JPS5843075B2 - Method for producing α-D-galactosidase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention involves culturing a microorganism belonging to the genus Elsinoe and having the ability to produce α-D-galactosidase (EC3,2,1,22) in a nutrient medium to produce α-D-galactosidase. The present invention relates to a method for producing α-D galactosidase.

α-D-galactosidase is an enzyme that acts on α-galactoside such as melibiose, raffinose, stachyose, etc., and decomposes it into dillic acid to liberate galactose.

To date, α-C-galactosidases are known from plants, bacteria, yeasts, and some higher plant organs, and among others α-D-galactosidases from filamentous fungi, actinobacteria, and bacteria.
Galactosidase has traditionally been used in the sugar beet industry to decompose raffinose contained in sugar solution extracted from sugar beets.

However, all of them have low α-D-galactosidase titers, and therefore their uses are limited to some research uses other than those mentioned above.

The present inventors investigated microorganisms that produce potent α-D-galactosidase.

As a result, it was discovered that a microorganism belonging to the genus Elsinoe can easily produce αD-galactosidase with high activity, and the present invention was completed.

Below, various properties of α-D-galactosidase of the present invention will be described.

(1) Action: In addition to oligosaccharides such as p-nitrophenyl-α-D-4-y')l-cyto, melibiose, raffinose, and stachyose, it also acts on guar gum and blood group B substances containing α-galactoside bonds. , liberating galactose.

(2) Substrate specificity p-nitrophenyl-α-D galactoside, melibiose, raffinose, and stachyose, respectively, as substrates,
Changes in the reaction rate of this enzyme were determined by changing the concentration, and Lineweaver-Burk'
Table 1 shows the Km and Vmax values for each substrate determined from the s plot.

In addition to the above-mentioned natural oligosaccharides, when guar gum and blood group B substances are also used as substrates, the amount of guar gum is 3.2% (calculated as galactose based on the total sugar content), and the amount of blood group B substances is 3.2% (calculated as galactose). It shows a decomposition rate of 23% based on the total amount of sugar.

Moreover, it does not act on methyl-α-D-galactoside.

(3) Optimal pH and stable pH range As shown in Figures 1 and 2, this enzyme
It has an optimum pH in the vicinity and is stable in the pH range of 4 to 10.

The stable pH range was determined by storing 20 μl of the enzyme in 200 μl of a buffer (0.1M) at a predetermined pH for 2 hours at 37°C, and then measuring the residual activity.

(4) Activity measurement method α-galactosidase activity is determined by p-nitrophenyl-α
-D-galactoside was used as a substrate for measurement.

That is, 0.1 ml of appropriately diluted enzyme solution, 0.01 ml of 50 mM substrate solution, 0.1 M acetate buffer (pH
4,s)o, ITL1, and 0.29 ml of distilled water were added to make a liquid volume of 0.5 rul, and the mixture was reacted at 37°C for 10 minutes.

Add 0.1MM acetate buffer pH 8,2) to the reaction solution at 3.0
- the reaction was stopped and the yellow color of the liberated p-tritrophenol was measured under a wavelength of 420 nm.

Under these measurement conditions, the amount of enzyme that can hydrolyze 1 μmol of substrate per minute is defined as 1 unit (U).

(5) Optimal temperature As shown in Figure 3, the optimal temperature when reacting at each temperature for 15 minutes is about 50°C.

(6) To 0.111 Ll of thermostable enzyme solution was added 0.1 M acetate buffer o, i rfL1 at pH 4,5, and after keeping it at a predetermined temperature for 1 hour, the residual activity was measured.

As shown in FIG. 4, the result is that it is stable at temperatures below 50°C, but gradually loses its activity at temperatures above that temperature.

(7) Inhibition, activation and stabilization of Ag+2Mg2+, Pb2+, Fc3+ especially p-C
MB is significantly inhibited by SDS, but Uu2+
, Zn2+, and Ca2+ have almost no influence.

Glucose and maltose show non-competitive inhibition.

Galactose and galacturonic acid at low concentrations (1-2mM)
shows competitive inhibition, but at high concentrations (5-10 mM) shows mixed inhibition.

The inhibition status by various metal salts and carbohydrates is shown in Tables 2 and 3.

In addition, the inhibition rate in Tables 2 and 3 refers to the percentage of α-D-glucosidase activity in the presence or absence of an inhibitor relative to the α-D-glucosidase activity in the absence of an inhibitor (control system). Indicated by

Furthermore, an activator and stabilizer for this enzyme have not yet been found.

(8) Purification method It can be purified in the same way as regular enzymes.

An example thereof is shown in Example 1.

(9) Molecular weight As shown in Figure 5, Sephadex G-150 (manufactured by Pharmacia, Sweden) and 4
A calibration curve was prepared using the various proteins, and the molecular weight of the enzyme was determined to be 220,000±20,000.

On the other hand, as shown in Figure 6, in SDS electrophoresis, 60,000±
5,000 and is estimated to be a tetramer.

(10) Others Since crystallization of this enzyme has not yet been successful, the crystal structure cannot be shown.

Therefore, we will describe the following physical and chemical properties in place of this.

(a) Ultraviolet absorption It shows an absorption band between 275 and 280 nm.

(0) It has an isoelectric point near pH 3.0 as measured by isoelectric focusing using isofocusing ampholine (LKB, Sweden).

Microorganisms belonging to the genus Yersinoe can be advantageously used as the microorganisms used in the method for producing α-D-galactosidase of the present invention.

For example, Jenkins, A, E, et al:
Arq.

In5t, Biol, S, Paulo, 417.67~
72 (1946) and Shigeo Takaya et al.: Tea Industry Technology Research No. 4
9, No. 79-88 (1975), etc., and has been deposited with the Agency of Industrial Science and Technology's Institute of Microbial Technology under FERM-PA3874.
oe 1eucospila) can be advantageously used in the production of ercinane of the present invention.

Similarly, Elsinoea ampelina (Elsinoea ampelina)
mpelina) IFo 5263, IFo 0635
9. Elsinoe ara
liae) IF06166, same IFO7162, Elsinoe fawcett
i) IFO6442, IFO8417, ATCC1
32001 Elsinoe
annonae) ATCC15027, Elsinoe ATCC11189, Elsinoe heveae ATC
C12570, Elsinoe
lepagei) ATCC13008, Elsinoe tiliae (Elsinoe tiliae) AT
Microorganisms belonging to the genus Elsinoe, such as CC24510,
It can be advantageously used in the production of this enzyme.

Microorganisms may be cultured by liquid culture, solid culture, etc. according to conventional methods.

In the case of liquid culture, static culture may be used, but shaking culture or aerated agitation culture is preferred.

As a medium, microorganisms belonging to the genus Elsinoe grow,
Any synthetic, semi-synthetic, or natural substance that produces α-D-galactosidase can be used.

In either case, it is known that α-D-galactosidase production is induced.

It is preferable to increase enzyme production by culturing in a medium containing galactose, lactose, raffinose, melibiose, sorbitol, maltitol, etc., or by adding these to the medium.

In particular, if sorbitol or maltitol is used as a carbon source for growing microorganisms, most of the produced α-D-galactosidase will be released outside the microbial cells, so the culture can be grown without removing mucilage. It is convenient because it can be used as it is as an enzyme solution.

Generally, the pH of the medium is adjusted to 5 to 8, microorganisms belonging to the genus Elsinoe are inoculated, and cultured at 20 to 35°C for 0.5 to 10 days.

The culture thus obtained can be used as it is as an enzyme preparation, but after separating the bacterial cells from the culture, the enzyme can be extracted from the bacterial cells and used as a crude enzyme solution.

In addition, known purification separation methods such as salting out, dialysis, filtration,
It can be easily purified and separated and collected by centrifugation, concentration, freeze-drying, etc.

Furthermore, if a high degree of purification is required, it is possible to further combine known protein purification methods such as adsorption/desorption to ion exchangers, gel filtration, affinity chromatography, isoelectric focusing, and electrophoresis. It is often possible to obtain α-D-galactosidase preparations of the highest purity.

The α-D-galactosidase produced by the method of the present invention can be used not only for various test purposes such as raffinose decomposition in sugar beet solution, quantitative enzyme agent, and reagent for determining the structure of compounds, but also for medical purposes such as digestive agents. It can also be advantageously used as a reagent for immunochemical research or as a modifier for physiologically active substances such as blood.

Although the α-D-galactosidase according to the present invention is stable as it is, it may be chemically modified by a known method to further stabilize the enzyme, or the enzyme can be stabilized by a known immobilization method. Measure and use repeatedly,
It is also free to use it more advantageously in the above applications.

The present invention will be described below with reference to a few examples.

Example 1 Sucrose 5w/v%, (NH4)2SO40,3vi/
v 1:l:, yeast extract 0.5 w/v%, peptone 0.5w/v%, K2HPO40.2w/v%,
Mg5O, -7H20o, 05 W/V%, Mn
SO4・4~6H200, OO2w/v%, FeSO4
- Medium 61 containing 200,001 w/v% of 7H was placed in a jar fermentor and kept at 120°C for 20 minutes to sterilize it.

After cooling, the initial pH of the medium was set to 6.7, and then inoculated with Elsinoe leukospira FERM-P 3874.
The cells were cultured at 300°C for 6 days under 61°C aeration.

The culture solution was centrifuged to collect bacteria, and sonicated (20KHz).
, 10 minutes), the enzyme was extracted from the bacterial cells.

20 g of DEAE-Sephadex A-50 ion exchange gel (manufactured by Pharmacia, Sweden) in which this crude enzyme solution was equilibrated with 0.05 MIJ acid buffer pH 6.0.
The mixture was stirred gently to adsorb the enzyme protein.

Elution was performed with the same 0.05 MIJ acid buffer pH 6.0 containing 0.1 M NaCl, and three elutions were performed to obtain a concentration of 96.
An activity recovery of 4% was obtained.

This eluate was then diluted with 0.05 MIJ acid buffer pH 6.
, 0, same <DEAE-Sephadex A-5
0 ion exchange gel column.

The α-D-galactosidase active fraction was concentrated using ultrafiltration (Toyo Roshi UHP-76 filter UK-10) and a collodion bag.

The concentrated solution was fractionated using a column filled with Sephadex G-100 gel (manufactured by Pharmacia, Sweden).

The active fraction was dialyzed against 0.05M acetate buffer, pH 4,5 for 5 hours.

The enzyme solution concentrated by the above method was then treated with a column packed with CM-cellulose (Whatman, UK), and α-D-galactosidase activity was recovered in the non-adsorbed fraction.

The above purification steps are listed in Table 4.

As is clear from this table, this purification step increases the specific activity by about 170 times, and the activity recovery rate at this time is about 4.
It was 8.1%.

Example 2 Culture was carried out in the same manner as in Example 1 except that a medium prepared using raffinose instead of sucrose was used to produce α-D-galactosidase.

After collecting and centrifuging the cells in the same manner as in Examples, the enzyme was extracted from the sonicated cells, and the α-D-galactosidase activity in the supernatant was measured.

The amount of α-D-galactosidase produced per ml of culture was approximately 2.1 units.

Example 3 Sorbitol 2 w/v%, yeast extract 0.5 w/vφ
, NaN0a O,2w/v %, K2HPO40,0
42 w/v %, KH2PO 40,018 w/v % was placed in a jar fermentor, and 1
It was sterilized by keeping it at 20°C for 20 minutes.

After cooling, Yersinoe arariae IF06166 was inoculated at an initial pH of 6.5 and cultured with aeration at 25° C. for 5 days.

After completion of the culture, the culture was centrifuged, and α-D-galactosidase activity in the supernatant was measured.

The amount of α-D-galactosidase produced per 1 rIL of culture was approximately 1.5 units.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the optimum pH. FIG. 2 is a diagram showing a stable pH region. FIG. 3 is a diagram showing the optimum temperature. FIG. 4 is a diagram showing a stable temperature region. FIG. 5 is a diagram showing a molecular weight calibration curve using Sephadex G-150 and the molecular weight of α-D-galactosidase. Figure 6 shows the molecular weight calibration curve and α-
It is a figure showing the molecular weight of D-galactosidase.

Claims (1)

[Claims] 1. A method for producing α-D-galactosidase, which comprises culturing a microorganism belonging to the genus Elsinoe and having the ability to produce α-D-galactosidase in a nutrient medium to produce α-D-galactosidase, and collecting the same.