JPS6351677B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel maltopentaose synthase and a method for producing the same. In recent years, research on maltooligosaccharides has been 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 maltotriose to maltohexaose have been discovered one after another, and it has become easier to produce various oligosaccharides from starch. For example, regarding maltopentaose
Enzymes described in Arch.Biochem.Biophys., 155 , 290 (1973) and in the abstracts of the 1981 conference of the Japanese Society of Agricultural Chemistry, page 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 synthesize maltopentaose. In the process, the inventors discovered that the desired maltopentaose synthase could be obtained by culturing a microorganism belonging to the genus Alcaligenes, 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 produces maltopentaose by acting on amylose, soluble starch, potato starch, sweet potato starch, rice starch, tapioca starch, corn starch, waxy corn starch, sago starch, etc. (2) The optimal pH for this enzyme is 6 to 7 at 45℃, and the pH
It is stable between 5.5 and 9. (3) The optimal temperature for this enzyme at pH 6.0 is 45°C, and it will be 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 82,000 (according to disk gel electrophoresis). (6) The isoelectric point of this enzyme is PH5.2 (according to ampholine electrophoresis). Second, the present invention involves culturing a microorganism capable of producing a novel maltopentaose synthase that belongs to the genus Alcaligenes and having the above-mentioned properties, accumulating the enzyme in the culture, and collecting the enzyme. This is a novel method for producing maltopentaose synthase. The novel maltopentaose synthase of the present invention is produced using a microorganism, and the producing microorganism may be any microorganism that belongs to the genus Alcaligenes and has the ability to produce an enzyme having the above-mentioned properties, such as Alcaligenes fuecalis IAM. -1015 etc. Although there are differences in production capacity,
Both produce the target maltopentaose synthase. In addition, microorganisms of the genus Achromobacter and Moraxida are only different from microorganisms of the genus Alcaligenes in terms of motility, catalase test, etc., and are considered to be microorganisms related to the genus Alcaligenes. It is presumed that some of them have the ability to produce the enzyme of the present invention. As mentioned above, the microorganisms used in the present invention may belong to the genus Alcaligenes 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 may be carried out using conventional methods, such as radioisotope (RI), ultraviolet light (UV), nitrosoguanidine, etc. Culture conditions for microorganisms for producing the novel maltopentaose synthase of the present invention were investigated. First, a base 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 ratio (value when maltose is set as 100) at this time is shown in Table 1. As is clear from the table,
Maltose is the best carbon source, and among starches, considerably high activity was obtained when rice starch and sweet potato starch were used. In addition, the activity ratio when using various types of powdered candy increased as DE increased, and high maltose syrup had the same level of activity as maltose.

[Table] Next, in order to consider the nitrogen source, meat extract
1% of each substance was added to a medium containing 0.7% maltose, 1% maltose, and 0.3% salt, 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.

【table】

[Table] Furthermore, as a result of examining the respective concentrations of maltose, ammonium sulfate, and meat extract, it was found that the optimal medium composition was one containing 0.8% maltose, 1% ammonium sulfate, and 0.8% meat extract. 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 produce the desired enzyme with good activity. Next, we examined changes in activity depending on the number of days of culture, and the results shown in FIG. 1 were obtained.
As shown in the figure, more than 70% activity was obtained within one day of culture, and the activity gradually increased until the third day. but,
After that, activity decreases. Therefore, culture for 1 to 3 days is appropriate for enzyme production, and usually 3 days.
After culturing for one day, the supernatant obtained by centrifuging and removing insoluble matter in the culture solution may 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, one example of which is as follows. As a method for purifying enzymes, a starch adsorption method using moist heat-treated starch is effective. That is, approximately 80% of the activity is adsorbed by simply adding moist heat-treated starch to the crude enzyme solution and stirring overnight at a low temperature of 4°C or less. Next, a partially purified enzyme can be easily obtained by collecting the enzyme-adsorbed starch 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.
DEAE - cellulose column chromatography,
It is possible to obtain a specimen that shows a single band on disk gel electrophoresis 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. (1) Action The course of the reaction when this enzyme is applied to soluble starch is shown in Figures 2 and 3.
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 membrane reactor, and the produced sugar is 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 G ₁ : Glucose, G ₂ : Maltose, G ₃ : Maltotriose --
G ₅ : Maltopentaose, --G ₃ : Maltooctaose. G ₈ ：〇－〇－〇－〇－〇－〇－〇－○／→G ₅ ＋G ₃ G ₇ ：〇－〇－〇－〇－〇－〇○／ →G ₅ ＋G ₂ G ₆ ：〇－ 〇―〇―〇―〇○／ →G ₅ ＋G ₁ G ₅ ：〇－〇－〇－〇○／ →G ₃ ＋G ₂ G ₄ ：〇－〇－〇－○／ →G ₂ ＋G ₂ G ₃ , G ₂ : No action From this mode of action, a mixture of G ₂ and G ₃ is produced,
_G2 is digested by yeast to produce _G3 ,
Moreover, a mixture of G ₂ and G ₃ 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, most of the remaining starch is converted into _G5 or _G2 and _G3 by the action of this enzyme. At this time, if a debranching enzyme such as pullulanase is coexisting, G ₅
yield can be improved. (2) Optimum PH and PH stability The reaction solution composition was: substrate (2% reduced soluble starch solution) 0.5ml, various pH buffers (100mM) 0.4ml, enzyme solution (8IU/ml) 0.1ml, The reducing power was measured by reacting at 45℃ for 30 minutes.
Figure 4 shows the results when the highest value is expressed as 100. As is clear from the figure, the optimal pH of this enzyme
is 6-7. Regarding PH stability, 1ml of this enzyme solution was mixed with 100mM buffer of various PH (PH4-6: acetate buffer,
PH6-8: Phosphate buffer, PH8-9: Tris-HCl buffer, PH9-10: Sodium carbonate buffer) 0.1ml
was added and left to stand at 45℃ for 60 minutes, then 0.1ml of each was taken and added with 0.4ml of 100mM acetate buffer (PH6.0) and 2.
Add 0.5ml of % substrate solution and react at 45℃ for 30 minutes.
Residual enzyme activity was measured. The results are shown in Figure 5.
As shown in FIG. 5, this enzyme is stable within the pH range of 5.5 to 9.0. (3) Enzyme titer measurement method Enzyme activity was determined by reducing soluble starch (manufactured by Merck & Co., Ltd., for analysis) and using it as a substrate, and measuring the reducing power by the Somogyi-Nelson method.
The amount of enzyme that cleaves a micromolar equivalent of glucosidic bonds was defined as 1 IU (international unit). (4) Optimum temperature for action and temperature stability The reaction solution composition was: substrate (2% reduced soluble starch solution) 0.5ml 100mM acetate buffer (PH6.0) 0.4ml enzyme solution (8IU/ml) 0.1ml. The reducing power was measured by reacting at various temperatures for 30 minutes, and the results are shown in Figure 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. Also, after leaving it at various temperatures for 15 minutes at PH6.0,
The reaction was carried out at ℃ and the residual activity was measured. The results are shown in FIG. As is clear from Figure 7, 55℃
Above that, it rapidly loses its activity. (5) Inhibition, activation and stabilization This enzyme is inhibited in 1mM mercuric parachlorobenzoate and monoiodoacetamide solutions, but the inhibition rate is not high at 10-20%. Next, regarding the influence of various metal ions (1mM concentration), mercury and silver have a high inhibition rate of over 80%, manganese, cobalt, zinc, and aluminum have a inhibition rate of less than 50%, barium, magnesium,
Iron, lithium, and copper account for 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 point The isoelectric point determined by ampholine electrophoresis is PH5.2. (8) Crystal structure and elemental analysis Although a crystal sample of this enzyme has not yet been obtained, amino acid analysis was performed on a purified sample 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 hydrochloric acid to the sample, sealing the sample under reduced pressure, and decomposing it at 110°C for 22 hours. In addition, tryptophan was determined by the colorimetric method of Mr. Spies, and cysteine was determined by the performic acid oxidation method. The results are shown in Table 3. Table 3 Amino acid moles Alanine 48 Valine 42 Leucine 53 Isoleucine 36 Proline 38 Phenylalanine 18 Tryptophan 29 Methionine 24 Glycine 78 Serine 41 Threonine 45 Cysteine 5 Tyrosine 39 Aspartic acid 82 Glutamic acid 65 Lysine 42 Arginine 28 Histidine 20 As shown above properties This enzyme has a completely different action from conventional enzymes, and is a novel enzyme that produces maltopentaose in large amounts. The present inventors named this enzyme 1,4-α-D-glucan maltopentaohydrolase. As mentioned above, this enzyme acts on amylose, soluble starch, and various starches to produce maltopentaose. Therefore, at least one of starch, a compositional fraction of starch, and a starch decomposition reaction product
Maltopentaose is produced and accumulated by allowing this enzyme to act on the seed material. When carrying out the reaction, conditions should be selected that maximize the amount of maltopentaose produced, taking into account the properties of the enzyme. Here, starch can be obtained from any raw material such as potato, sweet potato, corn, waxy corn, barley, wheat, rice, tapioca, or sago. 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 sweet gum; oxidized starch, low-viscosity modified (enzyme,
Modified starches such as starch (treated with acids, high-speed mechanical stirring, etc.); Starch derivatives such as starch ethers and starch esters, typified by phosphoric acid starch, acetic acid starch, etc.;
Physically treated starch, such as starch that has been irradiated with radiation or neutron beams, high frequency treatment, or moist heat treatment; α-
Examples include starch. These starches may be used alone or in combination of two or more. After the reaction is completed, the reaction is stopped by heating to inactivate the enzyme, and 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 that it will be used in various applications including food. be done. Maltopentaose has excellent solubility, lacks sweetness, and has a body texture, making it useful as an ingredient for confectionery.Since it is also easily digested and absorbed, it can be used as a nursing food for infants, the elderly, and patients. be. Next, the present invention will be explained by examples. Example 1 Alcaligenes fuecalis strain IAM-1015 was inoculated onto a slanted agar medium containing 0.8% meat extract, 1% ammonium sulfate, and 0.8% maltose, and cultured at 40°C for 2 days. The cells were transferred to a liquid medium (100 ml medium/500 ml 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 0.01 IU/ml. 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 collected. This was cultured at 45° C. for 3 days in a medium containing 0.8% meat extract, 1% ammonium sulfate, and 0.8% maltose, and the subsequent operations were the same as in Example 1. The activity of this crude enzyme solution was 0.64 IU/ml. Application example 1 Potato starch is liquefied using bacterial liquefying enzyme (BLA), and the iodine-starch reaction is deactivated with blue color.
Substrate concentration 10%, maltopentaose synthase (crude enzyme solution of Example 2) 1 IU/g substrate, pH 6.0, reaction at 45°C for 6 hours with stirring to obtain a reaction solution containing 30% maltopentaose. . Application Example 2 A liquefied potato starch solution was prepared as in Application Example 1, substrate concentration 20%, maltopentaose synthase (crude enzyme solution from Example 2) 1 IU/g substrate, pH 6.0, 45.
℃ using an ultrafilter [manufactured by Toyo Paper Co., Ltd., UHP-76 (membrane is
UK-10)] was reacted under pressure with nitrogen gas to obtain a sugar solution containing more than 80% maltopentaose. The yield was 60% of the starting substrate after 12 hours of reaction, and the resulting sugar solution could be concentrated to 20% using a reverse osmosis membrane. Application example 3 The procedure was the same as application example 2 except that pullulanase was added to 1IU/5g substrate, and a sugar solution containing more than 80% maltopentaose was produced in a yield of 65% in 12 hours of reaction.
I got it from

[Brief explanation of the drawing]

Figure 1 is a graph showing activity changes and pH changes depending on the number of days of culture, Figure 2 is a graph showing the reaction progress of maltopentaose synthase (1 IU/g substrate), and Figure 3 is a graph showing the reaction progress of maltopentaose synthase (1 IU/g substrate).
The figure is a graph showing the reaction progress of this enzyme (1IU/10mg substrate), Figure 4 is a graph showing the optimum pH of this enzyme,
FIG. 5 is a graph showing the PH stability of this enzyme, 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.

Claims (1)

[Claims] 1. A novel maltopentaose synthase having the following properties. (1) This enzyme produces maltopentaose by acting on amylose, soluble starch, potato starch, sweet potato starch, rice starch, tapioca starch, corn starch, waxy corn starch, sago starch, etc. (2) The optimal pH for this enzyme is 6 to 7 at 45℃, and the pH
It is stable between 5.5 and 9. (3) The optimal temperature for this enzyme at pH 6.0 is 45°C, and it will be 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 82,000 (according to disk gel electrophoresis). (6) The isoelectric point of this enzyme is PH5.2 (according to ampholine electrophoresis). 2. A novel malt which is characterized by culturing a microorganism that belongs to the genus Alcaligenes and has the productivity of a novel maltopentaose synthase having the following properties, accumulating the enzyme in the culture, and collecting the enzyme. Method for producing pentaose synthase. (1) This enzyme produces maltopentaose by acting on amylose, soluble starch, potato starch, sweet potato starch, rice starch, tapioca starch, corn starch, waxy corn starch, sago starch, etc. (2) The optimal pH for this enzyme is 6 to 7 at 45℃, and the pH
It is stable between 5.5 and 9. (3) The optimal temperature for this enzyme at pH 6.0 is 45°C, and it will be 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 82,000 (according to disk gel electrophoresis). (6) The isoelectric point of this enzyme is PH5.2 (according to ampholine electrophoresis). 3. The production method according to claim 2, wherein the novel maltopentaose synthetase producing bacterium belonging to the genus Alcaligenes is Alcaligenes faecalis. 4. A novel maltopentaose synthase-producing bacterium belonging to the genus Alcaligenes is cultured in a medium containing maltose, maltose-containing starch syrup, starch, or soluble starch as a carbon source. Production method.