JPS6054679A - Novel maltopentaose synthetase, its preparation and production of maltopentaose using said enzyme - Google Patents

Abstract

PURPOSE:To produce a novel enzyme capable of producing a large amount of maltopentaose from amylose, soluble starch and various other starches by culturing a novel microbial strain belonging to Alcaligenes genus, and to produce maltopentaose using said enzyme. CONSTITUTION:A novel microbial strain belonging to Alcaligenes genus and capable of producing a novel maltopentaose-synthetase, e.g. Alcaligenes faecalis IAM-1015 strain, is cultured in a nutrient medium, and the novel maltopentaose synthetase is separated from the cultured product. The enzyme is added to one or more kinds of materials selected from starch, fractionated component of starch, and decomposition product of starch to produce and accumulate maltopentaose.

Description

[Detailed description of the invention] The present invention is a novel maltopentaose synthase.

The present invention relates to a method for producing maltopentaose and a method for producing maltopentaose using the enzyme.

In recent years, research on maltooligosaccharides has been progressing, but maltose is the only one currently being industrially produced in large quantities. In addition to maltose, maltotriose is produced in small quantities for use as a reagent, and maltopentaose is produced in small quantities as a layer for 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, Mrch, 13iochem, B
iophys, 155.290 (1973) and the 178th page of the abstracts of the 1981 conference of the Japanese Society of Agricultural Chemistry. However, these enzymes produce deposits other than maltopentaose from the early stage of the reaction, and amylases that produce only maltopentaose are unknown.

The present inventors have conducted extensive research to search for an enzyme that can synthesize maltopentaose with high kinetics. During this process, the inventors discovered that the desired maltopentaose synthase could be obtained by culturing microorganisms belonging to alkaline earth metals, leading to the completion of the present invention.

The present invention first relates to a novel maltopentaose synthase having the properties shown below.

(1) This enzyme contains amylose, soluble starch, potato starch, sweet potato starch, rice starch, and tapioca starch! Last month, maltopentaose was produced in flour, corn starch, waxy corn starch, sago starch, etc.

(2) The optimum temperature for this enzyme is 6 to 7 at 45°C, and it is stable at 5.5 to 9.

(3) The optimal temperature for this enzyme is 45°C at 6°O, and it is inactivated if left at a temperature of 55°C or higher for 15 minutes.

(4) This enzyme is inhibited in 1 mM mercuric parachlorobenzoate and monoiodoacetamide solutions, but the inhibition rate is 10 to 20 Da.

(5) The molecular weight of this enzyme is approximately 82,000 (according to disk gel electrophoresis).

(6) Isoelectric lighting of this enzyme)! 15.2 (A/) by second in electrophoresis method).

The second invention is to cultivate a microorganism capable of producing a novel maltopentaose synthase, which belongs to alkaline earth metals and has the above-mentioned properties, to accumulate the enzyme in one culture, and to collect the enzyme. This is a novel method for producing maltopentaose synthase.

Furthermore, the present invention provides at least one of starch, a compositional fraction of starch, and a decomposition reaction product of starch as a third invention.
The present invention relates to a method for producing maltopentaose, which comprises producing a novel maltopentaose synthase having the above properties from a seed substance. The novel maltopentaose synthase of the present invention is produced using a microorganism, and the producing microorganism may be one that belongs to alkaline earth metals and has the ability to produce an enzyme having the above-mentioned properties.・7-engine caris ■AM-1015 etc. Although these have different production abilities, they all produce the target maltopentaose synthase. In addition, microorganisms of the genus Achromobacter and Morakykuda are only found to differ from microorganisms of alkaline earth metals in terms of motility, catalase test, etc., and are considered to be microorganisms related to alkaline earth metals. It is presumed that some strains belonging to this group have the ability to produce the enzyme of the present invention.

As mentioned above, the microorganisms used in the present invention may belong to alkaline earth metals and have the ability to produce enzymes having the above properties, and are not limited to the above-mentioned strains and their variants and mutants. 'Here, the mutation means may be any conventional method, for example, radioisotope (RI), ultraviolet@('ay), nitrosoguanidine, etc. may be used.

Culture conditions for microorganisms for producing the novel maltopentaose synthase of the present invention were investigated. 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. This medium was inoculated with the bacterial strain shown in Example 2, which will be described later, 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 is the best carbon source, and among starches, rice starch,
Considerably high activity was obtained when sweet potato starch was used. In addition, the activity ratio when using various types of powdered candy increased as the DB increased, and high maltose syrup had the same activity as maltose.

Table 1 Glucose 0.01 3,0 Fructose 8,02 6゜1 Syucrose 0.04 12゜1 Maltose 0033 Zo.

High maltose syrup 0.31 94 amylose (
Eggplant 100) 0.1 7 51.5 Soluble starch 0.1
8 55.6 Potato starch 0゜07 21゜2 Sweet potato starch 0.20 60.6 Rice starch 0.23 68.5 Tapioca starch 0.04 12゜1 Corn starch 0.04 12゜1 Waxy corn starch 0.07 21.2 Wheat starch 00 Sago starch 0.07 21. ．． 2. Cyclodextrin powder candy 0.12 36.4 Corn steep liquor - 0.0412, 1. Next, 1. To study the nitrogen source, 1. Meat processing, kiss 0.7 g, maltose 1. 1 salt 0.3 da. Add 1% of various substances to the medium,
Shaking culture was performed at 0°C for 3 days. The activity ratio at this time (value when ammonium sulfate is taken as 100) is the second
Shown in the table. As is clear from the table, significantly higher activity was obtained when ammonium sulfate or ammonium nitrate was used.

Table 2 Peptone 0°34 16.7 Polybeptone 0.18, 8.9 Casein 0.22 10.8 Casamino acid 0.09 4.4 Potato protein OO Ammonium acetate 00 Ammonium sulfate 2.03 100 Ammonium nitrate 1,58 77. 8 Urea 0 0 No additives 00 Furthermore, as a result of examining the respective concentrations of maltose, ammonium sulfate, and meat extract, the optimal medium composition was found to contain 0.8% maltose, 1% ammonium sulfate, and 0.8% meat extract. It turned out that it was. Therefore, with reference to the above findings, the culture medium used for culture should be selected to have a composition that allows the test strain to have good activity and produce the desired enzyme.

Next, we investigated the change in activity depending on the number of days of culture, and the results shown in FIG. 1 were obtained.

As shown in the figure, an activity of 70- or higher was obtained in cultured IEr, and the activity gradually increased until the third day. However, after 1, the activity decreases. Therefore, culturing for 1 to 3 days is appropriate for enzyme production, and usually after culturing for 3 days, the supernatant obtained by removing insoluble matter in the culture solution by centrifugation can be used as the crude enzyme. . Although culture conditions vary depending on the strain used, they 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.

As a method for purifying enzymes, a starch adsorption method using moist heat-treated starch is effective. In other words, by simply adding moist heat-treated starch to the crude enzyme solution and stirring overnight at a low temperature of 4°C or less, approximately 80%
activity is adsorbed. Next, a partially purified enzyme can be easily obtained by collecting the enzyme-absorbed Sm powder and washing with ice water. The adsorbed enzyme can act on the liquefied starch as it is, and it is practically advantageous to use this enzyme preparation for producing maltopentaose.

The adsorbed enzyme is desorbed by stirring in warm water at 45°C for 15 minutes.
I! - Cellulose column chromatography.

A specimen that shows a single band on disk gel electrophoresis can be obtained by purification by gel filtration chromatography or the like.

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

(The reaction process when this enzyme acts on soluble starch is the second
This is the case shown in FIG.

As is clear from the figure, this enzyme produces maltopentaose at the beginning of the reaction, and then hydrolyzes it 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 produced sugar is removed from the reaction system using an ultrafiltration membrane. It is desirable to adopt the method of

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

The abbreviation is G1 Nigel Course.

G! : Maltose + Gl'' Maltotriose-G, : Maltobentaose +-G@': Maltooctaose.

G8: →G, 10G.

G7: 0-aGooo → Qs + QtG6: αG00ノ →G, 10G.

GII: () (l) ◇ # - * 03 10G,.

G4: αG0 da → G, + G.

Gs, Gl: No action From this mode of action, a mixture of G2 and G is produced, and G is digested by yeast to produce G3, or G,
A mixture of G3 and G3 can also be used as a product.

When a substrate with a low degree of S polymerization is used, this enzyme exhibits an action as a so-called 1ifxo-type amylase, but for dextrin with a high degree of polymerization, Findo 9
Acts as amylase. Therefore, most of the starch is converted into GIl, G, and G3 by the action of this enzyme, without leaving anything uncut. At this time, if a debranching enzyme such as pullulanase is coexisting, 1. The yield of G can be improved.

(2) Optimal action point and circular stability The reaction solution composition was set, and the reducing power was measured after reacting at 45° C. for 30 minutes. The results are shown in FIG. 4, with the highest value being expressed as 100. As is clear from the figure, the optimum application of this enzyme is 6-7.

In addition, regarding stability, 1 bottle of each product is added to 1 bottle of this enzyme solution.
00 m, M buffer (states 4-6: acetate buffer.

Field 6-8 Niphosphate Buffer, State 8-9 Nidris-Carbonate Buffer, )! Add 0.1 d of 100 mM acetate buffer (46,0) and 0.1 d of 100 mM acetate buffer (46,0) and 2% substrate. 0.5 m of the solution was added and reacted at 45°C for 30 minutes, and the residual enzyme activity was measured. The results are shown in Figure 5. As shown in FIG. 5, this enzyme has a pH of 5.5 to 9.
It is stable in the range of 0.

(3) Enzyme titer measurement method Enzyme activity can be measured using soluble starch (manufactured by Merck & Co., Ltd. for analysis).
was reduced and used as a substrate, the reducing power was measured by the Sosogy-Nernon method, and the amount of enzyme that cleaves 1 micromole equivalent of glucoside bonds per minute at 45° C. was defined as I IU (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 30 minutes. The results are shown in FIG. 6, with the highest value being expressed as 100. As is clear from the figure, the optimal temperature for the action of this enzyme is 45°C.

After leaving it at various temperatures for 15 minutes at 6°O, it was heated to 45°C.
The reaction was carried out and the residual activity was measured. The results are shown in FIG. As is clear from FIG. 7, the activity is rapidly deactivated at temperatures above 155°C.

(5) Inhibition, Activation and Stabilization This enzyme is inhibited in 1 mM mercuric parachlorobenzoate and monoiodoacetamide solution, but the inhibition rate is not high at 10-20%.

'Next, regarding the effects of various metal ions (1mM concentration), mercury and silver have a high inhibition rate of over 80%, but manganese, cobalt, zinc, and aluminum have a inhibition rate of less than 50%, and barium and magnesium have an inhibition rate of less than 50%.

Iron, lithium and copper are 20-30%. Moreover, calcium ions increase the heat resistance of this enzyme by 2 to 3°C.

(6) Molecular Weight The molecular weight of this enzyme obtained by disk gel electrophoresis is approximately 82,000.

(7) Isoelectric focusing F determined by ampholine electrophoresis
! ('5.2.

(8) Crystal structure and elemental analysis Although a crystal preparation of this enzyme has not yet been obtained, amino acid analysis was performed on a purified preparation that showed a single band in electrophoresis.

Amino acid analysis was carried out using an automatic amino acid analyzer (Hitachi Model 835) after adding 6N sulfuric acid to the sample, vacuuming, sealing, and decomposing at 1110° C. for 22 hours. Note that tryptophan was determined by the colorimetric method of Mr. 8 Pies, and cis9ine was determined by the performic acid oxidation method. The results are shown in Table 3.

Table 3 Amino acid moles 42 ソ ソ 酸 イ イ イ イ イ イ イ イ イ イ イ イ イ イ イ イ ジ ジ ジ ジ ジ ジ ジ ジ ジ ジ ジ ジ ジ ジ ジ ジ ジ ジ42 Arginine 28 Histidine 20 or more The present enzyme, which has the properties shown above, is a novel enzyme that exhibits a completely different action from conventional enzymes and produces maltopentaose in large amounts. The inventors have one 13 HIE 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, a compositional fraction of starch, and a decomposition reaction product of starch. Conditions should be selected to maximize the amount of maltopentaose produced, taking into consideration the reaction conditions and the properties of the enzyme. Here, as starch, for example, potato lr
, Sweet 1 Jlli, ) Those obtained from arbitrary raw materials such as corn, waxy corn, barley, wheat, rice, tapioca, and sago can be used. In addition, as a composition fraction of starch, for example, amylose.

Examples include amylopectin, and starch decomposition reaction products include white dextrin, yellow dextrin,
(roasted dextrin such as pritish gum) IJn; modified starch such as oxidized starch, low-viscosity modified starch (by treatment with enzymes, acid, high-speed mechanical stirring, etc.); / Starch represented by phosphoric acid starch, acetic acid starch, etc. ether,
Examples include starch derivatives such as starch ester; physically treated starch such as starch irradiated with radiation or neutron beams, high frequency treatment, or moist heat treatment; α-starch. These starches may be used alone or in combination of two 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.

Maltopentaose is currently used as a substrate for measuring α-amylase activity in diagnostic agents, reagents, etc., and if this enzyme can be produced at low cost according to the present invention, it is expected to be used in various applications including food. . Maltopentaose has excellent solubility, sweetness, and body, making it useful as an ingredient for confectionery.Also, because it is easily digested and absorbed, it can be used as a nutritional food for infants, the elderly, and patients. Ami.

Next, the present invention will be explained with reference to Examples.Example 1 Alcaligenes faecalis XAM-1015 strain was prepared using 0.8% meat extract, 1% ammonium sulfate, 0.0% maltose,
After inoculating onto an 8% agar slant and culturing at 40°C for 2 days, one loopful was taken and inoculated into a liquid medium with the same composition (100%
The cells were transferred to a medium (150 Qm/erlenmeyer flask) and cultured in an aerated screw tube 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 a crude enzyme.The activity of this crude enzyme solution was 0.01 IU~.

Example 2 A small amount of culture solution of Alcaligenes faecalis strain IAM-1015 was taken and treated with RI, UV, and nitrosoguanidine in a conventional manner, followed by plate culture and colonies with high amylase activity were taken. This was grown at 45°C in a medium containing 0.8% meat extract, 1% ammonium sulfate, and 0.8% maltose.
The cells were cultured for 3 days, and the subsequent operations were the same as in Example 1. The activity of this crude enzyme solution was 0.64 IU/go.

Example 3 Potato starch was liquefied using bacterial liquefying enzyme (BLA),
The iodine-starch reaction was deactivated in blue and the substrate 11 was
0%, maltopentaose synthase (crude enzyme solution of Example 2) I NU/p substrate, 1.5. Q, 4. s
The reaction was carried out with stirring at ℃ for 6 hours to obtain a reaction solution containing 30% maltopentaose.

Example 4 A liquefied potato starch solution was prepared as in Example 3, and the substrate concentration was 20%. Filter [manufactured by Toyo Okishi Co., Ltd., UHP-76 (membrane is from UK)
-10)) was reacted under pressure with nitrogen gas to obtain a sugar solution containing 80% or more of maltopentaose. The yield was 60% of the starting substrate after 12 hours of reaction, and the obtained sugar solution could be concentrated to 201, J/C using a reverse osmosis membrane.

Example 5 The same procedure as in Example 4 was carried out except that pullulanase was added to the IIU15p substrate, and a sugar solution containing 80% or more of maltopentaose was obtained in a yield of 65% in 1 to 12 hours of reaction.

[Brief explanation of drawings]

Figure 1 is a graph showing changes in activity and voice depending on the number of days of culture, and Figure 2 is a graph showing the reaction progress of maltopentaose synthase (1
Fig. 3 is a graph showing the reaction progress of this enzyme (IIU/1owi substrate), Fig. 4 is a graph showing the optimum of this enzyme, and Fig. 5 is a graph showing the reaction progress of this enzyme (IIU/1owi substrate). FIG. 6 is a graph showing the optimum temperature of this enzyme, and FIG. 7 is a graph showing the temperature stability of this enzyme. Patent applicant Ministry of Agriculture, Forestry and Fisheries Food Research Institute Nagaki Suiko Refining Co., Ltd. Figure 1 0 1 2.545 Koyo 1. l1Table G1・1 G2・Φ. 3・ - St O510152030456090120St reaction opening/closing) 3rd meat 01・ ・ St O125103060120180360St anti-A-Ginma (ke) Fig. 4 45 7 9 Re57911 H Fig. 6 20 30 40 50 60 70 Warm &('C) procedure Written amendment (spontaneous) February 22, 1980 Kazuo Wakasugi, Commissioner of the Japan Patent Office 1, Indication of the case Patent application 1982-156273 2, Title of the invention: Novel maltopentaose synthase, method for producing the same, and use of the enzyme Method for producing maltopentaose 3, relationship with the case of the person making the amendment Patent applicant Ministry of Agriculture, Forestry and Fisheries Food Research Institute Director Shiomizuko Sugar Co., Ltd. 4 Agent ■104 Address IO 1-1 Kyobashi, Chuo-ku, Tokyo Nishikan Building 5th Floor Name (7407) Patent Attorney Kubo 1) Fujibe Telephone (27
5) No. 0721 No. 5, Column 6 of Detailed Description of the Invention of the Specification Subject to Amendment, Contents of the Amendment (1) In the first line of page 8 of the specification, "to create" is corrected to "to act." (2) RCA+c- on page 15, line 8 of the same page is rGZ+G
! Correct to J. (Continued from page 1) ■Int, CI,' Identification code Internal reference number 0 Inventor Kozo Hara 2 Namiki, Kanazawa-ku, Yokohama ≧-6-3-1
04

Claims (1)

[Claims] 1. A novel maltopentaose synthase having the following properties. (1) This enzyme contains amylose, soluble starch, potato starch,
Sweet potato starch, rice starch, tapioca starch, corn starch,
0 (2) This enzyme produces maltopentaose by acting on waxy corn starch, sago starch, etc. at 45℃! (6 to 7 is optimal,
11 (5,5-9 is stable. (3) This enzyme is F4 (6,0, the optimum temperature is 45
℃, and is inactivated if left at a temperature of 55℃ or higher for 15 minutes. (4) This enzyme is inhibited in 111 M mercuric parachlorobenzoate and monoiodoacetamide solutions;
The inhibition rate is 10-20%. (5) The molecular weight of this enzyme is approximately 82,000 (based on disk gel electrophoresis). (6) The isoelectric point of this enzyme is F! (5,2 (according to ampholine electrophoresis) A novel method for producing maltopentaose synthase, which is characterized by accumulating and collecting this enzyme. (1) This enzyme contains amylose, soluble starch, potato starch, sweet potato starch, rice starch, tapioca starch, corn +1
14) It acts on waxy corn starch, T-goose flour, etc. to produce maltopentaose. (2) The optimal temperature for this enzyme is 6 to 7 at 45°C, and F
! (It is stable at temperatures between 5, 5 and 9. (3) The optimal temperature for this enzyme is 45°C at 6.0, and it is inactivated if left at a temperature of 55°C or higher for 15 minutes◇ (4) This enzyme is inhibited in 1mM mercuric parachlorobenzoate and monoiodoacetamide solutions, but the inhibition rate is 10-20%. (5) The molecular weight of this enzyme is approximately 32,000 (according to disk gel electrophoresis). (6) The isoelectricity of the enzyme is 5.2 (as determined by electrophoresis). (5) A patent claim in which the producing bacterium of the novel maltopentaose synthase belonging to Alcaligenes iRK is Alcaligenes faecalis. The production method according to Scope 2. 4. A culture medium containing maltose, maltose-containing starch syrup, starch, or soluble starch as a carbon source, using a novel maltopentaose synthase-producing bacterium belonging to an alkaline earth metal. 5. A novel maltopentaose synthesis in which at least one substance selected from starch, a compositional fraction of starch, and a decomposition reaction product of starch has the following properties: A method for producing maltopentaose characterized by the action of an enzyme. (1) This enzyme can be used on amylose, soluble starch, potato starch, sweet potato starch, rice starch, tapioca starch, corn starch, waxy corn starch, sago starch, etc. (2) This enzyme is optimal at temperatures of 6 to 7 at 45°C, and is stable at temperatures of 5 and 5 to 9. (3) This enzyme produces maltopentaose at 45°C. The optimum temperature at 6.0 is 45℃
It is deactivated when left at a temperature of 55° C. or higher for 15 minutes. (4) This enzyme is inhibited in 1mM mercuric parachlorobenzoate and monoiodoacetamide solutions;
The inhibition rate is 10-20%. (5) The molecular weight of this enzyme is approximately 82,000 (according to disk gel electrophoresis). (6) The isoelectric point of this enzyme is [5,2 (according to ampholine electrophoresis). & The production method according to claim #15, wherein the starch composition fraction is amylose or amylopectin. 1. The production method according to claim 5, wherein the starch decomposition reaction product is a modified starch.