KR960007741B1 - Novel(alpha)-1,6-glucosidase and process for producing the same - Google Patents

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Description

Novel α-1,6-glucosidase and preparation method thereof

Figures 1 to 5 show the enzyme chemical properties of α-1,6-glucosidase according to the present invention, Figure 1 is a graph showing the optimum pH range, Figure 2 is 40 ℃ (○) and 50 ℃ (*) Is a graph showing the stable pH range, FIG. 3 is a graph showing the optimum temperature, Figure 4 is a graph showing the thermal stability, Figure 5 is a 55 ℃ (○) 60 ℃ (□) and 65 ℃ ( And enzyme stability at various pH values.

The present invention relates to a novel α-1,6-glucosidase and a method for producing the same, wherein α-1,6-glucosidase has an optimum pH of about 5.0 to 5.5 and an optimum temperature of about 55 ° C in more detail. Production of the novel strain Bacillus sectorramus (Bacillus sectorramus) characterized in that the new α-1,6-glucosidase obtained from the strain culture and the α-1,6-glucosidase from the culture of the strain described above One novel α-1,6-glucosidase is provided.

α-1,6-glucosidase is an enzyme capable of producing linear amylose by acting on α-1,6-glucosidase binding in starch. Maltose, glucose and high fructose corn syrup (HFCS: It is widely used in the saccharification industry producing high fructose corn syrup.

α-1,6-glucosidase has long been found in microorganisms, such as higher plants and yeasts, but recently it has been reported that Aerobactor aerogenes, Escherichia intermedia, Produced by various bacteria such as Pseudomonas amyloderamosa, Streptococcus mites, Cytophaga, Streptomyces and Flavochromogenes It turned out to be.

On the other hand, Bacillus cereus (IFO 300), Bacillus fermus (IFO 3330), Bacillus acidofluity among the α-1,6-glucosidase produced by the Bacillus strain. Those due to Cous (Bacillu acidopullulyticus) and the like are already known.

The most widely known α-1,6-glucosidase has a neutral or weakly alkaline optimal pH value and is not suitable for use in the saccharification industry where the saccharification process is performed under acidic conditions.

Α-1,6-glucosidase, which has an acidic optimal pH value, is already known to be produced by Pseudomonas amylodaemosa, Bacillus asidfluluticus, etc., but the former is impractical due to its poor temperature resistance. On the other hand, the latter is not economically desirable because it requires a long bacterial culture period of 60 to 73 hours.

The present inventors have therefore studied to find microorganisms having temperature resistance, acid optimal pH and height α-1,6-glucosidase productivity.

As a result, based on the findings that the newly discovered new Bacillus strains from soil microorganisms can produce α-1,6-glucosidase in the culture in a very short incubation period of 10 to 15 hours. The present invention has been accomplished by collecting the product.

Some of the present invention provide novel α-1,6-glucosidase having the enzymatic properties described below.

(1) Action: The enzyme acts on α-1,6-glucosidase binding to produce straight amylose.

(2) Substrate specificity: The enzyme has the following biological activity against the polysaccharide.

(3) Km value for Vulran and Vmax: The enzyme has a Km value of 0.14 mg / ml and a Vmax of 70.0 μmol / min / mg protein.

(4) Optimum pH: 5.0 to 5.5

(5) pH stability: The enzyme is stable at 40 ° C. for 30 minutes at a pH range of 4.5 to 6.5 and at 50 ° C. for 30 minutes at a pH range of 5.0 to 6.0.

(6) Optimal temperature: about 55 ℃.

(7) Thermal Stability: The enzymes were treated at different temperatures for 30 minutes at pH4.5 and the enzymes retained initial activity of 80% at 40 ° C and 10% at 60 ° C.

(8) Effect of Inhibitors: Enzymes are inhibited by more than 70% by p-chloromercuric benzoic acid or sodium dodecyl sulfate, but not by o-pinanthroline or potassium parisanide.

(9) Effect of metal salts: The enzyme is hardly affected by Ni ²⁺ , Ba ²⁺ , Zn ²⁺ , Ca ²⁺ or Mn ²⁺ , but is inhibited by Fe ³⁺ , and Hg ²⁺ or Ag Loses activity by ²⁺ and

(10) Molecular weight: about 95,500 (measured by SDS electrophoresis).

In another part of the present invention, Bacillus Sectoramus strain producing α-1,6-glucosidase is cultured in a nutrient vegetation to produce α-1,6-glucosidase, and α-1,6 A method for producing α-1,6-glucosidase, which is characterized by recovering α-1,6-glucosidase after accumulating -glucosidase.

The α-1,6-glucosidase-producing strain used in the present invention is a novel strain, which was named by Bacillus sectoramus.

According to the Budapest Convention on the International Approval of Microbial Deposits in accordance with the Patent Procedure, the strain is the Ministry of Trade, Industry and Energy of Japan, located in Higashi 1-chome 1-3, Yataba-machi, 305 Tsukuba, Japan The Institute of Microbiology and Technology Institute (FRI) has been deposited with the accession number FERM BP-1471 (original accession number: FERM P-8973, deposited on September 22, 1986), the following describes the strain as the present invention strain Shall be.

The strain of the present invention has the following bacteriological properties:

A. Form

(1) The shape of the cell: Fungus

(2) Cell size: 1.0-1.3 × 1.5-5.2 μm

(3) Mobility: None

(4) Spores: Exist

(5) Gram dyeing: Positive

(6) Acid-resistant strain: Negative

B. Growth in Various Media

(1) standard agar plate culture

Colonies form in a circle with complete surroundings.

The surface is smooth and translucent grayish white.

(2) Standard agar slope culture

Straight, buttery, glossy with a flat surface.

Medium growth.

(3) Standard liquid culture

Slightly cloudy with powdery precipitate.

No pigment is formed.

(4) Standard gelatin culture.

Straight, not liquefied.

(5) litmus milk

immutability.

C. litmus milk

(1) nitrate reduction: positive

(2) Denitrification: Positive

(3) Methyl rad test: positive

(4) Formation of Acetyl Methyl Carscale: Negative

(5) Formation of indole: negative

(6) formation of hydrogen sulfide: negative

(7) starch hydrolysis: positive

(8) citric acid assimilation: positive

(9) assimilation of inorganic nitrogen sources (nitrates and ammonium salts): positive

(10) Pigmentation: negative

(11) urease activity: negative

(12) Oxidase activity: negative

(13) catalase activity: positive

(14) Growth range:

pH 4.0-7.1

Temperature 45.5 ~ 15.0 ℃

(15) Oxygen composition: anaerobic

(16) Formation of deoxyacetone: negative

(17) Decomposition of Hypuric Acid: Negative

(18) Arginine Degradation: Negative

(19) Deamination of Phenylalanine: Negative

(20) Temperature resistance: positive

(85 ℃, 10 minutes)

(21) Sodium chloride resistance: 7% negative

3.5% positive

(22) Growth in Saburo Agar Bunny: Positive

(23) Growth in 0.001% lysozyme medium: positive

(24) Tyrosine Degradation: Positive

(25) Alkali Production in Citrate-Ammonium Agar: Positive

Casein Breakdown: Negative

(27) Gelatin breakdown: negative

(28) Positive growth in anaerobic medium

(29) Growth negative in McConkie medium

(30) Lecithinase reaction: negative

(31) Alkali Productivity in VP Medium: Negative

(32) A fairy tale and acid productivity of sugar: negative

(a) L-arabinose positive

(b) D-xylose positive

(c) D-glucose positive

(d) D-Mannorz positive

(e) D-protose positive

(f) D-galactose positive

(g) Maltose positive

(h) Sukurozu positive

(i) lactose voice

(j) Trehalose positive

(k) D-sorbitol negative

(l) D-mannitol negative

(m) inositol negative

(n) glycerin positive

(o) starch positive

(p) melibiose positive

(q) The voice of life

(r) ethanol positive

Buggy's Manual of Determinative Bacteriology. 8th edition, williams wilkins Co. (1974) and the International Journal of Systematic Bacteriology, 1974-1986. It has been shown to be classified as genus Bacillus due to Gram-positive, presence of spores and aerobic growth.

The strain of the present invention does not resemble any Bacillus strain, but is somewhat similar to Bacillus cereus or Bacillus megaterium in that the cells are relatively thick, about 1.0 to 1.3 mu.

However, the strains are distinguished from the strains of the present invention in the following aspects.

Bacillus cereus grows in 7% saline and shows positive results in yolk reaction, casein breakdown and gelatin breakdown, but the strain of the present invention does not grow in 7% saline and does not show yoke reaction, casein breakdown and Negative results on gelatin degradation.

In addition, Bacillus megaterium grows in 7% saline and shows positive results in casein and gelatin degradation, but the strain of the present invention does not grow in 7% saline and shows negative results in casein and gelatin degradation.

Furthermore, as summarized in Table 1 below, the strain of the present invention is also distinguished from the new Bacillus asidopuluriticus disclosed in Japanese Patent Laid-Open No. 43994/1986.

Thus, the strains of the present invention have been identified as new strains different from all known Bacillus strains and have been named as described above.

Hereinafter will be described with respect to the conditions for producing and accumulating α-1,6-glucosidase from the culture of the present invention strain.

First, suitable inorganic salts; Carbon sources such as starches such as soluble starch, waxy starch and potato starch and hydrolyzed starches such as maltose and dextrins; And suitable culture media containing nitrogen sources such as polypeptone, meat extract, yeast extract, corn steep liquor, hydrolyzed proteinyl such as diammonium phosphate, casein and soy protein have been shown to provide high pullulanase activity. .

The strain of the present invention was inoculated into the above-described culture medium, the pH was adjusted to 4.0 to 7.0, and then shaken under agitation or under aerobic conditions, or aerated, for 1 to 2 days at 20 to 45 ° C. Shows good results).

After incubation, the cells are removed from the culture broth, and the obtained supernatant fractions are concentrated to obtain crude pullulanase preparation.

The preparation was fractionated with ammonium sulfate and then subjected to affinity chromatography using γ-cyclodextrin-cephalos 6B (trademark, Pharmacia Chemicals) produced by coupling γ-cyclodextrin to active Sepharose 6B. , SDS-polyacrylamide gels were subjected to electrophoresis to purify single bands.

The following describes the enzymatic properties of purified α-1,6-glucosidase.

1.Action: The lake acts on α-1,6-glucosidine binding to produce straight amylose.

2. Km Value and Vmax for Substrates: The km value (mg / ml) and Vmax value (1 μmol / min / mg protein) for flulan, potato amylopectin and corn amylopectin of the enzyme of the present invention are shown in Table 2 below. .

3. Relative activity against polysaccharides: The relative activity of enzymes against soluble corn amylopectin, soluble starch, oyster glycogen, hare glycogen, corn amylopectin and potato amylopectin compared to pullulan activity is shown in Table 3 below.

Optimal pH: The optimal pH in 25 mM citric acid-50 mM disodium phosphate buffer (see Figure 1) was found to be 5.0-5.5.

5.pH stability: exposure to 40 ° C. in a range of pH 4.5 to 6.5 in 25 mM citric acid-50 mM disodium phosphate buffer for 30 minutes, and exposure to 50 ° C. in a range of pH 5.0 to 6.0 for 30 minutes. It was stable (see Figure 2).

6. Optimum temperature: In 50 mM acetic acid buffer (pH4.5), the enzyme showed an optimum temperature of about 55 ° C (see Figure 3).

Thermal stability: After 30 minutes of treatment at 50 ° C acetic acid buffer (pH4.5) at different temperatures, the enzyme maintained 80% initial activity at 40 ° C but 10% initial activity at 60%. See Figure 4.

8. Activity analysis: 4 ml of 0.5% pullulan solution (pH 4.5) is added to 1 ml of enzyme solution, and the reaction is carried out at 40 ° C for 30 minutes. Then 2 ml of sogyi solution is added and the mixture is heated for 20 minutes and then cooled. After cooling, 1 ml of ammonium argenomolybdenate is added and then water is added to bring the total volume to 25 ml. The absorbance at 500 nm of the solution was measured using a 1 cm thick film. Under the above conditions, the activity when 1 μmol of glucose and the corresponding reducing sugar is produced per minute is defined as 1 unit.

9. Inhibitor Effect: The enzyme is inhibited by more than 70% by p-cholomercuricbenzoic acid (PCMB) or sodium donesylsulfate (SDS), but not by o-phenanthroline or potassium ferricyanide. The effects of the various inhibitors are shown in Table 4 below, and these results show the residual activity after treatment for 30 minutes with the final concentration of the inhibitor as 1 mM in 50 mM acetic acid buffer (pH 4.5) at 40 ° C.

10.Effect of Metal Salts , Ba , Zn , Ca And Mn Hardly affected by, but Fe Suppressed by, Hg And Ag The effects of various metal salts are shown in Table 5 below. Indicates.

11.Molecular weight: about 95,000 (SDS electrophoresis)

The enzyme obtained according to the present invention is also clearly distinguished from the enzyme (hereinafter referred to as enzyme A) disclosed in Japanese Patent Application Laid-Open No. 174,089 / 1985.

1. The production strain of the present invention is Bacillus sectoramus, a novel strain belonging to the genus Bacillus, whereas the production strain of enzyme A is Bacillus asidoflulicus.

2. The optimum temperature of the enzyme according to the invention is 55 ° C., while the optimum temperature of enzyme A is 60 ° C.

3. Enzyme activity at various pH values and temperatures WHEREIN: The enzyme which concerns on this invention shows a very sharp difference in activity with temperature, as shown in FIG. In addition, in the effect of pH on enzymatic activity, the activity peaks at pH 5.0, and rapidly decreases at higher or lower pH. Enzyme A exhibits a completely different aspect from the enzyme according to the present invention.

4. In terms of optimal pH, the enzyme according to the invention has a narrow optimal pH range of pH 5.0 to 5.5 and a very specific specificity when compared to the pH range 3.5 to 5.5 of enzyme A.

Thus, it is concluded that the α-1, 6-glycosidase obtained according to the present invention is a novel enzyme different from enzyme A.

The novel α-1,6-glucosidase obtained according to the present invention may be added for the purpose of improving the yield when preparing glucose from starch using glucoamylase, or high maltose from starch. It can be used in combination with β-amylase for the purpose of producing in yield.

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

Example 1

Bacillus sectoramus (FERM BP-1471) was inoculated in a medium having the following composition:

Soluble Starch 1.5%

Meat Extract 0.5%

(NH) HPO0.3%

KHPO0.1%

MgSO, 7HO 0.1%

Initial pH 5.5

Shake culture in a Sakaguchi flask at 37 ° C. for 20 hours yields 8.1 μ / ml flulanase activity from the culture. The culture is centrifuged to remove the cytosolic solids and the resulting supernatant is concentrated using an ultrafiltration membrane. Subsequently, cold alcohol was added to a concentration of 80% to precipitate the enzyme, separated by centrifugation, and lyophilized to give a powdered floranase.

Example 2

Bacillus sectoramus (FERM BP-1471) was inoculated in a medium having the following composition:

Maltose 2%

Yeast extract 2%

KHPO0.2%

MgSO, 7HO 0.1%

Initial pH 4.5

After being placed in a 30 L jar fermenter, the culture was carried out under aeration conditions at 37 ° C. for 16 hours to obtain 13.6 μ / ml of the flulanase activity from the culture. The cells and solids were removed from the culture by centrifugation, and the resulting supernatant was concentrated to about 1/20 using an ultrafiltration membrane to obtain 200 µ / ml (pH4.5) of a crude liquid sample of flulanase. The sample was fractionated with ammonium sulfate, and then subjected to affinity chromatography using γ-cyclodextrin-cephalos 6B to obtain a purified enzyme sample 76.7 μg / mg protein.

Example 3

Bacillus sekkoramus (FERM BP-1471) was inoculated in a medium having the following composition:

Beeswax Starch 2%

Polypeptone 1%

KHPO0.2%

MgSO, 7HO 0.1%

Initial pH 5.0

After being placed in a 1000 L tank, 18.2 µ / ml flulanase activity was obtained from the culture by stirring under aeration conditions at 37 ° C. for 15 hours. The culture solution was treated in the same manner as in Example 2, whereby about 26 L of a crude liquid sample of fluranase having an activity of about 30 μ / ml was obtained.

Experimental Example 1

Substrate for saccharification: corn starch (33 g / dℓ.DE 8.0)

Glycozyme (Gluczyme.Amino Seiyaku Kabuki Kaisha) (2.5μ / g D.S.)

Enzyme according to the invention: liquid fluranase obtained in Example 3 (0.1 μg g D.S.)

Saccharification Temperature: 62 ℃

The saccharification process is performed under the above conditions, and high-performance liquid chromatography (HPLC) is performed at different times to measure the contents of monosaccharides (G1), disaccharides (G2), trisaccharides (G3), and polysaccharides (Gn). . As a control, also under the same saccharification process conditions except that the enzyme according to the invention is not used, the results are summarized in Table 6 below.

By incubating the new strain Bacillus sectoramus (FERM BP-1471) for a short time, the present invention enables the production of novel α-1, 6-glucosidase and consequently heat stable α-1, 6- It provides a large amount of glucosidase production and provides an economical way to contribute to the starch saccharification industry.

Claims (1)

Novel α-1,6-glucosidase having the following enzymatic properties: (1) Action: Enzymes act on α-1,6-glucoside bonds to produce straight amylose; (2) Substrate specificity: The enzyme has the following relative activity against the polysaccharide;

(3) km value for flurane and Vmax: enzyme has a km value of 0.14 mg / ml and a Vmax of 70.0 μmol / min / mg protein; (4) optimum pH: 5.0 to 5.5; (5) pH stability: The enzyme is stable at 40 ° C. for 30 minutes at a pH range of 4.5 to 6.5 and at 50 ° C. for 30 minutes at a pH range of 5.0 to 6.0. (6) The optimum temperature: Approximately 55 degrees Celsius (7) Thermal stability: After 30 minutes of treatment at different temperatures at pH 4.5, the enzyme was found to retain 80% at 40 ° C and 10% at 60 ° C. (8) Influence of inhibitors: Enzymes are inhibited by more than 70% by p-chloromercuric benzoic acid or sodium dodecyl sulfate, but not by o-phenanthroline or potassium ferricyanide. (9) Effect of metal salts: Enzymes are hardly affected by Ni ²⁺ , Ba ²⁺ , Zn ²⁺ , Cd ²⁺ and Mn ²⁺ , but are inhibited by Fe ³⁺ , and Hg ²⁺ or Ag Loses activity by ²⁺ ; And (10) Molecular weight: Approx. 95,500 (measured by SDS electrophoresis). 2. Bacillus sectorramus strain that can produce α-1,6-glucosidase is cultured to produce α-1,6-glucosidase in the nutrient medium, the α-1, And accumulating 6-glucosidase and then recovering α-1,6-glucosidase.

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