not limited to the above test examples and examples, but can be carried out by those skilled in the art by appropriately combining known methods.

In the above Test Examples and Examples, the β-galactosidase value, alkaline protease activity, β-galactosidase heat resistance, and protein quantitative value were each shown as measured values by the following methods.

(Method for measuring β-galactosidase value) ONPG substrate method was used.

That is, 3.5 mmol of substrate solution containing 5.7 mmol of 0-nitrophenyl-β-D-galactobiranondo (ONPG)
ml (pH 4,5) of enzyme solution 0.5fnI! Mix and react at 30°C for 10 minutes, then add 1 mol of lithium carbonate solution 1-
was added to stop the reaction. The amount of O-nitrophenol produced in this solution was determined by measuring the absorbance at 420 nm. The amount of enzyme that hydrolyzes 1 μmol of 0NPG per minute was defined as 1 unit (U).

(Method for measuring alkaline protease activity) Protease activity was measured in accordance with the modified Anson-Hagiwara method. That is, 0.5 m of enzyme solution containing 20 mmol of EDTA (
pH 7.0) at 40°C for 15 minutes to inactivate neutral protease.

2.5mj! 0.6% (W/V) casein, 50
Mix mmolar disodium phosphate solution (pH 7.0) and react at 30°C for 10 minutes, then add 2.5 ml of a mixture of 0.11 molar trichloroacetic acid, 0.22 molar sodium acetate, and 0.33 molar acetic acid. The reaction was stopped. 3
After standing at 0°C for 30 minutes, a portion of the reaction solution was thoroughly filtered. The increase in non-protein Folin reagent colored substance corresponding to tyrosine produced in the sap was quantified. The amount of tyrosine produced in reaction solution 1 in 1 minute was defined as 1 μmol tyrosine.

(Heat resistance test method for β-galactosidase) pH 4, 5,
After treatment at 60°C for 30 minutes, the residual activity ratio (correspondence) capable of decomposing the substrate at 30°C was determined.

(quantification of protein) By the Lowry method.

patent applicant Wakamoto Pharmaceutical Co., Ltd.

Claims (1)

[Claims] 1. A method for producing heat-stable β-galactosidase, which comprises culturing β-galactosidase-producing bacteria belonging to Aspergillus oryzae in a medium in which alkaline protease activity is suppressed.