JPH0714343B2 - Novel maltotetraose-forming enzyme and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel maltotetraose-forming enzyme and a method for producing the same.

[Conventional technology]

Recently, researches on maltooligosaccharides have been actively promoted, but maltose is the only one currently mass-produced industrially. In addition to maltose, maltotriose is only produced in small quantities for reagents and maltopentaose for amylase activity measurement.

[Problems to be solved by the invention]

However, maltotriose to maltohexaose malto-oligosaccharides have been recently discovered one after another, and various oligosaccharides can be easily produced from starch. For example, regarding maltotetraose, Agric-Biol, Chem., 46
(3), 639-646 (1982) JFRobyt and RJAckerman:
The enzymes described in Arch. Biochem. Biophys., 145 , 105-114 (1971) are known. This enzyme is produced by Pseudomonas Stutzeri, but its productivity is low (2-5 IU / ml. Other microorganisms are not yet known).

[Means for solving problems]

Therefore, the present inventors have conducted research to find a highly active enzyme capable of efficiently producing maltotetraose. As a result of searching various preserved strains in the process, it was found that the target maltotetraose synthase can be obtained by culturing Pseudomonas Saccharophila, and the present invention was completed.

The present invention firstly relates to a novel maltotetraose synthase having the following properties.

(1) This enzyme acts on amylose, soluble starch, potato starch, sweet potato starch, rice starch, tapioca starch, corn starch, waxy corn starch, sago starch and the like to produce maltotetraose.

(2) The optimum pH of this enzyme at 30 ° C is pH 5.5-
It is stable at 10.5.

(3) The optimum temperature of this enzyme is 50 to 55 ° C at pH 7.0, and it is inactivated when left at a temperature of 60 ° C or higher for 30 minutes.

(4) This enzyme was inhibited in 1mM mercuric parachlorobenzoate solution, the inhibition rate was 60-70%, Fe ³⁺ , Co ²⁺ was 94-99%, but Mg ²⁺ was completely Not hindered.

(5) The molecular weight of this enzyme is 62000 (according to the disc gel electropermanent method).

(6) The isoelectric point of this enzyme is pH 4.7 (according to ampoline electric permanence method).

Secondly, the present invention is a method for producing a novel maltotetraose-forming enzyme, which comprises culturing C. saccharophila, accumulating the novel maltotetraose-forming enzyme in the culture, and collecting the enzyme. .

The novel maltotetraose-forming enzyme of the present invention is produced by using a microorganism, and its producing bacterium may be any one having the ability to produce the enzyme having the above-mentioned properties, and examples thereof include C. saccharophila IAM1504. The microorganisms used in the present invention include this strain and its variants,
It is not limited to the mutant strain, and any strain having the ability to produce the above enzyme may be used. Here, as the mutation means, a conventional method may be used, such as radioisotope (RI),
It may be performed using ultraviolet rays (UV), nitrosoguanidine, or the like.

Next, the culture conditions of the microorganism for producing the novel maltotetraose-forming enzyme of the present invention were examined. First, as a basic medium, carbon source 1%, nitrogen source 1%, phosphoric acid 1
Using 0.1% potassium and 0.28% dipotassium phosphate (pH 7.0), the nitrogen source was fixed to polypeptone, and the carbon source was studied by adding 1% of each substance shown in Table 1. This medium was inoculated with C. saccharophila IAM1504 and subjected to agitation culture at 30 ° C. for 2 days. The activity ratios (values when the soluble starch is 100) at this time are shown in Table 1. As is clear from the table, soluble starch was the best as the carbon source, and when starch candy, various starches and maltose were used, considerably high activity was obtained.

Table 1 Carbon source IU / ml activity ratio No addition 0.3 2.1 Glucose 0 0 Syucrose 0 0 Maltose 6.5 45.5 Lactose 0.3 2.1 Commercial flour candy (DE22) 10.5 73.4 Soluble starch 14.3 100 Potato starch 9.5 66.4 Corn starch 7.5 52.4 Waxy corn starch 7.6 53.1 Next, in order to examine the nitrogen source, a carbon source was fixed to soluble starch, 1% of various substances shown in Table 2 and 2% of water were added, and shake culture was carried out at 30 ° C. for 2 days. The activity ratio (value when polypeptone is 100) at this time is shown in Table 2. As is apparent from the table, remarkably high activity was obtained when polypeptone was used.

Table 2 Nitrogen source IU / ml Activity ratio No addition 0.1 0.7 Peptone 9.3 65.0 Polypeptone 14.3 100 Oatmeal ^* 0.3 2.1 Fusuma ^* 0.6 4.2 Corn steep liquor ^* 1.1 7.7 Ammonium acetate 0.3 2.1 Ammonium sulfate 0.1 0.7 Add sodium nitrate 2.5 17.5 * 2% From the above examination results, the optimum medium composition is soluble starch 1
%, Polypeptone 1%, potassium 1 phosphate 0.1%, and potassium diphosphate 0.28%.
Therefore, the medium to be used for culture should be selected with reference to the above findings, so that the strain to be tested can produce the desired enzyme with good activity.

The culture conditions should be selected to maximize the production of the target enzyme, which is usually 30
Cultivation should be carried out at ℃ for 2 days. In order to collect and purify the enzyme from the culture solution, known methods may be combined appropriately. The supernatant obtained by centrifuging and removing insoluble matters in the culture solution can be used as a crude enzyme. The enzyme can be purified by various methods.
An example is as follows.

Ammonium sulfate was added to the crude enzyme solution at a low temperature of 4 ° C to give 0.3
Fractions that precipitated at ~ 0.5 saturation were collected and collected in 10 mM phosphate buffer (p
It dissolves in H7.0). This enzyme solution is dialyzed against the same buffer solution overnight, and the following operations are performed. The recovery rate in this salting out with ammonium sulfate is about 95%. Then, it can be purified by DEAE-Toyopearl 650M column chromatography, gel perchromatography, etc. to obtain a standard sample showing a single band on disk gel electrophoresis (first).
Figure).

The properties of this enzyme were examined using the purified enzyme thus obtained. The results are shown below.

(1) Action The reaction progress when this enzyme is allowed to act on soluble starch is the second
As shown in the figure. As is clear from the figure, this enzyme specifically produces maltotetraose from the early stage of the reaction.

Therefore, in order to efficiently produce maltotetraose, a method in which the enzyme is allowed to act on liquefied starch in a container such as a membrane reactor and the produced sugar is taken out of the reaction system using an ultrafiltration membrane. , Column chromatography can be used, and it can be combined with an immobilized enzyme.

The mode of action of this enzyme is as follows when an oligosaccharide of maltooctaose or less is used as a substrate. The abbreviation is
G ₁ : glucose, G ₂ : maltose, G ₃ : maltotriose,
G _5: maltopentaose, G _8: shows the maltooctaose Orth.

As described above, when a substrate having a low degree of polymerization is used, this enzyme acts as a so-called Exo-type amylase,
It acts as an Endo-type amylase on dextrin with a high degree of polymerization. Therefore, most of starch is converted to G ₄ by the action of this enzyme without leaving any residue. At this time, the coexistence of a debranching enzyme such as pullulanase can improve the yield of G ₄ .

(2) Optimal action pH and pH stability Fig. 3 shows the results when the reducing power was measured by reacting at 30 ° C for 15 minutes and the maximum value was expressed as 100. As is clear from the figure, the optimum pH of this enzyme is 6.7.

Regarding pH stability, 1 ml of this enzyme solution should be used at various pH levels of 10 m
M buffer (pH 4 to 6: acetate buffer, pH 6 to 8: phosphate buffer, pH 8
~ 9: Tris-HCl buffer, pH 9-10: Sodium carbonate buffer)
After adding 0.1 ml and standing at 30 ° C for 60 minutes, take 0.1 ml of each, add 0.4 ml of 100 mM phosphate buffer (pH 7.0) and 0.5 ml of 2% substrate solution, and add 30 ml at 30 ° C for 30 minutes. The reaction was carried out and the residual enzyme activity was measured. Results are shown in FIG. As shown in FIG. 4, this enzyme is stable in the pH range of 5.5 to 10.5.

(3) Method for measuring enzyme titer The activity of the enzyme was measured by reducing the soluble starch (manufactured by Merck & Co., Inc.) as a substrate and measuring the reducing power by the Somogy-Nelson method. The amount of enzyme that cleaves micromolar equivalents of glucosidic bonds was 1 IU (international unit).

(4) Optimum temperature of action and temperature stability And react for 15 minutes at various temperatures to measure the reducing power,
The results when the maximum value is expressed as 100 are shown in FIG. As is clear from the figure, the optimum action temperature of this enzyme is 50 ~
55 ° C.

Moreover, after leaving still at pH 7.0 for 15 minutes at various temperatures, the reaction was carried out at 30 ° C. to measure the residual activity. Results are shown in FIG. As is clear from FIG. 6, it deactivates rapidly at 55 ° C and above.

(5) Inhibition, activation and stabilization This enzyme is inhibited in a 0.4 mM mercuric parachlorobenzoate solution, and the inhibition rate is 60 to 70%.

Next, the effects of various metal ions (1 mM concentration) are
The inhibition rate by cobalt, copper and iron is as high as 90% or more, and it is 2 for aluminum, zinc and manganese.
It is 0,30,60%. Also, the activity is 115 in strontium.
Calcium ion increases the heat resistance of this enzyme by 5 ℃.
Increase.

(6) Molecular weight The molecular weight of this enzyme obtained by disk gel electrophoresis is 62000.

(7) Isoelectric point The isoelectric point obtained by the amphiline electrophoresis method is
pH is 4.7.

(8) Crystal structure and elemental analysis Although a crystalline standard has not been obtained for this enzyme, a purified standard showing a single band by electrophoresis has been obtained.

As shown above, this enzyme is similar to the conventional maltotetraose-forming enzyme in terms of action, but its molecular weight,
It is a novel enzyme that produces a large amount of maltotetraose, unlike the conventional ones in terms of properties such as isoelectric point and type of inhibition.

EXAMPLES Next, the present invention will be described with reference to examples.

Example 1 C. saccharophila IAM1504 was inoculated on a slant agar medium containing 1% of soluble starch, 1% of polypeptone, 0.1% of potassium phosphate 1 and 0.28% of potassium diphosphate, at 30.degree.
After culturing for 2 days, take 1 platinum loop and transfer to a liquid medium of the same composition (100 ml medium / 500 ml Erlenmeyer flask),
Aeration shaking culture was performed at 2 ° C for 2 days.

After the completion of the culture, the bacterial cells and insoluble materials in the culture were removed by centrifugation at low temperature to obtain a supernatant, which was used as a crude enzyme. The activity of this crude enzyme solution was 14.0 IU / ml.

Example 2 A small amount of a culture solution of C. saccharophila IMA1504 was taken, treated with RI, UV and nitrosoguanidine by a conventional method, and then plated, and a colony having a high amylase activity was picked. Soluble starch 1%, polypeptone 1%,
The cells were cultured at 30 ° C. for 2 days in a medium containing 0.1% potassium diphosphate and 0.28% potassium diphosphate. Subsequent operations are described in Example 1.
Same as. The activity of the obtained crude enzyme solution was 18.0 IU / ml.

〔The invention's effect〕

According to the present invention, a novel maltotetraose-forming enzyme can be efficiently produced in a large amount at low cost, and thus the application of this enzyme is expected to be expanded to various fields including food.

[Brief description of drawings]

FIG. 1 is a disk gel electrophoretic photograph of the purified enzyme, FIG. 2 is a chromatogram showing the reaction course of maltotetraose-producing enzyme (5 IU / g substrate), and FIG. 3 is a graph showing the optimum pH of this enzyme. Fig. 4 is a graph showing the pH stability of this enzyme,
FIG. 5 is a graph showing the optimum temperature of this enzyme, and FIG. 6 is a graph showing the temperature stability of this enzyme.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hitoshi Hashimoto 4-31-10 Imaizumidai, Kamakura City, Kanagawa Prefecture (72) Inventor Kozo Hara 2-6-3-104, Namiki, Kanazawa-ku, Yokohama City, Kanagawa Prefecture (56) Reference Literature Agric. Biol. Chem, 47 (8) (1983) P. 1761-1768

Claims (3)

[Claims] 1. A novel maltotetraose-forming enzyme having the following properties. (1) The optimum pH of this enzyme at 30 ° C is 6.7, pH 5.5-
It is stable at 10.5. (2) The optimum temperature of this enzyme is 50 to 55 ° C at pH 7.0, and it is inactivated when left at a temperature of 60 ° C or higher for 30 minutes. (3) This enzyme was inhibited in 1mM mercuric chlorobenzoate solution, the inhibition rate was 60-70%, and Fe ³⁺ , Co ²⁺ was 94%.
~ 99%, but Mg ²⁺ does not ^show any inhibition. (4) The molecular weight of this enzyme is 62000 (by the disc gel electropermanent method). (5) The isoelectric point of this enzyme is pH 4.7 (according to ampoline electric permanence method). 2. A method for culturing Pseudomonas Saccharophila to accumulate an enzyme having the following properties in the culture,
A novel method for producing a maltotetraose-forming enzyme, which comprises collecting this. (1) The optimum pH of this enzyme at 30 ° C is 6.7, pH 5.5-
It is stable at 10.5. (2) The optimum temperature of this enzyme is 50 to 55 ° C at pH 7.0, and it is inactivated when left at a temperature of 60 ° C or higher for 30 minutes. (3) This enzyme was inhibited in 1mM mercuric chlorobenzoate solution, the inhibition rate was 60-70%, and Fe ³⁺ , Co ²⁺ was 94%.
~ 99%, but Mg ²⁺ does not ^show any inhibition. (4) The molecular weight of this enzyme is 62000 (by the disc gel electropermanent method). (5) The isoelectric point of this enzyme is pH 4.7 (according to ampoline electric permanence method). 3. The method according to claim 2, wherein the C. saccharophila is C. saccharophila IAM1504.