JP3468626B2 - β-glucosidase and method for producing the same - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel β-glucosidase, a method for producing the same and a microorganism producing the β-glucosidase.

[0002]

BACKGROUND OF THE INVENTION Monoterpenes play an important role in the scent of grapes and wines, and in grapes, many of these compounds exist as unscented glycosides. Further, it is known that the β-glucoside bond between the terpene and the sugar is hydrolyzed by β-glucosidase to release the terpene. Therefore, there is much interest in whether β-glucosidase can be applied to increase the aroma component of wine, but there are problems in using β-glucosidase such as its substrate specificity and glucose inhibition.
In particular, β-glucosidase derived from plants, yeasts and molds is known to be inhibited by glucose. Saccharomyces cerevisiae is the only one showing glucose tolerance.
It was reported that β-glucosidase isolated from periplasm of Siae) was not inhibited at all even at a glucose concentration of 5% (280 mM) (Vign.
eVin. 1988, 22, 189).

[0003]

Therefore, the present invention provides a novel β-comprising glucose tolerance and ethanol tolerance.
It is to provide a glucosidase, a method for producing the same, and a microorganism that produces the β-glucosidase.

[0004]

Means for Solving the Problems The present inventors have conducted a screening for a microorganism capable of producing β-glucosidase having glucose tolerance and ethanol tolerance, and as a result, have found that Debaryomyces.
It was found that one strain belonging to s) produces a novel β-glucosidase, and completed the present invention.

As described below, the strain producing the enzyme of the present invention is characterized by debaryomyces (Deba) due to its mycological properties.
It was determined to be a single strain belonging to the genus Ryomyces), and was identified as a new strain belonging to Debaryomyces hansenii by comparison with other known strains. The present inventors have identified this strain as Debaryomyces.
Hansenii) Y-44 and named it to Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry, September 11, 1995.
Deposited under accession number FERM P-15168 by date.

Debaryomyces hansenii (Deba
The mycological properties of the Ryomyces hansenii) Y-44 strain are as follows. (1) Morphological properties The malt extract liquid medium growth form is multipolar sprouting growth, and the cells are spherical or short oval and the size is 2 to 7 ×.
It is 2.4 to 8.5 μm. Form precipitates and films.
A yellowish off-white colony forms on malt extract agar. Sexual reproduction occurs between a mother cell and a daughter cell in a heterozygote. One or very rarely two in the ascus. The color tone of the colony after ascus formation becomes brown.

(2) Physiological properties (2-1) Fermentable glucose-or very weak galactose-or very weak lactose-maltose-or very weak sucrose-or very weak (2-2) assimilation Galactose + erythritol + maltose + ribitol + sucrose + mannitol + cellobiose + inositol-trehalose + soluble starch + raffinose + succinic acid + xylose + nitrate-arabinose + (+: assimilate, 2-: do not assimilate) 3) Vitamin requirement Biotin and thiamine requirement.

The theeastes based on the above properties
/ A taxonomy study (third
revised and enriched edit
ion; edited by N.I. J. W. Krega
r-Van Rij), and the Y-44 strain was searched for Debaryomyces ha.
nsenii).

To obtain the β-glucosidase of the present invention using the Y-44 strain, this strain may be inoculated into an appropriate medium and cultured according to a conventional method. The microorganism used to obtain the enzyme of the present invention is not limited to the Y-44 strain, and any strain may be used as long as it produces the β-glucosidase of the present invention having the characteristics described below. .

As the medium used in the present invention, any medium can be used as long as the bacteria of the genus Devariomyces according to the present invention can grow and produce β-glucosidase. For example, glucose, xylose, cellobiose, glycerol, succinic acid and the like are usually used as the carbon source, and cellobiose is most suitable because it produces a large amount of β-glucosidase. As the nitrogen source, meat extract, peptone, defatted soybean powder, yeast extract, malt extract, corn steep liquor and the like are used, and inorganic salts such as phosphate, sodium salt, calcium salt and magnesium are added.

The culture is carried out under aerobic conditions, for example, the aeration and stirring method or the shaking culture method. The culturing temperature is 20 to 35 ° C., preferably 25 to 32 ° C. where growth is good. The initial pH is preferably 4.0-7.0, p
H is also preferably 4.0 to 7.0. Culture time is 12-30
It takes about time, and the culture may be terminated when the β-glucosidase activity reaches the maximum.

After culturing, the cells are collected by filtration, centrifugation or filtration, and the cells are disrupted by lysis enzyme treatment or ultrasonic treatment, mechanical disruption treatment such as French press, homogenizer, etc., and obtained by centrifugation. The supernatant obtained is used as a crude enzyme solution. The crude enzyme solution can be used as it is, but can be separated and purified according to a general enzyme separation and purification method. That is, a salting-out precipitation method in which salts such as ammonium sulfate are added to the separated liquid to precipitate proteins,
Alternatively, a precipitate is obtained by a solvent precipitation method in which a hydrophilic organic solvent such as ethanol or acetone is added to precipitate the protein,
It can also be used as a dry powder. Further, an enzyme preparation having a high activity purity can be prepared by further using a purification method such as ion exchange chromatography or gel filtration chromatography.

The activity of the enzyme of the present invention thus obtained is measured by the following method. 0.2 ml of the enzyme sample solution diluted with 100 mM citric acid-phosphate buffer (pH 5.0) was mixed with 0.2 ml of 5 mM p-nitrophenyl-β-D-glucopyranoside solution dissolved in the same buffer, and 30 React at 10 ° C for 10 minutes. Then, the reaction was stopped by adding 1.6 ml of 1 M sodium carbonate,
P released by measuring the absorbance at 0 nm
-Quantify nitrophenol. At this time 1.0 mMp
Calculated by taking the absorbance of the nitrophenol solution at 400 nm as 0.057. Further, one enzyme unit (U) showing the enzyme activity is 1 minute per minute from p-nitrophenyl-β-D-glucopyranoside at 30 ° C. and pH 5.0.
It was defined as the amount of enzyme that produced μmol of p-nitrophenol.

The properties of the enzyme of the present invention obtained by purifying the crude enzyme solution by combining various chromatographic methods are as follows. (1) Action: p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-β-D-xylopyranoside, p-nitrophenyl-α-L-arabinofuranoside or p-nitrophenyl-α-L- When rhamnopyranoside is used as a substrate, both substrates release p-nitrophenol. (2) Optimum pH: p-nitrophenyl-β-D-glucopyranoside as a substrate, 100 mM citric acid-phosphate buffer solution (pH 2.0 to 7.0), 100 mM as a buffer solution
Relative activity was measured according to the above-described enzyme activity measuring method except that a phosphate buffer (pH 8.0 to 9.0) and a 100 mM glycine-sodium hydroxide buffer (pH 10.0) were used. The results are shown in Fig. 1, which shows the optimum pH of the reaction.
Is around 7.0. (3) pH stability: 100 mM citric acid-phosphate buffer (pH 2.0 to 7.0), 100 mM phosphate buffer (p
H8.0-9.0), 100 mM glycine-sodium hydroxide buffer (pH 10.0), after holding for 30 minutes at 30 ° C. in each buffer, the residual activity was measured according to the above-mentioned enzyme activity measuring method. . The result is as shown in FIG. 2, and the stable pH range is pH 6.0 to 9.0.

(4) Optimum temperature: Relative activity was measured at various temperatures according to the method for measuring enzyme activity. The result is as shown in FIG. 3, and the optimum temperature is about 25 ° C. (5) Temperature stability: After maintaining in 100 mM citric acid-phosphate buffer (pH 5.0) for 30 minutes, the residual activity was measured according to the above-mentioned enzyme activity measuring method. As a result, as shown in FIG. 4, the present enzyme showed stable enzyme activity up to 30 ° C., and was rapidly inactivated beyond that. (6) Effects of various reagents: The effects of various reagents on this enzyme were examined. That is, various reagents shown in Table 1 were added to 100 mM.
It was dissolved in a citric acid-phosphate buffer solution (pH 5.0) to a concentration of 1 mM, the enzyme solution was mixed with this, and the residual enzyme activity after holding at 30 ° C. for 10 minutes was measured according to the enzyme activity measuring method. The results shown in Table 1 were obtained.

[0016]

[Table 1]

(7) Molecular weight: The purified enzyme was subjected to slab gel electrophoresis, and the result was 1 band. As a marker, catalase (molecular weight 232,000 dalton),
The molecular weight measured using lactate dehydrogenase (molecular weight 140,000 daltons) and bovine serum albumin (molecular weight 67,000 daltons) is approximately 95,000 daltons. The result of SDS-polyacrylamide gel electrophoresis was 1 band, and b-type phosphorylase (molecular weight 94,000 daltons) was used as a marker.
Bovine serum albumin (molecular weight 67,000 Dalton),
Ovalbumin (molecular weight 43,000 daltons), carbonic anhydrolase (molecular weight 30,000 daltons) and soybean trypsin inhibitor (molecular weight 2
The molecular weight measured using 0,100 daltons is about 2
It is 5,000 daltons. (8) Isoelectric point: Lentil lectin (ba) as a marker
sic: pI8.65), lentil lectin (midd)
le: pI 8.45), lentil lectin (acidi)
c: pI 8.15), equine myoglobin (basic: p)
I7.35), horse myoglobin (acidic: pI)
6.85), human carbonic anhydrolase B (p
I5.85), β-lactoglobulin A (pI5.2)
0), soybean trypsin inhibitor (pI4.55),
The isoelectric point measured with amyloglucosidase (pI3.50) is pI4.9.

(9) Glucose tolerance: The relative activity was measured in 100 mM citrate-phosphate buffer (pH 5.0) containing glucose at a concentration of 0 to 500 mM according to the above-mentioned enzyme activity measuring method. The results are shown in FIG. 5, and show a relative activity of 80% even at 500 mM glucose. (10) Ethanol resistance: 100 mM citric acid-phosphate buffer solution (pH containing 0 to 20% by volume of ethanol)
In 5.0), the relative activity was measured according to the enzyme activity measuring method described above. The results are shown in FIG. 6, and 80% relative activity is exhibited even in 20% by volume ethanol.

[0019]

INDUSTRIAL APPLICABILITY The enzyme of the present invention is a β-glucosidase of microbial origin whose enzymatic activity shows resistance to glucose and ethanol, and acts on monoterpene glycosides to release monoterpenes. , It opens the way for its application in improving the flavor, especially in the field of winemaking.

[0020]

EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified in the following description, the indicated concentration is% by weight.

Example 1: Effect of carbon source 2.0% of the carbon source shown in Table 2 was added to a YM basic medium consisting of 0.5% peptone, 0.3% yeast extract and 0.3% malt extract, The pH was adjusted to 4.0 with hydrochloric acid. Charge 50 ml of this to a 250 ml Erlenmeyer flask and
It was sterilized by autoclaving at 15 ° C for 15 minutes. Debaryomyces hansenii (Debaryomyces) which was previously cultured in YM basic medium containing 2.0% glucose
hansenii) Y-44 strain (FERM P-151
1 ml of the seed mother of 68) was inoculated and cultured with shaking at 28 ° C. and 200 rpm for 24 hours. Table 2 shows the β-glucosidase activity in each of the obtained culture solutions. Debaryomyces hansenii
i) It is found that cellobiose is suitable as a carbon source for β-glucosidase production of Y-44 strain. Further, glucose suppresses the expression of β-glucosidase.

[0022]

[Table 2]

Example 2 Culture of Strains A medium consisting of 2.0% cellobiose, 0.5% peptone, 0.3% yeast extract and 0.3% malt extract was added with hydrochloric acid to p.
The mixture was adjusted to H4.0, 100 ml was charged into a 500 ml Erlenmeyer flask, and autoclaved at 120 ° C. for 15 minutes. Debaryomyces hansenii prepared in the same manner as in Example 1 was added thereto.
ii) Inoculate 2 ml of the seed mother of Y-44 strain, and
The cells were cultured with shaking at 0 rpm for 24 hours. The obtained culture solution was centrifuged to collect the bacterial cells, and the cells were washed twice with 30 ml of 20 mM Tris-HCl buffer (pH 7.0). This was suspended in 60 ml of the same buffer and glass beads (diameter 0.45 to 0.55 mm) 2.5 times the weight of the cells were added to disrupt the cells. The disrupted cells were removed by centrifugation to obtain 50 ml of cell extract. This is ultracentrifuged (100,000 ×
(g, 60 minutes) and separated into a microsomal fraction and a supernatant fraction. When the β-glucosidase activity of these fractions was measured, all the activities were present in the supernatant fraction, so the supernatant fraction was subjected to the purification operation described below.

Example 3: Purification of enzyme 50 ml of the supernatant fraction obtained in Example 2 was added to 20 mM bis (2
-Hydroxyethyl) iminotris (hydroxymethyl) methane buffer (Bis / Tris buffer: pH 6.
0) equilibrated with DEAE Toyopearl (DEAE-To
yopearl 650M column (φ26mm × 80m
m: manufactured by Tosoh Corporation) and eluted with 500 ml of a sodium chloride linear gradient (0 to 0.5 M). The active fractions were collected and equilibrated with 20 mM Bis / Tris buffer (pH 6.0) containing 1.0 M ammonium sulfate (Butyl-Toyope).
arl) 650S column (φ9 mm × 63 mm: manufactured by Tosoh Corporation), and the adsorbed protein was eluted with 100 ml of an ammonium sulfate linear gradient (1.0 to 0 M). Active fractions were collected and collected by ultrafiltration (PM-1
0: manufactured by Amicon) and concentrated at 4 ° C. Repeat this with 20 mM Bis / Tris buffer (pH
DEAE Toyopearl equilibrated with 6.0) (DEAE-
Toyopearl 650M column (φ9mm × 50)
mm) and eluted with 80 ml of sodium chloride linear gradient (0 to 0.5 M) to obtain purified β-glucosidase. Table 3 shows the activity of the enzyme in each of the above purification steps.

[0025]

[Table 3]

Example 4 Substrate Specificity Using the purified enzyme obtained in Example 3, decomposition tests of various color-developing substrates shown in Table 4 were conducted. The decomposition test was carried out according to the method for measuring the enzyme activity, and the presence or absence of the activity was examined by the production of p-nitrophenol. The results are shown in Table 4.

[0027]

[Table 4]

From Table 4, this enzyme is p-nitrophenyl-
β-D-glucopyranoside, p-nitrophenyl-β-
D-xylopyranoside, p-nitrophenyl-α-L-
Arabinofuranoside and p-nitrophenyl-α-L
It can be seen that both substrates release p-nitrophenol by acting on rhamnopyranoside.

Example 5: Action on Glycosides Derived from Grape Juice 1000 ml of concentrated grape juice (three times concentrated: Muscat seed) adjusted to pH 7.0 with sodium hydroxide was used as a synthetic adsorption resin column (φ22 mm × 350 mm, Amberli).
te XAD-2: manufactured by Rohm and Haas Company). This was washed with 500 ml of distilled water to remove the water-soluble compound. Further pentane-ether (1: 1) 10
After passing through 00 ml to remove free terpenes, glycosides were eluted with 1000 ml of methanol. This fraction was concentrated to dryness under reduced pressure at 30 ° C. to obtain 200 mg of glycosides. Purified enzyme solution 2 obtained in Example 3
100 mg of the above glycosides was dissolved in 0 ml (3.2 U, 100 mM citric acid-phosphate buffer solution (pH 5.0)) and reacted at 22 ° C. for 3 days. The liberated terpenes were extracted twice with 20 ml of pentane-ether (1: 1), concentrated, and then analyzed using capillary gas chromatography under the following conditions. The results are shown in Table 5.

[0030]

[Table 5]

(Analysis conditions) Apparatus: Hewlett Packard 5980 Column: DB-WAX capillary column (φ0.25
mm × 30 m: manufactured by J & WS Scientific) Detector: FID Helium Flow rate: 2.0 ml / min Initial column temperature: 75 ° C. Column heating rate: 4 ° C./min Final column temperature: 220 ° C. Sample injection part temperature: 230 ° C. Detection part Temperature: 220 ° C

[Brief description of drawings]

[Figure 1] Debariomyces Hansenii (Debar
FIG. 3 is a graph showing the relationship between the action pH and the activity of β-glucosidase produced by the Yomyces hansenii) Y-44 strain.

[Figure 2] Debaryomyces Hansenii (Debar
FIG. 2 is a graph showing a stable pH range of β-glucosidase produced by Yomyces hansenii) Y-44 strain.

[Figure 3] Debaryomyces Hansenii (Debar)
FIG. 2 is a graph showing the relationship between the action temperature and the activity of β-glucosidase produced by the Yomyces hansenii) Y-44 strain.

[Figure 4] Debaryomyces Hansenii (Debar
FIG. 2 is a graph showing the action temperature stability of β-glucosidase produced by Yomyces hansenii) Y-44 strain.

Figure 5: Debaryomyces hansenii (Debar)
FIG. 2 is a graph showing the relationship between the activity of β-glucosidase produced by Yomyces hansenii) Y-44 strain and glucose concentration.

[Figure 6] Debaryomyces Hansenii (Debar
FIG. 3 is a graph showing the relationship between the activity of β-glucosidase produced by Yomyces hansenii) Y-44 strain and the concentration of ethanol.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI C12R 1: 645) (56) References J. Appl. Bacteriology, 1994, 77 (5), 519-527 Applied Microbiology and Biotechnology, 1987, 26 (6), 552-554 (58) Fields investigated (Int.Cl. ⁷ , DB name) BIOSIS / WPI (DIALOG)

Claims (4)

(57) [Claims] 1. A β-glucosidase having the following properties. (1) Action: p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-β-D-xylopyranoside, p-nitrophenyl-α-L-arabinofuranoside or p-nitrophenyl-α-L- When rhamnopyranoside is used as a substrate, both substrates release p-nitrophenol. (2) Optimum pH: The reaction has an optimum pH of about 7.0 in a method of reacting with p-nitrophenyl-β-D-glucopyranoside as a substrate and measuring the amount of released p-nitrophenol by absorbance at 400 nm. It is in. (3) pH stability: When kept at 30 ° C. for 30 minutes,
It exhibits stable enzyme activity at pH 6.0 to 9.0. (4) Optimum temperature: The optimum reaction temperature is about 25 ° C. in the method of reacting with p-nitrophenyl-β-D-glucopyranoside as a substrate and measuring the amount of released p-nitrophenol by absorbance at 400 nm. is there. (5) Temperature stability: It is a method of measuring the enzyme activity by keeping it at pH 5.0 for 30 minutes, and shows stable enzyme activity up to 30 ° C., and when it exceeds it, it is rapidly deactivated. (6) Inhibitors: mercury p-chlorobenzoate, Cu ²⁺ , Al
It is inhibited by ^{3+ and} hardly inhibited by ethylenediaminetetraacetic acid. (7) Molecular weight: The molecular weight measured by SDS-polyacrylamide gel electrophoresis is about 25,000 daltons. (8) Isoelectric point: pI 4.9. (9) Glucose tolerance: 80% relative activity is shown in 500 mM glucose. (10) Ethanol resistance: 80% relative activity in 20% by volume ethanol. 2. Debaryomy
ces), a β-glucosidase according to claim 1 is collected from a culture obtained by culturing a microorganism belonging to ces). 3. Debaryomy
The microorganism belonging to ces is cellobiose (cellob).
A method for producing β-glucosidase, which comprises collecting the β-glucosidase according to claim 1 from a culture obtained by culturing in a medium containing iose). 4. Debaryomyces hansenii (Deba) having the β-glucosidase-producing ability according to claim 1.
ryomyces hansenii) Y-44 strain (F
ERM P-15168).