JPH062057B2 - Novel β-galactosidase A and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel β-galactosidase A and a method for producing the same, more specifically, Cryptococcu.
s) A novel β-galactosidase A produced by a microorganism belonging to the genus and a method for producing the same.

In the present invention, “β-galactosidase A” refers to an enzyme having physicochemical properties described below.

[Prior Art] β-galactosidase is an enzyme that hydrolyzes a β-D-galactoside bond to release D-galactose, and is widely present in plants and animals. Some microorganisms also produce β-galactosidase, and up to now, enzymes such as yeast, mold and bacteria have been widely studied and used for foods and pharmaceuticals. This enzyme also has the ability to transfer galactoside bonds simultaneously with hydrolysis, and it is known that this ability is used to produce galactooligosaccharides (oligosaccharides having galactose residues) (- Eiji Shima "Food Industry and Enzymes", page 68, Asakura Shoten). Specifically, Aspergillus oryza
The lactose or the lactose-containing substance is treated with the β-galactosidase produced in e) to give a compound of the general formula Gal- (Gal)
A method for producing a galactooligosaccharide represented by n-Glc (wherein Gal represents a galactose residue, Glc represents a glucose residue, and n represents an integer of 1 to 4) is known (Japanese Patent Publication No. 58-20266). . This galacto-oligosaccharide attracts attention as a growth factor for Bifidobacterium and is expected to be used for the purpose of improving the intestinal flora.

Other β-galactosidases for producing galacto-oligosaccharides include Bacillus circulans.
s circulans) β-galactosidase is known (Kazuhiro Nakanishi et al., “Agricultural Kansai Branch 325th Annual Meeting Report”, 1982.
5.22) [Problems to be solved by the invention] In addition to the above, the production of galactooligosaccharides by the transfer action of β-galactosidase has been reported, but most of them have low yields of galactooligosaccharides. Since it is an extracellular enzyme, it lacks stability in temperature and pH, and is unsuitable for mass production of galactooligosaccharides.

In consideration of the above facts, the present invention provides β-galactosidase suitable for producing galactooligosaccharides, particularly β that produces galactooligosaccharides useful as a Bifidobacterium growth factor in good yield.
-To provide galactosidase and a method for producing the same.

[Means for Solving Problems] The present inventors have found that various βs having a galactose group transfer ability are used.
-As a result of extensive studies on galactosidase, a microorganism belonging to the genus Cryptococcus was found to be β
The present invention has been completed by finding that β-galactosidase A having a strong transposition action, which mainly produces a galacto-oligosaccharide having a -1,4-galactoside bond, is produced in cells.

The β-galactosidase A of the present invention is a novel β-galactosidase having the physicochemical properties described below, and particularly when lactose is used as a substrate, the galactose group produced by decomposition is transferred, and O-β-D- Galactopyranosyl- (1 → 4) -O-β-D-galactopyranosyl- (1
→ 4) It has an action of producing galacto-oligosaccharide mainly composed of -D-glucose. This novel β-galactosidase A is produced by culturing a β-galactosidase A-producing bacterium belonging to the genus Cryptococcus, and separating and collecting β-galactosidase A from the culture.

Hereinafter, the present invention will be described in detail.

(I) Microorganism used in the present invention The microorganism used in the present invention has a β-galactosidase A-producing ability and is a bacterial species belonging to the genus Cryptococcus. One example is Cryptococcus lauren.
entii var. laurentii) OKN-4 (hereinafter referred to as OKN-) is a fungal species discovered in the soil of Nasu-gun, Tochigi Prefecture by the present inventors, and is sent to the Institute of Microbiology and Industrial Technology, Institute of Industrial Science and Technology Deposited as No. 7629.

OKN-4 has the following mycological properties.

The tests for the mycological properties described below are described in J. Lodder; The Yeast (1970) Hiroshi Iizuka, Shoji Goto; Classification and identification method for yeast (1969) Takeharu Hasegawa; Classification and identification of microorganisms (1975). Lodder; The Yeast
(1970).

(A) Bacteriological properties of OKN-4 (a) Growth state in each medium MY liquid medium: cultured at 25 ° C for 3 days, the cell shape is spherical, elliptical, elongated size (3.0 to 5.3) × (4.0 to 5.3) μ Multipolar budding, islet-like film formation Medium forms a turbid precipitate MY agar medium: 25 ° C for 1 month, colonies are pale orange to yellowish brown, soft, and It is viscous.

 Slide culture: Hyphae and pseudohyphae do not form on potato-dextrose medium.

 (B) Ascospore formation: Not observed on normal sporulation medium.

 (C) Formation of shot spores: Not observed in MY agar plate culture.

 (D) Physiological properties (1) Optimal growth conditions: pH 6 to 7, temperature 30 ° C (2) Growth range: pH 3 to 9, temperature 20 to 40 ° C (3) Assimilation of nitrate: not assimilation (4) Decomposition of fat: No decomposition (5) Decomposition of urea: Decomposition (6) Liquefaction of gelatin: No liquefaction (7) Formation of carotenoid: No formation or very little formation (8) Formation of organic acid: No formation (9) Production of starch-like polysaccharides: Produce (10) Vitamin requirement: Does not grow in vitamin-deficient medium (11) Arbutin degradation: Decomposes (12) Cycloheximide resistance: Does not grow (13) At 37 ° C Growth: Grow (14) Growth in 50% glucose yeast extract medium: No growth (e) Assimilation to each carbon source 1) D-arabinose + 2) L-arabinose + 3) D-ribose + 4) D- Xylose + 5) D-Glucose + 6) D-Galactose + 7) L-Rhamnose + 8) L-Sorbo + 9) Maltose + 10) Sucrose + 11) Lactose + 12) Mellibiose + 13) Cellobiose + 14) Trehalose + 15) Raffinose + 16) Merezitose + 17) α-Methyl-D-glucoside + 18) Soluble starch ± 19 ) Inulin-20) Ethanol + 21) Adnit + 22) Erythritol + 23) Inosit + 24) D-Mannitol + 25) D-Sorbit + 26) Drusit + 27) Glycerin + 28) DL-Lactate -29) Amber Acid salt + 30) Citrate − 31) Salicin + (+: Well assimilated ±: Suspected assimilation −: Not assimilated) Note that there is no fermentability for sugars.

Based on the above bacteriological characteristics, this strain was identified as belonging to Cryptococcus laurentii variety lorentii.

OKN-4 is one example of the microorganism used in the present invention, and naturally and artificial mutants thereof as well as microorganisms belonging to the genus Cryptococcus having β-galactosidase A-producing ability are all used in the method of the present invention. Can be used.

(B) Culturing method For culturing the above-mentioned microorganism, either a solid medium or a liquid medium which is usually used may be used, but the liquid medium is preferable.
The medium used for culturing the microorganism can use a carbon source that the microorganism can assimilate as the carbon source, but preferably contains lactose or whole milk, lactose as one component such as skim milk. Materials, sorbit, lactit, etc. Examples of nitrogen sources include nitrogen compounds such as yeast extract, casein, corn steep liquor, soybean flour, cottonseed flour, wheat gluten, peptone, and meat extract, and (NH
₄ ) ₂ SO ₄ , NH ₄ Cl, inorganic nitrogen compounds such as urea,
As the inorganic salts, sodium salts, potassium salts, magnesium salts, phosphates and the like can be appropriately used. In addition, vitamins and trace metal salts can be added to improve the growth of the bacteria used.

The concentration of carbon source is in the range of 1-40% by weight, and the culture temperature is 20-
40 ℃, pH of the culture solution is in the range of 2-9, culture time is 1
~ About 6 days. Any of static culture, aeration stirring, and shaking culture can be used.

(II) β-Galactosidase A β-galactosidase A is produced in the cells of the above-mentioned microorganism, and is separated and collected from the culture of the microorganism. As a means for separating from the culture, a known enzyme purification method can be used. The microbial culture may be filtered and centrifuged to be used as a crude enzyme, or may be used as a purified enzyme using a purification means such as a salting-out method, a dialysis method, a carrier adsorption method, an electrophoresis method, a gel filtration method. You can also

β-galactosidase A is a novel enzyme having the following physicochemical properties.

<Physical and Chemical Properties> (1) Action and Substrate Specificity Lactose having a β-galactoside bond, O-nitrophenyl-β-D-galactopyranoside (hereinafter referred to as ONP
G) and other β-D-galactopyranosyl derivatives to hydrolyze the β-D-galactoside bond to decompose galactose and glucose, galactose and O-nitrophenol, etc. −
Transfers the galactose group.

When lactose is used as a substrate, the produced D-galactose group is transferred, and O-β-D-galactopyranosyl-
(1 → 4) -O-β-D-galactopyranosyl- (1 →
4) An oligosaccharide mainly containing -D-glucose is produced.

(2) Optimum pH and stable pH range As a result of reacting ONPG as a substrate at 30 ° C for 30 minutes at each pH, the optimum pH was pH 4.0-
It was 5.0. Moreover, the residual activity after incubation for 60 minutes at 30 ° C. for 60 minutes with ONPG as a substrate was measured, and as a result, as shown in FIG.
It showed a residual activity of 0% or more.

(3) Method of measuring titer (titer against ONPG) 1.0 mM of 5 mM ONPG, Maccllavin
e) 2.0 ml of buffer solution (pH 5.0) and 1.0 ml of enzyme solution were mixed and reacted at 40 ° C. for 30 minutes, then 1.0 M Na ₂ CO ₃ 1.0
The reaction was stopped by adding ml, and the produced O-nitrophenol was measured by absorption at a wavelength of 420 nm. The ability to decompose 1 μmol of lactose per minute was defined as 1 unit (U).

(4) Optimum temperature range for action As a result of reacting ONPG as a substrate at pH 5.0 for 30 minutes, the optimum temperature range for action is 50-60.
It was in the range of ° C.

(5) Thermal Stability As a result of heating ONPG as a substrate at pH 5.0 at each temperature, as shown in FIG. 4, it was stable up to 57.5 ° C. and deactivated at 60 ° C. by about 30% as shown in FIG.

(6) Inhibition It is inhibited by 0.05 to 0.5 mM mercury ion, silver ion, and 0.5 mM mercaptoethanol. It is also inhibited by glucose and maltose.

(7) Purification method The culture of the microorganism was disrupted, the disrupted product was treated with the cell wall lysing enzyme Zymolyase 20-T (trade name), and the supernatant of the treated solution was treated with DEAE-Sephadex.
ex) A-50 (trade name) and Toyo Pearl HW-55S
(Product name) Can be separated by adsorbing on a column.

(8) Molecular weight As a result of measurement by gel filtration using Toyopearl HW-55S, the molecular weight was about 200,000.

[Example] Below, the Example of this invention is shown.

(1) Production of bacterial cells 100 ml of a medium having the following composition was charged into a 500 ml Erlenmeyer flask, sterilized at 120 ° C. for 20 minutes and then cooled, and Cryptococcus
Lorenty variety Lorenti OKN-4 strain was inoculated and shake-cultured at 30 ° C. for 1 day to give an inoculum.

Medium composition Lactose 50g NH ₄ Cl 2g KH ₂ PO ₄ 0.8g Na ₂ HPO ₄ · 12H ₂ O 0.3g MgSO ₄ / 7H ₂ O 0.02g Yeast extract 3g Water 1pH 6.0 Main culture is the same as above 2.5 ml of
The culture was carried out at 0 ° C. for 2 days with shaking at 160 rpm. After the completion of the culturing, the cells were collected from 100 ml of the culture solution by centrifugation (5000 r.p.m for 10 minutes) to collect the cells, washed 5 times with distilled water and resuspended in 40 ml of water. The enzyme activity of this cell suspension was 0.08 U / ml using ONPG as a substrate.

(1) Purification of enzyme After thoroughly washing 200 g (wet weight) of the cells cultured by the above method with distilled water, 160 ml of ethyl acetate and 40 ml of 20 mM phosphate buffer (pH 5.8) were added and well suspended. Shake back and forth at 4 ° C for 24 hours. The obtained cells were thoroughly washed with distilled water and then suspended in distilled water to obtain 800 ml of a cell suspension. This was crushed (8,000 psi 10 solution) by a Manton-Gawling microbial cell crusher in ice water, and then centrifuged (5,000 rpm for 10 minutes) to obtain a microbial cell lysate. The cell wall fraction in the disrupted cell suspension was washed once with distilled water and then freeze-dried to 27.6 g.
A lyophilized product was obtained. The enzyme activity of this cell wall fraction is O
It was 10 U / g using NPG as a substrate.

Next, 27.6 g of the lyophilized product of the above cell wall fraction was suspended in 690 ml of McCluben's buffer (pH 6.0),
20-T (manufactured by Seikagaku Corporation) was added so as to be 0.01% by weight, and the mixture was shaken reciprocally at 30 ° C. for 3 hours to solubilize the enzyme.
After the reaction, the obtained solution was filtered through Toyo Roshi (trade name) No. 2 and the filtrate was dialyzed against distilled water for one day. After dialysis, freeze-drying gave 4.0 g of crude enzyme product. The activity of this crude enzyme was 25 U / g using ONPG as a substrate.

Furthermore, this crude enzyme product was added to 20 mM phosphate buffer (pH 5.8) 3
It was dissolved in 40 ml and adsorbed on a DEAE-Sephadex A-50 (50 mmφ × 740 mmH) column that had been equilibrated with the same buffer in advance, and then eluted with the same buffer (0 to 1.0 M NaCl gradient) at a flow rate of 70 ml / Hr. did. Active fraction 90
After dialyzing 0 ml of distilled water and freeze-drying, 1.75 g of crude enzyme was obtained. Further, add this to 20 mM phosphate buffer (pH 5.8) 2
Toyopearl HW-55 was dissolved in 4 ml, left to stand in ice water for 1 hour to remove white precipitates, and 4 ml of 24 ml of the supernatant was pre-equilibrated with 20 mM phosphate buffer (pH 5.8).
Inject it into the S column (22 mmφ x 750 mmH) for adsorption,
Then, the same buffer solution was eluted at 1.0 ml / min. This operation 6
After repeated 108 times to obtain a total of 108 ml of active fraction, this was dialyzed against distilled water and freeze-dried to obtain 9.0 mg of purified enzyme. This enzyme showed a single band on disk electrophoresis. The enzyme activity of the purified enzyme was 4.86 U / mg using ONPG as a substrate.

(3) Transfer action of enzyme Using 1 ml of a 2.5 wt% lactose solution as a substrate, 0.02 U of the enzyme purified by the above-mentioned method was added, and the pH was adjusted to 5.0 or 50.
The enzymatic reaction was performed at ° C. The change with time in the composition of the reaction solution is shown in FIG. In the figure, GO-1 is the following formula O-β-D galactopyranosyl- (1 →
4) -O-β-D Galactopyranosyl- (1 → 4) -D
-Glucose, which is useful as a bifidobacterial growth factor.

As can be seen from these results, the enzyme β-galactosidase A of the present invention hydrolyzes the β-D-galactoside bond to selectively produce a (1 → 4) -linked galactooligosaccharide at a high yield. Other β-galactosidases, such as K. fragillis, K. lactis, Asp.oryza, B.I. circu
β-galactosidase derived from lans and the like has a low yield of oligosaccharides produced and many types, and also has an optimum pH,
Since β-galactosidase A has different physicochemical properties such as optimum temperature and temperature stability, it can be said that β-galactosidase A is a novel enzyme.

[Effect of the Invention] As described above, the β-galactosidase A of the present invention
Is a novel enzyme, and by using this enzyme, galactooligosaccharide useful as a bifidobacteria growth factor can be obtained with high yield and high selectivity.
Since H stability and temperature stability are also high, it has a great effect on the industrial production of galactooligosaccharides.

[Brief description of drawings]

1 to 4 show β-galatoxidase A, respectively.
FIG. 5 is a graph showing the activity against pH, the stability against pH, the activity against temperature, and the stability against temperature, and FIG. 5 is a graph showing the change with time of the composition of the reaction solution of the enzymatic reaction by β-galactosidase A using lactose as a substrate.

Claims (3)

[Claims] 1. A β-galactosidase A having the following properties. Action and Substrate Specificity Acting on a β-D-galactopyranosyl derivative,
It hydrolyzes the D-galactoside bond and transfers the D-galactose group generated by the decomposition. When lactose is used as a substrate, oligos mainly containing O-β-D-galactopyranosyl- (1 → 4) -O-β-D-galactopyranosyl- (1 → 4) -D-glucose Produces sugar. Optimum pH and stable pH range Optimum pH is at pH 4.0 to 5.0 and stable pH is pH 2.7 to
9.3 (treated at 30 ° C for 60 minutes). It has an optimum working temperature in the optimum working temperature range of 50 to 60 ° C. Thermal stability It is stable up to 57.5 ℃ when treated for 10 minutes. Inhibition Inhibited by mercury ions, silver ions, mercaptoethanol, glucose and maltose. Molecular weight Approximately 200,000 as measured by gel filtration method. 2. A method for producing a novel β-galactosidase A, which comprises culturing a novel β-galactosidase A-producing bacterium belonging to the genus Cryptococcus, and separating and collecting the novel β-galactosidase A from the culture. . 3. A novel β-galactosidase A-producing bacterium belonging to the genus Cryptococcus is Cryptococcus lauren.
tii var. laurentii) OKN-4, The method for producing the novel β-galactosidase A according to claim (2).