JPS6019995B2 - Novel β-galactosidase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides Venicillium multicolor KU-○-13
The present invention relates to a novel 8-galactosidase produced by No. 2.

8-Galactosidase is an enzyme that hydrolyzes the lactose contained in the milk of mammals into glucose and galactose, and has recently become an enzyme that has attracted attention in the treatment of lactose intolerance and in the processing of dairy products. Some industrial development has been carried out, but there are many problems and nothing satisfactory has been achieved.

8-galactosidase produced by microorganisms includes those produced by bacteria such as lactic acid bacteria, and Saccharomyces fragilis (Sac
It is known that yeasts such as Charomyces fra js) are produced by yeasts, but these enzymes are intracellular enzymes and must be extracted and separated from the bacterial cells, so the preparation of the enzyme requires many steps and does not require high yield. Difficult to recover at a certain rate. In addition, exocrine 8-galactosidase is generally known to originate from filamentous fungi, such as Asbergillus niger (Hakama Kosho 491-21078), Asbergillus niger, and Aspergillus niger.・Olyse (ASpergmuSohizae) (Tokusei Sho 51-15
1 spot No. 5), Asbergillus awamori (Asperg.
In addition to those caused by Asbergillus sp.
7677), Sclerotium (No. 7677), Sclerotium
) genus (Tokusei 49-116291), Trichoderma (T
There are some produced by microorganisms such as the genus ricoderma (Samurai Kaiho 49-626). Among these, P-galactosidase produced by microorganisms of the genus Asbergillus niger, Asbergillus awamori, and Sclerotium is P-galactosidase.
Acidic 8 with an optimum pH on the extremely acidic side of H2-3.5
-It is a galactosidase and its uses are limited.

In addition, except for Asbergillus oryzae FERM-PI Sengongju, these enzyme-producing bacteria
production capacity is weak. Asbergillus oryzae FE
It is recognized that RM-P168 is a strain with relatively high productivity, but the enzyme it produces is
At 2.5 to 3.0, most of it is deactivated, and it depends on the stability on the acidic side. Therefore, when used for medicinal purposes, the pH in the stomach
2.5 to 3.0, it is easily deactivated. Furthermore, Benicillium citrinum and the like are known as microorganisms that produce 8-galactosidase (Japanese Patent Application Laid-Open No. 51-142593). In addition, 8-galactosidase generally has poor stability as an enzyme preparation, and to prevent this, lyophilization (freeze-drying) is required.
Special Publication No. 5039 of 1971) and Inocit are added (
An enzyme stabilization method such as Hakama Sekisho 51-9783) has been proposed. The present inventors have discovered that it is easy to produce industrially and
As a result of research into the production of 8-galactosidase that meets a wide range of purposes, Penicillium multicolor
The present invention was completed based on the discovery of new knowledge that allows the production of a novel 8-galactosidase with excellent enzymatic properties in large quantities. That is, the 8-galactosidase of the present invention can be obtained by culturing Benicillium multicolor in a medium and extracting 8-galactosidase from the culture.
- Consists of collecting galactosidase.

Venicillium multicolor is already a member of The Japanese Federation of Culture Collections of Microorganisms (THE JAPA).
NESE FEDERATION OFCULTURE
COLLECTIONS OF Hada C
ROORGANISMS) Catalog 19 Annual Edition 103
Benicillium multicolor KU-, which is a 3-galactosidase-producing bacterium used in the present invention, is described on page
○-132 is also a similar strain, but it has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under accession number 4375.

In carrying out the production of P-galactosidase of the present invention, the bacterial strain is cultivated aerobically in a solid or liquid column.

The composition of the medium used for culture is based on natural products such as rice bran, defatted soybeans, and cottonseed powder, and if necessary, carbon sources such as starch, glucose, arabinose, and xylose, ammonium sulfate, sodium nitrate, veptone, and malt. Nitrogen sources such as extracts, yeast extracts, etc., and inorganic salts such as inorganic phosphates, magnesium salts, calcium salts, etc. can be added. The culture temperature is 10~40oo, preferably 20~35qo,
It is desirable to culture aerobically for 1 to 8 days in an environment of pH 2.5 to 9, preferably pH 3.5 to 7.5. Generally, the production of 8-galactosidase reaches its maximum after 2 to 6 days of culture. The culture can be purified from the culture solution if it is a liquid culture, or from the culture extract if it is a solid culture, by a conventional enzyme purification method. That is, if necessary, the culture is concentrated by low-temperature vacuum concentration, membrane concentration, etc., salted with ammonium sulfate, etc., or precipitated with acetone, alcohol, etc., and collected separately. Of course, the culture can also be used as it is as an enzyme. For further purification, it is possible to carry out purification by conventional enzyme purification methods such as ion exchange method, gel filtration method, dialysis, carrier adsorption method, and protein precipitating agent method. The 8-galactosidase of the present invention is completely different from known enzymes.

The substrate specificity of the enzyme of the present invention is as follows. Lactose, o-nitrophenyl-PD-galactopyranoside,
Using p-nitrophenyl-6-D-galactopyranoside and phenyl-BD-galactoside as substrates, the activity ratio is:
When lactose is taken as 100, it is 224 and 180 respectively.
P-nitrophenyl-131 has no effect on the substrates of galactopyranoside, phenyl-Q-mannoside, phenyl-8-arabinoside, phenyl-Q-glucoside, phenyl-8-glucoside, and phenyl-8-xyloside.

Next, the enzymatic properties of the enzyme of the present invention will be compared with those of cougalactosidase produced by Benicillium citrinum and the like.

Benicillium nitrophenyl-6-D-galactosidase (1) Substrate specificity [glycoside] [activity] Lactose 100 100 Nitrophenyl-6-D-galacto 224 398
Pyranocyto P-nitrophenyl-8-D-galacto 186 288 Hiranoside (2) Molecular weight (gel filtration method) Approx. 120,000 Approx. 100,000 (3) Optimal PH 4.5 4~
5(4) Effect PH 2.5-7
35~8(5) Optimum temperature 55qC
50qC■ Action temperature Less than 750C PH stability less than 6pC (4CC24
2.5-8.0 3.5-8.0 (8) Temperature stability (PH6. Residual activity after 15 minutes treatment) 600C 95-100% 50% 650C
60% 0%7piC 25 more
○% 7son C ○Grandchild ○※ (9) Effect of metal ions (10-3 moM concentration) Iron
Slightly unstable Stable Stable Copper Stable
Extremely unstable 00) etc. Frost point 5.37 6.4
1 (Sequential migration of fanphorine) As mentioned above, clear differences can be seen.

In particular, there are clear differences in substrate specificity, temperature stability, influence of metal ions, etc. Furthermore, when 3-galactosidase produced by Venicillium citrinum is separated by precipitation from a solid culture extract using acetone, it is concentrated in the acetone concentration range of 45 to 70%, but the 3-galactosidase of the present invention
- Galactosidase can be separated and recovered by precipitating most of the B-galactosidase in the acetone concentration category of 45% or less, more precisely in the 30-40% category.

In addition to proving the physical difference, this fact has the advantage that the amount of organic solvent used can be reduced to 1'3 or less in industrial terms. Next, the enzyme lysis rates will be listed and compared.

Beninrium, Citriona, Honnada, etc. The next evening Iso 40% methanol water 0% 87% 50%
Methanol water 0 71 40% ethanol water 0% 82% 50% ethanol water
0 6440% acetone water 0%
91 grandchildren 50% acetone water 0
72 Furthermore, regarding the purification method, 8-galactosidase produced by Benicillium citrinum was DE
After passing through the AE-Sephadex column, the B-galactosidase according to the present invention is not adsorbed on the SP-Sephadex column unless the pH is 3.0 to 3.2.

In addition, in terms of enzyme production value, which is the most important point for industrial use, Benicillium multicolor of the present invention has an enzyme productivity that is 3 times stronger than the aforementioned Benicillium citrinum and the like.

That is, the amount of 8-galactosidase per several ta is 20 to 30 U' in Benicillium citrinum, whereas it is 1000 U' in the present invention. This 8-galactosidase productivity is higher than that of currently known enzyme productivity of strains of Asbergillus genus, Trichoderma genus, Sclerotium genus, etc. That is, as seen in the examples, Benicillium multicolor of the present invention produces about 1,000 peaks per few days in the case of solid culture, and about 1,000 peaks per few days in the case of liquid culture.
It shows a productivity of about 150 U per 1M of culture solution, and has a productivity of 10 to 3'' of the above-mentioned known producing bacteria.
The enzyme of the present invention is different from the 3-galactosidase produced by microorganisms such as Asbergillus oryzae, Macrophomina, and Trichoderma in terms of optimum temperature, optimum pH, stable temperature, stable wind, etc.

The enzyme of the present invention is stable and has a great feature in this respect, and the enzyme preparation has the feature of being able to be stored for a long period of time without any stabilization treatment. Next, the titer of 8-galactosidase was measured by the following commonly used method, which will be specifically explained with reference to Examples.

That is, 0 nitrophenyl-8-D-galactopyranoside 37 to 1 of the substrate solution containing 4.5,0
．． Mix two parts of 2M Macnvai buffer solution and one part of enzyme solution, react for 15 minutes at 55q0, then add 4 parts of IM sodium carbonate solution to stop the reaction, and perform colorimetry at a wavelength of 42 cm. The amount of 0-nitrophenol produced is determined from the 0-nitrophenol calibration curve. 1 under the same conditions
One unit (U) is defined as 1 mole of ○nitrophenol produced per minute. Example 1 Mix 10 pieces of defatted soybeans, 10 pieces of defatted soybeans, and 20 pieces of water, and
The mixture was placed in an Erlenmeyer flask, sterilized by autoclaving, and then inoculated with Benicillium multicolor KU-0-132 from a sloped base.

After culturing for 4 days under an environmental condition of 2:0, 8-galactosidase was extracted with 200 ml of water, and the solids were removed by furnace separation.

The 8-galactosidase titer of this extract was 8 U/'
Met. Example 2 Rice bran 3%, cotton seed flour 1%, tripotassium phosphate 0.25%,
Composition of disodium phosphate 0.15% liquid tower (within 6.
2) Put 100' into the 500'customer's Zhen Liao flask,
After sterilization with autoclave, Benicillium multicolor KU-0-132 was inoculated from the slope tower and incubated at 28℃ for 3 hours.
The koji was cultivated for several days.

The 8-galactosidase titer of the clear liquid obtained by blasting the culture solution was 140 U/nu. Example 3 Mix 1.5k9 of de-digitated soybeans, 1.5x9 cottonseed powder, and 3x3 of water uniformly, place in a large vat, sterilize with autoclave, and mix in several layers (number: water = 1:1). 15M seed koji of Benicillium multicolor KU-1132 cultured for 3 days was inoculated, and solid static culture was performed for 5 days under an environmental condition of 2C.

Claims (1)

[Scope of Claims] 1 β-galactosidase having the following physicochemical properties Physicochemical properties (1) Substrate specificity Lactose, o-nitrophenyl-β-D-galactopyranoside, p-nitrophenyl-β-D-galacto The activity ratio using pyranoside and phenyl-β-D-galactoside as substrates is 22 for each when lactose is 100.
It is 4,186,131. p-nitrophenyl-α-galactopyranoside, phenyl-α-mannoside, phenyl-β-arabinoside,
phenyl-α-glucoside phenyl-β-glucoside,
It has no effect on the substrate of phenyl-β-xyloside. (2) Molecular weight Molecular weight by gel filtration method is approximately 12,000
It is 0. (3) Optimal pH 4.5 (4) Working pH 2.5-7 (5) Optimum temperature 55°C (6) Working temperature less than 75°C (7) pH stability 24 at pH 2.5-8.0 Stable for time (4°C). (8) Temperature stability The residual activity after treatment at pH 6 for 15 minutes is as follows. 60°C 95-100% 65°C 60% 70°C 25% 75°C 0% (9) Effect of metal ions Slightly unstable with iron and stable with copper at 10^-^3 molar concentration. (10) Isoelectric point The result determined using ampholine was pH 5.37.
It is.