JPS6350994B2 - - Google Patents

Description

[Detailed description of the invention]

[1] Object 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 glycose, 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, and others to show residual activity of 40-50%. cannot be said to be sufficient in terms of heat resistance. The present inventors previously conducted various studies regarding a method for producing β-galactosidase by culturing an Aspergillus oryzae strain in order to find preferable culture conditions for the purpose of improving yield.
Surprisingly, we found that changing the culture conditions not only changes the enzyme productivity, but also the heat resistance of the enzyme produced by the same bacterial strain. The present invention is based on the results of various studies on culture conditions for Aspergillus oryzae for the purpose of creating β-galactosidase with better heat resistance. First, the present inventors changed the medium composition for culturing Aspergillus oryzae and measured the heat resistance of β-galactosidase produced therein, and at the same time measured the alkaline protease activity. It has been found that there is 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 is. Next, the present inventors used various media in which the expression of alkaline protease activity was suppressed,
The present invention was completed by culturing Aspergillus oryzae and confirming that the β-galactosidase produced therein has extremely excellent heat resistance. [2] Structure of the invention The scope of the claims of the present invention is as follows. (1) A medium containing β-galactosidase-producing bacteria belonging to Aspergillus oryzae with the addition of an alkaline protease inhibitor, a medium with an acidic buffer containing a nitrogen atom, and a medium with a combination of an acidic buffer and a nitrogen source substance. The heat-resistant β-
A method for producing heat-stable β-galactosidase, which comprises collecting galactosidase. (2) The method for producing thermostable β-galactosidase according to claim 1, wherein the medium is a solid medium. (3) Heat resistance according to claim 1, wherein the medium to which an acidic buffer containing nitrogen atoms is added is monoammonium phosphate or diammonium citrate in an amount of 40 to 150 mg per gram of medium raw material. Method for producing β-galactosidase. (4) A medium to which an acidic buffer and a nitrogen source substance have been added together is a medium in which 40 to 150 mg of an acidic buffer and 10 to 150 mg of a nitrogen source substance are added together per gram of the medium raw material. A method for producing thermostable β-galactosidase according to item 1. 〓 The β-galactosidase producing bacterium belonging to Aspergillus oryzae used in the present invention is not particularly limited, but in order to obtain an enzyme with particularly excellent heat resistance, it is a matter of course that the microorganism itself has a heat-resistant β-galactosidase. - It is more advantageous to use those with high galactosidase production ability. Aspergillus oryzae Y-22 mutant strain 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 discovered a wild-caught strain that produces β-galactosidase, which exhibits excellent heat resistance comparable to YU-22B. The strain was isolated from the soil on the premises of Wakamoto Pharmaceutical Co., Ltd. Aimira Oi Factory, and confirmed to be a strain belonging to Aspergillus oryzae. Deposited in. The deposit number is FEIB deposit number 7379. Furthermore, the present inventors created a mutant strain by irradiating the L-83 strain with ultraviolet rays, isolated a strain with low alkaline pyrotease production ability, cultured it in pure form, and used this strain as "Aspergillus oryzae U-8".
This was also deposited with the Microbial Technology Research Institute. The deposit number is Microtechnical Research Institute No. 7378. Next, Aspergillus oryzae L-83 and U
Explain the mycological properties of -8. [] Mycological properties of Aspergillus oryzae L-83 Strain used: Aspergillus oryzae L83 One platinum loop of conidia obtained by culturing the above strain on a malt extract agar medium was inoculated into the specified medium. . Media used: 3 types of malt extract agar, potato glucose agar, and Czapetsk agar in a plastic sieve with a diameter of 9 cm and a thickness of 25 cm.
It was used by dispensing it in ml portions. Morphological properties The results observed after culturing at 30°C for 87 hours are described. (1) Malt extract agar medium: Growth is vigorous. It grows abundantly with 1-2 mm velvety white aerial mycelia covered with pale yellow-brown conidia. No soluble dyes are observed. (2) Potato glucose agar medium: Growth is vigorous. With a growth condition similar to that of malt extract agar culture, aerial mycelia of 1 to 2 mm in size grow densely over the entire surface of the bacterial flora, forming a velvet-like shape, and the center swells by 2 to 3 mm. Conidia have brown edges. The underside of the bacterial flora is smooth and white.
No production of soluble pigment is observed. (3) Tuapetsk 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 dense white short (0.5-1 mm) aerial hyphae. 1 to 2 aerial hyphae with conidia
The length is mm. The back surface is smooth and pale yellow, but no soluble pigment is observed. Isolation The source of this bacterial strain was the site of Wakamoto Seiyaku Aimei Oi Factory (Kanete, Oimachi, Ashigarakami-gun, Kanagawa Prefecture).
It was isolated from soil collected from 378). Microscopic observation Malt extract agar plate culture (30℃, 87 hours)
We will describe the results of our observations on the bacteria. The tip of the conidiophore is flask-shaped and has a width of 25 to 50 μm.
The apical sac becomes the apical sac, and the apical sac bears (1 stage) a large number of spiky-shaped sycamores (10-20 μm in length x 5-8 μm in width), and conidia of 6-8 μm in size are produced from the tip. . The surface of conidia is smooth. Conidiophore (length around 300-1000 μm x
The surface (width 10-20 μm) is rough, especially near the apex. Angiosperms are not formed. Physiological properties (1) Optimal growth conditions: The optimal pH for growth is 4-11, the optimal temperature is 25-35°C, and it is aerobic. (2) Conditions for growth: wide PH range of 3 to 12, temperature 15 to 40
Can be grown at ℃. Based on the results of the observation of the above-mentioned mycological properties, the classification method described in Aspergillus Zazienus, pp. 357-404 (1965) and Food Hygiene, Vol. 13, pp. 1-11 (1972) A comparative study was conducted according to the following. As a result, this strain 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 was added to the specified medium. 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: Good growth. Abundant 1-2 mm velvety white aerial mycelia with white conidia attached to the entire surface are attached. No soluble dyes are observed. (2) Potato glucose agar medium: Good growth. The growth condition 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 swells by 2 to 3 mm. Conidia are pale yellow. The underside of the bacterial flora is smooth and white. No production of soluble pigment is observed. (3) Tuapetsk agar medium: Although growth is somewhat good, it is inferior to malt extract agar culture. Conidia are white. A characteristic of large settlements is that they are capitate-shaped. Aerial hyphae are extremely short (0.1-0.5 mm). The back surface is smooth and pale yellow, but no soluble pigment is observed. Isolation Conidia of Aspergillus oryzae L-83 strain were isolated from a strain that had been mutated by UV irradiation. Microscopic observation Malt extract agar plate culture (30℃, 87 hours)
We will describe the results of our observations on the bacteria. The tip of the conidiophore is flask-shaped and has a width of 10~
The apical sac has a diameter of 15 μm, and the apical sac has a relatively small number of sycamores (10 to 20 μm in length x 5 to 8 μm in width) attached to it (1 stage), and from the tip there are 6 to
Produces conidia of 8 μm. A deformed type of apical capsule is also seen. The surface of conidia is smooth. The surface of the conidiophore (around 150-500 μm in length x 7-10 μm in width) is somewhat rough. Angiosperms are not formed. Physiological properties (1) Optimal growth conditions: The optimal pH for growth is 4-11, the optimal temperature is 25-35°C, and it is aerobic. (2) Conditions for growth: wide PH range of 3 to 12, temperature 15 to 40
Can be grown at ℃. The present invention utilizes these β-galactosidase-producing bacteria belonging to Aspergillus oryzae in a medium supplemented with an alkaline protease inhibitor, a medium supplemented with an acidic buffer containing nitrogen atoms, and a combination of an acidic buffer and a nitrogen source substance. The present invention relates to a method for producing heat-stable β-galactosidase, which comprises culturing in a medium selected from the culture media and collecting heat-stable β-galactosidase accumulated in the medium. According to the present invention, the expression of alkaline protease activity in the medium is suppressed during the culture period, and thermostable β-galactosidase is accumulated in the medium without impairing the heat resistance of the origin β-galactosidase. Although it is effective to use the above-mentioned and medium alone, it is better and more effective to use them in combination. The microorganism to be used is a β-alkaline protease low titer mutant strain, such as the aforementioned Aspergillus oryzae U-8 (Feikoken Bacteria No. 7378).
The effects of the present invention will be even more effective if galactosidase-producing bacteria are used. As the alkaline protease inhibitor, known inhibitors such as potato extract and diisopropylphosphofluoridate can be used as appropriate. Examples of acidic buffers containing nitrogen atoms include phosphoric acid 1
Ammonium and diammonium citrate can be suitably used. It is generally preferable to add these in an amount of 40 mg or more, preferably 50 to 150 mg, per 1 g. As the acidic buffer, for example, inorganic and organic weak acid salts such as phosphate, citrate, borate, acetate, etc. can be used. Inorganic and organic nitrogen compounds can be used as nitrogen source substances. Typical examples include peptone, meat extract, amino acids, urea, ammonium nitrate, sodium nitrate, ammonium chloride, ammonium sulfate, and the like.
The amount of these additions is 13mg per 1g of urea.
In other cases, generally about 50
~150mg is appropriate. Next, Examples will be shown to explain the embodiments of the present invention in detail. Example 1 4.5g, 7.5ml water, (NH ₄ )H ₂ PO ₄ , 0.5g (
Conidia of Aspergillus oryzae L-83 strain were inoculated into a sterile medium consisting of 11.1%
The cells were statically cultured at ℃ for 60 hours. Add 60 ml of Matsukirbain buffer (PH
After adding 4.5) and shaking for 2 hours, the mixture was left at 4°C overnight. The crude enzyme was precipitated from this supernatant by 50-95% ammonium sulfate treatment, and this was resuspended in pine kirbain buffer (PH4.5), and the enzyme titer and protein quantification of the suspension was determined. I went. As a result, the amount of β-galactosidase produced per gram was 38.0 units, the amount of crude protein produced was 27.6 mg,
Therefore, the specific activity was 1.38 U/mg protein. This β-galactosidase has a pH of 4.5, 60℃,
It showed a residual titer of 68.9% after 30 minutes of heat treatment, and had significantly excellent heat resistance. [3] Effects of the invention Next, the effects of the invention will be explained using test examples. Test Example 1 Effect of alkaline protease activity of the medium on the heat resistance of β-galactosidase (a) Test method: The components shown in Table 1 were added to a sterilized medium containing 1.5 g and 2.5 ml of water, and Aspergillus oryzae L- 83 conidia were inoculated and cultured at 30°C for 50 hours. 15 ml of water was added to the culture and vigorously shaken at 30°C for 1 hour to extract the enzyme and obtain a liquid. The residue is
After adding 5 ml of Matsukirbain buffer (PH4.5) and stirring thoroughly, centrifugation was performed at 15,000 rpm for 10 minutes to obtain a supernatant, which was filtered (Toyo Shi No.
4) This was combined with the previous solution and used as a sample for measuring enzyme activity. β-galactosidase activity and alkaline protease activity were measured for each extract obtained from cultures in media of various compositions as described above, and the extracts were further incubated at pH 4.5 for 60 min.
The residual activity of β-galactosidase after heating at ℃ for 30 minutes was measured, and the residual activity rate was calculated to evaluate the heat resistance of β-galactosidase.

[Table] The results of this test are 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] Test Example 2 Effect of addition of alkaline protease inhibitor on the heat resistance of β-galactosidase (a) Test method 〓1.5g, cornstarch liquor 75mg, water
Aspergillus in sterile solid medium containing 2.5 ml
Conidia of Oryzae L-83 strain were inoculated and incubated at 30°C.
After culturing for 20 hours, 50 μ, 100 μ, 250 μ, and 500 μ of potato extract solution at a concentration of 200 mg/ml were added as an alkaline protease inhibitor, and the same amount of water was added as a control to prepare a culture. 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] As is clear from the results in Table 3, adding potato extract to the medium to inhibit alkaline protease activity in the medium significantly improved the heat resistance of β-galactosidase produced therein. . Next, we used Aspergillus oryzae L-83 strain and Aspergillus oryzae U-8 strain, a mutant strain with low alkaline protease activity obtained by irradiating this strain with ultraviolet light, and cultured both in a normal medium. The enzymes produced will be compared when culturing according to the method of the present invention, and it will be explained that the method of the present invention is effective regardless of the type of microorganism. Test example 3 Solid medium containing 1.5g and 2.5ml of water (normal medium)
and Aspergillus oryzae strain L-83 and Aspergillus oryzae U-8 in a medium (invention medium) containing 115 mg of monoammonium phosphate.
Conidia of each strain were inoculated and cultured stationary at 30°C 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 alkaline protease titer were measured. The results of this test are shown in Table 4.

[Table] As is clear from the results of the above test examples, the present invention provides a method for producing β-galactosidase with significantly superior heat resistance. In addition, in the above test examples and examples, β-
Galactosidase titer, alkaline protease activity, β-galactosidase heat resistance, and protein quantitative values were each shown as measured values by the following methods. (Method for measuring β-galactosidase titer) The ONPG substrate method was used. That is, 0.5 ml of enzyme solution was mixed with 3.5 ml of substrate solution (PH 4.5) containing 5.7 mmol of O-nitrophenyl-β-D-galactopyranoside (ONPG), and the mixture was heated at 30°C.
After reacting for 10 minutes, 1 ml of 1M sodium carbonate solution was added to stop the reaction. O- generated in this solution
The amount of nitrophenol was determined by measuring the absorbance at 420 nm. The amount of enzyme that hydrolyzes 1 μmol of ONPG per minute was defined as 1 unit (U). (Method for Measuring Alkaline Protease Activity) Protease activity measurement generally followed the modified Anson-Hagiwara method. i.e. 20 mmol EDTA
After treating 0.5 ml of enzyme solution (PH7.0) at 40℃ for 15 minutes to inactivate the neutral protease, 2.5 ml of enzyme solution containing
Mix 0.6% (W/V) dairy casein and 50 mmol disodium phosphate solution (PH7.0) and incubate at 30°C.
After 10 minutes reaction, 2.5 ml of 0.11 molar trichloroacetic acid,
The reaction was stopped by adding a mixture of 0.22M sodium acetate and 0.33M acetic acid. After standing at 30°C for 30 minutes, the reaction solution was filtered. The increase in colored non-protein phorin reagent corresponding to tyrosine produced in the solution was quantified. The amount of tyrosine produced in 1 ml of reaction solution per minute was defined as 1 unit. (Thermostability test method for β-galactosidase) After treatment at PH4.5 and 60°C for 30 minutes, the remaining activity percentage (%) capable of decomposing the substrate at 30°C was determined. (Quantification of protein) The Lowry method was used.

Claims (1)

[Scope of Claims] 1 β-galactosidase-producing bacteria belonging to Aspergillus oryzae are grown in a medium containing an alkaline protease inhibitor, a medium containing an acidic buffer containing nitrogen atoms, and a combination of the acidic buffer and a nitrogen source substance. A method for producing thermostable β-galactosidase, which comprises culturing in a medium selected from the added media and collecting thermostable β-galactosidase accumulated in the medium. 2. The method for producing heat-stable β-galactosidase according to claim 1, wherein the medium is a solid medium. 3. The heat-resistant β- medium according to claim 1, wherein the medium to which an acidic buffer containing nitrogen atoms is added is monoammonium phosphate or diammonium citrate in an amount of 40 to 150 mg per gram of the medium raw material.
Method for producing galactosidase. 4. Claim 1, wherein the medium to which an acidic buffer and a nitrogen source substance are added is a medium in which 40 to 150 mg of an acidic buffer and 10 to 150 mg of a nitrogen source substance are added in combination per 1 g of medium raw material. The method for producing the described thermostable β-galactosidase.