JPH0249707B2 - - Google Patents

Description

[Detailed description of the invention]

B. Object of the invention (1) Industrial application field The present invention relates to a novel thermostable β-galactosidase. β-galactosidase has been used in the food industry as an enzyme that decomposes lactose in dairy products, and is also widely used as a medicine or reagent to treat diarrhea caused by lactose intolerance. The novel thermostable β-galactosidase of the present invention is a commercially known β-galactosidase that has been commercially available in these fields.
Because it has better heat resistance than any other galactosidase, it is expected to have a wide range of uses. (2) Prior art As an example of increasing the heat resistance of β-galactosidase, Aspergillus oryzae Y-22 strain was treated with a mutagen to produce β-galactosidase, which has superior heat resistance compared to the original strain. It has been reported that the mutant strain Aspergillus oryzae YU-22 has been obtained. [Ogushi, S.et.al.: J.Ferment.Technol., 58 ,
115-122 (1980). ] Until now, there has been no known example of improving the heat resistance of β-galactosidase produced by improving culture conditions. We have completed an invention relating to a method for producing heat-stable β-galactosidase, which is characterized by culturing in a medium with low alkaline protease activity, and have applied for a patent. (Japanese Patent Application No. 58-244710) (3) Problems to be Solved by the Present Invention The present invention is based on the method for producing heat-stable β-galactosidase (Japanese Patent Application No. 58-244710), which was previously invented by the present inventors.
The purpose of this study is to isolate the enzyme produced as a pure substance (No. 244710) and clarify its physicochemical properties. B. Structure of the Invention (1) Means for Solving Problems The present invention provides a novel thermostable β-galactosidase having the following physical and chemical properties. (1) Action and substrate specificity A substrate having a β-D-galactoside bond is hydrolyzed to liberate D-galactose. (2) Optimal PH and stable PH range Optimum PH 4.5 Stable PH range 4.0 to 5.5 (3) Range of optimal temperature for action Optimum temperature for activation Approximately 60℃ (4) Conditions for inactivation due to PH, temperature, etc. Stable temperature range PH6 0 and below 60℃ Deactivation conditions by PH Inactivation by keeping at 37℃ for 1 hour in the range of PH2.5 or below or PH9.5 or above. (5) Inhibition, activation and stabilization Inhibited by silver nitrate and P-chloromercurybenzoic acid. Activated by methyl alcohol, ethyl alcohol, isopropyl alcohol, and n-propyl alcohol. (6) Molecular weight approximately 140,000 to 150,000 (Sephadex G-
(200 used, gel filtration method) (7) Isoelectric point PH4.7-4.3 (8) Elemental analysis C 46.40% H 6.43% N 12.01% S 0.36% (9) Infrared absorption spectrum Infrared absorption spectrum by KBr tablet method The absorption bands are 3300, 2950, 1690 (shoulder),
Located at 1650, 1530, 1450, 1390, 1240, 1060 cm ^-1 . The ratio of transmittance at wave numbers of 1530 cm ^-1 and 1060 cm ^-1 is 1:0.57. A group of novel β-galactosidases (hereinafter referred to as "the present enzymes") having the above-mentioned physicochemical properties produced by the present invention have pI values of isoelectric points of 4.68, 4.60,
It contains components 4.50, 4.42, and 4.37, and these individual components can be isolated by isoelectric focusing. Therefore, the present invention provides a group of novel β-galactosidases exhibiting the above-mentioned physicochemical properties, and the pI value of the isoelectric point separated from the β-galactosidases is 4.68.
Also provided is a single thermostable β-galactosidase exhibiting a value selected from 4.60, 4.50, 4.42 and 4.37. Next, a method for producing the novel thermostable β-galactosidase according to the present invention will be explained. β belonging to the genus Aspergillus used in the present invention
- As the galactosidase-producing bacterium, wild strains belonging to Aspergillus oryzae and natural or artificial mutant strains thereof are suitable. It is also expected that recombinant microorganisms incorporating these genes can be used in the same way. An example of a wild strain is Aspergillus oryzae strain L-83 (Feikoken Bacterium No. 7379), and an example of a mutant strain with low alkaline protease production is Aspergillus oryzae U-8.
The strain (Feikoken Bacillus No. 7378) can be mentioned. The mycological properties of these two strains were disclosed in the patent application.
58-244710 in detail. Cultivation is carried out by (1) adding an alkaline protease inhibitor to the medium, (2) adding an acidic PH buffer and a nitrogen source substance to the medium in combination, (3) using a low alkaline protease titer mutant strain. It is necessary to reduce the alkaline protease activity in the medium by performing these methods alone or in combination. Although liquid culture may be used for this culture, solid culture is usually preferred. As a method for collecting this enzyme from a culture, a conventional enzyme separation and purification method can be applied. For example, the present enzyme can be eluted and collected by suspending the culture in water and filling it in a column, and eluting a buffer solution of an appropriate composition or water from the top of the column down. This enzyme solution has a pH of 4.5
After adjusting to a range of ~5, salt out with 50~95% saturation of ammonium sulfate, or add 60% acetone, ethyl alcohol, or isopropyl alcohol.
(V/V) and the crude enzyme is recovered by precipitation. This is further followed by dialysis, centrifugation,
A purified sample of the present enzyme can be obtained by purification using an appropriate combination of filtration, extraction, desorption using various ion exchange resins or adsorbents, chromatography, etc. Next, Examples will be shown to specifically explain the method for producing the present enzyme. Example 1 〓12g, defatted soybean 3g, polypeptone 1.25g,
0.8 g of monoammonium phosphate, 0.3 g of glucose, and 27 ml of water were placed in a 500 ml Erlenmeyer flask and sterilized in an autoclave to prepare 120 flasks, each of which was inoculated with conidia of Aspergillus oryzae U-8 strain from a slant medium. Cultured at 30°C for 70 hours. The entire culture was collected and packed into a column, and the enzyme was extracted by flowing water 24 down to obtain extract 22.4. The crude β-galactosidase in this extract has a total activity of 73,000 U, a total protein of 7,690 mg, and a heat resistance of 56.0%.
It was hot. Example 2 (Production example of the present enzyme preparation) Using the crude β-galactosidase obtained in Example 1 as a starting material, (1) purification by ethyl alcohol fractionation, (2) purification by DEAE-Sephadex A-50, ( 3) Purification by Cephadex G-200, (4)
DEAE-Repurification by Cephadex A-50, (5)
Sequentially repurified using Sephadex G-200,
A sample of this enzyme was obtained. Each purification step will be described in detail below, and the results of comparing the total activity, total protein, specific activity, recovery rate, and heat resistance of the enzyme in each purification step will also be shown. (1) Purification by ethyl alcohol fractionation After concentrating 22.4 ml of the enzyme extract obtained in Example 1 to 3.25 using a holofiber ultrafiltration device, the pH was adjusted to 5.0 with 1N hydrochloric acid, and while stirring, the enzyme was extracted with cold ethyl alcohol. Alcohol was added to a final concentration of 10%, and the resulting impure protein precipitate was removed by centrifugation at 6000 rpm for 20 minutes. Add another 1N of supernatant.
Adjust the pH to 4.5 with hydrochloric acid, add cold ethyl alcohol while stirring until the final concentration is 60%, leave it at 4℃ overnight, and centrifuge the resulting enzyme at 6000 rpm for 15 minutes. It was collected and dissolved in 475 ml of pine kirbain buffer (PH4.5). Cold ethyl alcohol was added to this solution to a final concentration of 60%, and the resulting precipitate was collected by centrifugation at 6000 rpm for 40 minutes and dissolved in 170 ml of water. The enzyme in this solution has a total activity of 51900U and a total protein
1330 mg, specific activity 39.0 U/mg, recovery rate 71.1%, and heat resistance 55.3%. (2) Purification by DEAE-Sephadex A-50 After adjusting 170 ml of this enzyme solution obtained from the above ethyl alcohol fraction to pH 7.0 with 1N sodium hydroxide, 10 mM ammonium phosphate (PH 7.2)
Dialysis was performed overnight at 2. This enzyme solution was centrifuged at 17,000 rpm for 15 minutes, and the supernatant fraction (164 ml) was equilibrated with 10 mM ammonium phosphate (PH7.2) using DEAE-Sephadex A-50 (ion exchange manufactured by Pharmacia Fine Chemicals). column (diameter 6
x 36 cm) to adsorb this enzyme, and 60mM
After washing with 1.5 ml of 10 mM ammonium phosphate (PH 7.2) containing sodium chloride, it was eluted with 10 mM ammonium phosphate (PH 7.2) containing 330 mM sodium chloride. An eluate volume range of 360 to 935 ml was collected as a β-galactosidase active fraction (total volume 575 ml), and the pH was adjusted to 4.8 with 1N hydrochloric acid. Add cold ethyl alcohol to this solution to final concentration.
The mixture was added to 60%, and the resulting precipitate was collected by centrifugation at 5500 rpm for 30 minutes. After sufficiently removing the ethyl alcohol under reduced pressure, the enzyme
The enzyme was dissolved in 27 ml of 50 mM ammonium phosphate (PH5.0) containing 0.02% sodium azide and dialyzed overnight against Solution 1 having the same composition to obtain 30 ml of this enzyme. At this purification stage, this enzyme has a total activity of 32,600U,
Total protein was 579 mg, specific activity was 56.3 U/mg, recovery rate was 44.7%, and heat resistance was 52.6%. (3) Purification using Cephadex G-200 30 ml of this enzyme solution obtained by purification using the above DEAE-Sephadex A-50 was added to 50 mM ammonium phosphate (PH) containing 0.02% sodium azide.
A column (diameter 2.4 mm) of Cephadex G-200 (Super Fine, gel filtration agent manufactured by Pharmacia Fine Chemicals) equilibrated with
88.5 cm) at a flow rate of 20 ml/h. One fraction was 4.7 ml. Active fractions Nos. 58 to 70 were collected, adjusted to pH 4.8 with 1N hydrochloric acid, and cold ethyl alcohol was added to the final concentration of 60%. The resulting precipitate is
After centrifuging and collecting at 17,000 rpm for 30 minutes and thoroughly removing ethyl alcohol under reduced pressure,
It was dissolved in 12.5ml of 10mM ammonium phosphate (PH7.2) and dialyzed overnight against Solution 1 having the same composition to obtain 13ml of this enzyme solution. At this purification stage, this enzyme has a total activity of 26,100U,
Total protein was 285 mg, specific activity was 91.5 U/mg, recovery rate was 35.7%, and heat resistance was 54.4%. (4) Re-purification using DEAE-Sephadex A-50 using DEAE-Sephadex A-50, which was prepared by equilibrating 13 ml of the dialysate of this enzyme obtained by purification using the above-mentioned Cephadex G-200 with 10 mM ammonium phosphate (PH7.2). Column (diameter 3 x 22.5cm)
was filled. The adsorbed enzyme was 50-360mM contained in 2 x 500ml buffer with the same composition as the equilibration.
Elution was performed with a linear sodium chloride gradient system. The flow rate was 1.4 ml/min, and the volume of one fraction was 5 ml. Active fractions Nos. 72 to 87 were collected and adjusted to pH 4.8 with 1N hydrochloric acid. Cold ethyl alcohol was added to this collected fraction to a final concentration of 60%, and the resulting precipitate was centrifuged at 15,000 rpm for 30 minutes and collected. After sufficiently removing ethyl alcohol under reduced pressure, the precipitate was mixed with 100mM sodium chloride.
The enzyme solution was dissolved in 3 ml of 50 mM ammonium phosphate (PH 7.0) containing 0.02% sodium azide, and dialyzed against 650 ml of a solution of the same composition overnight at 4°C.
3.1 ml was obtained. At this purification stage, the enzyme had a total activity of 13200 U, total protein 111 mg, specific activity 119.0 U/mg, and recovery rate.
The heat resistance was 18.0% and the heat resistance was 57.6%. (5) Repurification using Cephadex G200 3.1 ml of the dialysate of this enzyme obtained by purification using the above DEAE-Sephadex A-50 containing 100 mM sodium chloride and 0.02% sodium azide.
Using a Sephadex G-200 (Super Fine) column (diameter 2.4 x 97 cm) equilibrated with 50 mM ammonium phosphate (PH7.0), the flow rate was
Gel filtration was performed at 11 ml/h. One fraction was 2.56 ml. No.108-117 with specific activity of 110 units/mg or more
was collected, the pH was adjusted to 4.8 with 1N hydrochloric acid, and cold ethyl alcohol was added to the final concentration of 60%.
The resulting precipitate was collected by centrifugation at 17,000 rpm for 30 minutes, and after sufficiently removing ethyl alcohol under reduced pressure, 50mM ammonium phosphate (PH
5.8) Dissolve in 5.5 ml and store at 4°C. The enzyme solution thus obtained was used as a standard of this enzyme. This standard enzyme contains 62 mg total protein and total activity.
8990U, specific activity 146.0U/mg, recovery rate 12.3%, and heat resistance 56.7%. Table 1 shows a comparison of the total protein, total activity, specific activity, recovery rate, and heat resistance of the present enzyme obtained in each purification step of Example 2 above.

[Table] In Table 1, the heat resistance value remains constant despite the purification of this enzyme to increase its specific activity. This suggests that only this enzyme with a certain high thermostability was produced. Example 3 0,125, 187.5, 250, 500 and 1 ammonium phosphate were added to a medium containing 7.5 g and 12.5 ml of water.
After adding 750 mg of each and autoclaving, conidia of Aspergillus oryzae L-83 strain were inoculated, and the mixture was statically cultured at 30°C for 50 hours. After culturing, the culture was packed into a column (diameter 2 x 40 cm), and the enzyme was eluted with water. The alkaline protease activity in this eluate was measured, and the produced β-galactosidase was separated and its activity and heat resistance were measured. That is,
The eluate was adjusted to pH 4.5 with 1N hydrochloric acid, and then centrifuged at 12,000 rpm for 30 minutes to collect the supernatant. Add cold alcohol up to 60% and remove the precipitate at 12,000 rpm.
It was centrifuged for 10 minutes and collected. After suspending this precipitate in pine kirbain buffer (PH4.5), cold ethyl alcohol was added again to 60%, and the resulting precipitate was suspended.
The mixture was centrifuged at 12,000 rpm for 10 minutes and collected. This enzyme was dissolved in 100mM potassium phosphate (PH5.8), and the activity of β-galactosidase was measured.
The heat resistance of β-galactosidase is determined by changing the pH of the enzyme solution.
After adjusting the temperature to 4.5 and incubating at 60°C for 30 minutes, the remaining O-nitrophenyl-β-D-galactopyranoside (hereinafter referred to as "ONPG") degrading enzyme activity ratio (%) was determined. The results are shown in Table 2.

[Table] As is clear from Table 2, the heat resistance of β-galactosidase increases in response to a decrease in the production titer of alkaline protease. When we performed slab SDS-polyacrylamide gel electrophoresis to measure the molecular weight of β-galactosidase enzymes with heat resistance of 52.5% and 24.2%, the former was 160,000 to 170,000,
The latter showed a value of 130,000 to 140,000, which is almost the same as that of a commercially available known enzyme. Example 4 〓6g, defatted soybean 1.5g, polypeptone 0.625
g, monoammonium phosphate 0.4g, glucose
Conidia of Aspergillus oryzae U-8 strain were inoculated into a sterilized medium containing 0.15 g and 13.5 ml of water, and cultured at temperatures of 27, 30, 33, and 36°C for 70 hours. After culturing, alkaline protease activity, β-galactosidase activity, and heat resistance were measured in the same manner as in Example 3. The results are shown in Table 3. The production of alkaline protease significantly increased when cultured at high temperature. Corresponding to the increase in alkaline protease production titer, the thermostability of β-galactosidase decreases. The Aspergillus oryzae U-8 strain is a low protease producing strain produced by using the Aspergillus oryzae L-83 strain as a parent strain and irradiating it with ultraviolet light (see JP-A-58-244710 specification).

[Table] Note that the activity of β-galactosidase in this specification is expressed as a value determined by measuring the hydrolysis power for ONPG, unless otherwise specified. i.e. 5.7mM ONPG solution adjusted to PH4.5 3.5
ml and 0.5 ml of the enzyme solution was kept at 30°C for 10 minutes, and then 1 ml of 1M sodium carbonate solution 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 ONPG per minute was defined as 1 ONPG unit. Next, the physicochemical properties and testing methods of this enzyme will be explained in detail. (1) Action and substrate properties D-galactose is released by hydrolyzing a substrate having a β-D-galactoside bond. Typical substrates include lactose or 4-O
-β-D-galactopyranosyl-D-glucopyranose, ONPG and P-nitrophenyl β
-D-galactopyranoside (hereinafter referred to as "PNPG"). (2) Optimum PH and stable PH range Optimum PH 4.5 (see Figure 1) Stable PH range 4.0 to 5.5 (see Figure 2) Optimum PH is measured by enzyme activity in Matskierbain buffer at various PHs. Calculated from the value, the stable PH range is
It was determined from the residual activity of the enzyme after it was treated for 1 hour at 37°C in pine kirbain buffer with various pH values. Each measured value was expressed as a ratio (relative activity rate and residual activity rate) to the maximum value, and the former is shown in FIG. 1 and the latter is shown in FIG. 2. However, PH
In measurements under conditions of 7.5 or higher, glycine-sodium hydroxide buffer was used instead of pine kirbain buffer. (3) Range of suitable temperature for action Optimum temperature for activity Approximately 60°C (see Figure 3) This value was determined from the enzyme activity values measured at various temperatures in Matskierbain buffer at PH4.5. Each measured value was expressed as a relative activity rate, with the measured value at 30°C set as 100, and is shown in Figure 3. (4) Conditions for deactivation due to PH, temperature, etc. Stable temperature range: 60℃ or less at PH6.0 (see Figure 4) Conditions for inactivation due to PH: 37℃, 1 hour at PH2.5 or less or PH9.5 or higher Deactivated by keeping it. (See Figure 2) The stable temperature range was determined from the residual activity of the enzyme stored at various temperatures for 30 minutes in a PH6.0 pine kirbain buffer. FIG. 4 shows the relationship between this residual activity rate and storage temperature. The conditions for inactivation due to PH are the above-mentioned "stable PH range"
It was determined from Fig. 2 in the measurement of . (5) Inhibition, activation and stabilization Inhibition: As shown in Table 4, especially AgNO ₃ ,
Inhibited by PCMB, FeSO ₄ .

[Table] The results in Table 4 are obtained by measuring the activity of this enzyme when various reagents are added at specified concentrations to the ONPG substrate reaction system, and comparing the values with the values when no additives are added.
It is expressed as a percentage of 100 (relative activity rate). Activation and Stabilization: As shown in Tables 5 and 6, the enzyme was significantly activated by adding 6.25% of methyl alcohol, ethyl alcohol, etc.

[Table] The results in Table 5 are the enzyme activity measured at 30°C when 6.25% (V/V) of various organic solvents were added to the ONPG substrate reaction system, and the values were compared to the values when no addition was made. It is expressed as a percentage of 100% (relative activity rate).

[Table] The results in Table 6 are the relative activity of ethyl alcohol, which is particularly effective among the various organic solvents mentioned above, measured at various temperatures when the amount added is varied. (6) Molecular weight: 140000-150000 Molecular weight (MW) is Sephadex G-200
Measurement was performed using a gel filtration method using a column (diameter 3 x 97 cm). Ovalbumin (MW45000), bovine serum albumin (MW68000), aldolase (MW158000), and catalase (MW240000) were used as samples with known molecular weights. Equilibration and elution with 50mM ammonium phosphate (PH7.0) containing 100mM sodium chloride.
The test was carried out at a flow rate of 13.7 ml/h at 4°C. (7) Isoelectric point: PH4.7-4.3 5% polyacrylamide gel as support,
Pharmalite pI2.5-5 and equal volume of Pharmalite pI4-6.5 (Carrier Amphorite manufactured by Pharmacia Huain Chemicals)
Gel electrophoresis was performed using (150 volts,
15 hours, 10℃). After electrophoresis, the gel was cut into 5 mm width sections and fractionated. The enzyme and formalite in each fraction were extracted with distilled water, and the enzyme activity and pH were measured. (8) Elemental analysis value C: 46.40%; H: 6.43%; N: 12.01%; S:
0.36% (9) Infrared absorption spectrum Figure 5 shows the infrared absorption spectrum obtained by the KBr tablet method. The absorption bands in the infrared absorption spectrum are 3300, 2950, 1690 (Shoulder),
It was observed at 1650, 1530, 1450, 1390, 1240, and 1060 cm ^-1 . The ratio of transmittance (% transmittance) at wave numbers of 1530 cm ^-1 and 1060 cm ^-1 is 1:0.57. Comparative test between the present enzyme and a known commercially available enzyme The physicochemical properties of the present enzyme and a known β-galactosidase derived from Aspergillus oryzae will be compared to clarify the reason why the present enzyme is novel. The commercially available β-galactosidase preparations used for comparison were products from different companies, with manufacturing numbers 73B6B, 914153, and BT14. Laczyme is a registered trademark), Wakamoto Pharmaceutical Co., Ltd.]
or simply "L", commercially available enzyme O [Oryzazyme (registered trademark): Yakult Pharmaceutical Co., Ltd.] or simply "O" and commercially available enzyme G [galantase powder (galantase is a registered trademark): Tokyo Tanabe Pharmaceutical Co., Ltd.] Or simply abbreviated as "G". The present inventors purified these enzymes by gel filtration and used them as control enzyme preparations in experiments. That is, using a Cephadex G-200 (diameter 2.4 x 91 cm) column, 50 mM ammonium phosphate (PH
7.0) at a flow rate of 7.13 ml/h, 4°C, and 2.5 ml/fraction, and the active fraction was collected to serve as a reference enzyme preparation. The specific activity is 156 units/mg for L and 152 units/mg for O.
mg, G was 43.6 units/mg. (b) Action and substrate specificity Km (Michaelis constant) and Vmax (maximum velocity) for ONPG, PNPG and lactose substrates are determined by Lineweaver-Burk plot (Basic Experimental Methods for Proteins and Enzymes, published by Nankodo, by Takeichi Horio and Nipei Yamashita. (ed., p. 387, 1981), and the results are shown in Table 7. The Km and Vmax of this enzyme were quite similar to the control enzymes L and O, but were quite different from G.

[Table] (b) Optimal PH and stable PH range The optimal PH was 4.5 for each enzyme. The stable PH range was almost the same for the present enzyme and control enzymes L and O, but G was narrower at PH4.3 to 5.3. (c) Optimum temperature for activity Figure 3 shows the changes in relative activity (%) with changes in temperature when ONPG was used as a substrate and reacted for 10 minutes at PH4.5 at various temperatures. The optimum temperature for activity of this enzyme is the highest at 60℃, and L
and O were both 58℃, and G was 52℃. (d) Stable temperature range: After keeping at each temperature for 30 minutes in Matsukielbain buffer with pH 6.0, dilute the enzyme solution 5 times and adjust the pH.
After adjusting to 4.5, PH4.5, 30℃, 10 minutes
Figure 4 shows the residual activity rate (%) for decomposing ONPG. The stable temperature range is the highest for this enzyme at 60℃, L at 59℃, and O at 57℃.
℃ and G were 51.5℃. (e) Inhibition, activation and stabilization AgNO ₃ , PCMB or
Table 8 shows the relative activity (%) of each enzyme when SDS was added. It is clear that the activity of this enzyme is less inhibited by these reagents than the control enzymes L, O and G. The degree of activation by organic solvents was similar to that of the present enzyme for L and O, but the difference was that G was not activated by n-propyl alcohol. Using ONPG as a substrate, the Ki (inhibition constant) and inhibition reaction format of galactose and P-aminophenyl 1-thio-β-D-galactopyranoside were determined according to the Lineweaver-Burk plot, and the results are shown in Table 9. It was shown to. Compared to these inhibitors, this enzyme has a higher Ki value than G, but a slightly lower Ki value than O and L.

【table】

[Table] (f) Molecular weight (a) Method by gel filtration (using Cephadex G-200 gel filtration agent, details as above):
This enzyme had the highest molecular weight. This enzyme: 150,000-140,000 L: 110,000-105,000 O: 110,000-105,000 G: 110,000-105,000 (b) Method using SDS-polyacrylamide gel electrophoresis: Slab SDS-polyacrylamide gel, electrophoresis (7.5% gel) Method of Michio Matsuhashi et al. [Chemotherapy, Vol. 27, 826-
840 pages (1979)] Yotsuta. Solubilization is 12.5%
The enzyme solution was kept at 37°C for 2 hours in a solution of glycerol, 0.5% 2-mercaptoethanol, 1% SDS, and 10mM sodium phosphate (PH7.0). Measuring the molecular weight of an enzyme is
SDS-PAGE marker I (RNA polymerase B, manufactured by Seikagaku Corporation, MW: 180000;
140000; 100000; 42000; 39000) and phosphorylase a (manufactured by Sigma, USA, MW:
94000) in parallel with each enzyme. This enzyme had the highest molecular weight. The protein with a molecular weight of 240,000 to 250,000 shown by commercially available enzyme G below is considered to be a dimer of the protein with a molecular weight of 130,000 to 140,000, and is thought to have been generated during solubilization with SDS. The molecular weights of each are as follows. This enzyme: 160,000-170,000 Commercially available enzyme L: 130,000-140,000 〃 O: 130,000-140,000 〃 G: 130,000-140,000 and 240,000-250,000 (published by Tokyo Kagaku Doujin, “Biochemistry Experiment Course Volume 1, Chemistry of Proteins” edited by the Japanese Biochemical Society,
306 pages, 1976). Gel is 5% polyacrylamide, carrier amphorite is pI2.5-5
1% formalite and 1% pI4-6.5 formalite, the electrode solution on the anode side is 0.1M sulfuric acid, the electrode solution on the cathode side is 0.1M histidine,
Electricity was applied for 15 hours at 150 volts and 10°C. After electrophoresis, the gel (length 10 cm) was cut into 0.5 cm pieces, formalite and enzyme were extracted with water, and the pH was measured using a PH meter to measure enzyme activity. Each enzyme preparation was composed of multiple enzymes with different pI values. The pI value, content (%), and thermal stability [H/N; residual activity rate (%) after being kept at 60°C for 30 minutes at pH 4.5] of the enzymes constituting each enzyme preparation were determined. Shown in Table 10. This enzyme is
It contained the enzyme with the highest pI value at the highest content ratio. In all enzyme preparations, among the constituent enzymes, those with pI values on the acidic side tended to have higher thermal stability. The molecular weight of each enzyme with different pI values in the enzyme preparation was measured by two-dimensional electrophoresis (first dimension: isoelectric focusing; second dimension: SDS-polyacrylamide gel electrophoresis). , were the same in each enzyme preparation. From the above findings, it is clear that the present enzyme preparation does not contain any enzyme molecules having the same physicochemical properties as the enzymes with different pI values that constitute the known enzyme preparations.

【table】 (H) Elemental analysis values Table 11 shows the elemental analysis values of each enzyme preparation.

[Table] (i) Infrared absorption spectrum The wave number (cm ^-1 ) of the absorption band in the infrared absorption spectrum of each enzyme was the same. The absorption bands at 3300 and 1060 cm ^-1 are considered to be stretching vibrations caused by polyoxyl groups, suggesting that each enzyme preparation has sugar chains. Absorption bands caused by the bending motion of the peptide bond are seen at 1650 and 1530 cm ^-1 . Therefore, in order to qualitatively examine the sugar content of each enzyme preparation, the ratio of transmittance at 1530 cm ^-1 and 1060 cm ^-1 was determined. Transmittance (1530 cm ^-1 ): Transmittance (1060 cm ^-1 ) This enzyme; 1:0.57 L; 1:0.43 O; 1:0.45 G; 1:0.63 The sugar content is highest in G, followed by this enzyme. , O and L are estimated to be the lowest. (J) Sugar content The sugar content was determined using D-mannose as a standard according to the phenol-sulfuric acid method [Analytical Chemistry, Vol. 28, p. 350 (1956)]. The results are shown in Table 12. Furthermore, the 12th
The total weight of the enzyme in the table was the sum of the weight determined by the Lowry method and the sugar weight determined by the phenol sulfuric acid method. As a result, G contained the most sugar, followed by this enzyme, O, and L, which decreased in that order.

[Table] (1) Immunological analysis The ability of rat antiserum against this enzyme to form precipitation lines with each enzyme preparation was investigated by two-dimensional double immunodiffusion method (Ochterlony method) [published by Tokyo Kagaku Doujin] Biochemistry Experiment Course Volume 1, Protein Chemistry”
Edited by the Japanese Biochemical Society, p. 343 (1976)]. Each enzyme preparation formed a completely fused single sedimentation line (fuse) against the enzyme antiserum. Therefore, immunologically, L, O, and G are all the same as this enzyme. (E) Solubility in water The enzyme preparation was dialyzed with distilled water until the external dialysate reached 1.7 μS/cm, and then the protein concentration was adjusted to 4.5 mg/ml (10 ml) and left at 4°C. Samples were collected at 0.5 cm below the water surface at each time point, and the amount of protein was measured using the Lowry method. Table 13 is
Relative protein concentration (%) for each time period is shown. This enzyme had a constant protein concentration and had the best solubility in water compared to other enzymes.

[Table] As is clear from the above physical and chemical properties, the β-galactosidase of the present invention does not contain any enzymes that are the same as known β-galactosidases (10th
Table) It is a new enzyme and has the highest optimal activity temperature (3rd temperature) compared to known β-galactosidase.
) and a stable temperature range (Figure 4), AgNO ₃ ,
It has extremely excellent properties as a medicine and reagent, such as being the least inhibited by PCMB and SDS (Table 8) and having the highest solubility in water (Table 13).

[Brief explanation of the drawing]

Figure 1 shows the influence of PH on the activity of this enzyme. Figure 2 shows the effect of PH on the stability of this enzyme. Figure 3 shows the effect of temperature on the activity of the present enzyme and commercially available enzymes (L, O and G). Figure 4 shows the effect of temperature on the stability of the present enzyme and commercially available enzymes (L, O and G). Figure 5 shows the infrared absorption spectrum of this enzyme obtained by the KBr tablet method.

Claims (1)

[Claims] 1. Novel heat resistance β having the following physical and chemical properties
- Galactosidase (1) Action and substrate specificity A substrate having a β-D-galactoside bond is hydrolyzed to liberate D-galactose. (2) Optimal PH and stable PH range Optimum PH 4.5 Stable PH range 4.0 to 5.5 (3) Range of optimal temperature for action Optimum temperature for activation Approximately 60℃ (4) Conditions for inactivation due to PH, temperature, etc. Stable temperature range PH6 0 and below 60℃ Deactivation conditions by PH Inactivation by keeping at 37℃ for 1 hour in the range of PH2.5 or below or PH9.5 or above. (5) Inhibition, activation and stabilization Inhibited by silver nitrate and P-chloromercurybenzoic acid. Activated by methyl alcohol, ethyl alcohol, isopropyl alcohol, and n-propyl alcohol. (6) Molecular weight: Approximately 140,000 to 150,000 (using Cephadex G-200,
(Gel filtration method) (7) Isoelectric point PH4.7-4.3 (8) Elemental analysis C 46.40% H 6.43% N 12.01% S 0.36% (9) Infrared absorption spectrum Absorption band in infrared absorption spectrum by KBr tablet method is 3300, 2950, 1690 (shoulder),
Located at 1650, 1530, 1450, 1390, 1240, 1060 cm ^-1 . The ratio of transmittance at wave numbers of 1530 cm ^-1 and 1060 cm ^-1 is 1:0.57.