JP3725148B2 - Maltose / trehalose converting enzyme, its production method and use - Google Patents

Description

  The present invention relates to a maltose / trehalose converting enzyme, a method for producing the same, and a use thereof. More specifically, the present invention relates to a novel maltose / trehalose converting enzyme that converts maltose to trehalose and converts trehalose to maltose, and a method for producing the same. The present invention relates to a microorganism, trehalose produced using this maltose / trehalose converting enzyme, or a saccharide containing the same, and a composition containing these saccharides.

  Trehalose (α, α-trehalose) has been known for a long time as a non-reducing carbohydrate having glucose as a constituent sugar, and its presence is described in Non-Patent Document 1, Non-Patent Document 2, and the like. In addition, it has a wide range of microorganisms, mushrooms, insects, etc., even in small quantities. Trehalose is a non-reducing saccharide, so it does not cause aminocarbonyl reaction with substances having amino groups such as amino acids and proteins, and does not damage amino acid-containing substances, so it can be used and processed without concern for browning and deterioration. Therefore, establishment of the industrial manufacturing method is desired.

  Examples of the method for producing trehalose include a method using a microorganism reported in Patent Document 1, a method of converting maltose by a combination of maltose phosphorylase and trehalose phosphorylase proposed in Patent Document 2. Are known. However, in the method using microorganisms, the microbial cells are used as a starting material, and the trehalose content contained therein is usually 15% by mass (hereinafter, unless otherwise specified,% by mass unless otherwise specified). In addition, the process of extracting and purifying this is complicated and unsuitable as an industrial production method. In addition, all methods using phosphorylase such as maltose phosphorylase and trehalose phosphorylase are via glucose phosphate, and it is difficult to increase the substrate concentration, and the reaction system generates the target product by an equilibrium reaction. The rate is low, and furthermore, it is difficult to keep the reaction system stable and allow the reaction to proceed smoothly, and it has not yet been realized as an industrial production method.

  On the other hand, starch partially decomposed products produced using starch as a raw material, for example, starch liquefied products, various dextrins, various maltooligosaccharides, etc. usually have a reducing group at the end of the molecule and have a large reducing power per solid. This is expressed as Dextrose Equivalent (DE). Those with large values generally have small molecules, low viscosity, and strong sweetness, but are highly reactive and easily cause aminocarbonyl reactions with substances having amino groups such as amino acids and proteins, browning, and foul odors. It is known that there is a property that the quality is easily deteriorated. Various characteristics of such a reduced starch partial decomposition product depend on the size of DE, and the relationship between the starch partial decomposition product and DE is extremely important. Traditionally, the industry has even believed that it was impossible to break this relationship.

  In relation to this, in the section of “Oligosaccharide” in Non-Patent Document 3, “A tremendous range of trehalose can be considered, but direct sugar transfer and hydrolysis reaction of this sugar from starch carbohydrates were used. Enzymatic production is said to be scientifically impossible at present. ”As described in the article, the production of trehalose using starch as a raw material by enzymatic reaction has traditionally been academic. Has also been considered impossible.

  However, the present inventors have overturned this common sense in the industry, and as disclosed in Patent Document 3 and Patent Document 4, the reduced starch partial decomposition product having a glucose polymerization degree of 3 or more produced using starch as a raw material. A non-reducing saccharide-forming enzyme that produces a non-reducing saccharide having a trehalose structure at the terminal and having a degree of polymerization of glucose of 3 or more to produce the non-reducing saccharide, and then glucoamylase, Alternatively, trehalose has been successfully produced enzymatically from a reduced starch partial degradation product by acting a trehalose releasing enzyme. However, in this trehalose production method using a non-reducing saccharide-forming enzyme, a relatively high-molecular starch saccharide having a glucose polymerization degree of 3 or more is used as a raw material, and its viscosity is relatively high. 2 or more are required, and the sugar composition of the reaction product obtained is relatively complex, and there is a concern about the high cost in the production of trehalose. Therefore, establishment of a new trehalose production method for converting maltose into trehalose, which is a starch partial decomposition product having a glucose polymerization degree of 2, which is industrially produced from starch and is stably supplied, is desired.

Japanese Patent Laid-Open No. 50-154485 JP 58-216695 A Japanese Patent Application No. 4-362131 Japanese Patent Application No. 5-156338 "Advances in Carbohydrate Chemistry", Vol. 18, pp. 201-225 (1963) Academic Press (USA) "Applied and Environmental Microbiology", Vol. 56, pp. 3213-3215 (1990) “Monthly Food Chemical”, August issue, pp. 67-72, “Current Status and Issues of Starch Development” (1992)

  The present invention relates to a maltose / trehalose converting enzyme that converts maltose that is industrially produced from starch and is stably supplied into trehalose, a new method for producing trehalose using the enzyme, or a sugar containing the same, and use thereof Is to provide.

  In order to solve the above-mentioned problems, the present inventors have sought a completely new converting enzyme that produces trehalose from maltose, and extensively searched for microorganisms that produce the enzyme. As a result, a novel microorganism R48 belonging to the genus Pimelobacter isolated from soil in Okayama City, Okayama Prefecture, a novel microorganism H262 belonging to the genus Pseudomonas isolated from soil in Nishinomiya City, Hyogo Prefecture, and a known thermus ( We found that a microorganism belonging to the genus Thermus produces a novel maltose / trehalose converting enzyme that converts maltose to trehalose, and established a method for producing trehalose that can act on this maltose-containing solution and a saccharide containing the same. In addition, the present invention was completed by establishing compositions such as foods, drinks, cosmetics and pharmaceuticals containing these carbohydrates.

  The maltose / trehalose converting enzyme of the present invention converts maltose into trehalose with a high conversion rate. Therefore, the establishment of the present invention opens up a completely new way to supply trehalose and carbohydrates containing it from a maltose derived from starch, an inexpensive and endless resource, industrially in large quantities and at low cost.

  Hereinafter, the identification test results of the microorganism R48 belonging to the genus Pymelobacter of the present invention and the microorganism H262 belonging to the genus Pseudomonas are shown. The identification test was performed in accordance with “Classification and Identification of Microorganisms” (Takeharu Hasegawa, Academic Publishing Center, 1985).

<Pimerobacter species R48 identification test results>
<A: Cell morphology>
(1) Meat broth agar culture, 27 ° C:
Usually 0.5-0.9 × 1.5-4.0 μm bacilli. A single, rarely V-shaped pair and linked cells are also observed. No mobility. No spores. Non-acidic. Gram positive.
(2) Yeast extract / malt extract agar culture, 27 ° C .:
The cell size of the culture day is 0.6 to 1.0 × 1.3 to 4.2 μm, and the culture day is 0.6 to 1.0 × 1.0 to 2.5 μm, which is close to the cocci Cells are also seen and polymorphism is observed. In addition, a single cell, rarely a V-shaped pair, is also observed.

<B: Culture characteristics>
(1) Meat broth agar plate culture, 27 ° C
Shape: Circular The size is 0.5 mm in 24 hours. 1.5 to 2 mm in 3 days.
Periphery: Wavy Bump: Half-lens Gloss: None Surface: Wrinkled Color: Opaque, creamy.
(2) Yeast extract / malt extract agar plate culture, 27 ° C.
Shape: Circular The size is about 1 to 1.5 mm in 3 days.
Periphery: Wavy Bump: Half-lens Gloss: None Surface: Rough Surface Color: Opaque, creamy.
(3) Meat broth agar slope culture, 27 ° C
Growth degree: Good Shape: Filament (4) Grape gelatin puncture culture, 27 ° C
: Liquefaction

<C physiological properties>
(1) Nitrate reducibility: positive (2) Denitrification reaction: negative (3) Methyl red test: negative (4) VP test: negative (5) Indole production: negative (6) Hydrogen sulfide production: positive ( 7) Hydrolysis of starch: Negative (8) Use of citric acid: Positive (9) Use of inorganic nitrogen source: Both ammonium salt and nitrate can be used.
(10) Pigment production: Negative (11) Urease: Negative (12) Oxidase: Negative (13) Catalase: Negative (14) Growth range: pH 5-9, temperature 15-40 ° C.
(15) Attitude toward oxygen: aerobic (16) Use of carbon source and presence or absence of acid generation
Usability Acid-producing ability D-glucose Utilization Negative D-Galactos Not utilized Negative D-Mannose Utilized Negative D-Fructose Utilized Negative L-arabinose Utilized Negative D-Xylose Utilized Negative L-Rhamnose Utilized Negative Maltose Utilized Negative Sucrose Used Negative Lactose Not Used Negative Trehalose Used Negative Raffinose Used Negative Mannitol Not Used Negative Dextrin Used Negative Dulzitol Not Used Negative (17) Decarboxylation test of amino acids: L-lysine, L-arginine, ornithine Also negative.
(18) Use of amino acids: Both sodium L-glutamate and sodium L-aspartate are used.
(19) DNase: negative (20) 3-ketolactose production: negative (21) GC content of DNA: 72%
(22) Cell wall main diamino acid: LL-diaminopimelic acid

  Based on the above bacteriological properties, “Bergey's Manual of Systematic Bacteriology”, Volume 2 (1986), and “Journal of General Applied” -Differences from known bacteria were examined with reference to "Microbiology (Journal of General Applied Microbiology)", Vol. 29, pp. 59-71 (1983). As a result, the present bacterium was a strain belonging to the genus Pimerobacter, and was found to be a novel microorganism that produces a novel maltose / trehalose converting enzyme.

  Based on these results, the present inventors named this microorganism Pimelobacter sp. R48 and traded in 1-3-1 Higashi 1-3-3, Tsukuba City, Ibaraki Prefecture as of June 3, 1993. Deposited at the Institute of Biotechnology, Institute of Industrial Science, Ministry of Industry, Patent Microorganism Depositary Center, and deposited under the deposit number FERM BP-4315.

<Results of Pseudomonas putida H262 identification test>
<A: Cell morphology>
(1) Meat broth agar culture, 27 ° C:
Usually, 0.5 to 0.7 × 1.0 to 2.0 μm of koji molds do not form spores. There is mobility by polar flagellum. Gram negative. Non-acidic.
(2) Yeast extract / malt extract agar culture, 27 ° C .:
The size of the cells in one day of culture is 0.6 to 0.8 × 2.0 to 4.0 μm

<B: Culture characteristics>
(1) Meat broth agar plate culture, 27 ° C
Shape: Circular, size is 1-2 mm in 24 hours. 3.5-4mm in 3 days
Periphery: Full edge Bump: Semi-lens Gloss: Wet light Surface: Smooth Color: Opaque, white to light yellow (2) Yeast extract / malt extract agar plate culture, 27 ° C.
Shape: Circular Size is about 4-5mm in 3 days
Periphery: Full edge Bump: Half-lens Gloss: Wet light Surface: Smooth Color: Opaque, white to cream (3) Meat broth agar slope culture, 27 ° C
Growth degree: Good Shape: String-like. The ridge is thin, the surface is smooth, moist light, opaque and slightly yellowish cream color (4) gravy gelatin puncture culture, 27 ° C
: Not liquefied

<C: Physiological properties>
(1) Nitrate reducibility: Positive (succinic acid medium)
(2) Denitrification reaction: Negative (3) Methyl red test: Negative (4) VP test: Negative (5) Indole production: Negative (6) Hydrogen sulfide production: Negative (7) Starch hydrolysis: Negative ( 8) Accumulation of poly-β-hydroxybutyrate: Negative (9) Decomposition of procatechuic acid: Ortho type (10) Use of citric acid: Positive (11) Use of inorganic nitrogen source: Both ammonium salt and nitrate can be used.
(12) Dye generation: Light yellow dye generation (13) Fluorescent dye generation: Positive (14) Urease: Positive (15) Oxidase: Positive (16) Catalase: Positive (17) Range of growth: pH 5 to 9 , Temperature 10 ~ 37 ℃
(18) Attitude toward oxygen: Aerobic (19) Utilization of carbon source and presence / absence of acid generation
Usability Acid-producing ability D-glucose Utilization Negative D-Galactos Not utilized Negative D-Mannose Utilized Positive D-Fructose Utilized Positive L-arabinose Utilized Positive D-Xylose Utilized Positive L-Rhamnose Not utilized Negative Maltose Not utilized Negative Not used Sucrose Not used Negative Lactose Not used Negative Trehalose Not used Negative Raffinose Not used Negative Mannitol Not used Negative Sorbitol Not used Negative Dullutol Not used Negative Glycerol Used Positive (20) Decarboxylation test of amino acids: L-lysine, L-ornithine Negative for this. Positive for L-arginine.
(21) Use of amino acids: L-sodium glutamate, sodium L-aspartate, L-arginine, L-histidine, L-valine, and D-alanine are all used. Do not use L-tryptophan.
(22) DNase: negative (23) 3-ketolactose production: negative (24) G-C content of DNA: 63%

  Based on the above bacteriological properties, with reference to “Bergey's Manual of Systematic Bacteriology”, Volume 1 (1984), the differences with known bacteria are described. investigated. As a result, the present bacterium was found to be a strain belonging to Pseudomonas putida.

  Based on these results, the present inventors named this microorganism Pseudomonas putida H262 and, as of February 23, 1994, are located in 1-3-1 Higashi 1-chome Tsukuba City, Ibaraki Prefecture. Deposited at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology, Patent Microorganism Depositary Center, and deposited under the deposit number FERM BP-4579.

  In the present invention, not only the strains described above, but also other strains belonging to the genera Pymelopacter and Pseudomonas and having the ability to produce maltose / trehalose converting enzyme, and mutants thereof can be used as appropriate.

  In addition, the microorganism used in the present invention is not limited to the above-mentioned novel microorganism, but also a microorganism belonging to a known genus of Thermus, for example, Thermus aquaticus ATCC 25104, Thermus aquaticus ATCC 27634, Thermus Aquaticus ATCC 33923, Thermus filiformis ATCC 43280, Thermus rubber ATCC 35948, Thermus sp. ATCC 43814, and Thermus sp.

  The medium used for culturing the microorganism of the present invention may be any medium that can grow microorganisms and produces the enzyme of the present invention, and may be either a synthetic medium or a natural medium. Any carbon source may be used as long as it can be assimilated by microorganisms. For example, sugars such as glucose, fructose, molasses, trehalose, lactose, sucrose, mannitol, sorbitol, partially decomposed starch, citric acid, succinic acid, etc. An organic acid such as or a salt thereof can also be used. The concentration of these carbon sources in the medium is appropriately selected depending on the type of carbon source. For example, the glucose concentration in the culture solution is desirably 40 w / v% or less, and preferably 10 w / v% or less from the growth and proliferation of microorganisms. Examples of the nitrogen source include inorganic nitrogen compounds such as ammonium salts and nitrates, and organic nitrogen-containing materials such as urea, corn steep liquor, casein, peptone, yeast extract and meat extract. Moreover, as an inorganic component, calcium salt, magnesium salt, potassium salt, sodium salt, phosphate, manganese salt, zinc salt, iron salt, copper salt, molybdenum salt, cobalt salt etc. are used suitably, for example.

  The culture is suitable for the temperature at which microorganisms grow and produce the enzyme of the present invention. Usually, the temperature is about 4 to 80 ° C., preferably about 20 to 75 ° C., pH is about 5 to 9, and preferably about pH 6 to 6. It is performed aerobically under the conditions selected from 8.5. The culture time may be a time during which microorganisms can grow, and is preferably about 10 to 100 hours. The dissolved oxygen concentration in the culture solution is not particularly limited, but usually about 0.5 to 20 ppm is preferable. For this purpose, means such as adjusting the aeration amount, stirring, using oxygen, and increasing the pressure in the culture tank are employed. The culture method may be either batch culture or continuous culture.

  In this way, after culturing the microorganism, the enzyme of the present invention is recovered from the obtained culture. This enzyme activity is observed in both the extracellular culture medium and the bacterial body of the culture, and the extracellular culture medium and the bacterial body may be recovered as a crude enzyme, and the whole culture is used as a crude enzyme. You can also. A normal solid-liquid separation means is employed for separation of the extracellular culture solution and the bacterial cells. For example, a means for centrifuging the culture itself as it is, a means for adding a filter aid to the culture or pre-coating for filtration separation, a means for membrane filtration separation using a flat membrane, a hollow fiber membrane, etc. are applied. Can do. The extracellular culture solution can be used as a crude enzyme solution as it is, but it is preferably concentrated by usual means. For example, an ammonium sulfate salting-out method, an acetone and alcohol precipitation method, a membrane concentration method using a flat membrane, a hollow fiber membrane, or the like is employed.

  The intracellular enzyme can be extracted from the bacterial body using a normal means and used as a crude enzyme solution. For example, an enzyme can be extracted from the cells by ultrasonic crushing, glass beads and alumina mechanical crushing, French press crushing, etc., and a clear crude enzyme solution can be obtained by centrifugation or membrane filtration. .

  Furthermore, the extracellular culture solution and the concentrate or the bacterial cell extract can also be immobilized by ordinary means. For example, a binding method to an ion exchanger, a covalent bond / adsorption method with a resin and a membrane, a comprehensive method using a polymer substance, and the like are employed. Moreover, although the microbial cell isolate | separated from the culture can be used as a crude enzyme as it is, you may use as an immobilized microbial cell. As an example, this is mixed with sodium alginate and dropped into a calcium chloride solution to form a gel. The granulated cells may be used as an immobilized enzyme that is further treated with polyethyleneimine or glutaraldehyde.

  The crude enzyme may be used as it is, but can also be purified by ordinary means. As an example, after dialysis of a crude enzyme preparation obtained by concentrating a cell disruption extract from ammonium sulfate, anion exchange column chromatography using DEAE-Toyopearl resin, followed by a hydrophobic column using butyl Toyopearl resin A single enzyme can be obtained electrophoretically by combining chromatography, anion exchange column chromatography using mono Q HR5 / 5 resin, gel filtration column chromatography using Toyopearl HW-55 resin, etc. .

The maltose / trehalose converting enzyme thus obtained has the following physicochemical properties.
(1) Action maltose is converted to trehalose and trehalose is converted to maltose.
(2) Molecular weight About 57,000 to 120,000 daltons as determined by SDS-gel electrophoresis.
(3) pI of about 3.8 to 5.1 by electrophoresis containing isoelectric point ampholine.
(4) Activity inhibition Inhibited with 1 mM Cu ⁺⁺ , Hg ⁺⁺ or 50 mM Tris-HCl buffer.
(5) An enzyme produced by the source microorganism.

The following is a specific example based on the difference in the source microorganism.
<Pantherobacter species R48-derived maltose trehalose converting enzyme>
(1) Action maltose is converted to trehalose and trehalose is converted to maltose. About 1 mole of trehalose or maltose is produced from 1 mole of maltose or trehalose, respectively.
(2) Molecular weight It is about 57,000-67,000 daltons by SDS-gel electrophoresis.
(3) An isoelectric point ampholine-containing electrophoresis method with a pI of about 4.1 to 5.1.
(4) Activity inhibition Inhibited with 1 mM Cu ⁺⁺ , Hg ⁺⁺ or 50 mM Tris-HCl buffer.
(5) Optimal temperature: pH 7.0, reaction at 60 minutes, around 20 ° C.
(6) Optimum pH
About 7.0 to 8.0 after reaction at 25 ° C. for 60 minutes.
(7) Temperature stability Stable up to around 30 ° C at pH 7.0, held for 60 minutes.
(8) pH stability
About 6.0 to 9.0 after holding at 20 ° C. for 60 minutes.

<Maltose trehalose converting enzyme derived from Pseudomonas putida H262>
(1) Action maltose is converted to trehalose and trehalose is converted to maltose. About 1 mole of trehalose or maltose is produced from 1 mole of maltose or trehalose, respectively.
(2) Molecular weight About 110,000 to 120,000 daltons by SDS-gel electrophoresis.
(3) An isoelectric point ampholine-containing electrophoresis method with a pI of about 4.1 to 5.1.
(4) Activity inhibition Inhibited with 1 mM Cu ⁺⁺ , Hg ⁺⁺ or 50 mM Tris-HCl buffer.
(5) Optimal temperature: pH 7.0, reaction at 60 minutes, around 37 ° C.
(6) Optimum pH
About 7.3 to 8.3 after reaction at 35 ° C. for 60 minutes.
(7) Temperature stability Stable up to around 40 ° C at pH 7.0, held for 60 minutes.
(8) pH stability: about 6.0 to 9.5 at 35 ° C. for 60 minutes.

<Maltose trehalose converting enzyme derived from Thermus aquaticus ATCC 33923>
(1) Action maltose is converted to trehalose and trehalose is converted to maltose. About 1 mole of trehalose or maltose is produced from 1 mole of maltose or trehalose, respectively.
(2) Molecular weight About 100,000 to 110,000 daltons by SDS-gel electrophoresis.
(3) pI of about 3.8 to 4.8 by electrophoresis containing isoelectric point ampholine.
(4) Activity inhibition Inhibited with 1 mM Cu ⁺⁺ , Hg ⁺⁺ or 50 mM Tris-HCl buffer.
(5) Optimal temperature: pH 7.0, reaction at 60 minutes, around 65 ° C.
(6) Optimum pH
About 6.0 to 6.7 after 60 minutes of reaction at 60 ° C.
(7) Temperature stability Stable up to about 80 ° C. at pH 7.0, held for 60 minutes.
(8) pH stability About 5.5 to 9.5 at 60 ° C. for 60 minutes.

  The activity of the maltose / trehalose converting enzyme of the present invention is measured as follows. 1 ml of enzyme solution is added to 1 ml of maltose 20 w / v% (10 mM phosphate buffer, pH 7.0) as a substrate, the reaction temperature is 25 ° C., 35 ° C. or 60 ° C., and the reaction is performed for 60 minutes. The reaction is stopped by heating for 10 minutes. This reaction solution was precisely diluted 11-fold with 50 mM phosphate buffer pH 7.5, and 0.1 ml of a trehalase-containing solution (1 unit / ml) was added to 0.4 ml of the diluted solution at 45 ° C. and 120 ° C. After incubating for minutes, the amount of glucose in the reaction solution is quantified by the glucose oxidase method. As a control, the same measurement is performed using an enzyme solution and trehalase previously inactivated by heating at 100 ° C. for 10 minutes. Using the above measurement method, the amount of trehalose produced by maltose / trehalose converting enzyme is determined from the increasing amount of glucose. One unit of activity is defined as the amount of enzyme that produces 1 μmole of trehalose per minute.

  The reaction temperature is 25 ° C. when the maltose / trehalose converting enzyme is derived from a microorganism belonging to the genus Pymelobacter, 35 ° C. when it is derived from a microorganism belonging to the genus Pseudomonas, and 60 ° C. when derived from a microorganism belonging to the genus Thermus. It was.

  Since the maltose / trehalose converting enzyme of the present invention can convert maltose to trehalose and convert trehalose to maltose, the substrate may be selected according to the purpose. For the purpose of producing trehalose, maltose may be used as a substrate.

  Maltose may be any maltose as long as the maltose / trehalose converting enzyme of the present invention acts to produce trehalose. In general, a maltose-rich material having a purity as high as possible, desirably a purity of 70% or more is used. . Commercially available maltose can be advantageously used, and maltose prepared by saccharifying starch according to a conventional method can be advantageously used.

  As a method for preparing maltose from starch, for example, maltose produced by reacting β-amylase with gelatinized or liquefied starch disclosed in JP-B-56-11437 and JP-B-56-17078 is disclosed. A method for separating maltose from a polymer dextrin and collecting a high content of maltose, or, for example, β-amylase in gelatinized or liquefied starch disclosed in Japanese Patent Publication No. 47-13089 and Japanese Patent Publication No. 54-3938 In addition, there is a method in which starch debranching enzymes such as isoamylase and pullulanase are allowed to act to collect high maltose content.

  Furthermore, for example, Japanese Patent Publication No. 56-28153, Japanese Patent Publication No. 57-3356, Japanese Patent Publication No. 56-28154, etc., in the contaminated saccharides such as maltotriose contained in the high maltose content obtained by these methods. Increase the maltose content by the action of the disclosed enzyme, or further, for example, by column chromatography using a salt type strongly acidic cation exchange resin disclosed in JP-A-58-23799 It is also advantageous to further increase the maltose content by methods such as removing water.

  The substrate concentration of the present invention is not particularly limited. For example, even when maltose is used as a substrate solution at 0.1% or 50%, the reaction of this enzyme proceeds to produce trehalose. Further, it may be a high concentration solution containing a substrate that cannot be completely dissolved in the substrate solution. The reaction temperature may be a temperature at which the enzyme is not inactivated, that is, up to about 80 ° C., but a range of about 0 to 70 ° C. is preferably used. The reaction pH is usually adjusted to a range of about 5.5 to 9.0, but is preferably adjusted to a range of pH about 6.0 to 8.5. The reaction time may be appropriately selected depending on the progress of the enzyme reaction, and is usually about 0.1 to 100 hours at an enzyme usage of about 0.1 to 100 units per gram of substrate solid matter.

  The reaction solution thus obtained was found to have a high conversion rate from the substrate maltose to trehalose, reaching a maximum of about 70 to 85%.

  The reaction solution may be filtered, centrifuged, etc. to remove insoluble matters, decolorized with activated carbon, desalted with H-type and OH-type ion exchange resins, concentrated, and made into a syrup-like product. It is optional to dry it into a powdered product or even into a crystalline product.

  If necessary, further advanced purification is optional. For example, high-purity trehalose can be purified by methods such as fractionation by ion exchange column chromatography, fractionation by activated carbon column chromatography, fractionation by silica gel column chromatography, and decomposition and removal of reducing sugars by alkali treatment. It is also easy to obtain a product. It is also possible to advantageously carry out a conversion reaction to trehalose by using maltose obtained by column chromatography or the like again as a substrate for the maltose trehalose converting enzyme of the present invention.

  The trehalose-containing saccharide of the present invention thus obtained is hydrolyzed with glucoamylase, α-glucosidase, etc., if necessary, or a starch partial decomposition product is added, and cyclomaltodextrin / glucanotransferase or glucosyltransferase is added. It is possible to adjust the sweetness, reducing power, viscosity, etc. by sugar transfer, etc., or to decompose and remove reducing sugars by alkali treatment, or to ferment and remove reducing sugars by yeast Furthermore, it is optional to perform further processing such as hydrogenation to make the reducing sugar a sugar alcohol so that the reducing power disappears. This can be purified by the above-described purification method, such as ion exchange column chromatography, and the fraction containing trehalose is collected and purified and concentrated to obtain a syrup-like product. Thus, supersaturation and crystallization to obtain trehalose hydrated crystals or anhydrous crystalline trehalose can be advantageously carried out.

  As ion exchange column chromatography, contaminating saccharides are removed by column chromatography using a salt type strongly acidic cation exchange resin disclosed in JP-A-58-23799 and JP-A-58-72598. Thus, a method of collecting a fraction containing high trehalose can be advantageously implemented. At this time, it is optional to adopt any of a fixed floor method, a moving floor method, and a simulated moving floor method.

  In order to produce a trehalose hydrous crystal, for example, a trehalose-containing liquid having a purity of 60% or more and a concentration of 65 to 90% is taken in an auxiliary crystal machine, and if necessary, in the presence of about 0.1 to 20% seed crystal, A mass kit containing trehalose hydrous crystals is produced by slow cooling with stirring at a temperature of 95 ° C. or lower, preferably 10 to 90 ° C. It is also possible to advantageously employ a continuous crystallization method in which crystallization is performed while concentrating under reduced pressure. As a method for producing a trehalose hydrated crystal or a honey-containing crystal containing the same from a mass kit, a known method such as a honey mash method, a block pulverization method, a fluid granulation method, or a spray drying method may be employed.

  In the case of the honey method, the mass kit is usually applied to a basket-type centrifuge to separate the trehalose hydrated crystals from the nectar (mother liquor), and if necessary, the crystals can be easily washed by spraying a small amount of cold water. And is suitable for producing higher-purity water-containing trehalose crystals.

  In the case of the spray drying method, usually, a mass kit having a concentration of about 60 to 85% and a crystallization rate of about 20 to 60% is sprayed from a nozzle with a high-pressure pump, and the temperature at which the crystal powder does not dissolve, for example, about 60 to 100 ° C. And then aged for about 1 to 20 hours with hot air of about 30 to 60 ° C., non-hygroscopic or hardly hygroscopic honey-containing crystals can be easily produced.

  In the case of the block pulverization method, usually, a mass kit having a water content of about 10 to 25% and a crystallization rate of about 10 to 60% is allowed to stand for several hours to 3 days to crystallize and solidify the whole into a block shape. If this is pulverized and dried by a method such as grinding or cutting, a non-hygroscopic or hardly hygroscopic honey-containing crystal can be easily produced.

  In order to produce anhydrous crystalline trehalose, it is also possible to dry and transform the water-containing trehalose crystal, but in general, a high concentration trehalose-containing solution having a water content of less than 10% is used as an auxiliary crystal machine, and seed crystals are obtained. A mass kit containing anhydrous crystalline trehalose is produced while stirring in the range of 50 to 160 ° C., preferably 80 to 140 ° C. in the presence of the mixture, and this is subjected to relatively high temperature drying conditions, for example, a block grinding method, fluid granulation It is produced by crystallization and pulverization by a method such as a method, a spray drying method, and an extrusion granulation method.

  The trehalose of the present invention thus produced has no reducing power, is stable, and can be mixed and processed with other materials, particularly amino acids, oligopeptides, proteins and other substances containing amino groups or amino groups. It is less likely to turn brown, generate off-flavors, or damage other mixed materials. Moreover, it has a good quality and elegant sweetness. Furthermore, since trehalose is easily decomposed into glucose by trehalase in the body, when it is taken orally, it is easily digested and absorbed and used as a calorie source. In addition, it can be used as a sweetener that is not easily fermented by caries-inducing bacteria or the like and hardly causes caries.

  Moreover, since it is a stable sweetener, in the case of a crystal product, it can be advantageously used as a sugar coating for tablets in combination with a binder such as pullulan, hydroxyethyl starch, polyvinylpyrrolidone and the like. It also has properties such as osmotic pressure controllability, shaping, irradiating properties, moisture retention, viscosity, anti-crystallization of other sugars, difficult fermentation, and starch anti-aging properties.

  Therefore, the trehalose of the present invention and the saccharide containing the same are used as sweeteners, taste improvers, quality improvers, stabilizers, excipients, etc. as various compositions such as foods, foods, foods, cosmetics, and pharmaceuticals. Can be advantageously used.

  The trehalose of the present invention and a saccharide containing the same can be used as a seasoning for sweetening as it is. If necessary, for example, flour, glucose, maltose, sucrose, isomerized sugar, honey, maple sugar, sorbitol, maltitol, lactitol, dihydrochalcone, stevioside, α-glycosyl stevioside, rebaudioside, glycyrrhizin, L-aspartyl-L -May be used in admixture with an appropriate amount of one or more other sweeteners such as phenylalanine methyl ester, saccharin, glycine, alanine, etc., and if necessary, such as dextrin, starch, lactose, etc. It can also be used as a mixture with other extenders.

  In addition, the trehalose of the present invention and the saccharide powder or crystalline product containing the same may be used as it is or mixed with a bulking agent, excipient, binder, etc. It is also optional to use it in various shapes such as plate, cube, and tablet.

  In addition, the sweetness of trehalose of the present invention and carbohydrates containing the same well matches various substances having other tastes such as acidity, salt to taste, astringency, umami, and bitterness, and has high acid resistance and heat resistance. Therefore, it can be advantageously used for sweetening and taste improvement of general foods and drinks and quality improvement.

  For example, soy sauce, powdered soy sauce, miso, powdered miso, moromi, hashio, sprinkle, mayonnaise, dressing, vinegar, three cups of vinegar, powdered sushi vinegar, Chinese element, tentsuyu, noodle soup, sauce, ketchup, takuanzuke, Chinese cabbage It can be advantageously used as various seasonings such as nori, yakiniku sauce, curry roux, stew, soup, dashi, compound seasoning, mirin, new mirin, table sugar, coffee sugar.

  In addition, for example, Japanese rice cakes such as rice crackers, hail, rice cakes, rice cakes, manju, oirou, chanterelles, sheep mushrooms, water sheep rice cakes, brocade balls, jelly, castella, rice cakes, bread, biscuits, crackers, cookies, pie , Pudding, butter cream, custard cream, cream puff, waffle, sponge cake, donut, chocolate, chewing gum, caramel, candy and other Western confectionery, ice cream, sherbet and other ice confectionery, fruit syrup pickled, syrup such as ice honey, flower Pastes such as paste, peanut paste, fruit paste, spread, fruit such as jam, marmalade, syrup pickles, sugar cane, processed foods of vegetables, pickles such as Fukujin pickles, bedara pickles, thousand pieces pickles, pickled pickles, hams, sausages, etc. Livestock meat products, fish Manufactured with fish products such as ham, fish sausage, kamaboko, chikuwa, tempura, various delicacies such as sea urchin, squid salted salt, vinegared konbu, sakisume, fugurin dried, seaweed, wild vegetables, sea urchin, small fish, shellfish, etc. Stewed food such as rutsuda stew, boiled beans, potato salad, konbu roll, dairy products such as milk drinks, yogurt, cheese, fish meat, livestock meat, fruits, bottled cans, canned goods, sake, synthetic liquor, liqueur, Western liquor, etc. Alcoholic beverages, tea, coffee, cocoa, juice, carbonated beverages, lactic acid beverages, lactic acid bacteria beverages and other soft drinks, pudding mixes, hot cake mixes, instant foods such as instant soups, baby foods, therapeutic foods, It can be advantageously used to improve taste for sweetening various foods such as drinks, and to improve quality.

  Moreover, it can also be used for the purpose of improving the palatability of feed, feed, etc. for domestic animals, poultry, and other domestic animals such as bees, cormorants and fish. In addition, tobacco, toothpaste, lipstick, lip balm, oral solution, tablets, troches, liver oil drop, mouth freshener, mouth fragrance, mouthwash, etc. It can be advantageously used as a sweetener for various compositions, as a taste improver, a taste corrector, and as a quality improver.

  As the quality improver and stabilizer, it can be advantageously applied to various physiologically active substances that easily lose active ingredients, activities, etc., health foods and pharmaceuticals containing the same. For example, interferon-α, interferon-β, interferon-γ, tsumore necrosis factor-α, tsumoa necrosis factor-β, macrophage migration inhibitory factor, colony stimulating factor, transfer factor, interleukin-2 and other lymphokine-containing liquids Such as insulin, growth hormone, prolactin, erythropoietin, egg cell stimulating hormone, placental hormone, etc., BCG vaccine, Japanese encephalitis vaccine, measles vaccine, polio vaccine, seedling, tetanus toxoid, hub antitoxin, human immunoglobulin, etc. Liquid containing biologics, liquid containing antibiotics such as penicillin, erythromycin, chloramphenicol, tetracycline, streptomycin, kanamycin sulfate, thiamine, riboflavin , L-ascorbic acid, liver oil, carotenoid, ergosterol, tocopherol and other vitamin-containing liquids, lipase, elastase, urokinase, protease, β-amylase, isoamylase, glucanase, lactase and other enzyme-containing liquids, ginseng extract, suppon extract , Chlorella extract, Aloe extract, propolis extract, and other bioactive substances such as viruses, lactic acid bacteria, yeast, and other biologically active substances such as royal jelly are stable and high-quality health without losing their active ingredients and activities. Foods and pharmaceuticals can be easily manufactured.

  The method of adding trehalose and a sugar containing the same to the various compositions as described above may be included in the process until the product is completed, for example, mixing, dissolving, melting, dipping, infiltration, spraying. A known method such as coating, coating, spraying, pouring, crystallization, and solidification is appropriately selected. The amount is usually 0.1% or more, preferably 1% or more. Next, the present invention will be described more specifically by experiments.

<Experiment 1: Production of enzyme>
Glucose 2.0 w / v%, polypeptone 0.5 w / v%, yeast extract 0.1 w / v%, dipotassium phosphate 0.1 w / v%, monosodium phosphate 0.06 w / v%, magnesium sulfate Liquid medium consisting of heptahydrate 0.05 w / v%, calcium carbonate 0.5 w / v%, and water is placed in 100 ml portions in a 500 ml Erlenmeyer flask, sterilized in an autoclave at 115 ° C. for 30 minutes, cooled, Pimelobacter sp. R48 (FERM BP-4315) was inoculated and cultured under rotation at 27 ° C. and 200 rpm for 24 hours as a seed culture.

  About 20 liters of medium having the same composition as in seed culture is placed in a 30 liter fermenter, heat-sterilized and cooled to a temperature of 27 ° C., then inoculated with 1 v / v% of the seed culture solution, temperature 27 ° C., pH 6 The culture was aerated and stirred for about 40 hours while maintaining the temperature at 0.0 to 8.0.

  The maltose / trehalose converting enzyme activity of the culture broth was 0.55 units / ml. A part of the culture solution is taken and centrifuged to separate the cells and the culture supernatant, and the cells are suspended in 50 mM phosphate buffer (pH 7.0), and the same solution as the original culture solution. Then, when the maltose / trehalose converting enzyme activity of the cell suspension and the culture supernatant was measured, the cell suspension had an activity of 0.5 units / ml in the culture supernatant. Activity of 0.05 units / ml was observed. The activity of this enzyme was measured at a reaction temperature of 25 ° C.

<Experiment 2: Purification of enzyme>
The culture solution obtained in Experiment 1 was centrifuged to collect cells having a wet mass of about 0.5 kg and suspended in 10 mM phosphate buffer (pH 7.0). About 5 l of this bacterial cell suspension was applied to Vibrogen Cell Mill (Edmond Buehler), the bacterial cells were crushed, and the crushed solution was centrifuged (15,000 G, 30 minutes) to obtain about 4.5 l. A supernatant was obtained. The supernatant was dissolved by adding ammonium sulfate to a saturation degree of 0.3, allowed to stand at 4 ° C. for 4 hours, and then centrifuged to collect the supernatant.

  Further, ammonium sulfate was added to the solution so that the degree of saturation was 0.8, and the mixture was allowed to stand at 4 ° C. overnight, and then centrifuged to collect ammonium sulfate salted-out.

  The obtained ammonium sulfate salted-out product was dissolved in 10 mM phosphate buffer (pH 7.0), dialyzed against the same buffer for 24 hours, and centrifuged to remove insolubles. The dialysate (400 ml) was divided into two portions and subjected to ion exchange column chromatography (gel amount: 300 ml) using DEAE-Toyopearl gel (manufactured by Tosoh Corporation).

  The maltose / trehalose converting enzyme of the present invention was adsorbed on DEAE-Toyopearl gel and eluted from the column with the same buffer containing sodium chloride. The eluted enzyme active fraction is collected, dialyzed against the same buffer containing 1M ammonium sulfate, the dialyzed solution is centrifuged to remove insoluble matters, and then butyl Toyopearl 650 gel (manufactured by Tosoh Corporation) ) Was used (hydrophobic column chromatography (gel amount: 300 ml)). The adsorbed maltose / trehalose converting enzyme was eluted from the column with an ammonium sulfate 1M to 0M linear gradient, and the enzyme activity fraction was recovered.

  Subsequently, ion-exchange chromatography (gel amount: 10 ml) using a mono Q HR5 / 5 column (Pharmacia LCB, Sweden) was performed, and the eluted enzyme activity fraction was recovered. Table 1 shows the amount of enzyme activity, specific activity, and yield in each step of purification.

  When the purity of the enzyme preparation was assayed by gel electrophoresis containing 7.5% concentration polyacrylamide, the purified enzyme preparation was a single and high purity preparation.

<Experiment 3: Properties of enzyme>
The purified maltose / trehalose converting enzyme preparation obtained by the method of Experiment 2 was subjected to SDS-polyacrylamide gel electrophoresis (gel concentration: 10%) and compared with a molecular weight marker (manufactured by Nippon Bio-Rad Laboratories Co., Ltd.). The molecular weight of the enzyme was measured and found to be about 57,000 to 67,000 daltons.

  The purified maltose / trehalose converting enzyme preparation was subjected to isoelectric polyacrylamide gel electrophoresis containing 2% ampholine (manufactured by Pharmacia El Kavy, Sweden), and after electrophoresis, the pH of the protein band and gel was measured. When the isoelectric point of the enzyme was determined, the isoelectric point was approximately 4.1 to 5.1.

The effects of temperature and pH on the enzyme activity were examined according to the activity measurement method. The results are shown in FIG. 1 (effect of temperature) and FIG. 2 (effect of pH). The optimum temperature of the enzyme was about 7.0 ° C. for reaction at pH 7.0 for 60 minutes, and the optimum pH was about 7.0 to 8.0 for reaction at 25 ° C. for 60 minutes. The temperature stability of the enzyme was determined by measuring the remaining enzyme activity after holding the enzyme solution (50 mM phosphate buffer, pH 7.0) at each temperature for 60 minutes, cooling with water. The pH stability was determined by maintaining the enzyme in 50 mM buffer at each pH at 20 ° C. for 60 minutes, adjusting the pH to 7.0, and measuring the remaining enzyme activity. The respective results are shown in FIG. 3 (temperature stability) and FIG. 4 (pH stability). The temperature stability of this enzyme was up to about 30 ° C., and the pH stability was about 6.0 to 9.0. The enzyme activity was inhibited by 1 mM Cu ⁺⁺ , Hg ⁺⁺ or 50 mM Tris-HCl buffer.

<Experiment 4: Effect on various carbohydrates>
Various sugars were used to test whether they could become substrates. Glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, soluble starch, amylose (average polymerization degree 18), trehalose, neotrehalose, gentiobiose, cordobiose, isomaltose, cellobiose, multi A solution of tall, sucrose, maltulose, tulanose, palatinose, trehalulose, or lactose, and glucose equivalent to α-glucose-1-phosphate, or glucose equivalent to β-glucose-1-phosphate A solution containing was prepared.

  To these solutions, 2 units of purified maltose / trehalose converting enzyme obtained by the method of Experiment 2 were added per gram of substrate solids, and the substrate concentration was adjusted to 5 w / v%. It was allowed to act at 0 for 24 hours. The reaction solution before and after the enzyme reaction was subjected to thin layer chromatography (hereinafter abbreviated as TLC) using Kieselgel 60 (Merck, aluminum plate, 20 × 20 cm) to confirm the presence or absence of enzyme action on each carbohydrate. did. TLC was developed once at room temperature using 1-butanol: pyridine: water = 6: 4: 1 (volume ratio) as a developing solvent. Coloring was performed by spraying a 20% sulfuric acid-methanol solution and heating at 110 ° C. for 10 minutes. The results are shown in Table 2.

  As is clear from the results in Table 2, the enzyme of the present invention acts only on maltose and trehalose among the various carbohydrates tested, and other carbohydrates, particularly α-glucose monophosphate and glucose. It is a novel enzyme, unlike the conventionally known phosphorylases such as maltose phosphorylase and trehalose phosphorylase, because it does not act on the system containing β-glucose 1-phosphate and glucose. There was found.

<Experiment 5: Product from maltose or trehalose>
Two units of the purified maltose / trehalose converting enzyme obtained by the method of Experiment 2 was added to a final 5% maltose aqueous solution per gram of substrate solids and allowed to act at 20 ° C. and pH 7.0 for 24 hours. The sugar composition of the enzyme reaction solution was analyzed by gas chromatography (hereinafter abbreviated as GLC). A part of the enzyme reaction solution was dried, dissolved in pyridine and then trimethylsilylated, and used as an analytical sample. The gas chromatograph is GC-16A (manufactured by Shimadzu Corporation), the column is a stainless steel column (3 mmφ × 2 m) packed with 2% silicon OV-17 / chromosolve W (manufactured by GL Sciences Inc.), and the carrier gas is Nitrogen gas was analyzed at a flow rate of 40 ml / min, and the column oven temperature was 160 ° C. to 320 ° C. at a heating rate of 7.5 ° C./min. For detection, a flame ion detector was used. The results are shown in Table 3.

  As is clear from the results in Table 3, it was found that a large amount of reaction product X was formed, and the retention time was consistent with that of commercial trehalose. In order to identify the reaction product X, the following confirmation test was conducted. That is, the enzyme reaction solution using maltose as a substrate was diluted with 20 mM acetate buffer, pH 4.5 so as to have a sugar concentration of 2%, and 0.5 ml of glucoamylase (Seikagaku Corporation) 0.1 The unit was added and reacted at 40 ° C. for 20 hours.

  Similarly, the enzyme reaction solution was diluted with 20 mM phosphate buffer, pH 7.0 so as to have a sugar concentration of 2%, 0.5 unit of trehalase was added to 0.5 ml, and the mixture was reacted at 40 ° C. for 20 hours. Analyzing and comparing the enzyme reaction solution with maltose as a substrate, its glucoamylase treatment solution and trehalase treatment solution by GLC, maltose is completely decomposed into glucose by the glucoamylase treatment, and the reaction product X remains without being decomposed. Was.

  On the other hand, maltose remained due to the trehalase treatment, but the reaction product X was completely decomposed into glucose. Considering the reaction characteristics of glucoamylase and trehalase, the oligosaccharide from maltose produced by the novel enzyme of the present invention is judged to be trehalose.

  Furthermore, when purified enzyme was allowed to act on trehalose as a substrate under the same conditions as in maltose and the reaction solution was similarly subjected to GLC analysis, it was found that maltose was produced from trehalose by the enzyme of the present invention. The above GLC analysis results are summarized in Table 4.

  As is clear from the results in Table 4, the enzyme of the present invention converts maltose to trehalose and converts trehalose to maltose. The equilibrium point is shifted to the trehalose side, and it has been found that the conversion rate from maltose to trehalose is high and is about 70% or more.

<Experiment 6: Effect of maltose concentration on trehalose production>
Purified maltose / trehalose converting enzyme obtained by the method of Experiment 2 at a maltose concentration of 2.5%, 5%, 10%, 20% or 40%, temperature 20 ° C., pH 7.0, 2 units per maltose gram In addition, the reaction solution was collected over time, and heated at 100 ° C. for 10 minutes to inactivate the enzyme.

  The total amount of sugar in this reaction solution was quantified in terms of glucose by the anthrone sulfate method and the amount of reducing sugar by glucose conversion by the Somogy-Nelson method, and the ratio of the amount of reducing sugar to the total amount of sugar was calculated as reducing power.

  Further, this reaction solution is diluted to a sugar concentration of about 1%, deproteinized with a small amount of ultrafilter Mulcat II LGC (manufactured by Nihon Millipore Limited), and then high performance liquid chromatography (hereinafter abbreviated as HPLC). ) To analyze the sugar composition. The HPLC apparatus is a CCPD system (manufactured by Tosoh Corporation), the analytical column is YMC-pack PA-03 (4.6 mmφ × 250 mm, YMC Corporation), and the eluent is acetonitrile: water = 78: 22 (volume ratio). Was detected with a differential refractometer at a flow rate of 1.2 ml / min. The results are shown in Table 5.

  As is clear from the results in Table 5, the conversion reaction from maltose to trehalose progressed well regardless of the maltose concentration of the substrate, and the conversion to trehalose was about 80%.

<Experiment 7: Effect of temperature on trehalose production>
At a maltose concentration of 20%, the pH is 7.0, and 2 units of the purified maltose / trehalose converting enzyme obtained by the method of Experiment 2 is added per maltose gram, and the temperature is 5 ° C, 10 ° C, 15 ° C, 20 ° C or 25 ° C. The reaction was collected over time and heated at 100 ° C. for 10 minutes to inactivate the enzyme. The sugar composition of this enzyme reaction solution was analyzed by HPLC in the same manner as in Experiment 6. Table 6 shows the trehalose content at each temperature and time.

  As is clear from the results in Table 6, the higher the reaction temperature, the higher the trehalose production rate, but the conversion reaction from maltose to trehalose progressed well even at a temperature of 5 ° C, and the conversion to trehalose was about 82%. did.

<Experiment 8: Preparation of trehalose from maltose>
Maltose (manufactured by Hayashibara Biochemical Laboratories Co., Ltd.) in 10 parts by mass of water is dissolved in 40 parts by mass of water, and the purified maltose / trehalose converting enzyme obtained by the method of Experiment 2 is used at a temperature of 15 ° C. and pH 7.0. Two units were added and reacted for 48 hours, and then heated at 100 ° C. for 10 minutes to inactivate the enzyme. This solution contained about 74% trehalose per solid. This solution is decolorized with activated carbon, purified by desalting with an ion exchange resin (H type and OH type), concentrated to a concentration of about 78%, and trehalose hydrous crystals as seed crystals, 0.1% per solid matter. When it was added and allowed to stand at room temperature overnight, crystals were precipitated. The obtained mass kit was honeyed and a small amount of water was sprayed on the crystal to wash the crystal, thereby obtaining about 3.0 parts by mass of extremely high-purity trehalose-containing crystal having a purity of 99.8%.

<Experiment 9: Production of enzyme>
Glucose 2.0 w / v%, ammonium sulfate 1.0 w / v%, dipotassium phosphate 0.1 w / v%, monosodium phosphate 0.06 w / v%, magnesium sulfate 0.05 w / v%, calcium carbonate 0 Liquid medium consisting of 3 w / v% and water was put into a 500 ml Erlenmeyer flask 100 ml each, sterilized by autoclaving at 115 ° C. for 30 minutes, cooled and inoculated with Pseudomonas putida H262 (FERM BP-4579). The seed culture was obtained by culturing for 24 hours at 27 ° C. and 200 rpm with shaking.

  About 20 liters of medium having the same composition as in seed culture is placed in a 30 liter fermenter, heat-sterilized and cooled to a temperature of 27 ° C., then inoculated with 1 v / v% of the seed culture solution, temperature 27 ° C., pH 6 The culture was aerated and agitated for about 20 hours while maintaining at .5 to 8.0.

  The maltose / trehalose converting enzyme activity of the culture broth was 0.12 units / ml. A part of the culture solution is taken and centrifuged to separate the cells and the culture supernatant, and the cells are suspended in 50 mM phosphate buffer (pH 7.0), and the same solution as the original culture solution. When the maltose / trehalose converting enzyme activity of the cell suspension and the culture supernatant was measured, the cell suspension had an activity of 0.11 unit / ml in the culture supernatant. Activity of 0.01 units / ml was observed. The activity of this enzyme was measured at a reaction temperature of 35 ° C.

<Experiment 10: Purification of enzyme>
The culture solution obtained in Experiment 9 was centrifuged to collect cells having a wet mass of about 0.45 kg and suspended in 10 mM phosphate buffer (pH 7.0). About 2 liters of this bacterial cell suspension is treated with an ultrahigh pressure bacterial cell disruption device minilab (sold by Dainippon Pharmaceutical Co., Ltd.) to disrupt the bacterial cells, and this disrupted solution is centrifuged (15,000 G, 30 minutes). This gave about 1.7 l of supernatant. Ammonium sulfate was recovered by adding ammonium sulfate to the supernatant so as to have a saturation degree of 0.7, leaving it to stand at 4 ° C. overnight, and then centrifuging it.

  The obtained ammonium sulfate salted-out product was dissolved in 10 mM phosphate buffer (pH 7.0), dialyzed against the same buffer for 24 hours, and centrifuged to remove insolubles. The dialysate (400 ml) was divided into two portions and subjected to ion exchange column chromatography (gel amount: 300 ml) using DEAE-Toyopearl gel (manufactured by Tosoh Corporation).

  The maltose / trehalose converting enzyme of the present invention was adsorbed on DEAE-Toyopearl gel and eluted from the column with the same buffer containing sodium chloride. The eluted enzyme activity fraction was collected, dialyzed against the same buffer, and again subjected to DEAE-Toyopearl ion exchange column chromatography (gel amount: 80 ml). The adsorbed maltose / trehalose converting enzyme was eluted from the column with a linear gradient of 0.1 M to 0.3 M of sodium chloride, and the enzyme activity fraction was recovered.

  Subsequently, gel filtration chromatography (gel amount: 400 ml) using Toyopearl HW-55S (manufactured by Tosoh Corporation) was performed, and the eluted enzyme activity fraction was recovered. Table 7 shows the amount of enzyme activity, specific activity, and yield in each step of purification.

  When the purity of the enzyme preparation was assayed by gel electrophoresis containing 7.5 w / v% polyacrylamide in the purified enzyme preparation, the protein band was a single and high purity preparation.

<Experiment 11: Properties of enzyme>
A purified molecular weight marker obtained by the method of Experiment 10 was subjected to SDS-polyacrylamide gel electrophoresis (gel concentration 7.5 w / v%), and simultaneously migrated molecular weight marker (Nippon Bio-Rad Laboratories, Inc.) The molecular weight of this enzyme was measured in comparison with that of the manufactured product, and the molecular weight was about 110,000 to 120,000 daltons.

  The purified maltose / trehalose converting enzyme preparation was subjected to isoelectric focusing polyacrylamide gel electrophoresis containing 2 w / v% ampholine (manufactured by Pharmacia El Kavy, Sweden). After electrophoresis, the protein band and the pH of the gel were measured. As a result, the isoelectric point of the enzyme was found to be about 4.1 to 5.1.

The effects of temperature and pH on the enzyme activity were examined according to the activity measurement method. The results are shown in FIG. 5 (effect of temperature) and FIG. 6 (effect of pH). The optimum temperature of the enzyme was about 37 ° C. for a reaction of pH 7.0 for 60 minutes, and the optimum pH was about 7.3 to 8.3 for a reaction of 35 ° C. for 60 minutes. The temperature stability of the enzyme was determined by measuring the remaining enzyme activity after holding the enzyme solution (50 mM phosphate buffer, pH 7.0) at each temperature for 60 minutes, cooling with water. The pH stability was determined by maintaining the enzyme in a 50 mM buffer solution at each pH at 35 ° C. for 60 minutes, adjusting the pH to 7.0, and measuring the remaining enzyme activity. The respective results are shown in FIG. 7 (temperature stability) and FIG. 8 (pH stability). The temperature stability of this enzyme was up to around 40 ° C., and the pH stability was about 6.0 to 9.5. This enzyme activity was inhibited by 1 mM Cu ⁺⁺ , Hg ⁺⁺ or 50 mM Tris-HCl buffer.

<Experiment 12: Effect on various carbohydrates>
Except that the reaction temperature was 35 ° C., the purified enzyme of Pseudomonas putida H262 obtained in Experiment 10 was allowed to act on various carbohydrates in accordance with the method of Experiment 4 to test whether it could be a substrate. As a result, the Pseudomonas putida H262 enzyme acted only on maltose and trehalose to convert maltose to trehalose and trehalose converted to maltose, as did the enzyme Pimelobacter species R48. The equilibrium point is shifted to the trehalose side, and it has been found that the conversion rate from maltose to trehalose is high and is about 70%.

<Experiment 13: Effect of maltose concentration on trehalose production>
At a maltose concentration of 5%, 10%, 20% or 30%, at a temperature of 35 ° C. and a pH of 7.0, the purified maltose / trehalose converting enzyme obtained by the method of Experiment 10 was added and reacted for 2 units per maltose gram. The reaction solution was collected over time and heated at 100 ° C. for 10 minutes to deactivate the enzyme.

  Using this reaction solution, the reducing power and sugar composition were measured in the same manner as in Experiment 6. The results are shown in Table 8.

  As is apparent from the results in Table 8, about 70% of trehalose was produced regardless of the maltose concentration of the substrate.

<Experiment 14: Preparation of trehalose from maltose>
10 parts by weight of maltose (sales by Hayashibara Biochemical Laboratories, Inc.) is dissolved in 40 parts by weight of water, adjusted to a temperature of 35 ° C. and a pH of 7.0, and 2 units of the purified maltose / trehalose converting enzyme of the present invention is added per maltose gram. The reaction was carried out for 48 hours, and then the enzyme was inactivated by heating at 100 ° C. for 10 minutes. This solution contained about 69% trehalose per solid. This solution is decolorized with activated carbon, purified by desalting with an ion exchange resin (H type and OH type), concentrated to a concentration of about 78%, and trehalose hydrous crystals as seed crystals, 0.1% per solid matter. When it was added and allowed to stand at room temperature overnight, crystals were precipitated. The obtained mass kit was honeyed and the crystal was washed by spraying a small amount of water to obtain about 2.3 parts by mass of extremely high purity trehalose crystals having a purity of 99.7%.

<Experiment 15: Production of enzyme>
Polypeptone 0.5 w / v%, yeast extract 0.1 w / v%, sodium nitrate 0.07 w / v%, disodium phosphate 0.01 w / v%, magnesium sulfate 0.02 w / v%, calcium chloride 0. A liquid medium consisting of 01 w / v% and water was adjusted to pH 7.5, and then 100 ml each was placed in a 500 ml Erlenmeyer flask, sterilized at 120 ° C. for 20 minutes in an autoclave, cooled, and thermus aquaticus ATCC 33923 was added. The seeded culture was inoculated and cultured with shaking at 60 ° C. and 200 rpm for 24 hours.

  About 20 liters of each medium having the same composition as in the case of seed culture was added to two 30 liter fermenters, heat-sterilized and cooled to a temperature of 60 ° C., and then seeded with 1 v / v% of the seed culture solution. The culture was aerated and stirred for about 20 hours while maintaining the temperature at 6.5 ° C. and 8.0 to 8.0.

  The maltose / trehalose converting enzyme activity of the culture solution was 0.35 units / ml. A portion of the culture solution is taken and centrifuged to separate the cells and culture supernatant, and the cells are further suspended in 50 mM phosphate buffer (pH 7.0), and the same volume as the original culture solution. Then, when the maltose / trehalose converting enzyme activity of the cell suspension and the culture supernatant was measured, the cell suspension had an enzyme activity of 0.33 units / ml, and the culture supernatant Was found to have an enzyme activity of 0.02 units / ml. The activity of this enzyme was measured at a reaction temperature of 60 ° C.

<Experiment 16: Purification of enzyme>
The culture solution obtained in Experiment 15 was centrifuged to collect cells having a wet mass of about 0.28 kg and suspended in 10 mM phosphate buffer (pH 7.0). About 1.9 l of this bacterial cell suspension was treated with an ultrasonic crusher model US300 (manufactured by Nippon Seiki Seisakusho Co., Ltd.) to crush the bacterial cells. The crushing solution was centrifuged (15,000 G, 30 minutes) to obtain about 1.8 l of supernatant. Ammonium sulfate was added to the supernatant so as to have a saturation degree of 0.7, dissolved, and allowed to stand at 4 ° C. overnight, and then centrifuged to recover ammonium sulfate salted-out.

  The obtained ammonium sulfate salted-out product was dissolved in 10 mM phosphate buffer (pH 7.0), dialyzed against the same buffer for 24 hours, and centrifuged to remove insolubles. The dialysate (1560 ml) was subjected to ion exchange column chromatography (gel amount 530 ml) using DEAE-Toyopearl 650 gel (manufactured by Tosoh Corporation) in three portions.

  The maltose / trehalose converting enzyme of the present invention was adsorbed on DEAE-Toyopearl gel and eluted from the column with the same buffer containing sodium chloride. The eluted enzyme active fraction is collected, dialyzed against the same buffer containing 1M ammonium sulfate, and then subjected to hydrophobic column chromatography using butyltoyopearl 650 gel (manufactured by Tosoh Corporation) (gel amount: 380 ml). Went. The adsorbed maltose / trehalose converting enzyme was eluted from the column with an ammonium sulfate 1M to 0M linear gradient, and the enzyme activity fraction was recovered.

  Next, gel filtration chromatography (gel amount 380 ml) using Toyopearl HW-55S (manufactured by Tosoh Corporation) was performed, and the eluted enzyme activity fraction was recovered.

  Subsequently, ion exchange chromatography (gel amount: 1.0 ml) using a mono Q HR5 / 5 column (manufactured by Pharmacia El Kavy, Sweden) was performed and eluted with a linear gradient of 0.1 M to 0.35 M of sodium chloride. The enzyme activity fraction was collected. Table 9 shows the amount of enzyme activity, specific activity, and yield in each step of purification.

  When the purity of the enzyme preparation was assayed by gel electrophoresis containing 5% w / v concentration polyacrylamide, the protein band was a single and high purity preparation.

<Experiment 17: Properties of enzyme>
A purified molecular weight marker obtained by the method of Experiment 16 was subjected to SDS-polyacrylamide gel electrophoresis (gel concentration 7.5 w / v%), and simultaneously migrated molecular weight marker (Nippon Bio-Rad Laboratories, Inc.) And the molecular weight of the enzyme was measured to be about 100,000 to 110,000 daltons.

  The purified maltose / trehalose converting enzyme preparation is subjected to isoelectric focusing polyacrylamide gel electrophoresis containing 2% w / v ampoline (manufactured by Pharmacia El Kavy, Sweden). After electrophoresis, the protein band and gel pH are measured. Then, the isoelectric point of the enzyme was determined, and the isoelectric point was about 3.8 to 4.8.

The effects of temperature and pH on the enzyme activity were examined according to the activity measurement method. The results are shown in FIG. 9 (effect of temperature) and FIG. 10 (effect of pH). The optimal temperature of the enzyme was around 65 ° C. for a reaction of pH 7.0 for 60 minutes, and the optimal pH was 6.0 to 6.7 for a reaction of 60 ° C. for 60 minutes. The temperature stability of the enzyme was determined by measuring the remaining enzyme activity after holding the enzyme solution (containing 50 mM phosphate buffer, pH 7.0) at each temperature for 60 minutes, cooling with water. The pH stability was determined by maintaining the enzyme in a 50 mM buffer solution at each pH at 60 ° C. for 60 minutes, adjusting the pH to 7.0, and measuring the remaining enzyme activity. The respective results are shown in FIG. 11 (temperature stability) and FIG. 12 (pH stability). The temperature stability of this enzyme was up to about 80 ° C., and the pH stability was 5.5 to 9.5. This enzyme activity was inhibited by 1 mM Cu ⁺⁺ , Hg ⁺⁺ or 50 mM Tris-HCl buffer.

<Experiment 18: Effects on various carbohydrates>
Except that the reaction temperature was 50 ° C., the purified enzyme of Thermus aquaticus ATCC 33923 obtained in Experiment 16 was allowed to act on various carbohydrates according to the method of Experiment 4 to test whether it could be a substrate. As a result, the enzyme of Thermus aquaticus ATCC 33923 acted only on maltose and trehalose to convert maltose to trehalose and to convert trehalose to maltose, like the enzyme of Pimerobacter species R48 or Pseudomonas putida H262. The equilibrium point is shifted to the trehalose side, and it has been found that the conversion rate from maltose to trehalose is high and is 70% or more.

<Experiment 19: Effect of Maltose Concentration on Trehalose Production>
Purified maltose trehalose converting enzyme of Thermus aquaticus ATCC 33923 obtained by the method of Experiment 16 at a maltose concentration of 2.5%, 5%, 10%, 20% or 40% at a temperature of 60 ° C. and a pH of 6.5 The reaction solution was collected at 72 hours and heated at 100 ° C. for 30 minutes to inactivate the enzyme. Using this reaction solution, the reducing power and sugar composition were measured in the same manner as in Experiment 6. The results are shown in Table 10.

  As is apparent from the results in Table 10, about 70% trehalose was produced regardless of the maltose concentration of the substrate.

<Experiment 20: Effect of temperature on trehalose production>
Purified maltose-trehalose converting enzyme of Thermus aquaticus ATCC 33923 obtained by the method of Experiment 16 at a maltose concentration of 20% was added at 2.5 units per maltose gram, and the temperature was 40 ° C, 50 ° C, 60 ° C. The reaction solution was collected over time and heated at 100 ° C. for 30 minutes to inactivate the enzyme. This reaction solution was analyzed for sugar composition by HPLC in the same manner as in Experiment 6. Table 11 shows the trehalose content at each temperature and time.

  As is clear from the results in Table 11, the conversion rate from maltose to trehalose was higher as the reaction temperature was lower, and about 80% was converted to trehalose at 40 ° C.

<Experiment 21: Production and properties of maltose / trehalose converting enzyme from other microorganisms>
Among known microorganisms, a specific microorganism having the ability to produce maltose / trehalose converting enzyme of the present invention was cultured in an Erlenmeyer flask for 48 hours according to Experiment 15. After examining the enzyme activity of the culture solution, according to the method of Experiment 16, the culture solution was applied to a crushing apparatus, and the supernatant was dialyzed to obtain a partially purified enzyme. The property was examined according to the method of Experiment 17. . The results are shown in Table 12.

  Using the partially purified enzymes derived from known microorganisms belonging to the genus Thermus shown in Table 12 and examining the action on various carbohydrates according to the method of Experiment 18, as in the case of the enzyme derived from Thermus aquaticus ATCC 33923. It was found that it acts only on maltose and trehalose and produces trehalose from maltose.

  Further, the maltose trehalose converting enzyme of Thermus louver ATCC 35948 was lower in the optimum temperature and the stable temperature than the enzyme of Thermus aquaticus ATCC 33923. It was found to have almost the same properties as the enzyme of, and to have high heat resistance.

<Experiment 22: Partial amino acid sequence of maltose / trehalose converting enzyme>
The purified enzyme preparation derived from Pimelobacter sp. R48 obtained by the method of Experiment 2, the purified enzyme preparation derived from Pseudomonas putida H262 obtained by the method of Experiment 10, and the thermus aquaticus obtained by the method of Experiment 16. After a portion of the purified enzyme preparation derived from ATCC 33923 was dialyzed against distilled water, about 80 μg of protein was used as a sample for N-terminal amino acid sequence analysis. The N-terminal amino acid sequence was analyzed using a protein sequencer model 473A (manufactured by Applied Biosystems, USA). The obtained N-terminal sequences are shown in Table 13.

That is, the enzyme derived from Pimerobacter sp. R48 is SEQ ID NO: 1 in the Sequence Listing, the enzyme derived from Pseudomonas putida H262 is SEQ ID NO: 2 in the Sequence Listing, and the enzyme derived from Thermus Aquaticus ATCC 33923 is SEQ ID NO: 3 in the Sequence Listing. It had the N-terminal amino acid sequence shown. As is clear from the results in Table 13, the enzyme derived from Pimelobacter sp. R48, the enzyme derived from Pseudomonas putida H262, and the enzyme derived from Thermus aquaticus ATCC 33923 have partial amino acid sequences with extremely high homology. Turned out to be. That is, the partial amino acid sequence from the 10th tryptophan to the 16th phenylalanine from the N-terminal side of the enzyme derived from Pymelobacter microorganisms and the 9th phenylalanine from the 3rd tryptophan from the N-terminal side of the Pseudomonas microorganism-derived enzyme The partial amino acid sequence is highly homologous and tryptophan-X ₁ -arginine-X ₂ -alanine-X ₃ -phenylalanine (where X ₁ means phenylalanine or proline, and X ₂ means threonine or proline) X ₃ means valine or alanine). In addition, the partial amino acid sequence from the 14th alanine to the 17th tyrosine from the N-terminal side of the enzyme derived from Pymelobacter microorganism and the partial amino acid from the 9th alanine to the 12th tyrosine from the N-terminal side of the enzyme derived from the Thermus genus The sequence is highly homologous and can be represented by alanine-valine-X ₄ -tyrosine (where X ₄ means phenylalanine or isoleucine).

<Experiment 23: Physicochemical properties of prepared trehalose>
The physicochemical properties were examined using a high-purity sample of trehalose prepared by the method of Experiment 8. The melting point was 97.0 ° C., the specific rotation was [α] 20D + 199 ° (c = 5), the heat of fusion was 57.8 KJ / mole, and the solubility was 77.0 g as an anhydride for water at 25 ° C. These physical property values agreed well with the values of commercial trehalose hydrated crystals (manufactured by Wako Pure Chemical Industries, Ltd.) measured simultaneously.

<Experiment 24: Utilization test in vivo>
According to the method reported by Koji et al., “Clinical Nutrition”, Vol. 41, No. 2, pages 200 to 208 (1972), a high-purity trehalose preparation (purity 99. 8%) 30 g of 20 w / v% aqueous solution, which was orally administered to 3 volunteers (healthy 26-year-old, 27-year-old, 30-year-old male) and collected blood over time to determine blood sugar level and insulin level. It was measured. As a control, glucose was used. As a result, trehalose behaved in the same manner as glucose, and both the blood glucose level and the insulin level showed a maximum value after about 0.5 to 1 hour after administration. Trehalose has been found to be easily digested, absorbed, and metabolized to become an energy source. Therefore, trehalose obtained by the method of the present invention and a saccharide containing the same are suitable as a sugar source for energy supply.

<Experiment 25: Acute toxicity test>
Using mice, an acute toxicity test was conducted by orally administering the high-purity trehalose preparation (purity 99.8%) prepared in Experiment 8. As a result, trehalose is a low-toxic substance, and no death was observed even at the maximum dose. Therefore, although it cannot be said to be an accurate value, its LD ₅₀ value was 50 g / kg or more.

  Hereinafter, the maltose / trehalose converting enzyme of the present invention, trehalose using the same, and a method for producing a saccharide containing the same are shown in Examples 1 to 14, and a composition containing trehalose and a saccharide containing the same is given in Example 15. Thru 37.

  Pimerobacter species R48 (FERM BP-4315) has the same medium composition as in Experiment 1 except that the glucose concentration in the medium components is 4.0 w / v%, and is about 60 hours with a fermenter according to the method of Experiment 1. The culture was aerated and stirred. The activity of maltose / trehalose converting enzyme in this culture broth was 0.75 units per ml of the culture broth. In addition, a part of the cells were separated into cells and culture supernatant by centrifugation, and the activity was measured. As a result, about 65% of the cells were detected and about 35% of the activity was detected in the culture supernatant. About 35 l of the culture solution containing the bacterial cells was treated with an ultrahigh pressure bacterial cell crusher minilab (manufactured by Dainippon Pharmaceutical Co., Ltd.) to disrupt the bacterial cells. This cell disruption suspension is centrifuged and the supernatant is recovered. The supernatant is further concentrated in a UF membrane to recover about 1.2 liters of concentrated enzyme solution having about 15 units of maltose / trehalose converting enzyme per ml. did. 10% potato starch milk (pH 5.5) was added α-amylase (trade name: Spitase HS, manufactured by Nagase Seikagaku Corporation) per gram of starch and heated to gelatinize and liquefy with stirring. Immediately, autoclave (120 C.) for 20 minutes and then adjusted to a temperature of 50 ° C. and a pH of 5.0. To this, β-amylase (manufactured by Nagase Seikagaku Corporation) was added at a rate of 20 units per gram of starch and isoamylase (produced by Hayashibara Biochemical Laboratories Co., Ltd.) at a rate of 500 units per gram of starch, and reacted for 24 hours. To obtain a sugar solution having a maltose content of about 92%. The reaction solution was heated at 100 ° C. for 20 minutes, adjusted to a temperature of 10 ° C. and pH 7.0, and maltose / trehalose converting enzyme prepared by the above method was added to the mixture so as to have a ratio of 1 unit per gram of solid matter. The reaction was performed for 96 hours. The reaction solution is kept at 95 ° C. for 10 minutes, cooled, decolorized and filtered with activated carbon according to a conventional method, purified by desalting with H-type and OH-type ion exchange resins, and further concentrated to a concentration of about 70%. Of syrup was obtained with a solids yield of about 95%. This product contains about 69% trehalose per solid, has a reducing power as low as DE18.2, has a mild sweetness, moderate viscosity, moisturizing, sweetener, taste improver, stabilizer, As excipients, it can be advantageously used in various compositions such as various foods, cosmetics, and pharmaceuticals.

In the maltose / trehalose converting enzyme reaction stop solution obtained by the method of Example 1, 10 units of glucoamylase (manufactured by Nagase Seikagaku Co., Ltd., trade name Gluocteam) per solid matter was added at pH 5.0 and 50 ° C. for 24 hours. In order to increase the trehalose content by using the sugar solution obtained by reacting, then heat inactivation, decolorization, and desalting and purification as a raw sugar solution, an alkali metal strongly acidic cation exchange resin (XT-1016, Na ⁺ type, Ion exchange column chromatography using a bridge degree of 4%, manufactured by Tokyo Organic Chemical Industry Co., Ltd. was performed. The resin was packed into four jacketed stainless steel columns with an inner diameter of 5.4 cm and connected in series to a total resin layer length of 20 m. While maintaining the temperature in the column at 60 ° C., the sugar solution is added to the resin at 5 v / v%, and the water is fractionated by flowing 60 ° C. warm water at SV 0.15 to remove glucose, and the trehalose-rich fraction Minutes were collected. Further purification, concentration, vacuum drying and pulverization gave a trehalose-enriched powder with a yield of about 55% per solid. This product contains about 97% trehalose, has extremely low reducibility, mellow and elegant sweetness. Various sweeteners, taste improvers, quality improvers, stabilizers, excipients, etc. It can be advantageously used for various compositions such as foods, drinks, cosmetics and pharmaceuticals.

  The trehalose-rich fraction obtained by the method of Example 2 was decolorized with activated carbon according to a conventional method, and the solution purified by desalting with an ion exchange resin was concentrated to a concentration of about 70%. About 2% of a trehalose hydrate crystal was added as a seed crystal and cooled slowly to obtain a mass kit having a crystallization rate of about 45%. This mass kit was sprayed at a high pressure of 150 kg / cm 2 from a nozzle on the drying tower. At the same time, 85 ° C hot air is blown from the top of the drying tower and collected on the transfer wire mesh conveyor provided at the bottom, while 45 ° C hot air is sent from the bottom of the conveyor and collected on the wire mesh conveyor. The powder was gradually moved out of the drying tower and taken out. The taken-out crystal powder is filled into an aging tower and aged for 10 hours while sending warm air, crystallization and drying are completed, and the trehalose-containing crystal powder is about Obtained in 90% yield. This product does not substantially exhibit hygroscopicity, is easy to handle, and can be advantageously used in various compositions such as various foods, cosmetics, and pharmaceuticals as sweeteners, taste improvers, stabilizers, and the like.

  The fraction containing high trehalose obtained by the method of Example 2 was purified in the same manner as in Example 3, then taken into an evaporation kettle and boiled under reduced pressure to obtain syrup having a water content of about 3.0%. Next, it was transferred to an auxiliary crystallizer, to which 1% of anhydrous crystalline trehalose was added as a seed crystal per syrup solid, stirred at 120 ° C., then taken out into an aluminum vat, and crystallized and matured at 100 ° C. for 6 hours. Block was prepared. Next, this block is pulverized with a cutting machine, fluidized and dried, and an anhydrous crystalline trehalose powder having a water content of about 0.3% is obtained at a yield of about 85% per solid relative to the raw trehalose-rich sugar solution. Got in. This product is not only used as a dehydrating agent for water-containing products such as foods, cosmetics, pharmaceuticals, raw materials, or processed intermediates, but also as a white powder sweetener with elegant sweetness. It can be advantageously used in the composition.

Pimerobacter species R48 (FERM BP-4315) was cultured in the same manner as in Experiment 1, and 100 g (about 800 units) of wet cells obtained by further centrifuging was dissolved in 10 mM phosphate buffer. The mixture was kneaded with 100 ml of sodium alginate (300 to 400 cp, manufactured by Wako Pure Chemical Industries, Ltd.). The slurry containing the cells was continuously dropped from a height of about 20 cm from the water surface into a 0.1 M CaCl ₂ solution stirred with a magnetic stirrer to form a spherical gelled product having a diameter of about 2 mm. The gelled product was kept in a CaCl ₂ solution at room temperature for about 2 hours, and then filtered on a Buchner funnel to recover alginic acid-immobilized cells. The immobilized cells were packed in a jacketed glass column having a diameter of 30 mm and a length of 200 mm, and the temperature was kept at 20 ° C. A maltose 40% solution having a pH of 6.8 was passed through the immobilized cell column in a downward flow at SV0.2 to obtain a sugar solution having a trehalose content of about 70%. The sugar solution was purified and concentrated according to the method of Example 1 to obtain syrup having a concentration of about 70% with a solid yield of about 95%. This product has low reducing properties, mild sweetness, and moderate moisturizing properties, and can be advantageously used in various compositions as in Example 1.

  After adding calcium carbonate to 33% corn starch milk so as to have a concentration of 0.1%, the pH was adjusted to 6.5, and α-amylase (trade name Termamil 60 L, manufactured by Novo) was added to the starch per gram of starch. The mixture was added to 2% and reacted at 95 ° C. for 15 minutes. The reaction solution was autoclaved (120 ° C.) for 30 minutes, then cooled to 55 ° C., and isoamylase (produced by Hayashibara Biochemical Laboratories Co., Ltd.) with 500 units per gram starch and β-amylase (Nagase Biochemistry). Kogyo Co., Ltd.) was added at a rate of 30 units per gram of starch and reacted for 48 hours to obtain a sugar solution having a maltose content of about 84%. The reaction was heated at 100 ° C. for 10 minutes, then cooled to 15 ° C. and maltose / trehalose converting enzyme prepared by the method of Example 1 was added at a rate of 1.5 units per gram of solids. Reacted for hours. The reaction solution was heated at 100 ° C. for 15 minutes to inactivate the enzyme, then decolorized and filtered with activated carbon according to a conventional method, purified by desalting with H-type and OH-type ion exchange resins, and further concentrated. A syrup concentration of about 70% was obtained with a solids yield of about 95%. This product contains about 64% trehalose per solid, has low reduction, mild sweetness, moderate viscosity, and moisturizing properties, and is advantageous for various compositions as in Example 1. Available to:

  The syrup obtained in the method of Example 5 was concentrated to a concentration of about 82% and placed in an auxiliary crystal machine. After mixing about 1% of seed crystals, the seed crystal was placed in a vat and allowed to stand at 20 ° C. for 4 days to crystallize and solidify. Subsequently, it was pulverized by a cutting machine and dried to obtain a hydrated trehalose hydrated crystal powder with a solid yield of about 95%. This product does not substantially exhibit hygroscopicity, is easy to handle, and is advantageous for various compositions as in the case of Example 1 as a sweetener, taste improver, quality improver, stabilizer and the like. Available to:

  The syrup obtained by the method of Example 6 was concentrated to a concentration of about 80% and placed in an auxiliary crystal machine. About 1% of a trehalose hydrous crystal powder was added as a seed crystal, and the mixture was gradually cooled and crystallized with stirring. Next, it was honeyed in a basket-type centrifuge, and the crystals were sprayed with a small amount of water and washed to obtain high-purity trehalose-containing crystals with a solid yield of about 20%. This product exhibits physicochemical properties similar to those in Experiment 23, and can be advantageously used as a sweetener, a taste improver, a quality improver, a stabilizer and the like in various compositions such as various foods, cosmetics, and pharmaceuticals. Further, it can be advantageously used for industrial reagents and chemical raw materials.

  Pseudomonas putida H262 (FERM BP-4579) was cultured with a fermenter in accordance with the method of Experiment 9 for about 20 hours with aeration and stirring. About 18 l of this culture solution was centrifuged to obtain cells having a wet mass of about 0.4 kg. This was suspended in 4 l of 10 mM phosphate buffer and treated with an ultrasonic crusher model US300 (manufactured by Nippon Seiki Seisakusho Co., Ltd.) to crush the cells. This cell disruption suspension was centrifuged, the supernatant was recovered, the liquid was further concentrated in a UF membrane, and about 400 ml of concentrated enzyme solution having 3.8 units of maltose / trehalose converting enzyme per ml was recovered. . 10% potato starch milk (pH 5.5) was added α-amylase (trade name: Spitase HS, manufactured by Nagase Seikagaku Corporation) per gram of starch and heated to gelatinize and liquefy with stirring. Immediately, autoclave (120 ° C) for 20 minutes, and then adjusted to a temperature of 50 ° C and a pH of 5.5. To this, pullulanase (produced by Hayashibara Biochemical Laboratories Co., Ltd.) was added at a rate of 500 units per gram starch and β-amylase (produced by Nagase Seikagaku Corporation) at a rate of 20 units per gram starch and allowed to react for 24 hours. A sugar solution having a maltose content of about 92% was obtained. After heating the reaction solution at 100 ° C. for 20 minutes, the temperature was adjusted to 40 ° C. and pH 7.0, and the maltose / trehalose converting enzyme prepared by the above method was added at a rate of 1.5 units per gram of solids. In addition, the reaction was allowed to proceed for 72 hours. The reaction solution is kept at 95 ° C. for 10 minutes, cooled, decolorized and filtered with activated carbon according to a conventional method, purified by desalting with H-type and OH-type ion exchange resins, and further concentrated to a concentration of about 70%. Of syrup was obtained with a solids yield of about 97%. This product contains about 65% trehalose per solid, has a reducing power as low as DE16.2, has a mild sweetness, moderate viscosity, moisturizing, sweetener, taste improver, stabilizer, As excipients, it can be advantageously used in various compositions such as various foods, cosmetics, and pharmaceuticals.

The reaction-terminated liquid obtained by the method of Example 9 with the maltose / trehalose converting enzyme was kept at 95 ° C. for 10 minutes to deactivate the enzyme, then adjusted to pH 5.0 and 55 ° C. to obtain glucoamylase (Nagase Biochemistry). Kogyo Co., Ltd., trade name Glucoteam) was added for 10 units per gram of solid matter and allowed to react for 24 hours. The reaction solution was heated according to a conventional method to deactivate the enzyme, decolorized, desalted and purified, and concentrated to 55% to obtain a sugar solution. Using this sugar solution as a raw sugar solution, column chromatography was performed in the same manner as in Example 2 using an alkaline earth metal type strongly acidic cation exchange resin (Dawex 99, Ca ⁺⁺ type, degree of cross-linking 6%, manufactured by Dow Chemical Company). The fraction with high trehalose content was collected. Furthermore, this is refined and continuously crystallized while concentrating. The resulting mass kit is honeyed in a basket-type centrifuge, and the crystals are sprayed with a small amount of water and washed to collect high-purity trehalose-containing crystals as a solid substance. Obtained at a rate of about 25%. This product exhibits the same physicochemical properties as in Experiment 23, and can be advantageously used in various compositions such as various foods, cosmetics, and pharmaceuticals, as well as industrial reagents and chemical raw materials, as in Example 8.

Pseudomonas putida H262 (FERM BP-4579) was cultured in the same manner as in Experiment 9 and centrifuged, and 100 g (about 400 units) of wet cells obtained by centrifugation was dissolved in 10 mM phosphate buffer to give 2.5%. It knead | mixed to 100 ml of sodium alginate (300-400 cp, Wako Pure Chemical Industries sale). The slurry containing the cells was continuously dropped from a height of about 20 cm from the water surface into a 0.1 M CaCl ₂ solution stirred with a magnetic stirrer to form a spherical gelled product having a diameter of about 2 mm. The gelled product was kept in a CaCl ₂ solution at room temperature for about 2 hours, and then filtered on a Buchner funnel to recover alginic acid-immobilized cells. The immobilized cells were packed in a jacketed glass column having a diameter of 30 mm and a length of 200 mm, and the temperature was kept at 35 ° C. A maltose 40% solution having a pH of 6.8 was passed through this immobilized cell column in a downward flow at SV0.1 to obtain a sugar solution having a trehalose content of 67%. This sugar solution was purified and concentrated according to the method of Example 9, which was crystallized and spray-dried to obtain a honey-containing trehalose hydrated crystal powder with a solid yield of about 90%. This product has low reducing properties, mild sweetness, and moderate moisturizing properties, and can be advantageously used in various compositions as in Example 9.

  Thermus aquaticus ATCC 33923 was cultured by aeration and agitation in a fermenter according to the method of Experiment 15 for about 20 hours. The activity of maltose / trehalose converting enzyme in this culture was 0.32 units per ml of culture. Bacteria having a wet mass of 0.18 kg collected from about 18 l of the culture broth were suspended in 10 mM phosphate buffer (pH 7.0). About 1.5 l of this bacterial cell suspension was treated with an ultrasonic crusher to crush the bacterial cells. This cell disruption suspension was centrifuged, the supernatant was collected, the solution was further concentrated in a UF membrane, and about 500 ml of a concentrated enzyme solution having about 10 units of maltose / trehalose converting enzyme per ml was collected. 15% corn starch milk (pH 5.5), α-amylase (trade name Spitase HS, manufactured by Nagase Seikagaku Co., Ltd.) was added in 2 units per gram of starch, and the mixture was gelatinized and liquefied with stirring. C.) for 20 minutes, and the temperature was adjusted to 55 ° C. and pH 5.0. To this was added isoamylase (produced by Hayashibara Biochemical Laboratories Co., Ltd.) at a rate of 300 units per gram starch and β-amylase (produced by Nagase Seikagaku Co., Ltd.) at a rate of 20 units per gram starch and reacted for 24 hours. To obtain a sugar solution having a maltose content of about 92%. After heating the reaction solution at 100 ° C. for 20 minutes, the temperature is adjusted to 50 ° C. and pH 7.0, and the maltose / trehalose converting enzyme prepared by the above method is added at a rate of 1.5 units per gram of solids. In addition, the reaction was allowed to proceed for 72 hours. The reaction solution is kept at 95 ° C. for 10 minutes, cooled, decolorized and filtered with activated carbon according to a conventional method, purified by desalting with H-type and OH-type ion exchange resins, and further concentrated to a concentration of about 70%. Of syrup was obtained with a solids yield of about 95%. This product contains about 64% trehalose per solid, has a reducing power as low as DE 18.0, has a mild sweetness, moderate viscosity, and moisture retention, sweetener, taste improver, stabilizer, As excipients, it can be advantageously used in various compositions such as various foods, cosmetics, and pharmaceuticals.

  The syrup obtained by the method of Example 12 was concentrated to a concentration of about 80% and placed in an auxiliary crystallizer, and crystallized and clarified in the same manner as in Example 8 to obtain a high-purity trehalose-containing crystal with a solid yield of about 20%. I got it. This product exhibits physicochemical properties similar to those in Experiment 23, and is advantageous for various compositions such as various foods, cosmetics, and pharmaceuticals, as well as industrial reagents, industrial raw materials, and chemical raw materials, as in Example 8. Available.

1. Thermus aquaticus ATCC 33923 was cultured in the same manner as in Experiment 15, and 50 g (about 1,500 units) of wet cells obtained by further centrifuging were dissolved in 10 mM phosphate buffer (pH 7.0). The mixture was kneaded with 100 ml of 5% sodium alginate (300 to 400 cp, manufactured by Wako Pure Chemical Industries, Ltd.). The slurry containing the cells was continuously dropped from a height of about 20 cm from the water surface into a 0.1 M CaCl ₂ solution stirred with a magnetic stirrer to form a spherical gelled product having a diameter of about 2 mm. The gelled product was kept in a CaCl ₂ solution at room temperature for about 2 hours, and then filtered on a Buchner funnel to recover alginic acid-immobilized cells. The immobilized cells were packed in a jacketed glass column having a diameter of 30 mm and a length of 200 mm, and the temperature was kept at 60 ° C. A maltose 40% solution with a pH of 6.5 was passed through this immobilized cell column in a downward flow at SV0.2 to obtain a sugar solution having a trehalose content of about 66%. The sugar solution was purified, concentrated and spray-dried according to a conventional method to obtain a trehalose-containing powder with a solid yield of about 90%. This product is low reducing and has a mild sweetness, and can be advantageously used in various compositions as in Example 12.

<Sweetener>
To 1 part by mass of the trehalose hydrous crystal powder obtained by the method of Example 8, 0.01 part by mass of α-glycosyl stevioside (trade name αG sweet, manufactured by Toyo Seika Co., Ltd.) and L-aspartyl-L-phenylalanine methyl ester (product) Nominal aspartame) 0.01 parts by mass was uniformly mixed and subjected to a granulating machine to obtain a granular sweetener. This product has excellent sweetness quality and has a sweetness level about 2.5 times that of sucrose, and the calorie per sweetness level is reduced to about 1 / 2.5 of sucrose. This sweetener has no degradation of high-intensity sweetener blended in it, has excellent stability, and as a low-calorie sweetener, low-calorie food and drink for obese people, diabetics, etc. who restrict caloric intake Suitable for sweetening products. In addition, the present sweetener is suitable for sweetening foods and the like that suppress dental caries because it produces less acid by caries-inducing bacteria and produces less insoluble glucan.

<Hard candy>
30 parts by mass of trehalose-containing syrup obtained by the method of Example 1 was heated and mixed with 100 parts by mass of a 55% sucrose solution, and then heated and concentrated under reduced pressure until the water content was less than 2%. To this, 1 part by mass of citric acid was added. In addition, an appropriate amount of lemon flavor and color were mixed and molded according to a conventional method to obtain a product. This product is a high quality hard candy that is crisp, tastes good and does not cause sucrose crystallization.

<Chewing gum>
3 parts by weight of the gum base is heated and melted to a degree of softening, and 3 parts by weight of crystalline maltitol powder and 4 parts by weight of the water-containing trehalose crystal powder obtained by the method of Example 3 are added thereto. Were mixed by a roll according to a conventional method, molded and packaged to obtain a product. This product is a chewing gum with good texture and flavor. In addition, this product is suitable as a low caries or hard caries chewing gum.

<Powder juice>
50 parts by mass of honey-containing trehalose hydrous crystal powder obtained by the method of Example 7, 10 parts by mass of sucrose, 0.65 parts by mass of anhydrous citric acid, malic acid with respect to 33 parts by mass of orange juice powder produced by spray drying 0.1 parts by weight, 0.1 parts by weight of L-ascorbic acid, 0.1 parts by weight of sodium citrate, 0.5 parts by weight of pullulan, and appropriate amount of powdered fragrance are mixed and stirred, pulverized into a fine powder, and flowed Prepared into a layer granulator, exhaust temperature 40 ° C., air volume 150 m3, sprayed with trehalose-containing syrup obtained in the method of Example 6 as a binder, granulated for 30 minutes, weighed and packaged. Obtained. This product is a powdered juice with a fruit juice content of about 30%, has no off-flavors and off-flavors, and maintains high quality for a long time.

<Lactic acid bacteria beverage>
175 parts by mass of skim milk powder, 130 parts by mass of trehalose-containing syrup obtained by the method of Example 9 and 50 parts by mass of high lactosucrose-containing powder disclosed in JP-A-4-281955 are dissolved in 1,150 parts by mass of water. Then, after sterilizing at 65 ° C. for 30 minutes and cooling to 40 ° C., 30 parts by mass of a starter of lactic acid bacteria was inoculated in accordance with a conventional method, and cultured at 37 ° C. for 8 hours to obtain a lactic acid bacteria beverage. This product is a lactic acid bacteria beverage with good flavor. In addition, this product contains oligosaccharides and not only stably retains lactic acid bacteria, but also has a bifidobacteria growth promoting action.

<Custard cream>
100 parts by mass of corn starch, 100 parts by mass of trehalose-containing syrup obtained by the method of Example 6, 80 parts by mass of maltose, 20 parts by mass of sucrose and 1 part by mass of sodium chloride, and 280 parts by mass of chicken egg were added and stirred. Gradually add 1,000 parts by weight of boiled milk to this, and continue to stir it over fire. When cornstarch is completely gelatinized and the whole becomes translucent, stop the fire and cool it down to the appropriate amount. Of vanilla flavoring, weighed, filled and packed to obtain a product. This product has a smooth luster, mild sweetness and deliciousness.

<Uiro no Moto>
Rice bran is produced by uniformly mixing 90 parts by mass of rice flour with 20 parts by mass of corn starch, 40 parts by mass of sucrose, 80 parts by mass of hydrated trehalose hydrous crystal powder obtained by the method of Example 11 and 4 parts by mass of pullulan. did. Uiro-no-Momo, an appropriate amount of Matcha and water were kneaded, and this was placed in a container and steamed for 60 minutes to produce Matcha Uiro. This product has good shine, mouthfeel, and good flavor. Moreover, the aging of starch is suppressed and the shelf life is good.

<Powder peptide>
40 parts of a soy peptide solution for food (produced by Fuji Oil Co., Ltd., trade name Hynewt S) was mixed with 2 parts by mass of the trehalose-containing hydrated crystal obtained by the method of Example 10, and placed in a plastic vat. It dried under reduced pressure at 50 degreeC and grind | pulverized and obtained the powder peptide. This product has a good flavor and can be advantageously used not only as a confectionery material such as premix and frozen confectionery but also as a baby food such as oral liquid food and tube liquid food, and a therapeutic nutrient.

<Powder miso>
3 parts by weight of anhydrous crystalline trehalose powder obtained by the method of Example 4 was mixed with 1 part by weight of red miso, poured into a metal plate provided with a number of hemispherical recesses, and allowed to stand overnight at room temperature to solidify. The mold was released to obtain about 4 g of solid miso per piece, and this was pulverized to obtain a powder miso. This product can be advantageously used as a seasoning for instant ramen and instant soup. The solid miso can be used not only as a solid seasoning but also as a miso confectionery.

<Powdered egg yolk>
The ratio of 4 parts by mass of anhydrous crystalline trehalose powder obtained by the method of Example 4 to 1 part by mass of liquid egg yolk obtained by sterilizing a yellow egg prepared from raw eggs at 60 to 64 ° C. with a plate-type heat sterilizer The mixture was transferred to a vat and allowed to stand overnight to convert it into trehalose hydrated crystals to prepare a block. The block was pulverized with a cutting machine to obtain powdered egg yolk. This product can be advantageously used not only as a confectionery material such as a premix, a frozen dessert, and an emulsifier, but also as a baby food such as an oral liquid food and a tube liquid food, and a therapeutic nutrient. Moreover, it can be advantageously used as a skin beautifying agent, hair restorer and the like.

<An>
In accordance with a conventional method, water was added to 10 parts by mass of raw azuki and boiled, astringent and extracted, and water-soluble impurities were removed to obtain about 21 parts by mass of red bean paste. Add 14 parts by mass of sucrose, 5 parts by mass of trehalose-containing syrup obtained by the method of Example 12 and 5 parts by mass of water to this raw bean curd, and add a small amount of salad oil to this so as not to break the crumbs. After kneading, about 35 parts by mass of the product was obtained. This product has no color burn, has a good texture, has a good flavor, and is suitable as an ingredient for bread, bun, dumpling, peach and ice confectionery.

<Bread>
100 parts by weight of wheat flour, 2 parts by weight of yeast, 5 parts by weight of sugar, 1 part by weight of trehalose-containing powder obtained by the method of Example 14 and 0.1 part by weight of inorganic food are kneaded with water according to a conventional method, Fermented at 26 ° C. for 2 hours, then aged for 30 minutes and baked. This product is a high quality bread with good hue and sudoku, moderate elasticity and mild sweetness.

<Ham>
The pork thigh is rubbed uniformly into 1,000 parts by mass of 15 parts by mass of sodium chloride and 3 parts by mass of potassium nitrate and deposited in a cold room for one day. This was dipped in a salted solution consisting of 500 parts by mass of water, 100 parts by mass of sodium chloride, 3 parts by mass of potassium nitrate, 40 parts by mass of water-containing trehalose crystal powder obtained by the method of Example 3 and an appropriate amount of spices in a cold room for 7 days, According to a conventional method, the product was washed with cold water, wound with a string, smoked, cooked, cooled and packaged to obtain a product. This product is a high quality ham with good color and good flavor.

<Sweetened condensed milk>
3 parts by mass of trehalose-containing syrup and 1 part by mass of sucrose obtained by the method of Example 5 are dissolved in 100 parts by mass of raw milk, sterilized by heating with a plate heater, then concentrated to a concentration of 70%, and canned in a sterile state. Got the product. This product has a mild sweet taste and good flavor, and can be advantageously used for seasoning infant foods, fruits, coffee, cocoa, tea and the like.

<Cosmetic cream>
2 parts by mass of polyoxyethylene glycol monostearate, 5 parts by mass of glyceryl monostearate self-emulsifying, 2 parts by mass of hydrated trehalose hydrous crystal powder obtained by the method of Example 7, 1 part by mass of α-glycosyl rutin, flow 1 part by weight of paraffin, 10 parts by weight of glyceryl trioctanoate and an appropriate amount of preservative are dissolved by heating according to a conventional method, and 2 parts by weight of L-lactic acid, 5 parts by weight of 1,3-butylene glycol and 66 parts by weight of purified water are added thereto. Then, the mixture was emulsified with a homogenizer, and an appropriate amount of a fragrance was added, followed by stirring and mixing to produce a cream. This product is excellent in stability and can be advantageously used as a high-quality sunscreen, skin beautifier, whitening agent and the like.

<Powdered ginseng extract>
After mixing 1.5 parts by mass of anhydrous crystalline trehalose powder obtained by the method of Example 4 with 0.5 parts by mass of medicinal carrot extract, the mixture was transferred to a vat and left to stand for 2 days to convert to trehalose hydrous crystals to prepare a block. . The block was pulverized with a cutting machine and classified to obtain a powdered ginseng extract. Subjected to granulation molding machine of this product with an appropriate amount of vitamin B ₁ and vitamin B ₂ powder was a vitamin-containing granule ginseng extract. This product can be advantageously used as a fatigue recovery agent, tonic, toughening agent and the like. It can also be used as a hair restorer.

<Solid formulation>
A natural human interferon-α preparation (manufactured by Hayashibara Biochemical Laboratories Co., Ltd., sold by Cosmo Bio Inc.) is applied to an immobilized anti-human interferon-α antibody column according to a conventional method, and the natural type contained in the preparation The trehalose-rich powder obtained by adsorbing human interferon-α, passing through and removing bovine serum albumin as a stabilizer, and then changing the pH to obtain natural human interferon-α by the method of Example 2. Elution was performed using 5% physiological saline. This liquid is microfiltered, added to about 20 times the amount of anhydrous crystalline maltose powder (trade name Fine Tooth, manufactured by Hayashibara Shoji Co., Ltd.), dehydrated and powdered, and tableted with a tableting machine. A tablet containing about 150 units of natural human interferon-α per about 200 mg) was obtained. About 1 to 10 adults per day are administered orally as sublingual tablets, etc., and this product can be advantageously used for the treatment of viral diseases, allergic diseases, rheumatism, diabetes, malignant tumors and the like. In particular, it can be advantageously used as a therapeutic agent for AIDS, hepatitis, etc., whose number of patients is rapidly increasing in recent years. In this product, maltose acts as a stabilizer together with trehalose and maintains its activity well for a long time even when left at room temperature.

<Sugar-coated tablets>
An uncoated tablet having a mass of 150 mg is used as a core agent, and 40 parts by mass of the trehalose hydrous crystal powder obtained by the method of Example 3, 2 parts by mass of pullulan (average molecular weight 200,000), 30 parts by mass of water, 25 parts by mass of talc, and oxidation Using an undercoat liquid consisting of 3 parts by mass of titanium, sugar coating is carried out until the tablet mass reaches about 230 mg, and then an overcoat liquid consisting of 65 parts by mass of the same trehalose hydrous crystal powder, 1 part by mass of pullulan and 34 parts by mass of water is used. Then, the sugar-coated tablet was further coated with a wax solution to obtain a sugar-coated tablet with an excellent glossy appearance. This product has excellent impact resistance and maintains high quality for a long time.

<Toothpaste>
Formulation
Dibasic calcium phosphate 45.0%
Pullulan 2.95%
Sodium lauryl sulfate 1.5%
Glycerin 20.0%
Polyoxyethylene sorbitan laurate 0.5%
Preservative 0.05%
Trehalose-containing powder obtained by the method of Example 14 12.0%
Maltitol 5.0%
Water 13.0%
The above materials were mixed according to a conventional method to obtain a toothpaste. This product has moderate sweetness and is particularly suitable as a toothpaste for children.

<Food edible solid formulation>
Trehalose hydrated crystals produced by the method of Example 8 500 parts by weight, powdered egg yolk 270 parts by weight, skim milk powder 209 parts by weight, sodium chloride 4.4 parts by weight, potassium chloride 1.8 parts by weight, magnesium sulfate 4 parts by weight, thiamine A blend consisting of 0.01 parts by weight, sodium ascorbate 0.1 parts by weight, vitamin E acetate 0.6 parts by weight and nicotinamide 0.04 parts by weight is prepared, and each 25 grams of the blend is moisture-proof laminated sachet. And heat sealed to obtain a product. This product is dissolved in about 150 to 300 ml of water to make a liquid food, and it is used orally or by tube use to the nasal cavity, stomach, intestine, etc., and is advantageous for supplementing energy to the living body. Available to:

<Infusion>
The high-purity trehalose-containing crystal produced by the method of Example 10 is dissolved in water to a concentration of about 10 w / v%, then subjected to microfiltration according to a conventional method to make pyrogen-free, and aseptically filled into a plastic bottle and plugged And got the product. This product is a stable infusion that does not change over time and is suitable for intravenous, intraperitoneal and other administration. This product is isotonic with blood at a concentration of 10 w / v%, and can be replenished with energy at twice the concentration of glucose.

<Infusion>
The high-purity trehalose water-containing crystal produced by the method of Example 13 and the amino acid compound having the following composition were mixed and dissolved in water so that the concentrations were 5 w / v% and 30 w / v%, respectively, and then the same as in Example 10. The product was purified to be pyrogen-free, and further filled into a plastic bag and plugged to obtain a product.
Composition of amino acid compound (mg / 100ml)
L-isoleucine 180
L-leucine 410
L-lysine hydrochloride 620
L-methionine 240
L-phenylalanine 290
L-threonine 180
L-tryptophan 60
L-Valine 200
L-arginine hydrochloride 270
L-histidine hydrochloride 130
Glycine 340
This product is a stable infusion with no changes over time because trehalose does not exhibit reducing properties despite the complex infusion of carbohydrates and amino acids, and is suitable for administration intravenously, intraperitoneally, etc. It is. This product can be advantageously used not only for energy supply to the living body but also for amino acid supply.

<Treatment for trauma>
50 parts by mass of methanol in which 3 parts by mass of iodine was added and mixed with 200 parts by mass of the trehalose-rich powder produced by the method of Example 2 and 300 parts by mass of maltose, and further 200 parts by mass of a 10 w / v% pullulan aqueous solution were added. To obtain a plaster for the treatment of trauma showing moderate elongation and adhesion. This product not only acts as a bactericidal action with iodine, but also acts as an energy replenisher to cells with trehalose, shortening the healing period and healing the wound surface cleanly.

  As is apparent from the above, the novel maltose / trehalose converting enzyme of the present invention converts maltose to trehalose with a high conversion rate. Trehalose obtained by this enzyme reaction and a saccharide containing the same are stable, have a good quality and a refreshing sweetness, and are digested and absorbed by oral ingestion, and are a saccharide that becomes a calorie source. These carbohydrates can be advantageously used in various compositions such as foods and drinks, cosmetics, and pharmaceuticals as sweeteners, taste improvers, quality improvers, stabilizers, excipients, and the like.

  Therefore, the establishment of the present invention opens up a completely new way to supply trehalose and carbohydrates containing it from a maltose derived from starch, an inexpensive and endless resource, industrially in large quantities and at low cost. The impact of this is not limited to academic fields such as starch science, enzymatic chemistry, and biochemistry, but also to industries, especially food, cosmetics, and pharmaceuticals, as well as agriculture, aquaculture, and chemical industries. And the industrial significance given to these industries is immeasurable.

It is a figure which shows the influence of the temperature which acts on the enzyme activity of the Pyrolobacter species R48 maltose trehalose converting enzyme. It is a figure which shows the influence of pH which acts on the enzyme activity of the maltose trehalose converting enzyme of Pimerobacter species R48. It is a figure which shows the influence of the temperature which acts on stability of the maltose trehalose converting enzyme of Pimerobacter species R48. It is a figure which shows the influence of pH which acts on the stability of the Pyrolobacter species R48 maltose trehalose converting enzyme. It is a figure which shows the influence of temperature on the enzyme activity of Pseudomonas putida H262 maltose trehalose converting enzyme. It is a figure which shows the influence of pH on the enzyme activity of Pseudomonas putida H262 maltose trehalose converting enzyme. It is a figure which shows the influence of temperature on stability of Pseudomonas putida H262 maltose trehalose converting enzyme. It is a figure which shows the influence of pH on the stability of Pseudomonas putida H262 maltose trehalose converting enzyme. It is a figure which shows the influence of the temperature which acts on the enzyme activity of the maltose trehalose converting enzyme of Thermus aquaticus ATCC 33923. It is a figure which shows the influence of pH on the enzyme activity of the maltose trehalose converting enzyme of Thermus aquaticus ATCC 33923. It is a figure which shows the influence of the temperature which acts on the stability of the maltose trehalose converting enzyme of Thermus aquaticus ATCC 33923. It is a figure which shows the influence of pH which acts on the stability of the maltose trehalose converting enzyme of Thermus aquaticus ATCC 33923.

Claims (7)

An enzyme formed by using an aqueous solution containing maltose as a substrate, converting the maltose-containing aqueous solution into trehalose using maltose as a substrate, and allowing an enzyme that does not use glucose 1-phosphate and glucose as a substrate to act. A method for producing a trehalose-containing saccharide, comprising a step of converting maltose into trehalose without passing through glucose phosphate by a reaction and a step of collecting the obtained trehalose-containing saccharide.   The method for producing a trehalose-containing saccharide according to claim 1, wherein the aqueous solution containing maltose as a substrate is obtained by allowing starch debranching enzyme to act on starch with β-amylase or β-amylase. Maltose is converted into trehalose as a substrate, and an enzymatic reaction in which glucose 1-phosphate and glucose are not used as substrates is carried out using any one of the following maltose and trehalose converting enzymes (1) to (3). A method for producing a trehalose-containing saccharide according to claim 1 or 2;
(1) has an action of converting maltose to trehalose and converting trehalose to maltose, and has tryptophan-phenylalanine-arginine-threonine-alanine-valine-phenylalanine as a partial amino acid sequence, and by SDS-gel electrophoresis A maltose / trehalose converting enzyme having a molecular weight of 57,000 to 67,000 daltons, which can be obtained from a microorganism belonging to the genus Pymelobacter,
(2) has an action of converting maltose to trehalose and converting trehalose to maltose, and has tryptophan-proline-arginine-proline-alanine-alanine-phenylalanine as a partial amino acid sequence, and by SDS-gel electrophoresis Maltose trehalose converting enzyme obtainable from a microorganism belonging to the genus Pseudomonas, having a molecular weight of 110,000 to 120,000 daltons,
(3) It has an action of converting maltose to trehalose and converting trehalose to maltose, and has a partial amino acid sequence of alanine-valine-isoleucine-tyrosine, and 100,000 to 110 by SDS-gel electrophoresis. A maltose / trehalose converting enzyme obtainable from a microorganism belonging to the genus Thermus having a molecular weight of 1,000 daltons.   The method for producing a trehalose-containing saccharide according to any one of claims 1 to 3, wherein the conversion rate of maltose to trehalose exceeds 60%.   The method for producing a trehalose-containing saccharide according to any one of claims 1 to 4, further comprising a step of allowing glucoamylase and / or α-glucosidase to act on the obtained trehalose-containing saccharide.   The method for producing a trehalose-containing saccharide according to any one of claims 1 to 5, comprising a step of subjecting the obtained trehalose-containing saccharide to column chromatography using a salt-type strongly acidic cation exchange resin to improve the trehalose content. The method for producing a trehalose-containing carbohydrate according to any one of claims 1 to 6, further comprising a step of crystallizing trehalose.

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