JP2530998B2 - A new strain belonging to the genus Thermus - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel strain belonging to the genus Thermus which produces a novel β-galactosidase.

[0002]

2. Description of the Related Art β-galactosidase is an enzyme that decomposes lactose (lactose) into glucose and galactose, and is used for producing foods such as lactose-decomposed milk or lactose-decomposed liquid. This β-galactosidase, including animals and plants, fungi, yeast, and microorganisms such as bacteria are widely present, but currently, enzymes used in the food industry are relatively low in thermostability mainly derived from mold or yeast. β-galactosidase. These β-galactosidases are disclosed in JP-B-53-24094 (Prior Art 1) and JP-A-52-44287 (Prior Art 2).

[0003] On the other hand, β-galactosidase having thermostability has been disclosed in Biotechnology and Bioengi-neerin.
g), 26, 1141, 1984 (Precedent 3), Journal of Applied Microbiology (Journal of Ap.
plied Microbiology), 2, 390 pages, 1980 (Precedent 4), Canadian Journal of Microbiology, 22, 8
17 pages, 1976 (Precedent 5), JP-A-56-154991 (Precedent 6) and Journal of Bacteriology (Journ)
al. of Bacteriology), Vol. 110, p. 691, 1972 (Precedent 7). However, as described below (see Table 2), each of these conventional β-galactosidases has one or more of low thermostability, narrow optimal pH range, or inhibition of enzymatic action by reaction products. .
Therefore, the fact is that it is not practically used.

[0004] As described above, β-galactosidase, which is currently used for food production on an industrial scale, is an enzyme having relatively low heat resistance. For this reason, the lactose decomposition process is generally performed at 55 ° C. or less, and because the raw material milk or lactose solution is a good nutrient source of bacteria,
Rotation due to bacterial contamination during treatment and immobilized β-galactosidase are a serious problem in production, such as insufficient washing and sterilization after production. In order to solve these problems, treatment of the target substance at a high temperature where bacteria are difficult to grow, or washing and sterilization using heat-resistant β-galactosidase is desired, and heat-resistant β-galactosidase that can be used industrially is desired. Development is strongly desired.

[0005] In order to meet such a demand, an attempt was made to search for thermostable β-galactosidase as described above, and various thermostable β-galactosidases were found. However, none of the conventionally obtained thermostable β-galactosidases having all of the enzymological properties described in the following (1) to (2), which are considered necessary for industrial use, does not exist. Has not reached. Sufficient heat resistance: Since thermal coagulation of milk and skim milk occurs at 78 to 80 ° C., it is desirable that the enzyme treatment is performed at a temperature below this temperature and as high as possible. Therefore, it is required that the heat stability of the enzyme at around 70 to 75 ° C is sufficient. Having an optimal temperature from neutral to acidic: raw materials to be processed by β-galactosidase include milk,
Skim milk, cheese whey, lactose liquid and the like are assumed. Since these are neutral (pH 6.5) to acidic (pH 4.5) raw materials, they are required to have sufficient enzymatic activity in that range. Low inhibition of enzyme activity by reaction products: β
-It is required that galactose and glucose, which are reaction products when galactosidase is allowed to act on lactose, decrease the enzyme activity little.

[0006]

DISCLOSURE OF THE INVENTION The present invention relates to a novel β-galactosidase which has high heat resistance, has an optimum pH in a neutral to acidic region, and has little reduction in enzyme activity due to reaction products. (Hereinafter referred to as the present enzyme).

[0007]

DISCLOSURE OF THE INVENTION The present inventors have aimed to create a β-galactosidase having the above-mentioned properties, and have pursued pure isolation of a microorganism producing such β-galactosidase from nature. Tried. as a result,
The present inventors have found that among a large number of microorganisms producing β-galactosidase, microorganisms belonging to the genus Thermus, which is a kind of highly thermophilic bacteria, produce β-galactosidase having the above-mentioned properties, and completed the present invention.

That is, the present invention has an optimum working temperature of 75-85 ° C., an optimum pH of 4.5-6.5, and a substantial enzymatic activity due to 50 mmol of galactose and glucose. Thermus sp. Is characterized by producing a novel β-galactosidase which does not degrade to a minimum and having at least the following mycological properties: (a) Morphological properties Shaped bacilli Motility-gram Staining-flagella-spores-acid-fast-size 0.4-0.6 × 2-7 μm (b) Growth on various media Agar flat media Circular or peripheral irregular, raised or flat, pale yellow to orange agar slant media Smooth, pale yellow to orange liquid medium Litmus milk with no sedimentation of growing cells No change 5% NaCl-containing liquid medium-2% NaCl-containing liquid medium + (C) Growth pH and temperature Growth pH 5.5-8.5 Growth temperature 40 Up to 80 ℃ (d) Biochemical properties Quality Reduction of nitrate + denitrification reaction ± VP test-formation of indole-formation of hydrogen sulfide-hydrolysis of starch ± use of citric acid-use of inorganic nitrogen source + formation of (NH ₄ ) pigment Generated (yellow, orange) ) Urease-oxidase + catalase + OF test O Oxygen requirement No growth under anaerobic conditions (e) assimilation of sugar D-glucose + D-mannose + D-fructose + maltose + sucrose + lactose + trehalose + Glycerin-starch-Specific examples of the microorganism include Thermus SP ZK
-001, THERMUS SP ZK-002, THERMUS SP ZK-003 and the like. These strains have been deposited in the Institute of Microbial Industry and Technology, National Institute of Advanced Industrial Science and Technology as No. 9184, No. 9185, No. 9186.

Hereinafter, the present invention will be described in detail. (1) Acquisition of microorganisms The present inventors have studied the use of Burgess Manual of Systematic Bacteriology (Be
Many microorganisms known to produce β-galactosidase were isolated according to the rgeys Manual of Systematic Bateriology, Volume 1 (hereinafter referred to as the manual). Then, those microorganisms were cultured to produce β-galactosidase, β-galactosidase was isolated from the medium, and its physicochemical properties were examined. As a result, three strains producing the desired β-galactosidase
When ZK-001, ZK-002 and ZK-003 were separated and identified according to the above manual, they were all aerobic aporous bacilli, had no motility, were Gram stain negative, catalase positive, and had a growth temperature of 40 to 80 ° C, optimal growth temperature 65-75 ° C, optimal growth
Since the pH is near neutrality, it is clear that the microorganism belongs to the genus Thermus, and differs from the microorganism belonging to the genus Thermus which is known from the growth in a liquid medium containing 2% NaCl and the assimilation of sugar. It was determined to be a new microorganism. The present inventors have deposited these microorganisms with the Research Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology (Thermus sp.).
No. 9184 for ZK-001 and No. 9 for Thermus sp. ZK-002
185 and Thermus sp. ZK-
003 was assigned a deposit number of No. 9186 of Microbiological Research Laboratory. The mycological properties of these microorganisms are as shown in Table 1.

[0010]

[Table 1]

*: Except for litmus milk, a medium having the following composition (pH 7.6) was used as a basal medium. 0.1% yeast extract, 0.1% tryptone, 10.0% inorganic salt solution (1L solution
1.0g nitrilotriacetic acid _{into, 0.6g CaSO 4 · 2H 2 O} , 1.0g Mg
_{SO 4 · 7H 2 O, 0.08g} NaCl, 1.03g KNO 3, 6.89g NaNO 3, 1.
11g Na ₂ HPO ₄ ), 1.0% ferric chloride solution (solution 1
0.28g FeCl ₃・ 6H ₂ O in L), 1.0% trace element solution
(0.5 ml H ₂ SO ₄ , 2.2 g MnSO ₄ .H ₂ O, 0.5 g in 1 L of solution
_{ZnSO 4 · 7H 2 O, 0.5g} H 3 BO 3, 0.016g CuSO 4 · 5H 2 O, 0.02
_{5g Na 2 M 0 O 4 ·} 2H 2 O, containing _{0.046g CoCl 2 · 6H 2 O)} , 0.001
% Thiamine hydrochloride, 0.001% nicotinamide, 0.001%
% Biotin, 0.001% para-aminobenzoic acid (2) Acquisition of the present enzyme The present enzyme-producing bacterium is inoculated into a medium described below, and is aerobically cultured at 50 to 80 ° C, more preferably at 65 to 75 ° C for 12 to 48 hours. I do.

The medium contains a carbon source, a nitrogen source and, if necessary, inorganic salts and trace nutrients. As the carbon source, various conventionally known materials can be used. For example, glucose, maltose, sucrose or soluble starch can be exemplified as typical examples. There is no particular limitation on the nitrogen source, and functional nitrogen such as yeast extract, peptone, meat extract, corn steep liquor, amino acid solution, and soybean meal, or nonfunctional nitrogen such as ammonium sulfate, urea, ammonium nitrate, and ammonium chloride are inexpensive. And it can be illustrated as what is easily available. It goes without saying that the organic nitrogen source is a carbon source. Further, in addition to such a carbon source and a nitrogen source, it is also possible to add various commonly used salts, for example, inorganic salts such as magnesium salts, potassium salts, phosphates, and iron salts, and vitamins. . Examples of suitable media are 0.2% glucose, 0.2% yeast extract, 0.2% tryptone, 10.0% inorganic salt solution (1.0 g nitrilotriacetic acid, 0.6 g CaSO _{4 in} 1 liter of solution).・ 2H ₂ O,
_{1.0g MgSO 4 · 7H 2 O,} 0.08g NaCl, 1.03g KNO 3, 6.89g Na
NO ₃ , 1.11g containing Na ₂ HPO ₄ ), 1.0% ferric chloride solution
(In solution 1L containing _{0.28g FeCl 3 · 6H 2 O)} , 1.0% of trace elements solution (in solution _{1L 0.5ml H 2 SO 4, 2.2g} MnSO 4 · H
_{2 O, 0.5g ZnSO 4 · 7H} 2 O, 0.5g H 3 BO 3, 0.016gCuSO 4 · 5H 2
O, 0.025g Na ₂ MoO ₄・ 2H ₂ O, 0.046g CoCl ₂・ 6H ₂ O included)
A liquid medium containing 0.001% thiamine hydrochloride, 0.001% nicotinamide, 0.001% biotin, and 0.001% para-aminobenzoic acid, the pH of which was adjusted to 7.6 with NaOH. The separation and purification of the present enzyme can be performed, for example, as follows. First, the cells in the culture solution are collected by centrifugation, filtration, or the like, and the obtained cells are disrupted by a conventional method in a buffer solution (for example, 10 mM phosphate buffer, pH 7.0) and subjected to extraction treatment. If the supernatant of the extract is taken and subjected to conventional ion exchange, gel filtration, etc. chromatography,
This enzyme is obtained.

A preferred method for obtaining the present enzyme is as follows. Thermus sp. ZK-0
01, for example, inoculated into 5 L of the above medium, and
12,000g of culture solution obtained by aerobically culturing for 20 hours at 20 ℃
And centrifuged for 30 minutes to collect the cells to obtain 55 g of cells. About 300 ml of phosphate buffer for extraction (10 mM, p
H7.0), and the mixture was ground with a mill to extract the enzyme. The extract was centrifuged at 40,000 g for 60 minutes, and the supernatant was collected. The precipitate was further added with 200 ml of the above-mentioned phosphate buffer for extraction, extracted again, centrifuged in the same manner, and the supernatant was collected. This was combined with the first supernatant to obtain 480 ml of a crude enzyme extract. 187 g of ammonium sulfate was added to the crude enzyme solution to make it 60% saturated, and the mixture was allowed to stand overnight to produce a precipitate. The precipitate was collected by centrifugation at 30,000 g for 60 minutes, and about 200 ml of 10 mM phosphate buffer (p.
H7.0) and dialyzed against the same buffer. After the precipitate in the dialyzed enzyme solution was removed by centrifugation at 40,000 g for 60 minutes, the enzyme solution was adsorbed to a DEAE-TOYOPEARL column equilibrated with 10 mM phosphate buffer (pH 7.0). Then 0
The enzyme was eluted by a concentration gradient method using 10 mM phosphate buffer (pH 7.0) containing 0.5 M NaCl. The eluted active fractions were collected, concentrated using an ultrafiltration membrane, and dialyzed overnight against a 10 mM phosphate buffer (pH 7.0) containing 0.1 M NaCl. This enzyme solution was added to a 10 mM phosphate buffer (pH 7.0) containing 0.1 M NaCl.
The mixture was applied to a gel filtration column of Sephacryl S-300 equilibrated in the above to separate three active fractions. The collected three active fractions were each dialyzed overnight using 10 mM phosphate buffer (pH 7.0) and then para-aminophenyl beta-dithiogalactopyranoside (p-amino phenyl-β) equilibrated with the same buffer. -D
-thiogalactopyranoside) was adsorbed on a Sepharose 4B column to which a covalent bond was attached. After thoroughly washing the column with 10 mM phosphate buffer (pH 7.0), the column was washed with 0.1 M borate buffer (pH 7.0).
10.0) was passed to elute the enzyme, and the active fraction was separated. The three active fractions thus obtained were each single in polyacrylamide gel disk electrophoresis (gel concentration 7.5%, pH 9.5). The obtained enzyme preparation was 3.4 mg in total of the three enzymes, and the activity yield was 12%. For the other two strains, the enzyme can be obtained in the same manner as described above with various changes in culture conditions. (3) Properties of the present enzyme The enzymatic chemical properties of the present enzyme produced by the method of the present invention are as follows. (a) Action: Decomposes lactose into galactose and glucose. (b) Substrate specificity: Decomposes lactose but not sucrose, melibiose, raffinose, maltose and gentiobiose. (c) Optimal pH and stable pH range: optimal pH is 4.5 to 6.5
It is stable within the range of pH 4.0 to 8.0 under the condition of holding at 55 ° C. for 24 hours (see the pH curve of the action of this enzyme in FIG. 1). (d) Stability to temperature: 100% activity remains after heating at 80 ° C for 1 hour at pH 7.0, and after heating for 1 hour at 85 ° C.
85% activity remains. (e) Range of suitable working temperature: It has an optimum working temperature of 75 to 85 ° C (see the working temperature curve of the present enzyme in FIG. 2). (f) Action of inorganic salts: 1 mM of ferric chloride, manganese chloride, calcium chloride and magnesium sulfate do not change enzyme activity, but 1 mM of zinc chloride and copper sulfate have 10% and 30% enzyme activity, respectively. Decrease. (g) Inhibition by the reaction product: The reduction of the enzyme activity by 50 mmol of galactose and glucose is 10% or less in each case (see FIG. 3 for the inhibition of the enzyme by the reaction product). (h) Molecular weight: The molecular weight of this enzyme was measured by gel filtration using Sephacryl S-300 column chromatography (1.6 × 100 cm), and found to be 55,000 ± 5,000 daltons, 11,000 ± 10,000 daltons, and 44,000 ± 50,000 daltons. Equivalent.

The physicochemical and enzymatic properties of this enzyme and β-galactosidase derived from a conventionally known microorganism (the enzymes of the above Examples 1 to 6) are compared in Table 2 below. still,
The method for measuring the activity of β-galactosidase and the method for indicating the activity are as follows. That is, 0.1 ml of the present enzyme solution was added to 2.4 ml of 0.1 M phosphate buffer (pH 6.5) containing 1.50% of O-nitrophenyl-β-D-galactopyranoside (hereinafter referred to as ONPG). Add 10% N after reacting at 70 ℃ for 10 minutes
The reaction is stopped by adding 2.5 ml of a ₂ CO ₃ solution. The amount of O-nitrophenol produced was determined from the absorbance at 420 nm,
The amount of the enzyme that releases 1 μmol of O-nitrophenol per minute was defined as one unit.

[0015]

[Table 2]

As shown in Table 2, the enzymatic and physicochemical properties of this enzyme are different from any of the known β-galactosidases, and particularly high heat stability and a wide range of pH
And the characteristics of the degree of inhibition by the reaction product are low, and have excellent characteristics not found in conventional enzymes.
Therefore, this enzyme is suitable for treating many lactose-containing foods such as milk, skim milk, cheese whey or lactose solution at a high temperature, and is an industrially useful β-galactosidase.

[0017]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited at all by the following examples. [Example 1] Thermus sp. ZK-001 (Thermus sp. ZK
-001: No. 9184, manufactured by Japan Fine Industrial Research Institute, Inc .; 0.2% glucose, 0.2% yeast extract, 0.2% tryptone, 10.0% inorganic salt solution (1.0 g nitrilotriacetic acid in 1 L of solution, 0.6g CaSO ₄
・ 2H ₂ O, 1.0g MgSO ₄・ 7H ₂ O, 0.08gNaCl, 1.03g KNO ₃ ,
6.89 g NaNO ₃ , 1.11 g Na ₂ HPO ₄ ), 1.0% ferric chloride solution (0.28 g FeCl ₃ .6H ₂ O in 1 liter of solution), 1.
0% trace element solution (0.5 ml H ₂ SO ₄ , 2.2 g Mn
SO ₄・ H ₂ O, 0.5g ZnSO ₄・ 7H ₂ O, 0.5g H ₃ BO ₃ , 0.016g CuS
_{O 4 · 5H 2 O, 0.025g} Na 2 MoO 4 · 2H 2 O, 0.046g CoCl 2 · 6H 2 O
, Containing 0.001% thiamine hydrochloride, 0.001% nicotinamide, 0.001% biotin, 0.001% paraaminobenzoic acid, inoculated in 10 L of a liquid medium adjusted to pH 7.6 with NaOH, and inoculated in 20 L. Aeration culture was performed at 70 ° C. for 24 hours in a jar fermenter. The enzymatic activity of the culture is
It was 0.84 units per ml of culture.

The culture was centrifuged at 12,000 g for 30 minutes at 20 ° C., and the cells were collected to obtain 107 g of the cells. About 600 ml of a phosphate buffer for extraction (10 mM, pH 7.0) was added to the cells, and the cells were ground by a mill to extract the enzyme. 40,000 extract
After centrifugation at g for 60 minutes, the supernatant was collected. The precipitate was further added with 400 ml of the above-mentioned phosphate buffer for extraction, extracted again, and then centrifuged in the same manner to collect the supernatant. This was combined with the first supernatant to obtain 975 ml of a crude enzyme extract. 380 g of ammonium sulfate was added to the crude enzyme solution to make it 60% saturated, and the mixture was allowed to stand overnight to form a precipitate. Next, the precipitate was collected by centrifugation at 30,000 g for 60 minutes, dissolved in about 30 ml of 10 mM phosphate buffer (pH 7.0), and dialyzed against the same buffer. The precipitate in the dialyzed enzyme solution was
After removing by centrifugation at 0,000g for 60 minutes,
DEAE-TOYOPEARL equilibrated with 0 mM phosphate buffer (pH 7.0)
The column was adsorbed (5 × 45 cm). Then 0-0.5M N
The enzyme was extracted by a concentration gradient method using 10 mM phosphate buffer (pH 7.0) containing aCl. 48.0 ml of the eluted active fraction was concentrated to about 10 ml using an ultrafiltration membrane (PM-10, manufactured by Amicon), and then overnight using a 10 mM phosphate buffer (pH 7.0) containing 0.1 M NaCl. Dialyzed. This enzyme solution is added to 0.1M NaCl containing 10M
Sephacryl S-30 equilibrated with mM phosphate buffer (pH 7.0)
The solution was passed through a gel filtration column (2.6 × 95 cm) of No. 0 to separate three active fractions. The three active fractions were combined, dialyzed overnight using 10 mM phosphate buffer (pH 7.0), and then equilibrated with the same buffer to give para-aminophenyl vader dithiogalactopyranoside (p-amino phenyl-β-). D-thiogalactopyr
Sepharose 4B column (1.0
× 15.0 cm). The column was thoroughly washed with 10 mM phosphate buffer (pH 7.0), and then washed with 0.1 M borate buffer (pH 1).
0.0) was passed to elute the enzyme, and the active fraction was separated. Thus, about 8.5 mg of the present enzyme was obtained.

[Embodiment 2] Thermus SP ZK-002 (T
hermus sp. ZK-002: Microtechnical Laboratory No. 9185) was inoculated to the medium of Example 1 in a volume of 10 L into a medium in which glucose was changed to 0.2% soluble starch under the same conditions as in Example 1. Culture,
A culture solution of 0.64 units / ml was obtained. Thereafter, purification was carried out in the same manner as in Example 1 to obtain about 6.8 mg of the present enzyme.

Embodiment 3 Thermus SP ZK-003 (T
hermus sp.ZK-003: No. 9186) was inoculated into 10 L of a medium having the composition described in Example 1 and cultured under the same conditions as in Example 1 to obtain a 0.68 unit culture solution. . Thereafter, purification was carried out in the same manner as in Example 1 to obtain about 7.3 mg of the present enzyme.

[0021]

According to the present invention, a β-galactosidase having high heat resistance, a wide pH range of action, and a low degree of inhibition by a reaction product, and a novel strain capable of producing the β-galactosidase are provided. Can be provided. The enzyme makes it possible to treat lactose-containing foods such as milk, skim milk, cheese whey and lactose at high temperatures at which bacteria are less likely to spoil. This makes the control of the manufacturing process at the time of processing these foods easier and is extremely significant in the food industry.

[Brief description of the drawings]

FIG. 1 is a diagram showing the pH curve of the action of the present enzyme.

FIG. 2 is a diagram showing an action temperature curve of the present enzyme.

FIG. 3 is a diagram relating to inhibition by the reaction product of the present enzyme.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical display location C12R 1:01) C12R 1:01)

Claims (1)

(57) [Claims] 1. It has an optimum working temperature of 75-85 ° C.,
To produce a novel β-galactosidase having an optimum pH of 6.5 and whose enzymatic activity is not substantially reduced by 50 mmol of galactose and glucose, and having at least the mycological properties described below. The feature is Thermus SP ZK-001, Thermus SP ZK-002 or Thermus SP ZK-003. (a) Morphological properties Shaped bacilli Motility-Gram stain-Flagella-Spores-Acid-fast-Size 0.4-0.6 x 2-7 μm (b) Growth on various media Agar flat media Round or peripheral Regular, raised or flat, pale yellow to orange agar slant medium Smooth, pale yellow to orange liquid medium No litmus milk with sedimentation of growing cells No change 5% NaCl-containing liquid medium-2% NaCl-containing liquid medium + ( c) Growth pH and temperature Growth pH 5.5-8.5 Growth temperature 40-80 ℃ (d) Biochemical properties Nitrate reduction + Denitrification reaction ± VP test-Indole formation-Hydrogen sulfide formation-Starch hydrolysis ± Use of citric acid-Use of inorganic nitrogen source + Formation of (NH ₄ ) pigment Generated (yellow, orange) Urease-Oxidase + Catalase + OF test O Oxygen requirement No growth under anaerobic conditions (e) Sugar Assimilation of D-gluco Scan + D- mannose + D- fructose + maltose + sucrose + lactose + trehalose + glycerin - starch -