JPH0634715B2 - Isomaltose degrading enzyme and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention specifically acts on isomaltose, and its α-
The present invention relates to a novel isomaltose degrading enzyme that hydrolyzes a 1,6-glucoside bond and a method for producing the same.

Isomaltose degrading enzyme is an enzyme that performs endo-hydrolysis of α-1,6-glucoside bond in dextrin or isomaltose produced from starch or glycogen by the action of α-amylase, and is useful in the starch industry or the brewing industry. The enzyme.

Conventionally, as the isomaltose-degrading enzyme, an enzyme obtained from Bacillus macerans has been known, but there are very few reports on this enzyme.

The enzyme of the present invention has a very wide working pH range of 5 to 10, and even when the concentration of glucose in the substrate solution is high, it selectively recognizes isomaltose and decomposes very well. In the above, it is a novel enzyme having characteristics not found in conventional isomaltose degrading enzymes.

The present invention will be described in detail below. First, the microorganism used in the present invention is a microorganism belonging to the genus Bacillus that grows well in the medium shown below, and has the ability to produce the isomaltose-degrading enzyme of the present invention. No. 1 to No. 5 are isolated from Mine hot spring in Shizuoka Prefecture, and No. 6 is isolated from soil in Chiyoda-ku, Tokyo, and the microorganism deposit numbers (FERM-P) are as follows.

The above microorganisms are referred to as F-2 bacteria, F-5 bacteria, and F-1 in the following order.
They are referred to as 4 bacteria, G-1 bacteria, G-4 bacteria and R-18 bacteria. The present inventors have found that each of the above microorganisms produces and accumulates a high unit of isomaltose degrading enzyme in the culture, and has completed the present invention.

The above microorganisms have the following mycological properties. In addition, the test of the mycological properties described below is "Bird AIDS
Manual of Definite Bacteriology 8th Edition ["Bergeys Manual of Determinative Bact
eriology "8 thedition (1974)] and" The Genus Bacillus "(19
3)], and unless otherwise stated, all sodium hydrogen carbonate (NaHCO ₃ )
Was used to adjust the pH to about 9.5.
This composition is shown in Table 1.

(1) Mycological properties of F-2 bacterium (a) Morphology (meat and agar medium) The size is 0.5 × 0.7 μ × 2 to 3 μ, and forms a hyphae long chain like actinomycetes at the initial stage of culture. Form spores and their size is
It has an oval shape of 0.5 × 0.7μ × 0.6 to 0.8μ. The sporangium is not inflated. Periflagellated and motile. Also, neither cell polymorphism nor acid resistance is present. Gram stain shows positive in the early stage of culture.

(b) Growth condition in medium Culture condition Growth medium broth agar plate culture The shape of the colony is circular, the surface of the colony is ridged, and the periphery of the colony is ears and thread-like. The color of the settlement is white, opaque and wrinkled, and the surface of the agar becomes purple.

Meat broth agar flat slope culture Feathery and agar surface turns purple.

Liquid broth culture Grows and becomes a thick-film pellicle. The bottom of the membrane turns purple.

Meat broth gelatin stab culture Only the surface grows, not the deep part. Liquefy gelatin.

Litmus milk Milk does not solidify and turns into peptone. Decolorize litmus.

(c) Physiological properties Reduction of nitrate Reduces.

No denitrification reaction MR test No change in MR under alkaline conditions VP test Positive No indole formation No hydrogen sulfide formation No hydrolysis of starch Decomposes.

Use of citric acid Grow slightly on Koser medium.

Grows well in Christensen medium.

Use inorganic nitrogen source.

Pigment formation Produces a water-insoluble pink to purple pigment (King A medium).

Urease positive oxidase positive catalase positive growth range pH 5-10 (optimal pH 7-9.5) 20-55 ° C (optimal temperature 40-50 ° C) attitude toward oxygen aerobic OF test aerobic, anaerobic, making acid together .

Utilization of saccharides (1) L-arabinose + (2) D-xylose- (3) D-glucose + (4) D-mannose + (5) D-fructose + (6) D-galactose- (7) maltose + (8) sucrose + (9) lactose − (10) trehalose + (11) D-sorbit − (12) D-mannitol − (13) inositol − (14) glycerin − (15) starch ± +: , Makes acid, but does not emit gas.

-: Do not use.

±: Use, but do not make acid.

Use of propionic acid It grows well.

Salt broth Grows with 10% salt.

Hydrolysis of casein Decomposes.

(2) Bacterial properties of F-5 bacterium (a) Morphology, (b) Growth state in medium, (c) Physiological properties are the same as those of the F-2 bacterium except for the following.

Utilization of saccharides (6) D-galactose + (12) D-mannitol + (3) F-14 fungal mycological properties (a) morphology, (b) growth state in medium, (c) physiological properties Same as the F-2 fungus except for the following.

(b) Growth state in medium Meat agar plate culture The shape of the colony is circular, the surface of the colony is ridge-like, and the periphery of the colony is ear-like and thread-like. The color of the village is white, opaque and wrinkled.

Meat broth agar flat slope culture Feather-like.

Liquid broth culture Grows and becomes a thick-film pellicle.

Litmus milk Coagulate milk.

Decolorize litmus.

(c) Physiological properties Pigment formation A small amount of water-insoluble pink pigment is formed (King A medium).

Utilization of saccharides (12) Mycological properties of D-mannitol + (4) G-1 bacterium (a) Morphology, (b) Growth state in medium, (c) Physiological properties except for the following F- Same as 14 bacteria.

(a) Morphology Size 0.4-0.6μ × 1.5-3μ Spores 0.4-0.5μ × 0.5-0.7μ (b) Growth condition in medium Litmus milk Milk does not coagulate but becomes peptone. Decolorize litmus.

(c) Physiological properties Pigment formation It produces a slight amount of color pigment in the medium (King A medium).

Utilization of saccharides (12) D-mannitol- (15) starch + (5) G-4 fungal properties (a) morphology, (b) growth state in culture medium, (c) physiological properties are as follows. Except for the above, it is the same as the G-1 bacterium.

(b) Physiological properties Pigment formation A slight amount of water-insoluble pink pigment is formed (King A medium). It produces a slight amount of color pigment in the medium (King A medium, King B medium).

Utilization of saccharides (12) D-mannitol + (15) starch ± (6) Mycological properties of R-18 bacterium (a) Morphology, (b) Growth state in medium, (c) Physiological properties are as follows. Except for the above, it is the same as the F-14 bacterium.

(b) Physiological properties Utilization of saccharides (12) D-mannitol-It is clear that each of the microorganisms used in the present invention belongs to the genus Bacillus since all of them are aerobic spore bacteria. Comparing these with known bacterial species, each of the 6 strains is close to Bacillus licheniformis in that it utilizes propionic acid, but considering that it does not grow under anaerobic conditions, Bacillus subtilis (Bacillus) subtilis).

However, compared to both Bacillus licheniformis and Bacillus subtilis producing acid from xylose and mannitol, 6 strains of xylose and F-2, F-5, and mannitol of F-2, F-5 and mannitol, respectively.
G-1 and R-18 bacteria could not even be assimilated.

Furthermore, the most characteristic difference is that in the case of Bacillus subtilis and Bacillus licheniformis, it grows only in the range of pH 5.0 to 8.0, whereas the microorganisms used in the present invention have a wide pH range of 5.0 to 10 respectively. It has been pointed out that it grows well and grows well even in a highly alkaline medium.
It is clearly distinguished from the comparative bacteria.

As described above in detail, the F-2 bacterium, F-5 bacterium of the present invention,
The F-14 bacterium, G-1 bacterium, G-4 bacterium, and R-18 bacterium cannot find a matching species among known bacterial species, and the Bacillus genus is judged from the characteristic mycological properties. It was concluded that it is appropriate to set it as a new strain of.

The microorganism used in the present invention is one that produces and accumulates a high unit of isomaltose-degrading enzyme in the cultured cells and culture medium, respectively, and in the culture thereof, glucose, sucrose, starch as a carbon source in the medium are used. Sugar sources such as, as the nitrogen source, it is preferable to use organic substances such as peptone, meat extract, corn stay liquor, defatted soybean,
Inorganic substances such as ammonium salts, phosphates and nitrates can also be used.

In addition, it is advisable to add a trace amount of inorganic metal salts, vitamins and growth promoting factors such as yeast extract. It is particularly desirable to add 0.5 to 1.5% of sodium hydrogen carbonate to the composition of the medium in order to bring the pH of the culture solution to around 9. Further, in culturing, it is preferable to carry out shaking or aeration stirring at around 30 to 50 ° C., and thus, the accumulation amount of the target isomaltose-degrading enzyme becomes maximum when the culturing time is about 15 to 45 hours. The culture solution is divided into bacterial cells and culture solution by a known method such as centrifugation, and the bacterial cells are treated by a known method such as ultrasonic treatment or French press to obtain a bacterial cell extract to obtain a crude enzyme solution. be able to. This crude enzyme solution may be used as it is, but a crude enzyme can be obtained by applying a known method such as an ammonium sulfate salting-out method, a solvent precipitation method, and a dialysis method in an appropriate combination. Alternatively, the product may be purified and crystallized by the above method to obtain a purified enzyme preparation.

The isomaltose-degrading enzyme preparation of the present invention is F-2, F-
No. 5, F-14, G-1, G-4, and R-18 bacteria can be obtained from both the bacterial cells and the culture broth.

Next, the physicochemical properties of the isomaltose-degrading enzyme A of the present invention (hereinafter referred to as “the present enzyme”) will be described.

(1) Action and substrate specificity: It acts specifically on isomaltose and hydrolyzes its α-1,6-glucoside bond extremely well.

(2) Working pH and optimum working pH: Working pH is in a wide range of pH 5 to 10, and optimum pH is pH.
6.5 to 7.0.

(3) Stable pH: It is stable at about pH 6 to 9 and relatively stable on the alkaline side rather than the acidic side.

[0.1M buffers (pH 3.0-5.0 is citrate buffer, pH
6.0-8.0 is phosphate buffer, pH 9.0-11.0 is glycine-N
aCl buffer, pH 120-13.0 is KCl-NaOH buffer) 1 m
After adding enzyme solution to the total volume of 2m, 60 at 40 ℃
After leaving it for a minute, it was cooled with water and the residual activity was measured immediately. (4) Working temperature and optimum working temperature: It works up to about 60 ° C, and the optimum working temperature is 40 to 50 ° C.

(In the enzyme activity measuring method (8) described later, the measurement was carried out by changing the temperature of the reaction at 40 ° C to each temperature.) (5) Thermal stability (inactivation condition): 0.05 M phosphate buffer The enzyme solution was added to (pH 6.8) and heated at each temperature for 60 minutes to measure the residual activity. As a result, almost no deactivation was observed by heating up to 45 ° C., and deactivation of about 30-40% by heating at 50 ° C. for 60 minutes,
About 80-90% deactivation by heating at 55 ° C for 60 minutes, 60
Almost completely deactivates by heating for 60 minutes at ℃.

(6) Inhibition: Strongly inhibited by Hg ⁺⁺ and Ag ⁺⁺ and also inhibited by p-chloromercury benzoic acid.

(7) Molecular weight: Cefacryl S-200 (“Sehacryl s-2”, fully equilibrated with 50 mM phosphate buffer (pH 6.8) containing 0.1 M NaCl.
When the gel was passed through a 00 "Swedish Pharmacia company, the molecular weight of this enzyme was 50,000-60,000.

(8) Enzyme activity measurement method: 0.1m of enzyme solution was added to 0.9m of 50mM phosphate buffer (pH6.8) containing 11.1mM isomaltose (3.8mg / m),
Incubate for 10 minutes at 40 ° C. Then, the increase in the sugar produced is measured by the "glycostat" method [Biochemical Experimental Method I, 1
40 pages (1978), Sakuzo Fukui, Academic Publishing Center; P.Trinder, Ann.Clin.Biochem., 6, 24 (196).
See 9)]. That is, 1 m of a glucostat reagent (manufactured by Fujisawa Pharmaceutical Co., Ltd.) was added to 0.5 m of the reaction solution or an appropriately diluted reaction solution, the mixture was heated at 37 ° C. for 10 minutes to develop color, and this was colorimetrically determined at a wavelength of 500 mn.

The unit of the enzyme titer is 1 unit of the amount of enzyme that decomposes 1 μmol of isomaltose per minute under the above-mentioned conditions (that is, produces 2 μmol of glucose).

Hereinafter, the present invention will be described in detail with reference to Examples.

Example 1 Soluble starch 1%, polypeptone 0.5%, yeast extract
Sterilize a medium containing 0.5%, K ₂ HPO ₄ 0.1%, MgSO ₄ · 7H ₂ O 0.02% by separate sterilization so that the final concentration is 0.5% and 1% respectively. The pH was adjusted variously.

500 prepared by previously sterilizing 100 m of the above medium
Place in an m-volume Erlenmeyer flask and add 1m of pre-culture liquid of each 6 strains
The cells were inoculated and cultivated at 37 ° C for 20 hours with rotary shaking.

After culturing, the culture solution was taken in a number of m, 1 to 2 drops of toluene was added, and the mixture was vigorously stirred and treated with toluene to obtain a crude enzyme solution.

The results of comparing the activities are shown in Table 2.

Example 2 5 g of soluble starch, 25 g of polypeptone, yeast extract
400 m of each medium containing 2.5 g, 5 g of K ₂ HPO ₄ and 0.1 g of MgSO ₄ .7H ₂ O was placed in a 2-volume pleated Erlenmeyer flask and sterilized by a conventional method. Separately sterilized 5% aqueous sodium hydrogencarbonate solution (100 m) was aseptically added to the medium to make the medium alkaline (pH 9.2).

Each medium was inoculated with the preculture liquid of each of the 6 strains used in Example 1, which had been precultured for 12 hours in the medium of the same composition, for 2.5 to 5 m, and cultured by shaking at 37 ° C. for 20 hours.

After culturing, it was centrifuged to separate into a supernatant and cells. The cells were washed 3 times with physiological saline, then suspended in physiological saline containing 50 mM phosphate buffer (pH 6.8), sonicated and centrifuged to obtain a cell extract.

Both the supernatant and the bacterial cell extract were dialyzed overnight in running tap water using a dialysis membrane tube.

After concentrated with polyethylene glycol 6,000, dialyzed against 50 mM phosphate buffer (pH 6.8) at 4 ° C for several hours, and the supernatant was adjusted to 1/5 volume of the medium volume and the cell extract to 1/10 volume. Then, it was used as a crude enzyme solution outside the bacterial cells and inside the bacterial cells, respectively.

The results of measuring the enzyme activity are shown in Table 3.

Example 3 0.1 containing 20 mM isomaltose, 40 mM glucose
When 0.5 m of the crude enzyme solution diluted to 100 mU / m was added to 0.5 m of the M phosphate buffer, reacted at 40 ° C. for 6 hours, and analyzed by high performance liquid chromatography.
Almost no residual isomaltose was confirmed in the crude enzyme reaction solution of each of the 6 strains used in.

The high performance liquid chromatography was performed using Shimadzu Corporation column SCR-101 under the conditions of 40 ° C. and distilled water of 0.6 m / min, and the isomaltose concentration was measured using a differential refractometer.

From the above results, it was found that the present enzyme specifically acts on isomaltose and hydrolyzes glucose very well even when glucose is present at a high concentration in the substrate solution.

Claims (8)

[Claims] 1. Isomaltose-degrading enzyme A having the following physicochemical properties: Action and substrate specificity: Acts specifically on isomaltose and hydrolyzes its α-1,6-glucoside bond. Working pH and optimum working pH: Working pH is pH 5-10, optimum working pH is pH 6.5-7.
It is 0. Stable pH: Stable pH is approximately in the range of pH 6-9. Working temperature and optimum working temperature: Working up to about 60 ° C, optimum working temperature is about 40-50 ° C
Is. Thermal stability (deactivation conditions): Stable at 45 ° C or lower, and almost completely deactivated at 60 ° C for 60 minutes. Inhibition: Strongly inhibited by Hg ⁺⁺ and Ag ⁺⁺ , and also inhibited by p-chloromercury benzoic acid. Molecular weight: The molecular weight is 50,000-60,000 (gel filtration method). 2. A method for producing isomaltose-degrading enzyme A, which comprises culturing an isomaltose-degrading enzyme A-producing bacterium belonging to the genus Bacillus and isolating and collecting isomaltose-degrading enzyme A from the culture. 3. An isomaltose-degrading enzyme A-producing bacterium belonging to the genus Bacillus is Bacillus sp. F-2 (Bacillus s).
p.F-2) (Ministry of Industrial Science, No. 7496), The method according to claim (2). 4. An isomaltose-degrading enzyme A-producing bacterium belonging to the genus Bacillus is Bacillus sp. F-5 (Bacillus s).
p.F-5) (Ministry of Microbiological Research, No. 7497), The method according to claim (2). 5. An isomaltose-degrading enzyme A-producing bacterium belonging to the genus Bacillus is Bacillus sp. F-14 (Bacillus).
sp. F-14) (Ministry of Industrial Science, No. 7498), The method according to claim (2). 6. An isomaltose-degrading enzyme A-producing bacterium belonging to the genus Bacillus is Bacillus sp. G-1 (Bacillus s).
p.G-1) (Ministry of Microbiological Research, No. 7499), The method according to claim (2). 7. An isomaltose-degrading enzyme A-producing bacterium belonging to the genus Bacillus is Bacillus sp. G-4 (Bacillus s).
p.G-4) (Ministry of Industrial Research, No. 7500). The method according to claim (2). 8. An isomaltose-degrading enzyme A-producing bacterium belonging to the genus Bacillus is Bacillus sp. R-18 (Bacillus).
sp. R-18) (Ministry of Microbiological Research, No. 7501), The method according to claim (2).