JPS63226295A - Method for producing maltopentaose using a novel maltopentaose-producing enzyme - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Object of the Invention] The present invention relates to a method for producing maltopentaose using a novel maltopentaose-producing enzyme.

[Prior art and problems to be solved by the invention] In recent years,
Research on malto-oligosaccharides is progressing, but maltose is the only one currently being industrially produced in large quantities. Other than maltose, maltotriose is produced for use as a reagent, and maltopentaose is produced for use in measuring amylase activity.

However, recently, amylases of microbial origin that specifically produce maltooligosaccharides of maltotriosulmaltohexaose have been discovered one after another, and it has become easier to produce various oligosaccharides from starch. For example, regarding maltopentaose, Arch, Biochem, B
iophys, 155.290 (1973) and
, and the enzymes described in the 1981 Conference Abstracts of the Japanese Society of Agricultural Chemistry, p. 178 are known. However, these enzymes produce various sugars other than maltopentaose from the early stage of the reaction, and amylases that produce only maltopentaose are not yet known.

The present inventors have conducted extensive research to search for an enzyme that can efficiently produce maltopentaose. In the process, they found that the target maltopentaose synthase was obtained by culturing a microorganism belonging to the genus Pseudomonas, and that maltopentaose could be obtained efficiently by using this enzyme, leading to the completion of the present invention. Ta.

That is, the present invention provides maltopentaose, which is characterized in that a novel maltopentaose-producing enzyme having the following properties is made to act on at least one substance selected from starch, a compositional fraction of starch, and a decomposition reaction product of starch. The present invention relates to a method for producing oath.

(1) This enzyme contains amylose, soluble starch, potato starch,
Sweet potato starch, rice starch, tapioca starch, corn starch, starch,
It acts on starch and silicon starch to produce maltopentaose.

(2) The optimal pH for this enzyme is 6 to 7 at 45°C, and
Stable at H6.5-9.

(3) This enzyme has an optimal temperature of 50~
The temperature is 55°C, and it is deactivated if left at a temperature of 55°C or higher for 15 minutes.

(4) This enzyme is inhibited in 0.4mM mercuric parachlorobenzoate and 1mM monoiodoacetamide solution, but the inhibition rate is 40-50%.

(5) The molecular weight of this enzyme is 72,500±2,500 (according to disk gel electrophoresis).

(6) The isoelectric point of this enzyme is pH 6.5 (according to ampholine electrophoresis).

The maltopentaose-producing enzyme used in the present invention is produced using a microorganism, and the producing microorganism may be one belonging to the genus Pseudomonas and having the ability to produce an enzyme having the above properties.
40 (FEBM P-7456) and its modified rice and mutant strains. Here, the mutation means may be any conventional method,
For example, radioisotope (R1), ultraviolet (UV)
, nitrosoguanidine, etc. may be used. The mycological properties of Pseudomonas KO-8940 were disclosed in JP-A-60
It is described in the publication No.-188065.

Next, we investigated the culture conditions for microorganisms to produce a novel maltopentaose-producing enzyme. First, a basic medium containing meat extract, polypeptone, salt, and a carbon source was used, and 1% of the various substances shown in Table 1 were used as the carbon source. Pseudomonas KO-
8940 (FERMP-7456) and cultured with shaking at 40°C for 3 days. The activity ratios (values when maltose is taken as 100) at this time are shown in Table 1. As is clear from the table, maltose was the best carbon source, and among starches, considerably high activity was obtained when rice starch and sweet potato starch were used. Furthermore, the activity ratio of various types of powdered candy increased as the DE increased, and high maltose syrup had an activity comparable to that of maltose.

Table 1 Glucose 0.01 1.5 Fructose 0.02 2.9 Sucrose 0.03 4.4 Maltose
0.88 100 High Maltose Syrup 0.67 98.5 Amylose (DP 100
) 0.24 35.3 Soluble starch
0.22 32.4 Potato starch 0
．． 15 22.1 Sweet potato starch Q, 30 44
．． 1 rice starch 0.42
61.8 Tapioca starch 0.1
0 14.7 Corn starch 0.10
14.7 Waxy corn starch 0.15
22.1 Wheat starch 00 Sago starch 0.14 20.6 Cyclodextrin powder candy 0.41 50.3 Corn stew liquor - 0,2333,8 Next, to examine the nitrogen source, meat extract 07%, maltose 1 1% of each substance was added to a medium containing 0.3% of sodium chloride, and cultured with shaking at 40° C. for 3 days. The activity ratio (value when ammonium sulfate is taken as 100) at this time is shown in Table 2.

As is clear from the table, significantly higher activity was obtained when ammonium sulfate or ammonium nitrate was used.

Pebuton
0.64 15.8 Polypeptone
0.35 8.6
N 0.48
11.9 Casamino acids 0.21
5.2 Potato protein 00 Ammonium acetate OO Ammonium sulfate 4.05 100 Ammonium nitrate 3.42 84.4 Urea 00 None
Furthermore, as a result of examining the concentrations of maltose, ammonium sulfate, and meat extract, the optimal medium composition was 0.8% maltose, 1% ammonium sulfate, and 0 meat extract.
．． It was found that it contained 8%. therefore,
With reference to the above findings, the medium used for culture should be selected to have a composition that allows the test strain to produce the desired enzyme with good activity.

Next, we examined the change in activity depending on the number of days of culture, and found that 70% or more activity was obtained on the first day of culture, and the activity gradually increased until the third day of culture. However, the activity decreases thereafter. Therefore, culturing for 1 to 3 days is appropriate for enzyme production, and usually after culturing for 3 days, insoluble matter in the culture solution can be removed by centrifugation, and the obtained supernatant crude enzyme can be used. .

The culture conditions should be selected so as to maximize the production of the desired enzyme. Furthermore, to collect and purify the enzyme from the culture solution, known methods may be appropriately combined.

Enzymes can be purified by various methods, but
An example of this is as follows.

Add ammonium sulfate to the crude enzyme solution at a low temperature of 4℃,
．． Both fractions precipitated at 2 to 0.5 saturation are collected and dissolved in 10 mM phosphate buffer (pH 7.5). 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 is about 80%.

Next, it can be purified by DEAE-cellulose column chromatography, gelcin filtration chromatography, etc. to obtain a specimen that shows a single band on disk gel electrophoresis.

The properties of the purified enzyme thus obtained were investigated. The results are shown below.

(1) Action When this enzyme acts on soluble starch, the reaction progress is as follows:
As shown in the figure and FIG.

As is clear from the figure, this enzyme produces maltopentaose at the beginning of the reaction, which is then thawed into maltose and maltotriose over time.

Therefore, in order to efficiently produce maltopentaose, this enzyme is allowed to act on liquefied starch in a container such as a Mempuran reactor, and the raw acid sugars are removed from the reaction system using an ultrafiltration membrane. It is desirable to adopt this method.

The mode of action of this enzyme is as follows when oligosaccharides below maltooctaose are used as substrates.

The abbreviations are GI nigercose, G2: maltose.

G3: maltotriose, G5: maltopentaose,
G8: indicates maltooctaose.

G8;
→ G 5 +G
.

G,: →G, +GyuG6
: →G5+GIG5:O(
>-0 Beark →G3+02G4: α-0
-0-◇ →G2+G2G3. G2: No action From this mode of action, a mixture of G2 and G is produced, G2 is digested by yeast to produce G3, or G2
A mixture of and 03 can also be made into a product.

Thus, when a substrate with a low degree of polymerization is used, this enzyme acts as a so-called Exo-type amylase, but with a dextrin with a high degree of polymerization, it acts as an Endo-type amylase. Therefore, due to the action of this enzyme, starch is mostly G5 or G2 and G3 without any uncut parts.
Convert to In this case, the yield of G5 can be improved by coexisting a debranching enzyme such as pullulanase.

(2) Effect principle p) I and pl (stability) The reducing power was measured by reacting at 45°C for 15 minutes with the reaction solution composition as ', and the results are shown in Figure 3 when the highest value is expressed as 100. As is clear from the figure, the optimum pH of this enzyme is 6-7.

Regarding p11 stability, 1mA+ of this enzyme solution was mixed with 10[0M buffers (pl (4-B: acetate buffer, pH 6-8 diphosphate buffer, pH 8-9: Tris-hydrochloric acid buffer, pH 6-8 diphosphate buffer) of various pH. pH9~1O: Sodium carbonate buffer) 0
．． After adding 1mi+ and leaving it for 60 minutes at 45℃,
．． Take 1ml each and add 100mM phosphate buffer (pH
6,5) 0.5 mJ of 0.4 mj+ and 2% substrate solution was added and reacted at 45°C for 300 minutes, and the residual enzyme activity was measured. The results are shown in Figure 4.

As shown in FIG. 4, this enzyme is stable in the pH range of 6.5 to 9.0.

(3) Enzyme titer measurement method Enzyme activity was determined by reducing soluble starch (manufactured by Merck & Co., Ltd. for 1-separation use) as a substrate and measuring the reducing power by the Somogyi-Nerzin method at 45°C for 1 minute. The amount of enzyme that cleaves 1 micromolar equivalent of glucoside bonds was defined as 1 10 (international units).

(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 the action of this enzyme is 50 to 55°C.

Further, after standing at various temperatures for 15 minutes at 9 116 , 5, the reaction was carried out at 45° C. 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 0.4mM mercuric parachlorobenzoate and 1mM monoiodoacetamide solution, but the inhibition rate is 40-50%, which is not high.

Next, regarding the influence of various metal ions (1 mM concentration), the inhibition rate by mercury, zinc, copper, and silver is as high as 80% or more, and it is 50% by iron. Additionally, calcium ions increase the heat resistance of this enzyme by 5°C.

(6) Molecular Weight The molecular weight of this enzyme obtained by disk gel electrophoresis is 72,500±2,500.

(7) Isoelectric point The isoelectric point determined by ampholine electrophoresis is pl(6,5).

(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.

The present enzyme, which has the properties shown above, exhibits an action completely different from that of conventional enzymes, and is a novel enzyme that produces a large amount of maltopentaose. The present inventors developed this enzyme with 1.4-α
-D-glucan maltopentaohydrolase.

As mentioned above, this enzyme acts on amylose, soluble starch, and various starches to produce maltopentaose. Therefore, maltopentaose is produced and accumulated by allowing the present enzyme to act on at least one substance among starch, the composition fraction of R flour, and the decomposition reaction product of starch.

When carrying out the reaction, conditions should be selected that will maximize the amount of maltopentaose produced, taking into account the properties of the enzyme. As the starch here, for example, those obtained from arbitrary raw materials such as potato, sweet potato, corn, waxy corn, barley, wheat, rice, tapioca, and sago can be used. Compositional fractions of starch include, for example, amylose and amylopectin, and starch decomposition reaction products include, for example, white dextrin, yellow dextrin, roasted dextrin such as British gum; oxidized starch, and low-viscosity modified (enzymatically modified) .

Modified starch such as N# powder by treatment such as acid 9 mechanical high speed stirring; starch conductors such as starch ethers and starch esters represented by phosphoric acid starch and acetic acid starch; irradiation with radiation or neutron beam, high frequency treatment or Examples include physically treated starch such as heat-treated starch; α-starch.

These starches may be used alone or in combination at 2 fm or more.

After the reaction is complete, heat to deactivate the enzyme and stop the reaction.
Maltopentaose can be obtained from the reaction solution by a conventional method.

[Example] Next, the present invention will be explained by examples.

Production example 1 Pseudomonas KO-1194 [1 (FERM P-7
45 [i) with 0.8% meat extract and 1% ammonium sulfate.
, inoculated onto slanted agar medium containing 0.8% maltose and incubated at 40°C.
After culturing for 2 days, one loopful was taken and transferred to a liquid medium of the same composition (100 mj + medium 1500 mg Erlenmeyer flask), and cultured with aeration and shaking at 45°C for 3 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 3.8117 mj+.

Production Example 2 Pseudomonas 0'-8940 (FEBM P-74
Take a small amount of the culture solution of 561 and use the usual method to prepare R1, LIV,
After treatment with nitrosoguanidine, plating was performed and colonies with high amylase activity were taken. This is 0.8% meat extract, 1% ammonium sulfate, and 0.8% maltose.
The cells were cultured in a medium at 45° C. for 3 days, and the subsequent operations were the same as in Production Example 1. The activity of Kigumi enzyme solution is 8. OIU/m
It was f.

Example 1 Potato starch was liquefied using bjM liquefiable enzyme (BLA), the iodine-starch reaction was deactivated in blue, and the substrate concentration was reduced to 10
%, maltopentaose producing enzyme (crude enzyme solution of Production Example 2) 1 1117g substrate, pH 6.0, reacted with stirring at 45°C for 6 hours to produce 30% maltopentaose
A reaction solution was obtained.

Example 2 A liquefied potato starch solution was prepared as in Example 1, substrate concentration 20%, maltopentaose-generating enzyme (crude enzyme solution of Production Example 2) 1 ru/g substrate, pH 6.0° 45°C.
Ultrafilter [manufactured by Toyoton Togami ■, Lll (P-711
(The membrane was UK-10)] was reacted under pressure with nitrogen gas to obtain a sugar solution containing 80% or more of maltopentaose. The yield is 60% of the starting substrate in a 12 hour reaction;
The obtained mucus could be concentrated to 20% using a reverse osmosis membrane.

Example 3 The same procedure as in Example 2 was carried out except that pullulanase was added to 15 g of the substrate, and 1! containing 80% or more of maltopentaose was reacted for 12 hours. Liquid i was obtained with a yield of 65%.

[Effects of the invention] Comaltopentaose is currently used as a substrate for measuring α-amylase activity in diagnostic agents and reagents, and if it is produced at low cost by the present invention, it is expected that it will be able to be used in various applications including food. be done. In addition, maltopentaose has excellent solubility, lacks sweetness, and has a body feel, so it is useful as a confectionery material.It is also easily digested and absorbed, so it is suitable for infants, the elderly, and patients. It can also be used as a J1-i meal.

Therefore, the method of the present invention, which can efficiently produce such maltopentaose from starch, is extremely useful industrially.

[Brief explanation of drawings]

Figure 1 shows the reaction progress of maltopentaose-generating enzyme (11
17g substrate), Fig. 2 is a graph showing the reaction progress of this enzyme (IIU/10mg substrate), Fig. 3 is a graph showing the optimum pH of this enzyme, and 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. Patent Applicant: Ministry of Agriculture, Forestry and Fisheries Ministry of Agriculture, Forestry and Fisheries Food Research Institute Director, engraving of drawings (no changes in content) Figure 1 St Q 5 10 15 20 3
0 45 60 90 +20 Stβ(fetal
Time %”l (61) Fig. 2 Fixed time (Intermediate J

Claims (1)

[Scope of Claims] 1) A novel maltopentaose-generating enzyme having the following properties is made to act on at least one substance selected from starch, a compositional fraction of starch, and a starch decomposition reaction product. A method for producing maltopentaose. (1) This enzyme contains amylose, soluble starch, potato starch,
Sweet potato starch, rice starch, tapioca starch, corn starch,
It acts on waxy corn starch, sago starch, etc. to produce maltopentaose. (2) The optimal pH for this enzyme is 6 to 7 at 45°C, and
Stable at H6.5-9. (3) The optimal temperature for this enzyme is 50-5 at pH 6.5.
5°C, and will be inactivated if left at a temperature of 55°C or higher for 15 minutes. (4) This enzyme is inhibited in 0.4mM mercuric parachlorobenzoate and 1mM monoiodoacetamide solution, but the inhibition rate is 40-50%. (5) The molecular weight of this enzyme is 72,500±2,500 (according to disk gel electrophoresis). (6) The isoelectric point of this enzyme is pH 6.5 (according to ampholine electrophoresis). 2) The production method according to claim 1, wherein the starch composition fraction is amylose or amylopectin. 3) The production method according to claim 1, wherein the starch decomposition reaction product is a modified starch.