KR19980042214A - Trehalose phosphorylase, preparation method and use thereof - Google Patents

Abstract

Thermally stable trehalose, obtained from microorganism belonging to the genus Themoanaerobium®, which hydrolyzes trehalose in the presence of inorganic phosphoric acid to produce D-glucose and β-D-glucose-1-phosphate. Phosphorylase. It can also be prepared by recombinant DNA technology of trehalose phosphorylase. The action of enzymes on β-D-glucose-1-phosphate as a sugar donor in the presence of other sugars provides an industrial scale for glucosyl transsaccharides, including glucosyl-D-galactosid, which is known but rarely available. It can be manufactured relatively inexpensively.

Description

Trehalose phosphorylase, preparation method and use thereof

The present invention relates to a novel trehalose phosphorylase, a preparation method and a use thereof, and more particularly, to inorganic phosphoric acid and / or salts thereof (hereinafter, unless otherwise noted), Through hydrolysis of trehalose in the presence of inorganic phosphoric acid through the D-glucose and β-D-glucose-1-yne and / or salts thereof (hereinafter, unless otherwise noted) new trehalose producing trehalose and inorganic phosphoric acid from β-D-glucose-1-phosphate and D-glucose; Containing a phosphorylase, a method for preparing trehalose phosphorylase, a glucosyl transition sugar prepared using the trehalose phosphorylase, and a sugar composition containing the sugar composition. Regarding the composition will be.

In recent years, oligosaccharides such as maltose and trehalose and their functions have been in the spotlight, and various and unique production methods thereof have been studied in various aspects. It is known that various phosphorylases such as phosphorylases such as maltose, trehalose, sucrose, trehalose and cellobiose phosphorylase can be used as the oligosaccharide preparation method described above.

In LR Marechal et al., The Journal of Biological Chemistry, Vol. 247, No. 10, pp. 3, 223-3, 228 (1972), Euglena gracilis is a trehalose phosphoryl. It has been reported that the enzyme is produced in cells, and S. Murao et al. Described the properties of this enzyme in Agriculture and Biological Chemistry, Vol. 49, No. 7, pp. 2, 113-2, 118 (1985). K. Aisaka et al., In Japanese Patent Laid-Open No. 59,584 / 95, describe the microorganisms belonging to the Catellatospora ferruginea species and the microorganisms belonging to the genus Kinosporia aurantiaca. Bacterial trehalose phosphorylase produced by H. Kizawa et al., Bioscience, Biotechnolgy and Biochemistry, Vol. 59, No. 10, pp. 1, 908-1, 912 (1995). Microorganisms belonging to the species Micrococcus varians also produced these enzymes. In addition, M. Yoshida et al. Reported in Plesiomonas sp. SH-35 in Oyo-Toshitsu-Kagaku, Vol. 42, No. 1, pp. 19-25 (1995). It is reported that the microorganism belongs to produce trehalose phosphorylase, among which microorganisms belonging to micrococcus varians, euglena gracilis and plecimonas sp. They have low thermal stability of less than 30 ° C., 40 ° C. and 45 ° C., respectively, resulting in relatively low reaction efficiency in large scale production and bacterial contamination during enzymatic reactions.

The present invention provides a novel trehalose phosphorylase having industrially advantageous thermal stability, a preparation method thereof, a sugar composition containing a glucosyl transition sugar prepared using the enzyme, and a use thereof. .

In order to solve the above problems and to produce trehalose phosphorylase having good thermal stability, which has not been known so far, the present inventors have searched extensively for microorganisms producing such enzymes, and thus, thermoanaerobium®. Thermoanaerobium brockii ATCC 35047 produced a novel trehalose phosphorylase and established the preparation method thereof. In addition, the present inventors completed the present invention by establishing a sugar composition containing a glucosyl transition sugar and a composition containing the sugar composition produced by contacting an enzyme with β-D-glucose-1-phosphate as a sugar donor in the presence of a sugar. It was.

1 is a graph showing the effect of temperature on the activity of trehalose phosphorylase of the present invention.

2 is a graph showing the effect of pH on the activity of trehalose phosphorylase of the present invention.

3 is a graph showing the effect of temperature on the stability of trehalose phosphorylase of the present invention.

4 is a graph showing the effect of pH on the stability of trehalose phosphorylase of the present invention.

5 is a restriction map of the recombinant DNA of the present invention. In the drawings, arrows indicate DNA that codes for trehalose phosphorylase of the present invention.

Trehalose phosphorylase according to the present invention hydrolyzes trehalose in the presence of enzymes derived from microorganisms belonging to the genus deeomoerobium and inorganic phosphoric acid to form D-glucose and β-D-glucose-1-phosphate. Contains enzymes that produce. This trehalose phosphorylase has the following action and physicochemical properties.

(1) action

(A) Hydrolysis of trehalose in the presence of inorganic phosphoric acid to produce D-glucose and β-D-glucose-1-phosphate.

(B) Trehalose and inorganic phosphoric acid are produced from D-glucose and β-D-glucose-1-phosphate, and the β-D-glucose-1-phosphate is used as a sugar donor to transfer glucose to other sugars. To facilitate the reaction.

(2) molecular weight

88,000 ± 5,000 Daltons on SDS-PAGE

(3) isoelectric point

PI 5.4 ± 0.5 in electrophoresis using amplolite

(4) optimum temperature

about 70 ° C. for 30 minutes at pH 7.0.

(5) optimum pH

7.0 ~ 7.5 when kept at 60 ℃ for 30 minutes

(6) thermal stability

Stable up to about 60 ℃ when kept at pH 7.0 for 1 hour

(7) pH stability

Stable at pH 6.0-9.0 when kept at 4 ° C for 24 hours

(8) activation and stabilization

Activated by 1 mM dithiodreitol

(9) deactivation

Inhibited by 1 mM Cu ⁺⁺ , Pb ⁺⁺ , Zn ⁺⁺ , Hg ⁺⁺ , Mg ⁺⁺ or Mn ⁺⁺

Trehalose phosphorylase is a partial amino acid sequence having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, SEQ ID NO: has the amino acid sequence of 4. If there are isolates of microorganisms producing the desired protein in the art, functional equivalents of the protein can be readily obtained by treating the microorganism with a suitable mutagen, or there is DNA that codes for the desired protein. If so, functional equivalents of proteins can be easily obtained by applying common recombinant DNA techniques to DNA. Of course, the trehalose phosphorylase of the present invention includes such functional equivalents. Functional equivalents have substantially the same activity as trehalose phosphorylase, where at least one amino acid is substituted with another amino acid and at least one amino acid is added to the N terminus and / or the C terminus or to an internal amino acid sequence. Refers to a protein having an amino acid sequence of an enzyme inserted and deleted at least one amino acid of the N terminal and / or C terminal region and at least one amino acid of the internal amino acid sequence. The trehalose phosphorylase of the present invention described above can be easily obtained by separating the enzyme from natural sources, such as cultures of microorganisms produced by these enzymes, their mutants obtained by treatment with mutagens, and the like. The enzyme may be artificially synthesized by applying recombinant DNA and peptide synthesis techniques.

The DNA of the present invention includes all of the above-described DNAs which code for trehalose phosphorylase of the present invention. Examples of such DNA include those having an amino acid sequence of SEQ ID NO: 4, which code for a nucleotide sequence of SEQ ID NO: 5, and functional equivalents thereof.

Functional equivalents refer to proteins having substantially the same action as trehalose phosphorylase, and having a nucleotide sequence of SEQ ID NO: 5 in which one or more bases are substituted for another base. It means DNA. In addition to the above-mentioned DNA, the DNA of the present invention may include a start codon initiator, a stop codon, a Shine-Dalgarno sequence, a nucleotide sequence coding a signal peptide, a recognition sequence by a suitable restriction enzyme, a promoter, an enhancer, and a terminator. And another DNA having a 5 'end and / or a 3' end linked to one or more DNA of the back.

The source and production method of these DNAs is not particularly limited in the present invention. Examples of natural sources of DNA include microorganisms belonging to the genus dermaeoerbium, including dermaerobium brochyi ATCC 35047. DNA containing the DNA of the present invention can be obtained by extracting DNA fractions from cell lysates of cultured microorganisms. The collected DNA itself can be used in the present invention, and fragments containing the DNA of the present invention can be introduced into a self-replicating vector to prepare extremely desirable recombinant DNA. That is, a gene library is obtained by applying a general recombinant DNA technique to the above-described DNA, and by applying a screening method such as hybridization method, the tre of the present invention is derived from a gene library based on a nucleotide sequence such as SEQ ID NO: 5 or the like. Recombinant DNA can generally be obtained by selecting the desired recombinant DNA that codes for halos phosphorylase. The recombinant DNA thus obtained is cultured and amplified by a transformant obtained by introducing into a suitable host such as a microorganism belonging to E. coli, and then, by applying a general alkali-SDS method to the culture, a desired amount of the DNA of the present invention can be easily obtained. You can get it. Using PCR as a template using DNA collected from the crushed cells or cells of the microorganism as a template and using a DNA synthesized chemically based on SEQ ID NO: 5 as a primer, or by applying the PCR method according to the conventional method or The DNA of the present invention can be easily obtained by chemically synthesizing the DNA containing SEQ ID NO :) 5. The functional equivalents of the DNA of the present invention may be, for example, by applying site-directed mutagenesis to the recombinant DNA described above, or using chemically synthesized DNA having a nucleotide sequence converted into a desired nucleotide sequence using the recombinant DNA as a template. It can be obtained by applying the PCR method using as a primer.

The DNA of the present invention includes those in the form of recombinant DNA introduced into a self-replicating vector. As described above, these recombinant DNAs are extremely useful for the preparation of the DNA of the present invention, and also for the preparation of trehalose phosphorylase of the present invention. Once the desired DNA has been obtained as described above, these recombinant DNAs can be obtained relatively easily by inserting the DNA into an appropriate vector by applying general recombinant DNA techniques. Examples of such vectors are those having replication properties in the appropriate host. The following vectors such as Bluescript II SK (+), pKK223-3 and λgt · λC; PUB110, pTZ4, pC194, ρ11, ø11 and ø105, which require microorganisms belonging to the genus Bacillus as hosts; And vectors such as pHY300PLK, pHV14, TRp7, YEp7 and pBS7, which require at least two types of hosts, can be advantageously used in the present invention. An example of a method for inserting the DNA of the present invention into a vector will be described. A DNA fragment and a vector fragment produced by digesting a suitable host, the present invention, or the DNA containing the DNA of the present invention with a restriction enzyme with a restriction enzyme are produced. Connect Examples of such restriction enzymes suitably used include Acc I, Alu I, Bam H I, Bgl II, Bst II, Eco R I, Hind III, Not I, Pst I, Sac I, Sal I, Sma I, Spe I, Xba I And Xho I and the like. When linking DNA fragments and vector fragments, chemically synthesized DNAs with suitable recognition sequences of restriction enzymes can be used, for example. The DNA and DNA are then annealed and then contacted with DNA ligase both intracellularly and extracellularly.

The DNA of the present invention includes those in the form of transformants into which DNA is introduced. Such transformants are extremely useful for obtaining trehalose phosphorylase and DNA of the present invention. For example, E. coli and Bacillus microorganisms, actinomyces and yeast can optionally be used as host microorganisms for transformants. This transformant can be obtained conventionally by introducing the above-mentioned recombinant DNA into a suitable host. When using microorganisms belonging to the genus Escherichia coli, microorganisms and recombinant DNA are incubated in the presence of calcium ions, whereas when using microorganisms belonging to the genus Bacillus, immunocytotoxicity and protoplasts are applied. The above-described methods for producing the DNA of the present invention are per se such as described in J. Sumbruck et al. in Molecular Cloning A Laboaratory Manual, 2nd edition, published by Cold Spring Harbor Laboratory Press (1989).

The method for producing trehalose phosphorylase of the present invention is characterized by culturing microorganisms producing enzymes and harvesting the produced enzymes from the culture. There are no particular limitations on the species, genus and culture method of such microorganisms used in the present invention. Examples of such microorganisms include those belonging to the genus of Dermaeoerobium, preferably Dermaeoerobium brokyi ATCC 35047 and transformants obtained by introducing the DNA of the present invention into a suitable host microorganism.

As long as the microorganism can grow and produce the enzyme of the present invention, any natural nutrient medium or synthetic nutrient medium can be used in the method of the present invention to culture the microorganism. As the carbon source used in the present invention, any microorganism can be used. Examples thereof include sugars such as maltose, trehalose, dextrin, and starch, sugars such as molasses, and natural products containing enzyme extracts. The concentration of these carbon sources in the medium is appropriately selected depending on the type. For example, the preferred sugar concentration is 20 w / v% or less, but 5 w / v% or less in consideration of the growth and growth of microorganisms. Examples of nitrogen sources used in the present invention include nitrogen-containing inorganic compounds such as ammonium salts and nitrates, and nitrogen-containing organic compounds such as urea, coon stiff liquor, casein, peptone, yeast extract, and meat extract. If desired, salts of inorganic compounds such as calcium, magnesium, potassium, sodium, phosphoric acid, manganese, zinc, iron, copper, molybdenum and cobalt can be used in the present invention.

The microorganisms are cultured anaerobicly under the conditions selected at a temperature of 50-80 ° C., preferably 60-70 ° C. and pH 5-8, preferably pH 6.5-7.5. As long as the microorganism can sufficiently grow, the present invention can be used at any culture time, preferably 10 to 50 hours. In the culture of the above-described transformants, the culture is usually incubated for about 1 to 6 days at a temperature of 20 to 65 ° C. and pH 2 to 9 under aerobic conditions.

After culturing the microorganism, the enzyme of the present invention is collected from the culture. In general, since enzyme activity is present in the cells, the cells or the treated cells can be obtained as coenzymes. The whole culture can also be used as coenzyme. Cells and nutrient media can be separated using conventional solid-liquid separation methods. For example, the method of direct centrifugation of a culture, a method of filtering a culture after adding a filtration aid to the culture or after precoating, and a method of filtering a culture by membranes such as flat filters and hollow fibers. There is this. As the coenzyme, the cells can be used as they are, and the treated cells themselves can be used, and these can also be produced as partial purified enzymes if necessary.

The types of cells to be treated include protein fractions of the cells, immobilized materials of the untreated and treated cells and drying treatments, lyophilization treatments and cells treated by methods such as surfactants, enzymes, ultrasonic waves, mechanical grinding, and mechanical pressure. do. The enzyme of the present invention can be used in crude or purified form, and the treated cells can be used in conventional methods such as salting out with ammonium sulfate, precipitation with acetone and alcohol, and flat membranes and hollow fiber. It can be further processed by the membrane concentration method / dialysis method.

The untreated and treated cells are immobilized by conventional methods such as bonding by ion exchange resins, covalent bonding / adsorption by resins and membranes, and enclosing by polymer materials.

The crude enzyme is used as it is or purified by conventional purification methods. For example, a crude enzyme is obtained by salting the treated cells with ammonium sulfate, followed by dialysis of the enzyme, followed by anion exchange column chromatography using DEAE-TOYOPEARL (cation exchange resin sold by Tosoh, Japan), CM-TOYOPEARL. Anion exchange column chromatography using (resin sold by Tosoh company in Japan), hydrophobic column chromatography using BUTYL-TOYOPEARL (hydrophobic resin sold by Tosoh company in Japan) and ULTROGEL AcA44 RESIN (Separor / in France) By continuous treatment by gel filtration column chromatography using IBF sa gel gel for gel filtration column chromatography), a protein band of a single enzyme is obtained electrophoretically.

The activity of trehalose phosphorylase of the present invention was measured as follows. That is, 0.2 ml of enzyme solution was added to 2 ml of 20 mM phosphate buffer (pH 7.0) containing 1.0 w / v% trehalose as a substrate, and the solution was incubated at 60 ° C. for 30 minutes to sample the reaction mixture in an amount of 0.5 ml. The sample is then incubated at 100 ° C. for 10 minutes to stop the enzyme reaction. 0.5 ml of D-glucose oxidase / peroxidase reagent was added to the heated sample and the mixture was stirred for 30 minutes at 40 ° C., 2.5 ml of 5-N hydrochloric acid was added to stir the mixture, and then the absorbance of the mixture was adjusted to a wavelength of 525 nm. Measure at One unit of enzymatic activity is defined as the amount of enzyme that produces 1 μmole of D-glucose per minute. The activity of maltose-phosphorylase and kojibiose-phosphorylase can be measured by the same method as described above, except that maltose and kojibiose are used instead of trehalose as a substrate, respectively.

Other suitable sugars, such as D-xylose, D-galactose, G-glucose, as receptors in the enzymatic reaction of the present invention to enhance the sugar composition containing glucosyl transsaccharide using trehalose phosphorylase Glucosyl-D-xyloxide by transferring enzymes to β-D-glucose-1-phosphate as a sugar donor together with monosaccharides such as D-fucose and L-fucose to transfer the glucosyl group to the reducing sugars described above. Glucosyl-D-galactoside, trehalose, glucosyl-D-fucoside and glucosyl-L-fucoside are produced.

Commercially available β-D-glucose-1-phosphate as a reagent can be used as a sugar donor in the present invention, which is prepared by contacting a suitable phosphorylase with a substrate sugar in the presence of inorganic phosphoric acid and / or its salt. Can be. Acting trehalose phosphorylase to trehalose, maltose phosphorylase to maltose, or cozibios phosphorylase to cozibioses, for example in the presence of inorganic phosphoric acid and / or salts thereof. It can be prepared by working. Any of the above-mentioned reactions of the phosphorylase to produce β-D-glucose-1-phosphate is carried out in the same reaction system using the trehalose phosphorylase of the present invention to produce glucosyl transition sugars. In this case, β-D-glucose-1-phosphate can be directly supplied to the system to reduce the manufacturing cost and simplify the number of process steps as an advantageous feature. The above-mentioned inorganic phosphoric acid includes orthophosphoric acid and condensed phosphoric acid. Orthophosphoric acid is usually used preferably. The salts of the inorganic phosphoric acid include compounds of the general phosphate ions derived from the inorganic phosphoric acid described above. Usually, sodium phosphate salts and potassium phosphate salts having a large water solubility are preferably used.

For example, the enzyme of the present invention can be used as the trehalose phosphorylase described above, and a commercial product of bacterial maltose phosphorylase can be used as it is. In the case of kojibiose phosphorylase, the enzyme disclosed in Japanese Patent Application No. 311,235 / 96 filed by the same applicant of the present invention (name of Kojibiose phosphorylase, its preparation method and use) can be used. Can be. Details are described in this specification. See, for example, seed cultures of Dermaeoerbium Brochyy ATCC 35047 in ATCC Catalog of BACTERIA AND BACTERIOPHAGES, 18th edition, pp. 452-456 (1992), inoculated in the nutrient medium for microorganisms described above, incubated at 65 ° C. under anaerobic conditions, and centrifuged to culture the cells, and the supernatant was collected. Get a fraction of the disturbance with

The concentration of the substrate used in the glucosyl transfer sugar production reaction using the trehalose phosphorylase of the present invention is not particularly limited. In general, preferably 1 to 20 w / w% of β-D-glucose-1-phosphate as a sugar donor (hereinafter, w / w% is simply expressed as% throughout the present specification unless otherwise specified) and receptors 1 to Use a solution containing 20%. As described above, when the β-D-glucose-1-phosphate production reaction by the action of a suitable phosphorylase is carried out in the same reaction system as the above-described glucosyl transfer sugar production reaction, it is preferable to carry out as follows. That is, when trehalose phosphorylase is applied to the substrate, in the presence of about 2 mM of phosphate such as sodium dihydrogen phosphate, about 1 to about 1 to about glucosyl transfer reaction substrate instead of β-D-glucose-1-phosphate. 20% trehalose solution is used. When other phosphorylase is applied to the substrate, maltose or kojibiose is used together with about 0.5 to 20 mM of phosphate such as sodium dihydrogen phosphate instead of β-D-glucose-1-phosphate as a substrate for glucosyl transfer reaction. About 1-20% coexist. Depending on the type of sugar used, it is preferable to co-exist maltose-phosphorylase or kojibiose-phosphorylase in an amount of about 0.1 to 50 units per gram of sugar.

The above reaction is carried out at a temperature at which the enzyme used in the presence of the substrate is not inactivated, that is, about 70 ° C. or lower, preferably about 15 to 65 ° C. The reaction pH is usually adjusted to a pH of about 4.0 to 9.0, preferably to a pH of about 5.0 to 7.5. The reaction time can be appropriately selected according to the enzyme reaction rate. When the enzyme is used in an amount of about 1.0 to 50 units per g of substrate on a solid basis, it is usually about 0.1 to 100 hours. As described above, when the β-D-glucose-1-phosphate production reaction by the action of different phosphorylases is carried out in the same reaction system as the glucosyl transfer sugar production reaction, the reaction temperature and pH depend on their thermal stability. It is desirable to set the phosphorylase used to a temperature and pH at which it does not deactivate.

The resulting reaction mixture thus contains a glucosyl transition sugar corresponding to the sugar used as the substrate. The yield of glucosyl transition sugars depends on the concentration of the substrate used in the enzyme reaction, the type of substrate and the reaction conditions. For example, when 10% trehalose and 5% D-galactose are used as substrates in the presence of inorganic phosphoric acid, glucosylgalactosidate is produced at a yield of about 30%. The yield of glucosyl transition sugars throughout this specification refers to the percentage of sugars produced relative to the total sugars in the reaction mixture of the solid sugar.

In order to increase the content of glucosyl transition sugars in the reaction mixture to the highest possible level, an enzyme source which decomposes and removes D-glucose produced in the reaction mixture is coexisted in the mixture to promote the sugar transfer reaction. When the above-mentioned β-D-glucose-1-phosphate production reaction using a suitable phosphorylase is carried out in the same reaction system as the glucosyl transfer reaction, and the sugar donor is directly supplied, the portion generated during the reaction process using this technique is used. Degradation and elimination of the organism D-glucose can promote the sugar transfer reaction.

Enzyme sources include microorganisms with D-glucose degradation activity, cultures of these microorganisms, untreated cells and treated cells and enzymes with D-glucose degradation activity. Any microorganism having a relatively high D-glucose degrading activity but no or substantially no action of degrading the resulting transsaccharide can be used, and yeast is preferred. Any enzymes such as glucose oxidase, catalase, pyranose oxidase, glucose dehydrogenase, glucokinase and hexokinase can be used, and among these enzymes, glucose oxidase and catalase are preferably used.

The reaction mixture containing the glucosyl transition sugar thus obtained is filtered in a conventional manner and centrifuged to remove impurities, followed by purification steps such as decolorization with activated carbon and desalting with H and OH ion exchange resins. Concentration gives a syrupy product. If necessary, this syrup-like product may be optionally dried by spray drying to obtain a powdery product.

By separating the sugar from the reaction mixture and purifying the obtained mixture, the sugar composition of the present invention containing glucosyl transition sugars can be processed into a high sugar content product. Examples of such a separation method include a method of separating and removing impurities, that is, a method of removing coarse sugars by fermentation or yeast fermentation using yeast, yeast fermentation, membrane filtration and column chromatography, and alkaline reaction pH. And a method of heating the mixture to decompose the reducing sugars. More specifically, high glucosyl transition sugars are required by removing coarse saccharides by column chromatography using strong acid cation exchange resins disclosed in Japanese Patent Laid-Open Nos. 23,799 / 83 and 72,598 / 83. A method of removing the fraction sugars from the fraction can be advantageously used on an industrial scale. In this case, the normal stationary phase method, the mobile phase method, and the pseudo mobile phase method can be advantageously used.

The impurities-free solution is filtered and centrifuged to remove insoluble materials, followed by purification by decolorization with activated carbon and desalting with H and OH ion exchange resins to obtain a syrupy product. . If necessary, the syrup-like product may be dried by spray drying or the like to obtain a powdery product.

The sugar composition of the present invention containing the glucosyl transition sugar thus obtained usually contains at least 5%, preferably at least 10%, of glucosyl transition sugar on a solid basis.

The sugar composition of the present invention containing glucosyl transition sugars has a taste, sweetness, osmotic pressure control, moisturizing, glossing, anti-crystallization and anti-aging, caries prevention, non-feed bacteria growth promoting and mineral absorption promotion We are satisfied with sex. Since these properties and functions are satisfactory, the sugar composition of the present invention can be widely used in various compositions such as food, tobacco, feed, pet food, cosmetics, pharmaceuticals, moldings, daily food and agricultural products, chemical industry reagents and products. Can be.

The sugar composition of the present invention containing glucosyl transition sugar can be used as a sweetening seasoning as it is. If necessary, the sugar composition of the present invention may be prepared by adding an appropriate amount of one or more other sweeteners such as powdered syrup, glucose, maltose, trehalose, sucrose, isomerized sugar, honey, maple sugar, sorbitol, maltitol, lactitol, dehydrochalcone. , Stevioside, al-glycosyl stevioside, rebaudioside, glycyrrhizin, L-aspartyl-L-phenylalanine methyl ester, saccharin, glycine and alanine, and fillers such as dextrin, starch, lactose and the like. .

The sugar composition of the present invention has a sweetness that harmonizes well with various substances having a sour taste, bitter taste, salty taste, astringent taste, and sweet taste, and has good acid resistance and heat resistance. Therefore, it can be advantageously used as a sweetener, a taste improver and a quality improving agent in general foods.

The sugar composition of the present invention is soy sauce, powdered soy sauce, miso, powdered miso, moromi (unfiltered liquor), hiso (unfiltered soy sauce), furikage (seasoned fish meat), mayonnaise, dressing, vinegar, sun bai- ( Sauce mixed with sugar, soy sauce, vinegar), hunmatsu-sushi-su (powdered vinegar for sushi), chuka-no-moto (seasoning seasoning for Chinese elution), tentsuyu (Japanese soup for soup), and mentsuyu ( Premixes for pickled foods, such as Japanese noodle sauce), sauces, Ketchup, Takuan-Tsuke-no-moto (prepared for pickled radish), Hakusai-Tsuke-no-moto (premix for Chinese cabbage), Yaguniku-no- Tare (Japanese barbecue source), Curry Roo, Instant Stew Mix, Instant Soup Mix, Dashi-no-moto (Instant Stock Mix), Complex Seasoning, Mirin (Synthesis), Shin Mirin (Synthetic Mirin), It can be used for various seasonings such as table syrup and coffee syrup.

Sugar composition of the present invention is senbei (rice crackers), Arare-mochi (cuboid rice cake), Okoshi (cake with millet and rice), mochi (rice cake), manju (red bean jam dumplings), Uiro (sweet jelly) ), Anza (red bean jam), yokan (sweet red bean jelly), Mizu-yokan (soft azuki red bean jelly), king yoku (a type of yokan), jelly, pao de castella and amedama (Japanese toffee) Japanese confectionery; Confections such as bread, biscuits, crackers, cookies, pies, puddings, butter creams, custard creams, cream puffs, waffles, sponge cakes, donuts, chocolates, chewing gums, caramels and candies; Ice creams such as ice cream and sherbet; Syrups such as kajitsu-no-syrup-zuke (pickled fruit syrup) and kolimits (sugar syrup for shaved ice); Pastes such as flower paste, pine nut paste, fruit paste, and spreads; Processed foods of fruits and vegetables, such as jam, marmalade, syrup-zuke (fruit pickles), and toka (sweets); Pickles and pickles such as Fukujin-zuke (red radish pickle), beta-zuke (thick radish pickle), senmai-zuke (pickled radish), rakyo-zuke (pickled green onion); Meat products such as ham and sausage; Fish meat products such as fish ham, fish sausage, fish paste, chikuwa, and tempura; Uni-no-Shiokara (sea urchin), Ika-no-Shiokara (squid), Su-Konbu (processed kelp), Saki-Suruma (dried squid), Fugu-no-mirin-boshi Various delicacies such as dried blowfish seasoned with; Tsukuda (soy stewed foods) such as laver, wild vegetables, dried squid, fish, shellfish; Ordinary foods such as nima mash (roasted soybeans), potato salad, and Konbu-maki (Tashima roll); Dairy products; Canned and bottled foods such as meat, fish, fruit and vegetables; Alcoholic beverages such as sake, synthetic liquor, wine, and liqueur; Soft drinks such as coffee, black tea, cocoa, juice, carbonated drinks, lactic acid drinks and lactic acid bacteria drinks; Instant foods such as instant pudding mix, instant hot cake mix, instant shiruko (instant mix of Azuki bean porridge and rice cake), instant soup mix and the like; And sweetening of various foods such as baby food, therapeutic food, drink, rice, noodles and frozen food, improving taste and improving quality of the food.

In addition, the sugar composition of the present invention can be used for the purpose of improving palatability, such as feed, feed for breeding animals such as livestock, poultry and fish. The sugar composition of the present invention may be applied to pastes and other liquid compositions such as tobacco, toothpaste, lipstick, lipstick, lip balm, oral medicine, tablets, troches, cod liver oil drops, dry coolants, dry fragrances, gargles, cosmetics, pharmaceuticals, etc. It can be used advantageously as a sweetener, a taste improver, a quality improver, etc. in this.

The sugar composition of the present invention can be used as a quality improving agent and stabilizer, as well as in various physiologically active substances which tend to lose active ingredients, activities and the like, as well as in health foods and pharmaceuticals containing the physiologically active substances. Examples of such physiologically active substances include α-, β- and γ-interferon, tumor necrosis factor-α (TNF-α), tumor necrosis factor-β (TNF-β), macrophage liner repressor, colony stimulation Lymphokines such as factor, transfer factor, interleukin 1, 2, 6, 12, 15 and 18; Hormones such as insulin, growth hormone, prolactin, erythropoietin, tissue plasminogen activator, lanpo stimulating hormone and placental hormone; Biologics such as BCG vaccine, Japanese encephalitis vaccine, measles vaccine, polio live vaccine, smallpox vaccine, tetanus toxoid, pyruvate antiserpent antitoxin and human immunoglobulin; Antibiotics such as penicillin, erythromycin, chloramphenicol, tetracycline, streptomycin and kanamycin sulfate; Vitamins such as thiamine, riboflavin, L-ascorbic acid, liver oil, carotenoids, ergosterol and tocopherol; Enzymes such as lipase, elastase, urokinase, protease, β-amylase, isoamylase, glucanase and lactase; Extracts such as medicinal ginseng extract, grow extract, gloella extract, aloe extract and propolis extract; Live fungi such as viruses, lactic acid bacteria and yeasts; And other various physiologically active substances such as royal jelly. By using the sugar composition of the present invention, it is possible to easily prepare the above various physiologically active substances with high stability and high quality health foods and medicines without losing their active ingredient and activity or being inactive.

As described above, the term composition refers to oral and parenteral foods, cosmetics and pharmaceuticals, as well as everyday products, agricultural and aquatic products, chemical industry reagents and products. As a method of incorporating the sugar composition of the present invention into the various compositions described above, for example, mixing, mixing, dissolving, melting, dipping, infiltrating, spraying, coating, coating, spraying, solidifying injection, and the like include known methods. The sugar composition of the present invention is usually contained in various compositions in an amount of at least 0.1%, preferably at least 0.5%.

The present invention will be described in more detail by the following experiment.

Experiment 1: Preparation of trehalose phosphorylase

ATCC Catalog of BACTERIA AND BACTERIOPHAGES, 18th edition, pp. 452-456 (1992), the medium was prepared using 0.5 w / v% trehalose instead of 0.5 w / v% glucose as a carbon source, and the medium was prepared according to the method for preparing the medium for aeromonerbium broki. 100 ml of the solution was placed in a 100 ml pressurized container, and then inoculated with aeromoerbium broccoli ATCC 35047, and left at 60 ° C. for 48 hours to obtain a seed culture.

Take approximately 10 liters of the same fresh nutrient medium used for the production of the seed culture, place it in an 11 liter stainless steel bottle, heat it and sterilize it, cool it to 60 ° C and inoculate the seed culture with 1v / v% of 60 It was left incubated for about 40 hours at ℃.

About 40 L of the obtained culture was centrifuged to obtain 92 g of wet cells, then suspended in 10 mM phosphate buffer, sonicated and centrifuged to obtain supernatant of the crushed cell suspension. The activity of this supernatant was 0.3 unit / ml of trehalose phosphorylase.

Experiment 2: Trehalose Phosphorylase Purification

The supernatant of Experiment 1 was concentrated to UF membrane to give about 360 ml of enzyme concentrate with about 30 units / ml of trehalose phosphorylase activity.

300 ml of this enzyme concentrate was dialyzed against 10 mM phosphate buffer (pH 7.0) for 24 hours and centrifuged to remove insoluble material. 380 ml of the obtained supernatant was subjected to ion exchange column chromatography using 380 ml of DEAE-TOYOPEARL 650 GEL (gel for ion exchange column chromatography sold by Tosoh, Japan).

The trehalose phosphorylase of the present invention was eluted by adsorbing to this gel and feeding sodium chloride which increased from 0M to 0.5M under a linear gradient. An enzymatic activity fraction eluting from about 0.1 M sodium chloride was collected and collected together to purify the enzyme in the solution as follows. That is, the solution was dialyzed against the same fresh buffer containing 1.5 M ammonium sulfate, the dialysate was centrifuged to remove insolubles, and the supernatant was subjected to hydrophobic column chromatography using 100 ml of BUTYL-TOYOPEARL 650 GEL. Trehalose phosphorylase adsorbed on the gel was eluted under a linear gradient of ammonium sulfate decreasing from 1.5M to 0.5M to obtain an enzyme activity fraction.

These fractions were collected and subjected to gel filtration chromatography using 300 ml of ULTROGEL AcA44 RESIN (gel for gel filtration column chromatography sold by Sepracor / IBF s.a. of France), followed by enzymatically active fractions.

The yield of purified enzyme obtained in the above purification step was about 25% relative to the enzymatic activity of the supernatant of the crushed cell suspension. The specific activity of this enzyme was 78.2 units per 1 mg of protein. Proteins were quantified by Lowry method using bovine serum albumin as standard protein.

The purity of this sample was measured by gel electrophoresis using 7.5w / v% polyacrylamide and found to be a relatively high purity protein detected as a single protein band.

Experiment 3 Properties of Trehalose Phosphorylase

The trehalose phosphorylase sample in Experiment 2 was subjected to SDS-PAGE treatment using a 10w / v% gel. In parallel with this, the molecular weight of the enzyme was measured in comparison with the electrophoretic marker protein (sold by Japan Bio-Rad Laboratories, Japan). The molecular weight was 88,000 ± 5,000 Daltons, and TSKGel G4000SW ( The gel filtration using a column of 7.5 mm in diameter and 600 mm in length filled with a gel filtration gel sold by Tosoh Co., Ltd. in Japan was found to have a molecular weight of 190,000 ± 10,000 Daltons.

Purified trehalose phosphorylase was subjected to polyacrylamide gel electrophoresis using 2w / v% AMPHOLINE (Ampolite, sold by Pharmacia LKB Biotechnology AB, Sweden), and then the protein bands and gel pH were measured. As a result, the pI of this enzyme was 5.4 ± 0.5.

The effect of temperature and pH on the activity of trehalose phosphorylase of the present invention was investigated according to the enzyme activity assay. The enzyme was reacted at about 50-85 ° C. instead of 60 ° C. used in the enzyme test to investigate the effect of temperature. When examining the effect of pH, the enzyme was reacted at a pH of about 4-9 instead of the pH of the buffer used for the enzyme activity measurement. In both cases, the enzyme reaction was stopped and the produced glucose was quantified as in the case of the enzyme test. These results are shown in FIG. 1 and FIG. 2, which are shown as relative to the maximum as data on the effects of temperature and pH, respectively. The optimum temperature of the enzyme was about 70 ° C. for 30 minutes at pH 7.0 and the optimum pH was about 7.0 to 7.5 for 30 minutes at 60 ° C. The thermal stability of the enzyme was determined by dissolving the enzyme in 10 mM phosphate buffer (pH 7.0), holding it at about 40 to 85 ° C. for 1 hour, cooling and measuring the activity of the remaining enzyme according to the enzyme test. The pH stability of this enzyme was determined by dissolving the enzyme in buffers of different pHs of about 4 to 10, maintaining each enzyme solution at about 4 ° C for 24 hours, adjusting the pH of each solution to 7.0, and then remaining enzyme according to the enzyme assay. It was determined by measuring the activity of. These results are shown in FIGS. 3 and 4, which are shown relative to the maximum as data on the thermal and pH stability of the enzyme, respectively. The thermal stability of the enzyme was about 60 ° C. or lower and the pH stability was about 6.0-9.0. The activity of this enzyme was inhibited by 1 mM Cu ⁺⁺ , Pb ⁺⁺ , Zn ⁺⁺ , Hg ⁺⁺ , Mg ⁺⁺ or Mn ⁺⁺ .

Experiment 4: Partial amino acid sequence of trehalose phosphorylase

[Experiment 4- (1): N-terminal amino acid sequence]

A part of the purified enzyme prepared by the method of Experiment 2 was dialyzed against distilled water, and about 40 μg of the dialysis enzyme was used as a sample for N-terminal amino acid sequence analysis by protein weight. Analyzes were carried out from the N terminus to five amino acid residues using PROTEIN SEQUENCER MODEL 473A (apparatus sold by Applied Biosytems, USA). The partial amino acid sequence analyzed was SEQ ID NO: 1. More precise analysis using the same fresh enzyme label revealed that the enzyme had an amino acid sequence of SEQ ID NO: 6 at the N-terminus.

[Experiment 4- (2): Internal partial amino acid sequence]

A portion of the purified enzyme preparation prepared by the method of Experiment 2 was dialyzed against 10 mM Tris-HCl buffer (pH 9.0) and the dialysate was diluted with the same fresh buffer to a protein concentration of 1 mg / ml. To 1 ml of the obtained solution, 10 µg of lysyl endopeptidase (an enzyme product sold by Wako Pure Chemical Industries, Ltd., Japan) was added, followed by enzymatic reaction at 30 ° C. for 22 hours to generate a peptide. μBONDASPHERE C-18 COLUMN (diameter 2.1 mm, length 150 mm) (column for reverse phase HPLC sold by Waters Chromatography Div. of MILLIPORE, USA), flow rate: 0.9 ml / min, at room temperature and in 0.1 v / v% trifluoroacetic acid Peptides were separated by reverse phase HPLC (high performance liquid chromatography) under 120 min treatment conditions under a linear gradient of acetonitrile increasing from 0v / v% to 48v / v%.

Peptides eluted from the column were detected by measuring absorbance at 210 nm. Two peptides clearly separated from each other, TP10 with 66 minutes retention time and TP14 with 86 minutes retention time, were separately collected and dried in 200 μl of a solution of 0.1v / v% trifluoroacetic acid and 50v / v% acetonitrile. Dissolved. Each peptide was analyzed in a proutine sequencer to identify up to five amino acid residues from the N terminus. TP10 and TP14 represent the amino acid sequence of SEQ ID NO: 2 and SEQ ID NO: 3, respectively. As a result of a more precise analysis of the same fresh enzyme standard by the same assay, TP14 showed SEQ ID NO: 7 at its N terminus.

[Experiment 5: Substrate Specificity for Sugar Hydrolysis by Trehalose Phosphorase]

D-glucose, maltose, sucrose, lactose, trehalose, neotrehalose, cellobiose, melibiose, kojibiose, isomaltose, sophorose, gentiose, nigerose and laminaribiose, maltose A 10-per-g unit of saccharides, based on solids, is purified trehalose phosphorylase obtained by the method of Experiment 2 in an aqueous solution of one sugar selected from pentaose and 4-O-α-D-glucosyltrehalose. And maintained at 60 ° C. and pH 7.0 in the presence of 5 mM sodium hydrogen phosphate for 24 hours.

The concentration of sugar in each reaction solution was 2w / w%. The pre-reaction solution and the post-reaction mixture were subjected to thin layer chromatography (hereinafter simply referred to as TLC) using KIESELGEL 60 (20 x 20 cm) (aluminum plate for TLC sold by Merck, USA). Using 1-butanol / pyridine / water (= 7: 3: 1, volume ratio) as the developing solvent system, each sample was developed once at room temperature in the plate, and then sprayed with a 20v / v% sulfuric acid / methanol solution on the plate. And developed by heating at 110 ° C. for about 10 minutes. The spots of the solution and the mixture detected on each plate were compared to check whether the enzyme acted on each saccharide. The results are shown in Table 1.

TABLE 1

(Note) +: hydrolyzed by the action of trehalose phosphorylase of the present invention.

-: Not hydrolyzed by the action of trehalose phosphorylase of the present invention.

As shown in Table 1, the trehalose phosphorylase of the present invention exhibited strong substrate specificity for trehalose and acted on it to produce D-glucose and β-D-glucose-1-phosphate. It can be seen that it did not work on other sugars.

[Experiment 6: Specificity of Receptor in Glucosyl Transferring Sugar Reaction by Trehalose Phosphorylase]

Tablet trehalo obtained by the method of Experiment 2 in an aqueous solution in which β-D-glucose-1-phosphate as a sugar donor and one of monosaccharides, oligosaccharides and sugar alcohols in the same weight as the receptor shown in Table 2 were dissolved in dry weight. The os phosphorylase was mixed with 10 units per 1 g of β-D-glucose-1-phosphate and enzymatically reacted at 60 ° C. and pH 7.0 for 24 hours. As in Experiment 5, the pre-reaction solution and the post-reaction mixture were subjected to TLC treatment and the plate was then developed. The spots of each sample of the solution and the mixture detected on the plate were compared to determine whether the production of transition sugars was based on the newly detected spot of the mixture after the reaction. By visually observing the color development of the newly detected spots, the yield of transition sugars was relatively evaluated. The results are shown in Table 2.

TABLE 2

(Note)-: No production of glucosyl transition sugars.

+: Produces relatively small amounts of glucosyl transition sugars.

++: Relatively high amounts of glucosyl transsaccharide.

+++: Produces significant amounts of glucosyl transfer sugars.

As shown in Table 2, trehalose phosphorylase of the present invention is derived from D-xylose, D-galactose, D-glucose and 2-deoxy- from the sugar donor β-D-glucose-1-phosphate. It has been found to produce glucosyl transsaccharides by effectively transferring glucosyl groups towards reducing sugars as receptors such as D-glucose, D-fucose, L-fucose, glucosamine and N-acetyl glucosamine. Given the substrate specificity of the enzymes of the present invention, these glucosyl transition sugars were determined to be non-reducing sugars. When α-D-glucose-1-phosphate was used as a sugar donor instead of β-D-glucose-1-phosphate, no glucosyl transsaccharide was produced.

Some of the glucosyl transition sugars found to be produced through the sugar transfer reaction by the trehalose phosphorylase of the present invention in Experiment 6 will be described in detail with reference to Experiments 7 and 8 below. do.

[Experiment 7: Glucosyl Transsaccharide from D-glucose and β-D-glucose-1-phosphate]

A part of the reaction mixture obtained using D-glucose and β-D-glucose-1-phosphate as substrates in Experiment 6 was analyzed by gas chromatography (hereinafter simply referred to as GLC). A portion of the reaction mixture was diluted with 10 mM phosphate buffer (pH 7.0) to a concentration of 1%, and 0.5 ml of a trehalose product (Sigma Chemical, USA) was added thereto, followed by enzymatic reaction at 45 ° C. for 20 hours. . A portion of the reaction mixture, treated or untreated with trehalose, was dried and dissolved in pyridine to trimethylsilylated. The obtained product was subjected to nitrogen gas as a carrier gas using a stainless steel column 3 mm in diameter and 2 m in length filled with 2% of SILICONE OV-17 / CHROMOSOLB W (resin for GLC sold by GL Sciences of Japan). Gas chromatography (hereinafter, simply referred to as GLC) analysis was performed under conditions of a temperature of 160 to 320 ° C and a temperature increase rate of 7.5 ° C / min. The sugar component was detected by a hydrogen salt ionization detector.

As a result, the retention time of the peak of the glucosyl transition sugar produced from D-glucose and β-D-glucose-1-phosphate by the action of trehalose phosphorylase of the present invention is that of pure trehalose. A match was found, and it was found that the peak and the produced D-glucose were extinguished by treatment with trehalose. Considering the substrate specificity of trehalose, this glucosyl transition sugar was assumed to be trehalose.

Experiment 8: Glucosyl-D-Galactoside

In order to confirm the glucosyl transfer sugar produced through the sugar transfer reaction to D-galactose by trehalose phosphorylase of the present invention, glucosyl transfer sugar was prepared and separated, and then the structure was confirmed. The method is as follows. That is, an aqueous solution containing 5% trehalose, 2.5% D-galactose and 5 mM sodium dihydrogen phosphate was prepared, the aqueous solution was adjusted to pH 5.0, and the solution was then purified by the method of Experiment 2 15 units of 1 g of β-D-glucose-1-phosphate was added thereto and reacted at 60 ° C. for 72 hours, and the reaction mixture was heated at 100 ° C. for 10 minutes to inactivate remaining enzymes. GLC analysis was performed on the sample obtained according to the method of Experiment 7. As a result, it was confirmed that the reaction mixture contained a relatively large amount of substances having a residence time different from that of trehalose, D-galactose, D-glucose and β-D-glucose-1-phosphate. It was assumed to be sugar. The yield of sugar was about 30% based on this GLC data. The remaining reaction mixture was adjusted to pH 7.0 and the remaining trehalose was added to 25 units / g, followed by enzymatic reaction at 45 ° C. for 20 hours to decompose the remaining trehalose in the reaction mixture. The product was heated at 100 ° C. for 10 minutes to inactivate remaining trehalose, decolorized with activated carbon, filtered, desalted and purified with H-type and OH-type ion exchange resins, and concentrated to a concentration of about 50%. After grafting, high fractions of glucosyl transition were collected.

The resin used for the fraction was XT-1016 (alkali metal strong acid type cation exchange resin, Na type, crosslinking degree 4% sold by Tokyo Organic Chemical Industries, Japan). The resin was suspended in water and filled into four jacketed stainless steel columns having a diameter of 3 cm and a length of 1 m, and then connected in series so that the total gel layer depth was about 4 m. While maintaining the internal temperature of the column at 40 ° C, the sugar solution was supplied to the column at a volume of 5v / v% to the resin, and 40 ° C hot water was passed through the column at a flow rate of SV (space velocity) of 0.15 to fractionate the sugar solution. , High glucosyl sugar fraction was collected.

Each fraction was collected, desalted, purified, and concentrated to give a concentration of approximately 40%, followed by chromatography on a column filled with YMC-Pack OSD (octadecyl silica gel sold by YMC, Japan) to transfer glucosyl. A fraction containing sugar was collected. These fractions were collected together, concentrated to a concentration of about 40%, and the above-mentioned column chromatography was performed again. The obtained glucosyl transition sugar high content solution was desalted, purified, concentrated and dried under vacuum to obtain a sugar-containing powder product at a yield of about 20% based on solids relative to the raw sugar used in the enzymatic reaction. GLC analysis of the powder product according to the method of Experiment 7 revealed that it contained about 98% of glucosyl transition sugar on a solid basis.

GLC analysis of the high-glucose transition sugar-containing powder product with acid showed that the sugar produced D-glucose and D-galactose in a molar ratio of about 1: 1 when it was degraded to acid. The powdered product was methylated, hydrolyzed with acid, reduced and acetylated to give partial methylhexitol acetate, followed by GLC analysis to give 2,3,4,6-tetra-O-methyl-1,5-di -O-acetylglucitol and 2,3,4,6-tetra-O-methyl-1,5-di-O-acetylglucitol were detected. From this data, the glucosyl transition sugar is composed of 1: 1 molar ratio of D-glucose and D-galactose, and the OH group of C-1 of D-glucose and the C-1 OH group of D-galactose between these sugars It was confirmed that it is involved in binding.

In order to confirm the structure of glucosylgalactoside in more detail, specific light intensity and ¹³ C-NMR spectrum of the saccharide were measured. As a result, the following measurements were obtained: specific light intensity [α] ^D ₂₀ = + 223 ° (c = 0.97, H ₂ O); ¹³ C-NMR spectra (100 MHz, D ₂ O): sigma ppm from TSP: 96.16, 95.97, 75.37, 74.94, 74.14, 73.90, 72.53, 72.11, 71.80, 70.76, 64.03 and 63.37.

These data were in close agreement with the true data of the chemically synthesized compound α-D-galactopyranosyl α-D-glucopyranoside, from which the glucosylgalactoside was derived from glucosyl-D-galactoside, namely α- As D-galactopyranosyl α-D-glucopyranoside, it was confirmed that D-glucose and D-galactose were disaccharides bound to each other through α-1,1 binding.

[Experiment 9: Acute Toxicity Test]

Glucosyl-D-galactosid powdery product prepared by the method of Experiment 8, Glucosyl-D-xyloxide high powdery product prepared by the method of Example A-12, method of Example A-14 Acute toxicity test of glucosyl-D-fucoside-containing powdery product prepared by the method and glucosyl-L-fucoside-containing powdery product prepared by the method of Example A-15 was orally administered to 7-week-old dd mice, respectively. Was tested. As a result, there was no death of the mouse even when the maximum dose of 50 g / kg of mouse body weight was administered. These data show that these sugars are extremely low in toxicity.

Example A describes a method for preparing a sugar containing trehalose phosphorylase of the present invention, a DNA that codes for this enzyme, and a glucosyl transition sugar prepared using this enzyme. Does not limit the invention.

Example A-1 Enzyme Solution

The Dermaeoerbium brokyi ATCC 35047 was incubated for about 30 hours using a fermenter under anaerobic conditions according to the method of Experiment 1 with the temperature set to 65 ° C. in the same fresh medium used in Experiment 1. The obtained culture was centrifuged to obtain the cells, which were then crushed and centrifuged. The supernatant was fed to a column packed with DEAE-TOYOPEARL GEL, adsorbed to the gel, eluted from the column by feeding an aqueous solution under a linear gradient of sodium chloride from 0M to 0.5M, and then eluted with about 0.1M sodium chloride. Halose phosphorylase active fractions were collected. By collecting these fractions together and concentrating on an ultrafiltration membrane, an enzyme solution containing about 20 units / ml of trehalose phosphorylase was obtained in a yield of about 40% with respect to the total activity of the raw material culture.

Example A-2 DNA Preparation

In accordance with the method of Experiment 1, the dermoererobium brokyi ATCC 35047 was inoculated into the same fresh nutrient medium as Experiment 1 and incubated at 60 ° C. for 24 hours. Proliferated cells were separated from the culture by centrifugation and suspended in an appropriate amount of Tris-EDTA-saline buffer (hereinafter simply referred to as TES buffer), followed by adding lysozyme 0.05w / v% to the cell suspension and at 37 ° C. Incubate for 30 minutes. Subsequently, the enzyme-treated mixture was frozen at -80 ° C for 1 hour, and then TES buffer (pH 9.0) was added thereto, followed by addition of TES buffer-phenol mixture at 60 ° C, followed by sufficiently stirring and cooling to centrifugation. Was collected. Two times the volume of cold ethanol was added thereto, the resultant precipitate was taken, dissolved in an appropriate amount of SSC buffer (pH 7.1), and then mixed with 7.5 μg of ribonuclease and 125 μg of protease, and maintained at 37 ° C. for 1 hour. A mixture of chloroform and isoamyl alcohol was added to the obtained mixture, which was then left to stir and the resulting upper layer was collected. Cold ethanol was added thereto, and the resulting precipitate was collected, washed with 70v / v% cold ethanol, and dried under vacuum to obtain DNA. This DNA was dissolved in SSC buffer (pH 7.1) to a concentration of about 1 mg / ml and frozen at -80 ° C.

Example A-3 Preparation of Transformant and Recombinant DNA

1 ml of the DNA solution of Example A-2 was placed in a container, and about 20 units of restriction enzyme Alu I was added thereto and held at 37 ° C. for 30 minutes to partially digest the DNA. The resulting mixture was ultrafiltered with sucrose density to collect DNA of about 2,000 to 5,000 base pairs. In parallel, Bluescript II SK (+) (plasmid vector sold by Stratagene Cloning Systems, Inc., USA) was completely digested with restriction enzyme Sma I, and 0.3 μg of the digested vector and about 3 μg of DNA fragment were digested with DNA LIGATION KIT (Takara Shuzo, Japan). Corp.) was connected according to the method attached to this kit. Using the obtained recombinant DNA, 100 μg of EPICURIAN COLI XL1 BLUE (microorganism belonging to Escherichia coli sold by Stratagene Cloning Systems, Inc., USA) was transformed by conventional immunocytometry to obtain a gene library.

10 g / l of tryptone, 5 g / l of yeast extract, 5 g / l of sodium chloride, 75 mg / l of ampicillin sodium salt and 5-bromo-4-chloro- Inoculate agar plates containing 50 mg / l of 3-indolyl-β-galactoside (pH 7.0), incubate for 18 hours at 37 ° C, and then approximately 5,000 white colonies on the plates are HYBOND-N + (Amersham, USA). Nylon membrane). Based on amino acids 9-15 of the N-terminal region of SEQ ID NO: 6 found in Experiment 4, an oligonucleotide having a nucleotide sequence represented by 5'-TAYCCNTTYGARGAYTGGGT-3 'was chemically synthesized and [γ- ³² P ] Labeled with ATP and T4 polynucleotide kinase to obtain synthetic DNA as primary probe. Of the colonies immobilized on the nylon membrane, the primary probe and three colonies strongly hybridized were selected by conventional colony hybridization method. These three colonies were fixed on the nylon membrane as described above. Based on the amino acid sequence of SEQ ID NO: 7 found in Experiment 4, an oligonucleotide having the nucleotide sequence represented by 5'-AAYTAYGAYTAYTAYGARCC-3 was chemically synthesized and [γ- ³² P] ATP and T4 polynucleotide kinase Labeling to obtain a synthetic DNA as a secondary probe. Of the three colonies immobilized on the nylon membrane, one colony strongly hybridized with a secondary probe was selected by conventional colony hybridization and named TTP4 as a transformant.

Transformant TTP4 was inoculated conventionally in L-broad (pH 7.0) containing 100 μg / ml of the sodium salt of ampicillin and incubated at 37 ° C. under rotary shaking for 24 hours. After the incubation, the culture was centrifuged to obtain the cells, and then the recombinant DNA was extracted by treating with a conventional alkali-SDS method. Conventional dideoxy analysis of recombinant DNA revealed that DNA having a nucleotide sequence of SEQ ID NO: 8 in 3,345 base pairs derived from ATCC 35047. As shown in SEQ ID NO: 8, the nucleotide sequence of base pairs 596-2,917 of SEQ ID NO: 8 was found to be an amino acid sequence of 774 amino acids.

A comparison was made between the amino acid sequence obtained from the nucleotide sequence and the N-terminal and internal amino acid sequences identified in Experiment 4, that is, SEQ ID NO: 1 water 3 and SEQ ID NO: 6-7. (SEQ ID NO :) 1-3 correspond to amino acids 2-6, 308-312, and 633-637 of SEQ ID NO: 8, respectively, while SEQ ID NO: 6-7 And amino acids 2 to 31 and 633 to 647 of SEQ ID NO: 8, respectively.

From this data, the trehalose phosphorylase of the present invention has an amino acid sequence of SEQ ID NO: 4, and the enzyme of Dermaeoerobium brochyy ATCC 35047 is SEQ ID NO: 5 It can be seen that it is coded by DNA having a nucleotide sequence of. Recombinant DNA prepared by the method described above and found to have its nucleotide sequence was named pTTP4. As shown in Figure 5, the recombinant DNA is located downstream of the recognition site by restriction enzyme Pst I.

Example A-4: Preparation of Trehalose Phosphorase by Transformation Agent

100 ml of an aqueous solution containing 16 g / l polypeptone, 10 g / l yeast and 5 g / l sodium chloride was placed in a 500 ml Erlenmeyer flask, autoclaved at 121 ° C. for 15 minutes, cooled, and aseptically adjusted to pH 7.0. 10 mg of sodium salt of ampicillin was aseptically added thereto to obtain liquid nutrient medium. The transformant TTP4 of Example A-3 was inoculated into this medium and cultured at 37 ° C. under aeration for about 20 hours to obtain a seed culture. According to the production method of the seed culture, 7 liters of the same fresh nutrient medium used for the seed culture was placed in a 10 liter fermenter, and then inoculated with 70 ml of the seed culture and incubated for about 20 hours under aeration. The obtained culture was centrifuged in the conventional manner to collect the cells, which were then suspended in 10 mM phosphate buffer (pH 7.0) and sonicated to disrupt the cells, and then centrifuged to remove impurities and the supernatant was recovered. The supernatant was dialyzed against 10 mM phosphate buffer, and the trehalose phosphorylase activity in the supernatant was measured, resulting in about 500 units of enzyme per liter of culture.

As a primary control, Escherichia coli XLI-Blue strains were cultured by inoculation in nutrient medium as in the hemoglobin convertor culture, except that ampicillin was not added to the medium in this case. The grown cells were disrupted and the supernatant was collected and dialyzed. As a second control, the dermoererobium brokyi ATCC 35047 was incubated at 60 ° C. in a nutrient medium containing the same ingredients, except that ampicillin was not used in this case. As in the case of the transformants, the cells in the culture were disrupted and the supernatant was collected and dialyzed. No activity of this enzyme was detected in the dialysate as the primary control. The dialysate as a secondary control showed about 2 units of enzymatic activity per liter of culture, which was much lower than for transformant TTP4.

According to the method of Experiment 2, the dialysate of Example A-4 was purified by column chromatography using DEAE-TOYOPEARL 650 GEL and ULTROGEL AcA44 RESIN, and the purified enzyme was analyzed according to the method of Experiment 3, Molecular weight is 88,000 ± 5,000 Daltons, molecular weight is 190,000 ± 10,000 Daltons by gel filtration chromatography, isoelectric point by isoelectric point fractionation using polyacrylamide gel is 5.4 ± 0.5, optimum temperature is about 70 ℃, optimum pH is about 7.0∼ 7.5, thermal stability below 60 ° C., and pH stability of about 6.0 to 9.0, all of which were substantially the same as for the enzymes prepared in Experiments 1 and 2. From these results, it can be seen that the trehalose phosphorylase of the present invention can be sufficiently produced by recombinant DNA technology, and thus the yield of enzyme can be significantly increased.

Example A-5: Enzyme solution

The transformant TTP4 of Example A-3 was cultivated in a nutrient medium by the method of Example A-4, the cells obtained by centrifugation of the culture were crushed by ultrasound, and then trestled to the supernatant in the crushed cell suspension. Haloose phosphorylase activity was measured and found to be about 0.7 units per liter of culture. The supernatant was concentrated to an ultrafiltration membrane and the concentrate was dialyzed to obtain an enzyme solution of about 10 units / ml of trehalose phosphorylase activity in a yield of about 70% relative to the total enzymatic activity of the culture.

Example A-6 Trehalose-Containing Sugar Solution

Trehalose force prepared by the method of Example A-1 with 5 units of bacterial maltose phosphorylase, commercially available in 25 mM dihydrogen potassium phosphate-citrate buffer containing 5% maltose (pH 6.0), per 1 g of maltose. 50 units of 1 g of polylase maltose were added, and then incubated at 30 ° C. for 120 hours. The reaction mixture was heated at 100 ° C. for 30 minutes to inactivate and cool the remaining enzyme, decolorize with activated carbon in the usual way, desalter with H-type and OH-type ion exchange resins after filtration, and concentrate by concentrating. One 75% syrup phase sugar solution was obtained with a yield of about 95% relative to the solid sugar raw material.

This product contains about 45% of trehalose per solid and has good sweetness and moderate viscosity and moisturizing properties. Therefore, it is a sweetener, taste improver, stabilizer, bifid bacteria growth accelerator and It can be advantageously used as a mineral absorption promoter.

Example A-7 Trehalose-High Powder

The sugar solution containing about 45% of trehalose per solid prepared by the reaction and purification in Example A-6 was used as a raw material, so that the concentration was about 20% per solid, followed by glucose per 1 g of solid. 5 units of amylase were added and maintained at pH 4.5 and 40 占 폚 for 16 hours to decompose the remaining maltose. The reaction mixture was heated at 100 ° C. for 30 minutes to stop the enzyme reaction and concentrated to about 40% solution. In order to increase the trehalose content, a water suspension of XT-1016 (alkali metal strong acid type cation exchange resin, Na type, crosslinking degree 4% sold by Tokyo Organic Chemical Industries, Japan) was filled with a diameter of 3 cm and a length of 1 m. Connect four jacketed stainless steel columns in series to make the total gel layer depth of about 4m. Pass the concentrate through 5v / v% of this resin, and supply 40 ℃ hot water to the column at a flow rate of SV 0.15. The solution was fractionated and then a trehalose-rich fraction was collected. Each fraction was combined, concentrated, dried under vacuum and powdered to give a trehalose-containing powder in about 40% yield relative to the raw material per solid.

This product contains about 95% of kojibiose per solid and has good sweetness and moderate moisturizing properties.It is a sweetener, taste improver, stabilizer, bifid bacteria growth accelerator and mineral absorption accelerator in food, cosmetics, medicine and various moldings. It can be used to advantage.

Example A-8 Glucosyl-D-Galactoside-Containing Sugar Solution

An aqueous solution containing 5% trehalose, 5% D-galactose and 5 mM sodium dihydrogen phosphate was adjusted to pH 5.0 and trehalose phosphorylase obtained by the method of Example A-1 was treated with trehalose. 10 units per 1 g of loose were added and subjected to enzymatic reaction at 60 ° C. for 72 hours. The reaction mixture was heated at 90 ° C. for 30 minutes to inactivate and cool down the remaining enzyme, decolorized with activated carbon in a conventional manner, filtered, desalted, purified with H type and OH type ion exchange resins, and concentrated again to glucosyl sorbose. A 75% syrup phase sugar solution containing was obtained in a yield of about 95% relative to the raw material of the solid sugar.

This product contains about 22% of trehalose per solid, has good sweetness and moderate viscosity and moisturizing properties, so it is a sweetener, taste improver, stabilizer, bifid bacteria growth accelerator, in foods, cosmetics, medicines and various moldings. It can be advantageously used as a mineral absorption promoter.

Example A-9 Glucosyl-D-galactose-containing Sugar Solution

An aqueous solution containing 10% trehalose, D-galactose and 5 mM sodium dihydrogen phosphate was adjusted to pH 6.0 and trehalose the trehalose phosphorylase obtained by the method of Example A-1. 30 unit per 1g was added, and the enzyme reaction was carried out at 60 degreeC for 96 hours. The reaction mixture was heated at 90 DEG C for 30 minutes to inactivate and cool down the remaining enzyme, and to this, 5% of commercially available baker's yeast was added by wet weight, 1N sodium chloride was added to adjust the pH to 5-6, and the reaction temperature was adjusted. D-glucose in the reaction mixture was assimilated while maintaining at 27 ° C. for 6 hours. The reaction mixture was centrifuged to remove yeast, and the obtained supernatant was decolorized with activated carbon, filtered, desalted with H type and OH type ion exchange resins, purified, and concentrated to concentrate 75% syrup per solid to a raw material per solid. About 65% yield.

This product contains about 40% of glucosyl-D-galactoside per solid and has a high quality sweetness, moderate viscosity and moisturizing properties, so it is a sweetener, taste improver, stabilizer, and non-feed in food, cosmetics, pharmaceuticals and various moldings. It can be advantageously used as a bacterial growth promoter and a mineral absorption promoter.

Example A-10 Glucosyl-D-Galactoside High Powder

The sugar solution containing about 22% of glucosyl-D-galactoside per solid prepared by the reaction and purification in Example A-8 was used as a raw material to have a concentration of about 45% per solid. Diameter filled with water suspension of XT-1016 (alkali metal strong acid type cation exchange resin, Na type, 4% crosslinking degree) sold by Tokyo Oranic Chemical Industries of Japan in order to increase the content of glucosyl-D-galactoside After connecting four stainless steel columns of 3 cm in length and 1 m in series, the total gel layer depth was about 4 m, and 5v / v% of solution was passed through the resin. Hot water at 40 ° C. was supplied to the column at a flow rate of SV 0.15 to fractionate the solution, and then a high glucosyl-D-galactosid fraction was collected. Each fraction was combined, concentrated, dried under vacuum and powdered to give a glucosyl-D-galactosid high powder in about 25% yield relative to the raw material per solid.

This product contains about 90% of glucosyl-D-galactoside per solid and has a high quality sweetness, moderate viscosity and moisturizing properties, so it is a sweetener, taste improver, stabilizer, and bifeed in food, cosmetics, pharmaceuticals and various moldings. It can be advantageously used as a bacterial growth promoter and a mineral absorption promoter.

Example A-11 Glucosyl-D-Xycloside-Containing Sugar Solution

An aqueous solution containing 5% trehalose, 2.5% D-xylose and 5 mM sodium dihydrogen phosphate was adjusted to pH 5.0, to which trehalose phosphorila prepared by the method of Example A-1. The enzyme was added to 15 units of 1 g of trehalose and subjected to enzymatic reaction at 60 ° C. for 72 hours. The reaction mixture was heated at 90 ° C. for 30 minutes to inactivate and cool down the remaining enzymes, and after decolorization with activated carbon in a conventional manner, filtered, desalted with H-type and OH-type ion exchange resins, and purified and concentrated to about 75% syrup. Was obtained in about 95% yield relative to the raw material per solid.

This product contains about 20% of glucosyl-D-xyloxide per solid, and has high quality sweetness, moderate viscosity and moisturizing property, so it can be advantageously used for food, cosmetics, medicine and various moldings.

Example A-12 Glucosyl-D-Xycloside High Powder

The sugar solution containing about 20% of glucosyl-D-xyloxide per solid prepared by the reaction and purification in Example A-11 was used as a raw material to have a concentration of about 45% per solid. The solution was prepared according to the method of Example A-10 except that DOWEX 50WX4 (Ca type) (alkaline earth metal strong acid type cation exchange resin sold by Dow Chemical, USA) was used to increase the content of glucosyl-D-xyloxide. Column chromatography was performed to obtain a high glucosyl-D-xyloxide fraction. Each fraction was combined, purified, concentrated, dried in vacuo and powdered to give a glucosyl-D-xyloxide high powder containing about 25% yield per solid.

This product contains about 60% of glucosyl-D-xyloxide per solid and has high quality sweetness, moderate viscosity and moisturizing property, so it can be advantageously used for food, cosmetics, medicine and various moldings.

Example A-13: Glucosyl-D-fucoside-containing sugar solution

An aqueous solution containing 5% trehalose, 2.5% D-fucose and 5 mM dihydrogen phosphate was adjusted to pH 5.0, and the trehalose phosphorylase obtained by the method of Example A-1 was treated with tre 20 units per 1 g of haloose were added and subjected to enzymatic reaction at 60 ° C. for 72 hours. The reaction mixture was heated at 90 DEG C for 30 minutes to inactivate and cool down the remaining enzyme, decolorize and filter in a conventional manner, purify with H-type and OH-type ion exchange resins, and concentrate again to obtain approximately 75% syrup as a raw material per solid. Obtained in about 95% yield.

This product contains about 20% of glucosyl-D-fucoside per solid, and has high quality sweetness, moderate viscosity and moisturizing property, so it can be advantageously used for food, cosmetics, medicine and various moldings.

Example A-14 Glucosyl-D-fucoside High Powder

An aqueous solution containing 5% trehalose, 2.5% D-fucose and 5 mM dihydrogen phosphate was adjusted to pH 5.0, and the trehalose phosphorylase obtained by the method of Example A-1 was treated with tre 20 units per 1 g of haloose were added and subjected to enzymatic reaction at 60 ° C. for 72 hours. The reaction mixture was heated and cooled at 100 ° C. while maintaining an alkaline pH of pH 10 or higher, filtered after decolorization with activated carbon in a conventional manner, desalted with H-type and OH-type ion exchange resins, purified and concentrated again to glucosyl-D A powder containing fucoside was obtained in a yield of about 60% relative to the raw material per solid.

This product contains about 50% of glucosyl-D-fucoside per solid, and has high quality sweetness, moderate viscosity and moisturizing property, so it can be advantageously used for food, cosmetics, medicine and various moldings.

Example A-15 Glucosyl-L-fucoside-containing Sugar Solution

An aqueous solution containing 5% trehalose, 2.5% D-fucose and 5 mM sodium dihydrogen phosphate was adjusted to pH 6.0 and tre Treose phosphorylase obtained by the method of Example A-1 15 units per 1 g of haloose were added and subjected to enzymatic reaction at 60 ° C. for 72 hours. The reaction mixture was heated at 90 ° C. for 30 minutes to inactivate and cool down the remaining enzyme, and then, after decolorization with activated carbon in a conventional manner, filtered, desalted, purified with H-type and OH-type ion exchange resins, and concentrated again to obtain glucosyl-. Powder containing L-fucoside was obtained in about 95% yield relative to the raw material per solid.

This product contains about 20% of glucosyl-L-fucoside per solid, and has high quality sweetness and moderate moisturizing property, so it can be advantageously used for food, cosmetics, medicine and various moldings.

Example A-16 Trehalose-Containing Sugar Solution

Trehalose force prepared by the method of Example A-5 with 5 units of commercially available bacterial maltose-phosphorylase in 25 mM hydrogen dipotassium potassium phosphate-citrate buffer containing 5% maltose (pH 6.0) and maltose 50 unit per 1 g of maltose was added, followed by enzymatic reaction at 120C for 120 hours. The reaction mixture was heated at 100 ° C. for 30 minutes to inactivate and cool down the remaining enzyme, decolorize with activated carbon in the usual manner, desalter with H-type and OH-type ion-exchange resins after filtration, and concentrate again to concentrate about 75% syrup. A yield of about 95% was obtained for raw materials per solid.

This product contains about 45% of trehalose per solid and has a high quality sweetness, moderate viscosity and moisturizing properties, so it is a sweetener, quality improver, stabilizer, bifid bacteria growth accelerator, in food, cosmetics, pharmaceuticals and various moldings. It can be advantageously used as a mineral absorption promoter.

Example A-17: Glucosyl-D-fucoside-containing sugar solution

An aqueous solution containing 5% trehalose, 2.5% D-fucose and 5 mM sodium dihydrogen phosphate was adjusted to pH 5.0 and tre Treose phosphorylase obtained by the method of Example A-5 was added to tre 20 units per 1 g of haloose were added and subjected to enzymatic reaction at 60 ° C. for 72 hours. The reaction mixture was heated at 90 ° C. for 30 minutes to inactivate and cool down the remaining enzyme, and to decolorize with activated carbon in the usual manner, desalted with H-type and OH-type ion exchange resins after filtration, and then purified and concentrated to give about 75% syrup. A yield of about 95% was obtained for raw materials per solid.

This product contains about 20% of glucosyl-D-fucoside per solid, and has high quality sweetness, moderate viscosity and moisturizing property, so it can be advantageously used for food, cosmetics, medicine and various moldings.

Example B below describes the sugar composition of the present invention containing glucosyl transition sugars.

Example B-1 Sweetener

0.05 parts by weight of αG SWEET (α-glycosyl stevioside sold by Toyo Seiki Co., Ltd., Japan) was added to 1 part by weight of the high glucosyl-D-galactosid powder prepared by the method of Example A-10. Powdered sweeteners were prepared by uniformly mixing.

It is a high-quality sweetener with about twice the sweetness of sucrose and about half the calories as sucrose. Therefore, it is a low-calorie sweetener that can be used satisfactorily to sweeten low-calorie foods for people who have limited calorie intake, such as obese people and diabetics. In addition, since the product produces less acid by caries-inducing bacteria and less insoluble glucan, it can be used for sweetening foods that suppress tooth decay.

Example B-2 Hard Candy

30 parts by weight of a sugar solution containing glucosyl-D-galactoside prepared in Example A-9 was added to 80 parts by weight of hydrogenated maltose starch with a water content of 25%, mixed and dissolved, and the resulting solution was water-containing. Concentrated under reduced pressure until less than about 2%, mixed with 1 part by weight of citric acid and an appropriate amount of lemon flavor and colorant, and then kneaded and molded the mixture to prepare a hard candy. This product has high quality sweetness, low moisturizing property and is good at biting without dissolving.

Example B-3: Chewing Gum

To 4 parts by weight of the high glucosyl-D-xyloxide powder prepared by the method of Example A-12, 2 parts by weight of a gum base and 3 parts by weight of glucose, which were softened and melted, were added, and an appropriate amount of peppermint perfume was added thereto. The chewing gum was then prepared by kneading in a roll to mold the mixture. This product has good texture and flavor.

Example B-4: Chocolate

40 parts by weight of cacao paste, 10 parts by weight of cacao butter, 10 parts by weight of sucrose and 15 parts by weight of skim milk were mixed with 15 parts by weight of a high glucosyl-D-fucoside powder prepared by the method of Example A-14. The mixture was passed through a refiner to refine the particle size, and then the mixture was placed in a cone and 0.5 parts by weight of lecithin was kneaded at 50 ° C. for two days. The kneaded mixture was molded and solidified in a molding machine to prepare chocolate. The product is whitening of fat and sugar, has a good taste and flavor, and melts well on the tongue.

Example B-5: Cuddard Cream

500 parts by weight of corn starch, 500 parts by weight of maltose and 5 parts by weight of salt were added to 400 parts by weight of the high glucosyl-L-fucoside powder prepared by the method of Example A-15, and the mixture was passed through a sieve and thoroughly mixed. Then, 1,400 parts by weight of eggs were added and stirred, and 5,000 parts by weight of boiled milk was slowly added thereto, and stirring was continued while heating. Custard creams were prepared by stopping heating, cooling and adding a small amount of vanilla flavor when the corn starch became fully translucent and translucent. It has a smooth surface and a good taste with no strong sweetness.

Example B-6: Uiro (starch paste)

90 parts by weight of glucosyl-D-fucoside-containing sugar solution prepared by the method of Example A-13 was added with 90 parts by weight of rice flour, 20 parts by weight of corn starch, 20 parts by weight of sugar, 1 part by weight of green tea powder, and an appropriate amount of water. After kneading, steamed for 60 minutes in a container to prepare a powdered green tea iro. This product has good gloss and bite, good flavor and taste. In addition, starch aging is also well suppressed and stable for a long time.

Example B-7: Betara-zuke (beef pickle)

1 part by weight of glucosyl-D-galactoside-containing sugar solution prepared by Example A-8, 3 parts by weight of maltose, 0.05 parts by weight of licorice preparation, 0.008 parts by weight of malic acid, 0.07 parts by weight of sodium glutamate, and 0.03 parts of potassium sorbate By weight and 0.2 parts by weight of pullulan evenly mixed to prepare a beta-zuke raw material. 30 kg of radishes were first marinated in a conventional manner and then marinated in sugar, and then beta-zuke was prepared by seasoning with 4 kg of beta-zuke raw material. This product has good color, gloss and fragrance, moderate sweetness and good bite.

Example B-8: Lactic Acid Bacteria Beverage

130 parts by weight of the glucosyl-D-xyloxide-containing sugar solution prepared by the method of Example A-11, 175 parts by weight of skim milk, and 50 parts by weight of NYUKAOLIGO (Lactose sucrose high powder sold by Japan Corporation). It melt | dissolved in 1,150 weight part, this solution was sterilized for 30 minutes at 65 degreeC, it cooled to 40 degreeC, 30 weight part of lactic acid bacteria as a starter was inoculated by normal method, and the required product was manufactured by incubating at 37 degreeC for 8 hours. This product is a beverage containing lactic acid bacteria and has good taste and aroma. This product contains oligosaccharides that stabilize bacteria and promote growth.

Example B-9 Skin Cream

2 parts by weight of polyoxyethylene glycol monostearate, 5 parts by weight of self-emulsifying glycerin monostearate, 2 parts by weight of α-glycosyl rutin, to 4 parts by weight of high trehalose-containing powder prepared by the method of Example A-7 Part, 1 part by weight of liquid paraffin, 10 parts by weight of glycerol trioctanoate and an appropriate amount of preservative were added, and the mixture was dissolved by heating in a conventional manner. 5 parts by weight of 1,3-butylene glycol and 66 parts by weight of purified water were added thereto, emulsified with a homogenizer, and an appropriate amount of perfume was mixed to prepare a skin cream. This product is a cream that has good spreadability and can be advantageously used as a sun sooty agent, skin cleanser, and skin whitening agent.

Example B-10: Toothpaste

45 parts by weight of calcium hydrogen phosphate, 1.5 parts by weight of sodium lauryl sulfate, 25 parts by weight of glycerin, 0.5 parts by weight of polyoxyethylene sorbitan laurate, 0.02 parts by weight of saccharin and 0.05 parts by weight of preservative, and 13 parts by weight of water. Toothpaste was prepared by mixing 15 parts by weight of the trehalose-containing sugar solution prepared by the method of 6. This product is suitable for use as toothpaste because of its excellent gloss and cleansing power.

Example B-11: Landscape Nutrients

A composition consisting of the following components was prepared: 80 parts by weight of the high glucosyl-D-galactosid powder prepared by the method of Example A-8, 190 parts by weight of dried egg yolk, 209 parts by weight of skim milk, sodium chloride 4.4 A composition consisting of parts by weight, 1.85 parts by weight of potassium chloride, 4 parts by weight of magnesium sulfate, 0.01 part by weight of thiamine, 0.1 part by weight of sodium ascorbate, 0.6 part by weight of vitamin E acetate, and 0.04 part by weight of nicotinamide was prepared. Each 25 g of this composition was put in a laminate made of laminated aluminum and heat sealed to prepare a desired product. This product is dissolved in about 150-300ml of water and used as a nutrient replenishing solution for administration to the nasal cavity, esophagus, stomach, etc. by landscape methods.

Example B-12 Strawberry Jam

150 parts by weight of fresh berries, 60 parts by weight of sucrose, 20 parts by weight of maltose, 40 parts by weight of trehalose-containing sugar solution prepared by the method of Example A-16, 5 parts by weight of pectin and 1 part by weight of citric acid, and boil in a pot Got the product of the disturbance. This product has a good taste, aroma and color.

Example B-13 Sweetened Condensed Milk

1 part by weight of sucrose and 3 parts by weight of glucosyl-D-fucoside-containing sugar solution prepared by the method of Example A-17 were dissolved in 100 parts by weight of fresh milk, and the solution was sterilized by heating in a plate heater, and then the concentration was about 70%. Concentrated and packed aseptically in cans to obtain the desired product. This product has a mild sweetness, aroma and taste, so it can be advantageously used for seasoning of baby food, fruits, coffee, cocoa and tea.

As is apparent from the above description, the present invention is based on the discovery of novel trehalose phosphorylase with higher optimum temperature and thermal stability than that of conventional trehalose phosphorylase. Trehalose phosphorylase according to the present invention has a relatively wide range of pH stability in which an optimal pH is present. Trehalose phosphorylase can be prepared using microorganisms capable of producing this enzyme satisfactorily in high yield. Thus, when the trehalose phosphorylase of the present invention is acted on β-D-glucose-1-phosphate as a sugar donor in the presence of other sugars, glucosyl-D-galactoside, which has been known in the art but has been hardly obtained, is known. Glucosyl transition sugars can be prepared on an industrial scale relatively inexpensively.

Glucosyl transition sugars and sugar compositions containing them can be used in foods, cosmetics, medicines and moldings as relatively high quality sweeteners, taste improvers, quality improvers, excipients, viscosity modifiers, humidity regulators, glossing agents and supplements. have. Due to these excellent features of the present invention, as well as in the fields of food, cosmetics and pharmaceuticals, it contributes to agriculture, fishery, animal husbandry and chemical industry.

[Array table]

(1) Information of array ID (SEQ ID NO :) 1:

(Iii) Features of the array:

(A) Array length: 5 amino acids

(B) Array type: amino acid

(D) form: straight

(Ii) Type of arrangement: peptide

(Iii) Fragment: N terminal fragment

(Ⅹⅰ) Array number (SEQ ID NO :) 1:

(2) Information of sequence ID (SEQ ID NO :) 2:

(Iii) Features of the array:

(A) Array length: 5 amino acids

(B) Array type: amino acid

(D) form: straight

(Ii) Type of arrangement: peptide

(Iii) Fragment type: internal fragment

(Iii) Sequence ID (SEQ ID NO :) 2:

(3) Information of sequence ID (SEQ ID NO :) 3:

(Iii) Features of the array:

(A) Array length: 5 amino acids

(B) Array type: amino acid

(D) form: straight

(Ii) Type of arrangement: peptide

(Iii) Fragment type: internal fragment

(Iii) Array ID (SEQ ID NO :) 3:

(4) Information of sequence ID (SEQ ID NO :) 4:

(Iii) Features of the array:

(A) Length of array: 773 amino acids

(B) Array type: amino acid

(D) form: straight

(Ii) Type of arrangement: peptide

(Iii) Sequence ID (SEQ ID NO :) 4:

(5) Information of sequence ID (SEQ ID NO :) 5:

(Iii) Features of the array:

(A) Array length: 2319 base pairs

(B) Type of array: Nucleic acid

(C) The number of chains: Two prints

(D) form: straight

(Iii) Sequence ID (SEQ ID NO :) 5:

(6) Information of sequence ID (SEQ ID NO :) 6:

(Iii) Features of the array:

(A) Array length: 30 amino acids

(B) Array type: amino acid

(D) form: straight

(Ii) Type of arrangement: peptide

(Iii) Fragment: N terminal fragment

(Iii) Sequence ID (SEQ ID NO :) 6:

(7) Information of sequence ID (SEQ ID NO :) 7:

(Iii) Features of the array:

(A) Array length: 15 amino acids

(B) Array type: amino acid

(D) form: straight

(Ii) Type of arrangement: peptide

(Iii) Fragment type: internal fragment

(Iii) SEQ ID NO: 7:

(8) Information of sequence number (SEQ ID NO :) 8:

(Iii) Features of the array:

(A) Array length: 3345 base pairs

(B) Type of array: Nucleic acid

(C) The number of chains: Two prints

(D) form: straight

(Ii) Type of sequence: Genomic DNA

(Iii) Origin:

(A) Biometric: Thermoanaerobium brokii

(F) State name: ATCC 35047

(Ⅸ) Features:

(A1) Characteristic symbols: 1-595 5'-UTR

(C1) how to determine features: E

(A2) Characteristic symbol: 596-2917 mat peptide

(C2) How to determine the feature: S

(A3) Symbol indicating features: 2916-3345 3'-UTR

(C3) How to determine the feature: E

(Iii) SEQ ID NO: 8:

Claims (26)

D-glucose and β-D-glucose-1-phosphate and / or its salts are obtained from microorganisms belonging to the genus Themoanaerobium® and hydrolyzed trehalose in the presence of inorganic phosphoric acid and / or salts thereof. To produce trehalose phosphorylase. The process according to claim 1, wherein trehalose and inorganic phosphoric acid and / or salts thereof are produced from D-glucose and β-D-glucose-1-phosphate and / or salts thereof, and the β-D-glucose-1-phosphate and Trehalose phosphorylase, which promotes the transfer of glucosyl groups to other sugars by using the salt as a sugar donor. The trehalose phosphorylase of claim 1, which is stable up to a temperature of about 60 ° C. when maintained at pH 7.0 for 1 hour. The trehalose phosphorylase of claim 1 having the following physicochemical properties: (1) molecular weight 88,000 ± 5,000 Daltons on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (2) optimum temperature 70 ℃ for 30 minutes at pH 7.0 (3) optimum pH 7.0 ~ 7.5 when kept at 60 ℃ for 30 minutes (4) thermal stability Stable up to about 60 ℃ when kept at pH 7.0 for 1 hour (5) pH stability Stable at pH 6.0-9.0 when kept at 4 ° C for 24 hours The method according to claim 1, trehalose phosphorylase having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2 and 3 as a partial amino acid sequence; Array number: 1 Array number: 2 Array number: 3 The trehalose phosphorylase of claim 1, having an amino acid sequence of SEQ ID NO: 4. 4. Array number: 4 The trehalose phosphorylase of claim 1, obtained by expressing a gene. DNA that codes for trehalose phosphorylase of claim 1. The DNA of claim 8, wherein the DNA has a nucleotide sequence selected from the group consisting of SEQ ID NO: 5 and its complementary sequence. Array number: 5 The DNA of claim 8, wherein one or more bases are substituted with other bases for degeneracy of the genetic code without altering the amino acid sequence coded by the DNA. The DNA of claim 8 introduced into a self-replicating vector. The DNA of claim 8 introduced into a suitable host. A method for producing trehalose phosphorylase, wherein the microorganism producing trehalose phosphorylase of claim 1 is cultured in a nutrient medium, and the resulting trehalose phosphorylase is collected from the culture. The method according to claim 13, wherein the microorganism is a kind of Thermoanaerobium 屬. The method according to claim 14, wherein the microorganism is a kind of thermoanaerobium brockii '. The method according to claim 13, wherein the microorganism is a transformant prepared by introducing a DNA coding for the trehalose phosphorylase of claim 1 into a suitable host. The method according to claim 13, the resulting trehalose phosphorylase dialysis, salting out, gel filtration, concentration, fractional precipitation, ion exchange chromatography, gel filtration chromatography, adsorption chromatography, hydrophobic chromatography, reverse phase chromatography, A process for harvesting by one or more techniques selected from the group consisting of affinity chromatography, gel electrophoresis and isoelectric point fractions. D-glucosyl, comprising acting on trehalose phosphorylase of claim 1 on β-D-glucose-1-phosphate and / or its salts and other saccharides to produce D-glucosyl transsaccharide Method for producing a transsaccharide-containing sugar composition. 19. The method of claim 18, wherein β-D-glucose-1-phosphate and / or its salts act upon trehalose phosphorylase on trehalose in the presence of inorganic phosphoric acid and / or salts thereof, And / or by acting maltose-phosphorylase on maltose in the presence of salts or by acting on cogibisose-phosphorylase on cozibioses in the presence of inorganic phosphoric acid and / or salts thereof. The method according to claim 18, wherein the other saccharide is one selected from the group consisting of D-xylose, D-galactose, D-glucose, D-fucose and L-fucose. The method according to claim 18, wherein the method is selected from the group consisting of fermentation methods using various column chromatography and yeast as a method for removing impurities other than glucosyl transition sugars and / or remaining glucosyl transition sugars in the reaction mixture. Manufacturing method comprising a. In addition to maltose-phosphorylase in the presence of inorganic phosphoric acid and / or salts thereof, trehalose phosphorylase of claim 1 is reacted with maltose to produce trehalose, or of inorganic phosphoric acid and / or salts thereof A method for preparing trehalose, comprising the step of reacting with cozibiose-phosphorylase in combination with trehalose phosphorylase to cogibis to produce trehalose. Sugar composition containing a glucosyl transition sugar prepared by the method of claim 22. The method of claim 23, wherein the glucosyl transsaccharide is glucosyl-D-xyloxide, glucosyl-D-galactoside, trehalose, glucosyl-D-fructoside and glucosyl-L-fucoside. Sugar composition of a kind selected from the group consisting of. A composition containing the sugar composition of claim 23. The composition of claim 25 in the form of a food, cosmetic, pharmaceutical or molding.