JPS61202687A - Novel maltotetraose-producing enzyme and production thereof - Google Patents

Abstract

PURPOSE:To obtain a large amount of maltotetraose, by culturing Pseudomonas saccharophila and separating novel maltotetraose-producing enzyme from the culture product. CONSTITUTION:A microbial strain capable of producing novel maltotetraose- producing enzyme, e.g. Pseudomonas saccharophila IAM 1504, etc. is cultured in a medium containing soluble starch, polypeptone, monopotassium dihydrogen phosphate, dipotassium hydrogen phosphate, etc., and the maltotetraose-producing enzyme is separated from the culture liquid by salting-out, chromatography, electrophoresis, etc. The enzyme has the following characteristics. Optimum pH, 6.7 at 30 deg.C; stable at 5.5-10.5pH; inhibited in 1mM mercuric p- chlorobenzoate solution at an inhibition ratio of 60-70% and in Fe<3+> or Co<2+> solution at an inhibition ratio of 94-99%, and resistant to Mg<2+>; molecular weight, 62,000 (disk-gel electrophoresis); isoelectric point, 4.7pH.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a novel maltotetraose-generating enzyme and a method for producing the same.

[Conventional technology]

In recent years, research on malto-oligosaccharides has been actively promoted, but maltose is the only one currently being industrially produced in large quantities. Other than maltose, maltotriose is produced as a reagent, and maltopentaose is produced as an amylase activity agent in small quantities.

[Problem that the invention seeks to solve]

However, recently, maltotriose (maltotriose-sulmaltohexaose) has been discovered one after another, and it has become possible to easily produce various olico-9 sugars from starch. For example, for maltotetraose, AS'ric-Bi
ol, Chem. , 46 (31,639-646 (19
82) J, F, Robyt and R, J, Ack
erman: Arch.

Biochem, Biophys, 145, 10
5-114 (1971) is known.

This enzyme is derived from Pseudomonas e stutscheri (Ps eu
domonas 5 tutzeri), but the productivity is low (2-5 IU/ml. Other microorganisms are not yet known.

[Means for solving problems]

Therefore, the present inventors conducted extensive research in order to search for a highly active enzyme that can efficiently produce maltotetraose. In the process, we searched for various preserved bacterial strains and found that F'seudomonas Saccha
The present inventors have discovered that the desired maltotetraose synthase can be obtained by culturing S. rophi la), and have completed the present invention.

The present invention first relates to a novel maltotetraose-producing enzyme having the properties shown below.

(1) This enzyme acts on amylose, soluble starch, potato starch, sweet potato starch, rice starch, tapioca starch, corn starch, waxy corn starch, soot starch, etc. to produce maltotetraose.

(2) This enzyme has an optimal pH of 6.7 at 30°C, and is stable at a pH of 5.5 to 10.5.

(3) The optimal temperature for this enzyme is 50 at pH 7.0.
~55°C, and is deactivated if left at a temperature of 60°C or higher for 30 minutes.

(4) This enzyme is inhibited in 1mM mercuric parachlorobenzoate solution, but the inhibition rate is 60-70%.
”, Co2+c is 94-99%, but M22+ is not inhibited at all.

(5) The molecular weight of this enzyme is 62,000 (according to disk gel electrophoresis).

(6) The isoelectric point of this enzyme is pH 4.7 (according to ampholine electrophoresis).

Second, the present invention provides a novel method for producing a maltotetraose-generating enzyme, which comprises culturing Pseudomonas Sacca a filla, accumulating the novel maltotetraose-generating enzyme in the culture, and collecting the novel maltotetraose-generating enzyme. It is.

The novel maltotetraose-producing enzyme of the present invention is produced using a microorganism, and the producing microorganism is as follows.

Any enzyme can be used as long as it has the ability to produce an enzyme having the above properties, such as Pseudomonas saccharophyla I.
There are AM1504, etc. The microorganisms used in the present invention are not limited to the present strain and its variants and mutants, but may be any microorganism as long as it has the ability to produce the above-mentioned enzymes. Here, the mutation means may be any conventional method, for example, radioisotope (RI), ultraviolet light (tJV), nitrosoguanidine, etc. may be used.

Next, culture conditions for microorganisms for producing the novel maltotetraose-producing enzyme of the present invention were investigated. First, as a basic medium, 1 carbon source, 1 potassium thiophosphate 0.1%, and 0628% dipotassium phosphate (p
The nitrogen source was fixed to polypeptone, and 1% of the various substances shown in Table 1 were added as a carbon source. This medium was inoculated with Pseudomonas saccharophyllae AM15.04, and cultured at 30° C. for 2 days with shaking.

The activity ratio at this time (value when soluble starch is 100) is shown in Table 1. As is clear from the table, soluble starch was the best carbon source, and a significantly higher activity was obtained when powdered candy, various starches, and maltose were used.

Table 1 Carbon source Elm flattened active ratio No additives
0.3 2.1 glucose
o.

Sucrose OQ Maltose 6.5 45.5 Lactose 0.3 2.1 Commercially available powdered candy (DE22) 10.5 7
3.4 Soluble starch 14.3 1QQ
Potato starch 9.5 66.4 Maize starch 0" 1 ts of the various substances shown in the table and 2 tsp of water were added, and the mixture was shaken and cultured for 5 days at 30°C.The activity ratio at this time (value when polypeptone is taken as 100) was
Shown in the table. As is clear from the table, significantly higher activity was obtained when polypeptone was used.

No additives 0.1 0.7 Pezoton° 9.3 65.0 Polypeptone 14.3 100 Oatmeal 0.32.
1 Nefusma 0.6 4.2 Corn Steel Delica - 1,17,7 Ammonium Acetate 0.3 2.1 Ammonium Sulfate 0.1 0.7 Sodium Nitrate 2.5 17.5* 2 Added to Chi From the above study results, The optimal medium composition was found to be one containing 1 part soluble starch, 1 part polypeptone, 0.1% monopotassium phosphate, and 0.281% dipotassium phosphate. Therefore, the culture medium to be used for culture should be selected with a composition that allows the test strain to produce the desired enzyme with good activity, with reference to the above findings.

Culture conditions should be selected to maximize the production of the desired enzyme;
℃ for 2 days.

Furthermore, in order to collect and purify the enzyme from the culture solution, known methods may be appropriately combined. Incidentally, a supernatant obtained by centrifugation removal of insoluble matter in the culture solution can be used as the crude enzyme. Enzymes can be purified by various methods, one example of which is as follows.

Ammonium sulfate was added to the crude enzyme solution at a low temperature of 4°C.
Both fractions that precipitate at 3 to 0.5 saturation are collected and dissolved in 10 mM phosphate buffer (pH 7.0). This enzyme solution was dialyzed against the same buffer overnight and the subsequent operations were performed. The recovery rate in this ammonium sulfate salting out was about 95 cm.

Next, it is purified by DEAE-Toyopearl 650M column chromatography, gel f perchromatography, etc. to obtain a specimen that shows a single band on disk gel electrophoresis (Figure 1).

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

(1) Action The reaction process when this enzyme acts on soluble starch is as follows.
This is the case 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, this enzyme is allowed to act on liquefied starch in a container such as a membrane reactor, and the produced sugar is removed from the reaction system using an ultrafon membrane. Methods such as column chromatography and column chromatography can be used, and combinations with immobilized enzymes can also be used.

The mode of action of this enzyme is as follows when an oligosaccharide below maltooctaose is used as a substrate. The abbreviation is G.
1 nigercose, G2: maltose, G3: maltotriose, Gs: maltopentaose, G8: maltooctaose.

G8: →G
4+G4G7:
→G4+G3G6:
→To 102GS: 0-O-〇-〇-0→G4+G Engineering G
4* G31 G2: No effect When a substrate with a low degree of polymerization is used, this enzyme acts as a so-called Exo-type amylase, but on dextrin with a high degree of polymerization, it acts as an Endo-type amylase. .

Therefore, most of the starch is converted to 04. by the action of this enzyme, without leaving anything uncut. At this time, the yield of G4 can be improved by coexisting a debranching enzyme such as pullulanase.

(2) Optimal action and stability The reaction solution composition was adjusted, and the reducing power was measured after reacting at 30° C. for 15 minutes. The results are shown in FIG. 3, with the highest value being expressed as 100. As is clear from the figure, the optimum value for this enzyme is 6.7.

- Regarding stability, add various P to 1 ml of this enzyme solution.
H010mM buffer (PF (4-6: acetate buffer, pH
6-8 Niphosphate buffer, pH 8-9 Nidris-hydrochloric acid buffer, pH 9-10: Soda carbonate buffer) O, l ml
were added and left to stand at 30°C for 60 minutes, then 0.1 rn
100mM phosphate buffer (pH 7.0)
Add 0.5 ml of 0.4d and 2t substrate solution and heat at 30°C.
The reaction was carried out for 300 minutes, and the residual enzyme activity was measured. The results are shown in Figure 4. As shown in Figure 4, this enzyme
It is stable in the range of 5.5 to 10.5.

(3) Enzyme titer measurement method Enzyme activity was determined by reducing soluble starch (2-minute Shingetsu manufactured by Merck & Co., Ltd.) as a substrate and measuring the reducing power by the Somogyi-Nernon method. The amount of enzyme that cleaves 1 micromole equivalent of glucoside bonds per minute was defined as IIU (international unit).

(4) Optimum temperature for action and temperature stability The reaction solution composition was determined, and the reducing power was measured by reacting at various temperatures for 15 minutes. The results are shown in FIG. 5, with the highest value being expressed as 100. As is clear from the figure, the optimum temperature for action of this enzyme is 5.
The temperature is 0 to 55°C.

In addition, after leaving it at various temperatures for 15 minutes at pH 7.0,
The reaction was carried out at ℃ and the residual activity was measured. The results are shown in Figure 6. As is clear from FIG. 6, the activity is rapidly deactivated at temperatures above 55°C.

(5) Inhibition, activation and stabilization This enzyme is inhibited in a 0.4 mM mercuric parachlorobenzoate solution, and the inhibition rate is 60-7°.

Next, regarding the influence of various metal ions (1mM concentration), the inhibition rate of mercury, silver, cobalt, copper, and iron was higher than 90%, and that of aluminum, zinc, and manganese was 20% and 30%, respectively.

It's 60 inches. In addition, strontium has an activity of 115
Calcium ions increase the heat resistance of this enzyme to 5°C.
enhance

(6) Molecular Weight The molecular weight of this enzyme obtained by disk gel electrophoresis is 62,000.

(7) Isoelectric point The isoelectric point determined by ampholine electrophoresis is PH4.7.

(8) Crystal structure and elemental analysis Although a crystal sample of this enzyme has not yet been obtained, a purified sample that shows a single band in electrophoresis has been obtained.

As shown above, this enzyme is similar to conventional maltotetraose-generating enzymes in terms of action, but has a molecular weight of 1.
It is a novel enzyme that produces large amounts of maltotetraose, with different properties such as isoelectric point and inhibition type from conventional enzymes.

〔Example〕

Next, the present invention will be explained using examples.

Example 1 Pseudomonas φ Satsurofila IAM 1504 was mixed with 1 t of soluble starch, 1% polypezotone, 1 potassium phosphate, 0
．． After inoculating a slanted agar medium containing 1% dipotassium phosphate and 0.281 g of dipotassium phosphate and culturing at 30'O for 2 days, one platinum loopful was removed and a liquid medium of the same composition (100ml medium 15ml) was inoculated.
The cells were transferred to an Erlenmeyer flask (OOMZ Erlenmeyer flask), and cultured for 5 days with aeration at 30°C for 2 days.

After completion of the culture, the cells and insoluble matter in the culture were removed by centrifugation at low temperature to obtain a supernatant, which was used as crude enzyme.

The activity of this crude enzyme solution was 14.0 IU/+++l.

Example 2 A small amount of culture solution of Pseudomonas saccharofila IAM 1504 was taken and treated with pR1, UV and di-longanidine in a conventional manner, followed by plate culture and colonies with high amylase activity were taken. Add this to 1% soluble starch
, 1% polypeptone, 1potassium phosphate 0.1%,
The cells were cultured at 30°C for 2 days in a medium containing 0.28% dipotassium phosphate. The subsequent operations were the same as in Example 1. The activity of the obtained crude enzyme solution was 18.0 IU/ml.

〔Effect of the invention〕

According to the present invention, the novel maltotetraose-generating enzyme can be produced efficiently in large quantities and at low cost, so it is expected that the use of the present enzyme will expand to various fields including foods.

[Brief explanation of the drawing]

Figure 1 is a disk gel electrophoresis photograph of the purified enzyme, Figure 2 is the reaction progress of maltotetraose-forming enzyme (5 IU/f
Chromatogram showing the substrate), Figure 3 shows the optimal
FIG. 4 is a graph showing the 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. Patent applicant: Ministry of Agriculture, Forestry and Fisheries Food Research Institute Director Shiomizu Port Sugar Refining Co., Ltd. Agent: Kubota, Fujibe (Fig. 2) f 2 5 10
3:) 60 120 Sit/F
7//F 9H Fig. 4 H Fig. 5 ``iL Good ('C) Fig. 6 ML degree ('C)

Claims (1)

[Claims] 1. A novel maltotetraose-generating enzyme having the following properties. (1) The optimal pH for this enzyme is 6.7 at 30°C, and
It is stable at H5.5 to 10.5. (2) The optimal temperature for this enzyme is 50-5 at pH 7.0.
5°C, and will be inactivated if left at a temperature of 60°C or higher for 30 minutes. (3) This enzyme is inhibited in 1mM mercuric parachlorobenzoate solution, and the inhibition rate is 60-70%, Fe^3^+
, is 94-99% for Co^2^+, but for Mg^2^
+ means no inhibition at all. (4) The molecular weight of this enzyme is 62,000 (according to disk gel electrophoresis). (5) The isoelectric point of this enzyme is pH 4.7 (according to ampholine electrophoresis). 2. A novel method for producing a maltotetraose-producing enzyme, which comprises culturing Pseudomonas satucarophylla, accumulating an enzyme having the following properties in the culture, and collecting the enzyme. (1) The optimal pH for this enzyme is 6.7 at 30°C, and
It is stable at H5.5 to 10.5. (2) The optimal temperature for this enzyme is 50-5 at pH 7.0.
5°C, and will be inactivated if left at a temperature of 60°C or higher for 30 minutes. (3) This enzyme is inhibited in 1mM mercuric parachlorobenzoate solution, and the inhibition rate is 60-70%, Fe^3^+
, is 94-99% for Co^2^+, but for Mg^2^
+ means no inhibition at all. (4) The molecular weight of this enzyme is 62,000 (according to disk gel electrophoresis). (5) The isoelectric point of this enzyme is pH 4.7 (according to ampholine electrophoresis). 3. The method according to claim 2, wherein the Pseudomonas satucharofila is Pseudomonas satucharofila IAM1504.