JP4171761B2 - Kojibiose phosphorylase, its production method and use - Google Patents

Description

  The present invention relates to a novel enzyme cordobiose phosphorylase, a method for producing the same, and uses thereof. More specifically, inorganic phosphate and / or a salt thereof (hereinafter referred to as “inorganic phosphate” throughout the present specification unless inconvenience occurs). In the presence of D-glucose and β-D-glucose-1-phosphate and / or a salt thereof (hereinafter referred to as “β-D-glucose unless otherwise noted”). -1 phosphate ")), and conversely, a novel enzyme cordobiose phosphorylase which has the action of producing cordobiose and inorganic phosphate from β-D-glucose-1 phosphate and D-glucose. And its production method, glucosyltransferase-containing saccharides produced using this cordobiose phosphorylase, and these saccharides are contained. It relates to a composition.

  In recent years, oligosaccharides such as maltose and trehalose and their functions have attracted attention, and various production methods of these oligosaccharides have been widely studied from various fields. As methods for producing these oligosaccharides, utilization of various phosphorylases such as maltose phosphorylase, trehalose phosphorylase, sucrose phosphorylase, cellobiose phosphorylase, laminaribiose phosphorylase is known. Various microorganisms are known as a source of these phosphorylases.

  Non-Patent Document 1 summarizes these phosphorylases, their actions, and the source microorganisms. However, phosphorylase capable of producing cordobiose is not yet known, and its provision is desired.

"Enzyme Handbook" Asakura Shoten (1982)

  The present invention provides a novel enzyme, cordobiose phosphorylase, as well as a method for producing the enzyme, a glucosyltransferase-containing saccharide using the enzyme, and a use thereof.

  In order to solve the above-mentioned problems, the present inventors searched for an unknown cordierbiose phosphorylase and searched for microorganisms that produce the enzyme. As a result, the microorganism Thermoanaerobium brokii (ATCC 35047) belonging to the genus Thermoanaerobium has been found to produce a novel enzyme, cordobiose phosphorylase, and a method for producing this enzyme Established. Furthermore, it contains a novel and / or known glucosyl transfer saccharide-containing saccharide obtained by allowing this enzyme to act in the presence of various saccharides using β-D-glucose-1-phosphate as a sugar donor, and contains the saccharide A method for producing the caulked composition was established to complete the present invention.

  The present invention is based on the discovery of a phosphorylase capable of producing cordobiose that has not been known so far, that is, cordobiose phosphorylase. The cordobiose phosphorylase of the present invention has a high temperature in the optimum temperature and temperature stability, a wide pH range in the pH stability, and the optimum pH is within the pH range, and further enzyme production from the producing microorganism The amount is also high. Therefore, if β-D-glucose-1-phosphate is used as a sugar donor and the enzyme of the present invention is allowed to act in the presence of various sugars, Cojibiose, which has been conventionally known but extremely difficult to obtain, Various glucosyl transfer saccharides such as glucosyl sorbose and the transfer sugar-containing saccharides can be produced in large quantities and at low cost.

The cordobiose phosphorylase of the present invention includes all enzymes having the action of degrading cordobiose in the presence of inorganic phosphate to produce D-glucose and β-D-glucose-1 phosphate, The origin and origin are not questioned. Examples of such cordobiose phosphorylase include enzymes that exhibit the following actions and have physicochemical properties.
(1) Action (a) Cozybiose is decomposed in the presence of inorganic phosphate to produce D-glucose and β-D-glucose-1-phosphate.
(B) Cozybiose and inorganic phosphoric acid are produced from β-D-glucose-1-phosphate and D-glucose, and β-D-glucose-1 phosphate is used as a sugar donor, and other carbohydrates have glucosyl groups. Catalyzes the transfer of
(2) Molecular weight 83,000 ± 5,000 daltons as determined by SDS-gel electrophoresis.
(3) Isoelectric point pI of 4.4 ± 0.5 by an ampholine-containing electrophoresis method.
(4) Optimum temperature: pH 5.5, around 65 ° C. for 30 minutes reaction.
(5) Optimum pH
The pH was around 5.5 after reaction at 60 ° C for 30 minutes.
(6) Temperature stability Stable up to around 65 ° C at pH 5.5 and maintained for 1 hour.
(7) pH stability The pH is about 5.5 to 10.0 at 4 ° C. for 24 hours.
(8) Inhibition This enzyme activity is inhibited by 1 mM Hg ⁺⁺ .

  More specifically, such a cordobiose phosphorylase includes, for example, the amino acid sequence shown in SEQ ID NO: 1, 2, or 3 in the sequence listing as a partial amino acid sequence, and the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing as a whole Examples thereof include proteins and functional derivatives thereof. As used herein, the functional derivative means a substance obtained by substituting one or more of the amino acids in the amino acid sequence with another amino acid within a range that does not substantially lose the action as the above-described cordobiose phosphorylase, the amino acid sequence In which one or more amino acids are added to the N-terminal and / or C-terminal, in which one or more amino acids are inserted in the middle in the amino acid sequence, and in the N-terminal and / or C-terminal in the amino acid sequence It means one in which one or more amino acids have been deleted and one in which one or more amino acids in the middle of the amino acid sequence have been deleted. The above-described cordobiose phosphorylase of the present invention is a mutant obtained by treating the microorganism with a mutagen or the like, even if it is isolated from a natural source such as a culture of the microorganism producing the enzyme. It may be separated from the culture or may be artificially synthesized by applying a recombinant DNA technique or a peptide synthesis method. As will be described in detail later, according to the production method of the present invention, the cordobiose phosphorylase can be obtained in a favorable manner both from a natural source and when recombinant DNA technology is applied. .

  The DNA of the present invention includes all of the DNAs encoding the above-described cordobiose phosphorylase of the present invention. Examples of such DNA include DNA containing the base sequence shown in SEQ ID NO: 5 in the sequence listing, which encodes the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing, and functional derivatives thereof. As used herein, the functional derivative means that one or more of the bases in the base sequence are replaced with other bases as long as the protein encoded by the DNA does not substantially lose its action as the above-mentioned cordobiose phosphorylase. In which 1 or more bases are added to the 5 ′ end and / or 3 ′ end of the base sequence, in which 1 or more bases are inserted in the middle of the base sequence, in the base sequence Those with one or more bases at the 5 'and / or 3' ends deleted, those with one or more bases in the base sequence deleted, and those with a base sequence complementary to these base sequences Means. Such functional derivatives include, for example, a base sequence in which one or more of the bases are substituted with other bases in the base sequence shown in SEQ ID NO: 5 in the sequence listing without changing the amino acid sequence encoded by the base sequence. The DNA consisting of The DNA of the present invention includes not only the DNA as described above, but also DNA other than the base sequence encoding the cordobiose phosphorylase at the 5 ′ end and / or 3 ′ end of the DNA, such as a start codon. In addition, DNA comprising one or more selected from a termination codon, a Shine-Dalgarno sequence, a base sequence encoding a signal peptide, a recognition sequence by an appropriate restriction enzyme, a promoter, an enhancer, a terminator, and the like is also included.

  In the present invention, the source and preparation method of such DNA are not limited. For example, natural sources of such DNA include microorganisms of the genus Thermoanaerobium, including Thermoanaerobium brokkii (ATCC 35047), and bacteria obtained by culturing the microorganisms by a conventional method If the DNA fraction is collected from the crushed material, DNA containing the DNA of the present invention can be obtained. The DNA thus obtained can be used in the present invention by itself, but when the DNA of the present invention is obtained as a recombinant DNA obtained by inserting a fragment containing the DNA into a vector capable of autonomous replication. This is extremely advantageous in practicing the present invention. The recombinant DNA is prepared by, for example, applying a general recombinant DNA technique to the DNA obtained as described above to produce a gene library, and the base sequence of the DNA encoding the cordobiose phosphorylase of the present invention, For example, it can be obtained by applying a hybridization method or the like that selects a target recombinant DNA from the gene library based on the base sequence shown in SEQ ID NO: 5 in the sequence listing. The recombinant DNA thus obtained is amplified when a transformant obtained by introduction into an appropriate host such as Escherichia coli is cultured, and if a general alkaline-SDS method or the like is applied to the culture, the present invention The desired amount of DNA can be obtained very easily. On the other hand, using the crushed cell of the microorganism or the DNA recovered therefrom as a template, using the DNA chemically synthesized based on the nucleotide sequence shown in SEQ ID NO: 5 in the sequence listing as a primer, and applying the PCR method according to a conventional method Alternatively, the DNA of the present invention can also be easily obtained by chemically synthesizing a DNA containing the base sequence shown in SEQ ID NO: 5 in the sequence listing. In order to obtain the DNA of the present invention as the above-mentioned functional derivative, for example, a site-directed mutagenesis method is applied to the recombinant DNA, or the recombinant DNA is used as a template and converted into a desired sequence. It can be obtained by applying a PCR method using a chemically synthesized DNA containing the nucleotide sequence as a primer.

  Further, the DNA of the present invention includes a recombinant DNA inserted into an autonomously replicable vector. As described above, such a recombinant DNA is not only extremely useful for obtaining the DNA of the present invention, but also very useful for producing the cordobiose phosphorylase of the present invention as described in detail later. . Such a recombinant DNA is relatively easy to obtain by inserting the DNA into a desired vector once the target DNA is obtained as described above, and usually by applying a general recombinant DNA technique. It is. The vector into which the DNA of the present invention can be inserted may be any vector as long as it has the property of autonomous replication in an appropriate host. For example, pUC18, Bluescript II SK (+), pKK223 using Escherichia coli as a host. PHY300PLK, pHV14, TRp7, YEp7, pBS7, etc. using two or more kinds of hosts such as pUB110, pTZ4, pC194, ρ11, φ1, φ105, etc. using Bacillus subtilis as a host, such as -3 and λgt · λC be able to. An example of a method for inserting the DNA of the present invention into such a vector will be described. First, the DNA of the present invention obtained as described above or a DNA containing the DNA and an appropriate vector are cleaved with a restriction enzyme, Next, the generated DNA fragment and the vector fragment are ligated. Examples of restriction enzymes used here include Acc I, Bam HI, Bgl II, Bst XI, Eco RI, Hind III, Not I, Pst I, Sac I, Sal I, Sma I, Spe I, Xba I, Any of Xho I and the like can be advantageously used. Further, when ligating the DNA fragment and the vector fragment, it is optional to intervene chemically synthesized DNA having an appropriate restriction enzyme recognition sequence or the like, if necessary. For DNA ligation, DNA ligase may be allowed to act inside or outside the cell after annealing both.

  Furthermore, the DNA of the present invention includes a form of a transformant introduced into an appropriate host microorganism. Such a transformant is extremely useful for obtaining the cordobiose phosphorylase of the present invention and the DNA of the present invention. As the host microorganism of such a transformant, for example, any of Escherichia coli, Bacillus subtilis, actinomycetes, yeast and the like can be advantageously used. Such a transformant is usually obtained by introducing the recombinant DNA obtained as described above into an appropriate host. Specifically, when the host is Escherichia coli, the host is treated with the recombinant DNA and calcium. The culture may be performed in the presence of ions. On the other hand, when the host is Bacillus subtilis, a competent cell method or a protoplast method may be applied. Each of the individual methods for obtaining the DNA of the present invention described above is conventional in the art. For example, J. Sambrook et al., “Molecular Cloning a Laboratory Manual”, 2nd edition, 1989. It is described in detail in the issue of Cold Spring Harbor Laboratory.

  The method for producing Kojibiose phosphorylase according to the present invention is characterized by culturing a microorganism capable of producing the enzyme and collecting the enzyme from the culture. It doesn't matter. Examples of such microorganisms include, for example, microorganisms belonging to the genus Thermoanaerobium, more preferably Thermoanaerobium brokki (ATCC 35047), and transformants obtained by introducing the DNA of the present invention into an appropriate host microorganism. Can be mentioned.

  In the production method of the present invention, the medium used for culturing the microorganism may be any medium that can grow the microorganism and produces the enzyme of the present invention, and may be either a synthetic medium or a natural medium. As the carbon source, any substance that can be assimilated by microorganisms may be used. For example, sugars such as maltose, trehalose, dextrin, starch, molasses, and sugar-containing substances such as yeast extract may be used. it can. The concentration of these carbon sources in the medium is appropriately selected depending on the type of carbon source. For example, the sugar concentration in the culture solution is desirably 20 w / v% or less, and usually 5 w / v% or less is preferable from the growth and proliferation of bacteria. Examples of the nitrogen source include inorganic nitrogen compounds such as ammonium salts and nitrates, and organic nitrogen-containing materials such as urea, corn steep liquor, casein, peptone, yeast extract and meat extract. In addition, as the inorganic component, for example, calcium salt, magnesium salt, potassium salt, sodium salt, phosphate, manganese salt, zinc salt, iron salt, copper salt, molybdenum salt, cobalt salt, etc. are used as necessary. It is done.

  For the culture, the conditions under which the microorganisms used for production grow well can be appropriately selected and applied. For example, when using the aforementioned microorganism belonging to the genus Thermoanaerobium, the temperature is usually 50 to 80 ° C., preferably 60 to 70 ° C., pH 5 to 8, preferably pH 6.5 to 7.5, Done anaerobically. The culture time may be a time during which the microorganism can grow, and is preferably 10 to 50 hours. On the other hand, when the above-mentioned transformant microorganism is used, although depending on the microorganism species, the culture time is usually maintained under aerobic conditions such as aeration stirring while maintaining the temperature at 20 to 65 ° C. and pH 2 to 9. It may be about 1 to 6 days.

  In this way, after culturing the microorganism, the enzyme of the present invention is recovered from the obtained culture. This enzyme activity is usually observed in the microbial cells of the culture, and the microbial cells or the treated microbial cells may be recovered as a crude enzyme, or the entire culture can be used as a crude enzyme. A normal solid-liquid separation means is employed for separation of the extracellular culture solution and the bacterial cells. For example, a means for centrifuging the culture itself as it is, a means for adding a filter aid to the culture or pre-coating for filtration separation, a means for membrane filtration separation using a flat membrane, a hollow fiber membrane, etc. are adopted. Is done. The bacterial cells or the processed bacterial cells can be used as a crude enzyme solution as they are. Further, if necessary, it can be used as a partially purified enzyme. For example, in the case of a transformant using Bacillus subtilis or the like as a host, the enzyme may be produced in the medium used for the culture depending on the type of recombinant DNA used for the transformation. In such a case, the culture supernatant can be used as a crude enzyme.

  Examples of the treated cells include dried cells, freeze-dried products, surfactant-treated products, enzyme-treated products, ultrasonic crushed materials, mechanically ground products, mechanical pressure-treated products, and bacterial cell protein fractions. Minute, microbial cells or immobilized microbial cells. As the enzyme, either a crude enzyme or a purified enzyme is used, and the processed bacterial cells are usually used for the purification of the enzyme, for example, ammonium sulfate salting out method, acetone and alcohol precipitation method, flat membrane, hollow fiber membrane, etc. Membrane concentration and dialysis methods are used.

  Furthermore, the bacterial cells and the treated bacterial cells can be immobilized by ordinary means. For example, a binding method with an ion exchanger, a covalent bond / adsorption method with a resin and a membrane, a comprehensive method using a polymer substance, and the like are employed.

  The crude enzyme may be used as it is, but can also be purified by ordinary means. As an example, after dialysis of a crude enzyme preparation obtained by subjecting a cell-treated product to ammonium sulfate salting out and concentration, anion exchange column chromatography using DEAE-Toyopearl resin (manufactured by Tosoh Corporation), followed by CM-Toyopearl resin Cation exchange column chromatography using Tosoh Corp., hydrophobic column chromatography using butyl Toyopearl resin (Tosoh Corp.), gel filtration using Ultrogel AcA44 resin (Sepracol, France) A single enzyme can be obtained electrophoretically by column chromatography.

  The activity of the cordobiose phosphorylase of the present invention is measured as follows. After adding 0.2 ml of the enzyme solution to 2 ml of 20 mM McKilvein buffer (pH 5.5) containing 1.0 w / v% cordobiose as a substrate and reacting at 60 ° C. for 30 minutes, 0.5 ml of the reaction solution was added at 100 ° C., 10 ° C. Heat for minutes to stop the reaction. To this reaction stop solution, 0.5 ml of D-glucose oxidase / peroxidase reagent is added and stirred, and allowed to stand at 40 ° C. for 30 minutes, then 2.5 ml of 5N hydrochloric acid is added and stirred, and the absorbance at 525 nm is measured. One unit of enzyme activity is defined as the amount of enzyme that produces 1 μmol of D-glucose per minute under the above reaction conditions. In addition, the activities of maltose phosphorylase and trehalose phosphorylase can be measured by the same procedure, except that Kojibiose as a substrate is used in place of maltose and trehalose, respectively.

  The enzyme reaction for producing a glucosyl transfer sugar-containing saccharide using the cordobiose phosphorylase of the present invention is usually performed by using β-D-glucose-1-phosphate as a sugar donor as a substrate and other suitable saccharides. For example, monosaccharides such as D-glucose and L-sorbose, disaccharides such as maltose, cordobiose, trehalose and sucrose, and oligosaccharides composed of trisaccharides such as maltotriose, maltotetraose and maltopentaose. The enzyme is allowed to act as a receptor under conditions as described below. As a result, depending on the receptor used, for example, Codybiose, glucosyl sorbose, Codybiosyl glucose, Cody triose, Codybiosyl glucoside, Codybiosyl fructoside, Codybiosyl maltose, Codybiosyl maltotriose and Various glucosyl transfer sugars including Kojibiosyl maltotetraose can be produced. Of these transfer sugars, glucosyl sorbose and kojibiosyl fructoside are novel carbohydrates first discovered in the present invention.

  For β-D-glucose-1-phosphate as a sugar donor, a commercially available reagent may be used as it is, or an appropriate phosphorylase is used as a sugar in the presence of inorganic phosphate and / or a salt thereof. It may be prepared by acting on quality. Specifically, for example, in the presence of inorganic phosphoric acid and / or a salt thereof, Codybiose phosphorylase is allowed to act on Codybiose, Maltose phosphorylase is allowed to act on maltose, or Trehalose phosphorylase is allowed to act on trehalose. It may be prepared. Furthermore, when any of the β-D-glucose-1-phosphate production reaction by the action of these phosphorylases is carried out in the same reaction system as the glucosyl transfer sugar production reaction using the cordobiose phosphorylase, Since β-D-glucose-1-phosphate can be directly supplied to the transfer sugar production reaction system, the production cost can be reduced and the production process can be simplified, which is extremely advantageous. As a receptor, it is also possible to advantageously use two or more of the above saccharides simultaneously. In addition, although inorganic phosphoric acid here includes not only orthophosphoric acid but condensed phosphoric acid, it is usually desirable to use orthophosphoric acid. In addition, the salt herein includes all phosphate ion compounds derived from the above-mentioned inorganic phosphoric acid. Of these, sodium salts and potassium salts with high water solubility are usually used. Is desirable.

  The substrate concentration in the glucosyl transferase production reaction using the cordobiose phosphorylase of the present invention is not particularly limited. Generally, β-D-glucose-1-phosphate as a sugar donor is 1 to 20 w / w% (hereinafter, unless otherwise specified, w / w% is abbreviated as%). It is preferable to use the receptor as an aqueous solution of about 1 to 20%. On the other hand, as described above, when the β-D-glucose-1-phosphate production reaction by the action of an appropriate phosphorylase is carried out in the same reaction system as the glucosyl transfer sugar production reaction, the following is desirable. That is, in the case of the action of cordobiose phosphorylase, instead of β-D-glucose-1-phosphate which is a substrate for the glucosyl transfer reaction, an aqueous solution of about 1 to 20% cordierbiose is used, and further 0.5 to 20 mM. A phosphate such as sodium dihydrogen phosphate to the extent is allowed to coexist. In the case of other phosphorylase action, instead of β-D-glucose-1-phosphate as a substrate of the glucosyl transfer reaction, for example, about 1 to 20% maltose or trehalose and about 0.5 to 20 mM are used. It is desirable that a phosphate such as sodium dihydrogen phosphate coexists with each other, and that maltose phosphorylase or trehalose phosphorylase be coexistent with each other in an amount of about 0.1 to 50 units per gram of the sugar based on the sugar used. .

  The reaction temperature may be a temperature at which the enzyme is not inactivated in the presence of the substrate, that is, up to about 70 ° C., preferably in the range of about 15 to 65 ° C. The reaction pH is usually adjusted to a range of about 4.0 to 9.0, preferably a pH of about 5.0 to 7.5. The reaction time may be appropriately selected depending on the progress of the enzyme reaction, and is usually about 0.1 to 100 hours at a use amount of about 0.1 to 50 units per gram of the substrate solid. In addition, as described above, when the β-D-glucose-1-phosphate production reaction by the action of other phosphorylases is carried out in the same reaction system as the glucosyl transfer sugar production reaction using the cordobiose phosphorylase. Is preferably adjusted to a reaction temperature or reaction pH at which any phosphorylase used is not inactivated, depending on the stability of the phosphorylase to be coexisted.

  Thus, glucosyl transfer sugars are produced in the reaction product depending on the carbohydrate used as the substrate. The production rate varies depending on the substrate concentration of the enzyme reaction, the type of substrate used, the reaction conditions, and the like. For example, when β-D-glucose-1-phosphate with a concentration of 5% and sorbose with a concentration of 25% are used as substrates, the production rate of glucosyl sorbose is about 30%. Throughout this specification, the production rate means the percentage of the transferred sugar mass produced with respect to the total carbohydrate mass in the reaction solution.

  In addition, in order to increase the glucosyl transfer sugar content in the reaction product as much as possible, an enzyme source having an activity of decomposing and removing D-glucose produced in the reaction solution is coexistent to promote the transfer reaction. it can. In this method, when the β-D-glucose-1-phosphate production reaction by the appropriate phosphorylase described above is carried out in the same system as the glucosyl transfer reaction and a sugar donor is directly supplied, D-by-product generated in the reaction process is produced. It can be particularly advantageously applied to decompose and remove glucose and promote the transfer reaction.

  Examples of the enzyme source having D-glucose degrading activity include microorganisms having D-glucose degrading activity, cultures of microorganisms, microbial cells or treated cells, or enzymes having D-glucose degrading activity. As the microorganism having D-glucose degrading activity, any microorganism can be used as long as it has a high D-glucose degrading activity and has little or no transfer sugar degrading activity. Preferably, yeast is used. As the enzyme having D-glucose degrading activity, for example, glucose oxidase, catalase, pyranose oxidase, glucose dehydrogenase, glucokinase, hexokinase and the like can be used. Glucose oxidase or catalase is preferably used.

  The reaction solution in which the glucosyl transfer sugar is produced as described above is subjected to conventional methods such as filtration and centrifugation to remove insoluble matters, followed by decolorization with activated carbon and desalting with H-type and OH-type ion exchange resins. After the purification process, etc., it is concentrated to make a syrup-like product. If necessary, it is optional to further dry it by a method such as spray drying into a powdered product.

  In addition, the glucosyl transfer saccharide-containing carbohydrate of the present invention can be separated from the enzyme reaction solution and purified to obtain a high content of the glucosyl transfer saccharide. As the method, for example, a method for removing monosaccharides by fermentation using yeast (yeast fermentation method), a method for separating and removing contaminating carbohydrates by membrane filtration, column chromatography and the like can be appropriately employed. In particular, the objective glucosyl is obtained by removing contaminating carbohydrates by column chromatography using a salt-type strongly acidic cation exchange resin disclosed in JP-A-58-23799 and JP-A-58-72598. The method of collecting a fraction having a high transfer sugar content can be advantageously carried out on an industrial scale. At this time, it is optional to employ any of the known fixed floor method, moving floor method, and simulated moving floor method.

  Thus, the liquid from which the contaminating saccharide has been separated is subjected to a purification process such as decolorization with activated carbon, desalting with H-type or OH-type ion exchange resin, after removing insoluble matter by filtration, centrifugation, etc., by a conventional method. After that, it is concentrated to a syrup product. If necessary, it is optional to further dry it by a method such as spray drying into a powdered product.

  The glucosyl transfer sugar-containing carbohydrate of the present invention thus obtained usually contains 5% or more, preferably 10% or more, of glucosyl transfer sugar per solid.

  The glucosyl transfer sugar-containing saccharide produced by the cordobiose phosphorylase of the present invention thus obtained has a good taste and sweetness, and also has osmotic pressure control, moisture retention, illumination, and crystallization prevention. It has properties such as anti-cariogenic properties, bifidobacteria growth promoting properties, and mineral absorption promoting properties, and is widely used in food, beverages, foods, feeds, feeds, cosmetics, and pharmaceuticals. It is advantageously used for various compositions such as articles, molded articles, and other daily necessities, agricultural, forestry and fishery products, reagents, chemical industrial products and the like.

  Glucosyl transfer sugar-containing saccharide can be used as a seasoning for sweetening as it is, but if necessary, for example, flour, glucose, maltose, trehalose, sucrose, isomerized sugar, honey, maple sugar, One or more other sweeteners such as sorbitol, maltitol, lactitol, dihydrochalcone, stevioside, α-glycosyl stevioside, rebaudioside, glycyrrhizin, L-aspartyl-L-phenylalanine methyl ester, saccharin, glycine, alanine, etc. It may be used by mixing with an appropriate amount of, and if necessary, it can also be used by mixing with a bulking agent such as dextrin, starch, lactose and the like.

  In addition, the taste of saccharides containing glucosyl transfer sugar is well harmonized with various substances having other tastes such as acidity, salt to taste, astringency, umami, bitterness, etc. It can be advantageously used for sweetening foods, improving taste and improving quality.

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

  In addition, for example, Japanese rice cakes such as rice crackers, hail, rice cakes, rice cakes, manju, oirou, chanterelles, sheep mushrooms, water sheep rice cakes, brocade balls, jelly, castella, rice cakes, bread, biscuits, crackers, cookies, pie , Pudding, butter cream, custard cream, cream puff, waffle, sponge cake, donut, chocolate, chewing gum, caramel, candy and other Western confectionery, ice cream, sherbet and other ice confectionery, fruit syrup pickled, syrup such as ice honey, flower Pastes such as paste, peanut paste, fruit paste, spread, fruit such as jam, marmalade, syrup pickles, sugar cane, processed foods of vegetables, pickles such as Fukujin pickled, bedara pickled, Senzuke pickled, ryaky pickled, ham, sausage, etc. Livestock meat products, fish Manufactured with fish products such as ham, fish sausage, kamaboko, chikuwa, tempura, various delicacies such as sea urchin, salted squid, vinegared konbu, suki-shime, fugumi-rin, dried fish, shellfish, small fish, shellfish, etc. Stewed food such as rutsuda stew, boiled beans, potato salad, konbu roll, dairy products, fish meat, livestock meat, fruits, bottled vegetables, canned foods, sake, synthetic liquor, liqueur, Western liquor, tea, coffee, cocoa , Juices, carbonated drinks, lactic acid drinks, soft drinks such as lactic acid bacteria drinks, pudding mixes, hot cake mixes, instant foods such as instant soups, instant soups, baby food, therapeutic foods, drinks, cooked rice, noodles, frozen foods It can be advantageously used for sweetening various foods such as food, improving taste, and improving physical properties.

  Moreover, it can also be used in order to improve the palatability of feed, feed, etc. for domestic animals such as livestock, poultry and fish. In addition, tobacco, toothpaste, lipstick, lip balm, oral solution, tablets, troches, liver oil drops, mouth fresheners, mouth fragrances, gargles, etc. It can be advantageously used as a sweetener for various compositions, as a taste improver, a taste corrector, and as a quality improver.

  As a quality improver and stabilizer, it can be advantageously applied to various physiologically active substances that tend to lose the activity of active ingredients or health foods and pharmaceuticals containing the same. For example, interferon-α, interferon-β, interferon-γ, tsumore necrosis factor-α, tsumore necrosis factor-β, macrophage migration inhibitory factor, colony stimulating factor, transfer factor, interleukin 1, interleukin 2, Cytokin-containing fluids such as interleukin 6, interleukin 12, interleukin 15 and interleukin 18; hormone-containing fluids such as insulin, growth hormone, prolactin, erythropoietin, tissue plasminogen activator, egg cell stimulating hormone, placental hormone, BCG Vaccine, Japanese encephalitis vaccine, measles vaccine, polio live vaccine, seedling, tetanus toxoid, hub antitoxin, liquid containing biologics such as human immunoglobulin, penicill , Erythromycin, chloramphenicol, tetracycline, streptomycin, kanamycin sulfate and other antibiotic-containing liquids, thiamine, riboflavin, L-ascorbic acid, liver oil, carotenoids, ergosterol, tocopherol, vitamin-containing liquids, lipase, elastase, Enzyme-containing liquids such as urokinase, protease, β-amylase, isoamylase, glucanase, and lactase, ginseng extract, suppon extract, chlorella extract, aloe extract, propolis extract and other extracts, live bacteria such as viruses, lactic acid bacteria, and yeast, Various physiologically active substances such as royal jelly can easily produce stable and high-quality health foods and pharmaceuticals without losing the activity of the active ingredients.

  As described above, the composition referred to in the present invention includes not only foods and beverages, cosmetics, and pharmaceuticals used orally or parenterally, but also other items such as daily necessities, agricultural, forestry and fishery products, reagents, chemicals, and the like. It has a wide range of uses such as industrial products.

  In addition, the method of incorporating the glucosyl transfer sugar-containing carbohydrate of the present invention into these compositions may be included in the process until the product is completed, for example, mixing, dissolution, dipping, infiltration, spraying, application Well-known methods such as spraying, pouring, and solidification are appropriately selected. The amount to be contained varies depending on the composition, but generally, an amount of 0.1% or more, desirably 0.5% or more is suitable as the glucosyl transfer sugar.

  Next, the present invention will be described more specifically by experiments.

<Experiment 1: Production of Cozybiose Phosphorylase>
“ATCC Catalog of Bacteria and Bacteriophages, 18th Edition” (American Type) except that 0.5 w / v% trehalose was used instead of 0.5 w / v% glucose as the carbon source In accordance with the preparation method of Thermoanaerobium brokki medium described in Culture Collection (1992), pages 452 to 456, 100 ml each of the medium was prepared in a 100 ml pressure-resistant bottle and then inoculated with Thermoanaerobium brokki ATCC35047. The seed culture was obtained by static culture at 60 ° C. for 48 hours.

  About 10 l of medium with the same composition as in the case of seed culture is placed in 4 stainless steel bottles with a volume of 11 l, heat-sterilized and cooled to a temperature of 60 ° C., then seeded with 1 v / v% of the seed culture solution per volume of the medium Then, static culture was performed at a temperature of 60 ° C. for about 40 hours.

  About 40 l of the culture solution was centrifuged to obtain 92 g of cultured cells. The bacterial cells were suspended in 10 mM phosphate buffer, sonicated, and then centrifuged to obtain a microbial disrupted supernatant. The activity of cordobiose phosphorylase was 0.1 unit / ml in terms of per culture broth.

<Experiment 2: Purification of Kojibiose Phosphorylase>
The cell disruption supernatant obtained in Experiment 1 was concentrated in a UF membrane, and about 360 ml of a concentrated enzyme solution having about 10 units of cordobiose phosphorylase per ml was recovered.

  Of the resulting concentrated enzyme solution, 300 ml was dialyzed against 10 mM phosphate buffer (pH 7.0) for 24 hours and centrifuged to remove insolubles. The dialysate supernatant (380 ml) was subjected to ion exchange column chromatography (gel amount: 380 ml) using DEAE-Toyopearl 650 gel (manufactured by Tosoh Corporation).

  The cordobiose phosphorylase of the present invention was adsorbed on DEAE-Toyopearl 650 gel and eluted from the column with a linear gradient from 0 M saline to 0.5 M saline. The enzyme activity fraction eluted with about 0.2M sodium chloride was collected and further purified by the following method. Dialyze the same buffer containing 1.5M ammonium sulfate, centrifuge the dialyzed solution to remove insolubles, and then use hydrophobic column chromatography using Butyl Toyopearl 650 Gel (manufactured by Tosoh Corporation) ( Gel amount 100 ml). The adsorbed cordobiose phosphorylase was eluted from the column with a linear gradient from 1.5 M ammonium sulfate to 0 M ammonium sulfate, and the enzyme activity fraction was recovered.

  Subsequently, gel filtration chromatography (gel amount: 300 ml) using Ultrogel AcA44 (Sepracol, France) was performed, and the eluted enzyme activity fraction was collected.

  The recovery rate of the purified enzyme preparation obtained by the above purification means was about 20% based on the cell disruption supernatant in terms of activity. The specific activity of the purified enzyme preparation was 71.4 units per mg of protein. The protein was quantified using the bovine serum albumin as a standard according to the Raleigh method.

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

<Experiment 3: Properties of Cozybiose Phosphorylase>
The Kojibiose phosphorylase preparation obtained in Experiment 2 was subjected to SDS-polyacrylamide gel electrophoresis (gel concentration: 10 w / v%) and compared with a molecular weight marker (manufactured by Nippon Bio-Rad Laboratories Co., Ltd.). The molecular weight of the enzyme was measured and found to be 83,000 ± 5,000 daltons. The molecular weight was measured by gel filtration using a TSKgel G4000SW column (φ7.5 mm × 600 mm, manufactured by Tosoh Corporation). As a result, the molecular weight was 500,000 ± 30,000 Dalton.

  This enzyme is subjected to isoelectric focusing polyacrylamide gel electrophoresis containing 2% w / v ampholine (manufactured by Pharmacia El Kavy, Sweden) with purified cordozyme phosphorylase. The isoelectric point was found to be 4.4 ± 0.5.

The effects of temperature and pH on the cordobiose phosphorylase activity of the present invention were examined according to the activity measurement method. That is, when investigating the influence of temperature, the reaction is carried out at any temperature of about 40 ° C. to about 80 ° C. instead of the reaction temperature of 60 ° C. in the activity measuring method, and when examining the influence of pH, the activity measurement is performed. In place of the buffer solution used in the method, the reaction is carried out using a buffer solution having a buffer capacity at a pH of about 4 to about 8, and then the reaction is stopped in the same manner as in the activity measurement method to produce glucose. The amount was measured. The results of the measurement results expressed as relative values with respect to the maximum value are shown in FIG. 1 (effect of temperature) and FIG. 2 (effect of pH). The optimum temperature of the enzyme was pH 5.5 at a reaction of 30 minutes and around 65 ° C., and the optimum pH was around 5.5 at a reaction of 60 ° C. and 30 minutes. The temperature stability of this enzyme is determined by maintaining the enzyme activity remaining after 10 mM McKilbain buffer solution (pH 5.5) in which the enzyme is dissolved is kept at a temperature of about 40 ° C. to 80 ° C. for 1 hour and cooled with water. It was determined according to the activity measurement method. Further, the pH stability is adjusted to pH 5.5 after dissolving the enzyme in a buffer solution having a buffer capacity at any pH of about 3.5 to about 10 and holding at 4 ° C. for 24 hours, The remaining enzyme activity was determined according to the activity measurement method. The results of the measurement results expressed as relative values with respect to the maximum values are shown in FIG. 3 (temperature stability) and FIG. 4 (pH stability). The temperature stability of this enzyme was up to around 65 ° C., and the pH stability was about 5.5 to 10.0. The enzyme activity was inhibited by 1 mM Hg ⁺⁺ .

<Experiment 4: Partial amino acid sequence of Cozybiose phosphorylase>
(1) N-terminal amino acid sequence After a portion of the purified enzyme preparation obtained by the method of Experiment 2 was dialyzed against distilled water, about 40 μg of protein amount was used as a sample for N-terminal sequence analysis. The N-terminal sequence was analyzed from the N-terminal to 5 residues using a protein sequencer model 473A (Applied Biosystems, USA). The sequence obtained from the N-terminus was the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. Furthermore, when the same sample was used and analyzed in more detail by the same method, the enzyme was found to contain the amino acid sequence shown in SEQ ID NO: 6 in the sequence listing at the N-terminus.

(2) Intermediate partial amino acid sequence A portion of the purified enzyme preparation obtained by the method of Experiment 2 was dialyzed against 10 mM Tris / HCl buffer (pH 9.0), and then 1 mg / ml with the same buffer. Dilute to a concentration of. 10 μg of lysyl endopeptidase (sold by Wako Pure Chemical Industries, Ltd.) was added to this sample solution (1 ml), and the mixture was reacted at 30 ° C. for 22 hours for peptide formation. Reverse phase HPLC was performed to isolate the resulting peptide. A microbonder sphere C18 column (diameter 2.1 mm × length 150 mm, manufactured by Waters, USA) was used, and a flow rate of 0.9 ml / min from a 0.1 v / v% trifluoroacetic acid-0% acetonitrile solution at room temperature was reduced to 0. The measurement was performed under a linear gradient condition of 120 minutes up to a 1 v / v% trifluoroacetic acid-48 v / v% acetonitrile solution. The peptide eluted from the column was detected by measuring the absorbance at a wavelength of 210 nm. Two peptides, KP15 (retention time of 66 minutes) and KP23 (retention time of 96 minutes), which were well separated from other peptides, were collected and dried in vacuo, and then 200 μl of 0.1 v / v% trifluoroacetic acid-50 v Dissolved in / v% acetonitrile solution. These peptide samples were subjected to a protein sequencer, and amino acid sequences from the N-terminal to 5 residues were analyzed. From peptide KP15, the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing was obtained, and from peptide KP23, the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing was obtained. Furthermore, when analyzed in more detail by the same method using the same sample, peptide KP15 and peptide KP23 contain the amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8 in the N-terminal part thereof, respectively. There was found.

<Experiment 5: Substrate specificity in the glycolytic reaction of Cozybee phosphorylase>
D-glucose, maltose, sucrose, lactose, trehalose, neotrehalose, cellobiose, melibiose, cordierbiose, isomaltose, sophorose, gentiobiose, nigerose and laminaribiose Codybiose phosphorylase was added in an amount of 10 units per gram of saccharide solid, and allowed to act at 60 ° C. and pH 5.5 for 24 hours in the presence of 5 mM sodium dihydrogen phosphate. The sugar concentration in each reaction solution was 2 w / v%. The reaction solution before and after the enzyme reaction was subjected to thin layer chromatography (hereinafter abbreviated as “TLC”) using Kieselgel 60 (Merck, Inc .; aluminum plate, 20 × 20 cm). TLC was developed once at room temperature using 1-butanol: pyridine: water = 7: 3: 1 (volume ratio) as a developing solvent, and then sprayed with a 20 v / v% sulfuric acid-methanol solution, and about 10 at 110 ° C. Color was developed by heating for minutes. The spots derived from both reaction solutions were compared to determine the presence or absence of enzyme action on each carbohydrate. The results are shown in Table 1.

  As shown in Table 1, it was found that the cordobiose phosphorylase of the present invention acts on cordobiose with extremely high specificity to produce D-glucose and β-D-glucose-1-phosphate. On the other hand, it was found that it does not act on other carbohydrates.

<Experiment 6: Receptor specificity in glucosyl transfer sugar formation reaction of Cozybee phosphorylase>
In an aqueous solution containing one of various monosaccharides, oligosaccharides and sugar alcohols shown in Table 2 as an acceptor and β-D-glucose-1-phosphate as a sugar donor dissolved in an equal amount in a solid substance amount Purified cordobiose phosphorylase obtained by the method of Experiment 2 was added in units of 10 units per gram of β-D-glucose-1-phosphate, and reacted at 60 ° C. and pH 5.5 for 24 hours. The concentrations of the acceptor and sugar donor in the reaction solution were both 1 w / v%. The reaction solution before and after the enzyme reaction was subjected to TLC in the same manner as in Experiment 5 and then developed. The spots derived from both reaction solutions were compared, and it was determined from the newly generated spots after the reaction whether or not transfer sugars were generated. Further, by visually observing the color intensity of the spot determined to be a transfer sugar, the generation rate of the transfer sugar was relatively evaluated. The results are shown in Table 2.

  As shown in Table 2, the cordobiose phosphorylase of the present invention comprises β-D-glucose-1 phosphate as a sugar donor, monosaccharides such as D-glucose and L-sorbose, maltose, cordobiose, trehalose, It has been found that glucosyl groups are well transferred to disaccharides such as sucrose and oligosaccharides such as maltotriose, maltotetraose, and maltopentaose to produce trisaccharose. When α-D-glucose-1-phosphate was used as a sugar donor instead of β-D-glucose-1-phosphate, no glucosyl transfer sugar was obtained.

  The detailed structures of some of the glucosyltransferases that have been clarified to be produced by the transglycosylation reaction using the cordobiose phosphorylase of the present invention by Experiment 6 will be described with reference to Experiments 7 to 12 below. .

<Experiment 7: Glucosyl transfer sugar from β-D-glucose-1-phosphate and D-glucose>
The sugar component of the enzyme reaction solution using β-D-glucose-1-phosphate and D-glucose as substrates as obtained in Experiment 6 was analyzed by gas chromatography (hereinafter abbreviated as “GLC”). A part of the enzyme reaction solution was dried, dissolved in pyridine, and then trimethylsilylated as an analytical sample for GLC. The GLC column is a stainless steel column (3 mmφ × 2 m) packed with 2% silicon OV-17 / chromosolve W (manufactured by GL Sciences Inc.), the carrier gas is nitrogen gas at a flow rate of 40 ml / min, and the column oven temperature. Was analyzed from 160 ° C. to 320 ° C. at a heating rate of 7.5 ° C./min. For detection, a flame ion detector was used.

  As a result, the retention time of the peak of glucosyl transfer sugar from β-D-glucose-1-phosphate and D-glucose by the cordobiose phosphorylase of the present invention was consistent with that of known carbohydrate cordobiose. The glucosyl transfer saccharide from β-D-glucose-1-phosphate and D-glucose by the cordobiose phosphorylase of the present invention is judged to be cordobiose.

<Experiment 8: Glucosylsorbose>
In order to confirm the glucosyltransferase produced by the transglycosylation reaction to L-sorbose by the cordobiose phosphorylase of the present invention, the glucosyltransferase was produced, isolated, and the structure was examined. First, an aqueous solution containing 2.5% of β-D-glucose-1-phosphate and 2.5% of L-sorbose was adjusted to pH 5.0, and the purified cordobiose phosphorylase obtained by the method of Experiment 2 was added thereto. 10 units per gram of β-D-glucose-1-phosphate were added, reacted at 60 ° C. for 48 hours, and then heated at 100 ° C. for 10 minutes to deactivate the enzyme. In accordance with the method shown in Experiment 7, a part of this reaction completed solution was analyzed by GLC. As a result, it was confirmed that a component having a different retention time was produced in both the β-D-glucose-1-phosphate and L-sorbose in the reaction completion solution, and this component is considered to be the glucosyl transferase. It was. Moreover, the production rate of the glucosyl transferase in this reaction obtained from the analysis result by GLC was about 24%. The remaining amount of the reaction solution is decolorized with activated carbon, filtered, purified by desalting with H-type and OH-type ion exchange resins, concentrated to about 50%, and subjected to column chromatography described below. A fraction containing a high amount of glucosyl transfer sugar was collected.

  As the fractionation resin, an alkali metal type strongly acidic cation exchange resin (manufactured by Tokyo Organic Chemical Industry Co., Ltd., trade name “XT-1016”, Na type, cross-linking degree 4%) is used, and the inner diameter is 3 cm and the length is 1 m. Four jacketed stainless steel columns were filled with water suspension and connected in series so that the total length of the resin layer was about 4 m. While maintaining the temperature in the column at 40 ° C., the sugar solution is added to the resin at 5 v / v%, and the water is fractionated by flowing warm water at 40 ° C. at a flow rate of SV0.15. Minutes were collected.

  The high sugar-containing fraction is desalted, purified, concentrated to about 40%, and chromatographed using a column packed with octadecyl silica gel (YMC Corporation, trade name “YMC-Pak ODS”). The glucosyl transfer sugar fraction was collected. Concentrate the collected fraction to a concentration of about 40% and repeat the operation for column chromatography using octadecyl silica gel again. The collected glycosyl transfer sugar-rich solution is desalted, purified, concentrated and vacuum dried. Thus, the high glucosyl transfer sugar-containing powder was obtained at a yield of about 20% per saccharide solid substance subjected to the reaction. When this powder sample was analyzed by GLC according to the method shown in Experiment 7, this sample contained about 98% of the glucosyl transferase per solid.

  Next, the high glucosyl transfer sugar-containing powder was acid-decomposed and subjected to GLC analysis. As a result, it was found that the glucosyl transferase produced D-glucose and L-sorbose at a molar ratio of about 1: 1 by acid decomposition. Further, the methyl glycosyl transfer sugar-rich powder was methylated and then hydrolyzed with acid, followed by reduction and acetylation. Tetramethyl-1,5-diacetylglucitol was observed. As for the above, the said glucosyl transfer sugar is what D-glucose and L-sorbose couple | bonded by the molar ratio of 1: 1, Furthermore, OH group of 1-position of D-glucose is concerned in the coupling | bonding. Suggests that No methylated derivative of sorbose was observed, but this was probably due to decomposition by acid.

In order to investigate the structure of the glucosyl transfer sugar in more detail, a ¹³ C-nuclear magnetic resonance spectrum was measured. As a result, ¹³ C-NMR spectrum (100 MHz, D ₂ O): σ ppm from TSP 101.2, 100.4, 99.5, 99.3, 77.1, 75.6, 74.9, 74.5, The values of 74.0, 73.2, 72.2, 66.3, 63.2, 62.1 were obtained. From this analysis data, the glucosyl transferase is glucosyl sorbose, which is a disaccharide in which the 1-position of D-glucose is linked to the 5-position of L-sorbose in the α-form, that is, α-D-glucosyl (1 → 5) -L- It turned out to be sorbose. This substance is a novel carbohydrate that has not been known so far.

<Experiment 9: Kojibiosyl glucose>
In order to confirm the glucosyltransferase produced by the transfer reaction to maltose by the cordobiose phosphorylase of the present invention, the glucosyltransferase was produced, isolated, and the structure was examined. First, an aqueous solution containing 5% β-D-glucose-1-phosphate and 10% maltose was adjusted to pH 6.0, and the purified cordierbiose phosphorylase obtained by the method of Experiment 2 was added to β-D-glucose- 20 units were added per gram of phosphoric acid, reacted at 60 ° C. for 48 hours, and then heated at 100 ° C. for 10 minutes to deactivate the enzyme. In accordance with the method shown in Experiment 7, a part of this reaction completed solution was analyzed by GLC. As a result, it was confirmed that a component having a different retention time was produced in both the β-D-glucose-1-phosphate and maltose in the reaction end solution, and this component was considered to be the glucosyl transferase. Moreover, the production rate of the glucosyl transferase in this reaction determined from the analysis result by GLC was about 40%. The remaining total amount of this reaction solution was fractionated and purified in the same manner as in Experiment 7, concentrated, and vacuum dried to obtain the glucosyl transfer sugar-rich powder with a yield of about 20% per saccharide solid substance subjected to the reaction. It was. This powder preparation contained about 98% of the glucosyl transfer sugar per solid.

  Next, after methylating the said glucosyl transfer sugar high content powder, it hydrolyzed with an acid, and the partial methyl hexitol acetate obtained by reducing and acetylating subsequently was used for the GLC analysis. As a result, 2,3,4,6-tetramethyl-1,5-diacetylglucitol and 3,4,6-trimethyl-1,2,5-triacetylglucitol and 2,3,6-trimethyl were obtained. -1,4,5-triacetylglucitol was found in a ratio of about 1: 1. This is because the glucosyl transferase is composed of glucose in which only the 1-position OH group is involved in binding, and both the 1-position and 2-position OH groups are involved in binding, or only the 2-position OH group is involved. Glucose involved in binding and glucose in which both 1-position and 4-position glucose are involved in binding, or glucose in which only the 4-position OH group is involved in binding are in a ratio of 1: 1 to 1, respectively. This suggests that they are oligosaccharides bonded to each other.

In order to investigate the structure of the glucosyl transfer sugar in more detail, a ¹³ C-nuclear magnetic resonance spectrum was measured. As a result, ¹³ C-NMR spectrum (100 MHz, D ₂ O): σ ppm from TSP 99.5, 99.4, 99.1, 98.6, 94.6, 79.2, 78.8, 78.6 78.0, 77.6, 77.3, 77.0, 76.1, 75.6, 75.3, 74.7, 74.2, 74.1, 73.8, 72.7, 72. The values of 2, 72.1, 63.6, 63.5, 63.2, 63.1 were obtained. From this analysis data, the structure of the present glucosyl transferase is a cordybiosyl glucose, which is a trisaccharide in which D-glucose is α-type and 1,2-linked to the non-reducing terminal glucose residue of maltose, that is, α-D-glucosyl (1 → 2) It was found to be α-D-glucosyl (1 → 4) -D-glucose.

<Experiment 10: Koji Triose>
In order to confirm the glucosyl transferase produced by the transglycosylation reaction to cordobiose by the cordobiose phosphorylase of the present invention, the glucosyl transferase was produced, isolated, and the structure was examined. First, 5 mM sodium dihydrogen phosphate dissolved in 20% cordobiose was adjusted to pH 5.5, and 10 units of purified cordierbiose phosphorylase obtained by the method of Experiment 2 was added per 1 g of cordierbiose, and the mixture was heated at 60 ° C. for 48 hours. Then, the enzyme was inactivated by heating at 100 ° C. for 10 minutes. In accordance with the method shown in Experiment 7, a part of this reaction completed solution was analyzed by GLC. As a result, it was confirmed that a component having different retention times was produced in both the cordobiose and the D-glucose and β-D-glucose-1-phosphate in the reaction completion solution. it was thought. Moreover, the production rate of the glucosyltransferase in this reaction determined from the analysis result by GLC was about 30%. The entire remaining amount of this reaction solution was fractionated and purified in the same manner as in Experiment 8, concentrated, and vacuum-dried to obtain the glucosyl transfer sugar-rich powder with a yield of about 15% per solid of cordobiose subjected to the reaction. . This powder preparation contained about 98% of the glucosyl transfer sugar per solid.

  Next, after methylating the said glucosyl transfer sugar high content powder, it hydrolyzed with an acid, and the partial methyl hexitol acetate obtained by reducing and acetylating subsequently was used for the GLC analysis. As a result, 2,3,4,6-tetramethyl-1,5-diacetylglucitol and 3,4,6-trimethyl-1,2,5-triacetylglucitol were in a ratio of about 1: 2. Admitted. This is because the glucosyl transferase is composed of glucose in which only the 1-position OH group is involved in binding, and both the 1-position and 2-position OH groups are involved in binding, or only the 2-position OH group is involved. This suggests that the glucose involved in the binding is an oligosaccharide bound to each other at a ratio of 1: 2. Furthermore, taking into account the possibility of glucose binding mode, this indicates that the glucosyl transfer saccharide has both glucose in which only the 1-position OH group is involved in binding and the 1- and 2-position OH groups. Strongly suggesting that the glucose involved in the binding and the glucose in which only the 2-position OH group is involved in the binding are oligosaccharides bound to each other at a ratio of 1: 1 to 1: 1, respectively. Yes.

In order to investigate the structure of the glucosyl transfer sugar in more detail, a ¹³ C-nuclear magnetic resonance spectrum was measured. As a result, ¹³ C-NMR spectrum (100 MHz, D ₂ O): σ ppm from TSP 99.3, 99.0, 98.3, 97.6, 96.5, 92.2, 81.3, 78.8, 78.6, 78.5, 77.2, 77.1, 75.7, 75.6, 74.7, 74.6, 74.5, 74.3, 74.2, 74.1, 74. 0, 73.9, 72.6, 72.3, 72.2, 72.1, 72.0, 63.6, 63.4, 63.2, 63.1, 63.1, 63.0 Each value was obtained. From this analytical data, the structure of the present glucosyl cordobiose is cordierose, which is a trisaccharide in which D-glucose is α-linked in the form of 1,2-linked to the non-reducing terminal glucose residue of cordobiose, that is, α-D-glucosyl (1 → 2) It was found to be α-D-glucosyl (1 → 2) -D-glucose.

<Experiment 11: Kojibiosyl glucoside>
In order to confirm the glucosyl transferase produced by the transfer reaction to trehalose by the cordobiose phosphorylase of the present invention, the glucosyl transferase was produced, isolated, and the structure was examined. First, an aqueous solution containing 5% β-D-glucose-1-phosphate and 10% trehalose was adjusted to pH 5.5, and the purified cordierbiose phosphorylase obtained by the method of Experiment 2 was added to β-D-glucose- 10 units were added per 1 g of phosphoric acid, reacted at 60 ° C. for 48 hours, and then heated at 100 ° C. for 10 minutes to deactivate the enzyme. In accordance with the method shown in Experiment 7, a part of this reaction completed solution was analyzed by GLC. As a result, it was confirmed that a component having a different retention time was produced in both the β-D-glucose and trehalose in the reaction completion solution, and this component was considered to be the glucosyl transferase. Moreover, the production rate of the glucosyl transferase in this reaction obtained from the analysis result by GLC was about 75%. The remaining total amount of this reaction solution was fractionated and purified in the same manner as in Experiment 8, concentrated, and vacuum dried to obtain the glucosyl transfer sugar-rich powder with a yield of about 65% per saccharide solid substance subjected to the reaction. It was. This powder preparation contained about 98% of the glucosyl transfer sugar per solid.

  Next, after methylating the said glucosyl transfer sugar high content powder, it hydrolyzed with an acid, and the partial methyl hexitol acetate obtained by reducing and acetylating subsequently was used for the GLC analysis. As a result, 2,3,4,6-tetramethyl-1,5-diacetylglucitol and 3,4,6-trimethyl-1,2,5-triacetylglucitol were found in a ratio of 2 to 1. It was. This means that the glucosyltransferase has a two-to-one relationship between glucose in which only the 1-position OH group is involved in binding and glucose in which both the 1- and 2-position OH groups are involved in binding. This suggests that the oligosaccharides are bound to each other in proportion.

In order to investigate the structure of the glucosyl transfer sugar in more detail, a ¹³ C-nuclear magnetic resonance spectrum was measured. As a result, ¹³ C-NMR spectrum (100 MHz, D ₂ O): σ ppm from TSP 98.6, 96.2, 93.3, 77.3, 75.8, 75.4, 75.1, 74.8, The values of 74.6, 74.6, 74.0, 73.9, 72.5, 72.3, 72.2, 63.4, 63.4, 63.3 were obtained. From this analytical data, the structure of the present glucosyl trehalose is cordierbiosyl glucoside (also known as ceraginose), which is a trisaccharide having D-glucose 1 or 2 linked to trehalose, that is, α-D-glucosyl (1 → 2) α-D- It was found to be glucosyl (1,1) α-D-glucoside.

<Experiment 12: Kojibiosyl fructoside>
In order to confirm the glucosyl transferase produced by the transfer reaction to sucrose by the cordobiose phosphorylase of the present invention, the glucosyl transferase was produced, isolated, and the structure was examined. First, an aqueous solution containing 5% β-D-glucose-1-phosphate and 10% sucrose was adjusted to pH 5.5, and the purified cordierbiose phosphorylase obtained by the method of Experiment 2 was added to β-D-glucose- 10 units were added per 1 g of phosphoric acid, reacted at 60 ° C. for 48 hours, and then heated at 100 ° C. for 10 minutes to deactivate the enzyme. In accordance with the method shown in Experiment 7, a part of this reaction completed solution was analyzed by GLC. As a result, it was confirmed that a component having a different retention time was produced in both the β-D-glucose-1-phosphate and sucrose in the reaction end solution, and this component was considered to be the glucosyltransferase. Moreover, the production rate of the glucosyl transferase in this reaction determined from the analysis result by GLC was about 70%. The remaining total amount of this reaction solution was fractionated and purified in the same manner as in Experiment 8, concentrated, and vacuum dried to obtain the glucosyl transfer sugar-rich powder with a yield of about 50% per saccharide solid substance subjected to the reaction. It was. This powder preparation contained about 98% of the glucosyl transfer sugar per solid.

  Next, the high glucosyl transfer sugar-containing powder was acid-decomposed and subjected to GLC analysis. As a result, it was found that the glucosyl transfer sugar produced D-glucose and D-fructose at a molar ratio of about 2 to 1 by acid decomposition. Further, when methyl of the glucosyl transfer sugar-rich powder was hydrolyzed with acid, and subsequently reduced and acetylated, the partial methyl hexitol acetate obtained by GLC was analyzed by GLC, 2,3,4,6-tetra Methyl-1,5-diacetylglucitol and 3,4,6-trimethyl-1,2,5-triacetylglucitol were found in a 1: 1 ratio. From the above, the glucosyl transferase is a combination of D-glucose and D-fructose in a molar ratio of 2 to 1, and the D-glucose constituting the glucosyl transferase is OH at the 1-position. This suggests that the group in which only the group is involved in bonding and the group in which both the 1-position and 2-position OH groups are involved in bonding exist in a molar ratio of 1: 1. In addition, although the methylated derivative of D-fructose was not recognized, it seems that this was decomposed | disassembled by the acid.

In order to investigate the structure of the glucosyl transfer sugar in more detail, a ¹³ C-nuclear magnetic resonance spectrum was measured. As a result, ¹³ C-NMR spectrum (100 MHz, D ₂ O): σ ppm from TSP107.0, 99.3, 92.5, 84.0, 79.1, 78.5, 76.5, 75.7, The values of 74.9, 74.7, 74.1, 74.0, 72.2, 72.1, 64.9, 64.6, 63.2, 63.1 were obtained. From this analysis data, the structure of the present glucosyl sucrose is cordierbiosyl fructoside, which is a trisaccharide in which D-glucose is α-linked and 1,2-linked to the glucose residue of sucrose, that is, α-D-glucosyl (1 → 2). It was found to be α-D-glucosyl (1 → 2) β-D-fructoside. This substance is a novel carbohydrate that has not been known so far.

<Experiment 13: Acute toxicity>
Using 7-week-old dd mice, the glucosyl sorbose-rich powder, the cordierbiosyl-glucose-rich powder, the cordierioose-rich powder, the cordierbiosylglucoside-rich powder and the cordiobio obtained in Experiments 8 to 12 When acute toxicity tests were carried out by orally administering each of the powders containing a high content of silfrucside, no deaths were observed at the maximum dose that could be administered, that is, the body weight of the mouse of 50 g / kg. Accordingly, all of these transfer sugars of the present invention are extremely toxic substances.

  Hereinafter, the cordierbiose phosphorylase of the present invention, the DNA of the present invention encoding the cordobiose phosphorylase, and a method for producing a glucosyltransferase-containing saccharide using the same will be described in Examples 1 to 16, and Compositions containing saccharides are shown in Examples 17 to 32.

<Enzyme solution>
Thermoanaerobium broccii ATCC 35047 was seed-cultured by the method shown in Experiment 1 and then added to a medium having the same composition prepared in an anaerobic fermenter according to the method shown in Experiment 1, with 1 v / v% per volume of the medium. The seed culture solution was inoculated and cultured at a culture temperature of 65 ° C. for about 30 hours. The bacterial cells obtained by centrifuging the culture broth were subjected to ultrasonic disruption, and the Codybiose phosphorylase activity of the supernatant of the disrupted solution was measured. When this activity was converted per 1 ml of the culture solution, it was 0.08 unit. The lysate supernatant is concentrated with an ultrafiltration membrane, and the concentrated solution is dialyzed to obtain an enzyme solution having about 8 units of cordobiose phosphorylase activity per ml with respect to the total activity of the original culture solution. % Yield.

<Preparation of DNA>
Thermoanaerobium brokkii (ATCC 35047) was cultured at 11 ° C. in the same composition as that shown in Experiment 1 for 24 hours at 60 ° C. according to the method shown in Experiment 1. The cells are separated from the culture by centrifugation, suspended in an appropriate amount of Tris-EDTA-saline buffer (hereinafter abbreviated as “TES buffer”) (pH 8.0), and lysozyme is suspended in the cell suspension. After adding 0.05% (w / v) to the volume of the solution, it was incubated at 37 ° C. for 30 minutes. Thereafter, the treated product was kept at −80 ° C. for 1 hour to freeze, and then a TES buffer / phenol mixed solution in which a TES buffer solution (pH 9.0) was previously added and heated to 60 ° C. was then added. In addition, the mixture was sufficiently stirred, and the upper layer formed by centrifugation after cooling with ice was collected. A precipitate formed by adding 2 volumes of cold ethanol to this upper layer was collected, dissolved in an appropriate amount of SSC buffer (pH 7.1), 7.5 μg ribonuclease and 125 μg protease were added, and 1 ° C. at 37 ° C. Incubated for hours. A mixed solution of chloroform / isoamyl alcohol was added thereto and stirred, and then the upper layer formed by allowing to stand was collected, and a precipitate formed by adding cold ethanol to the upper layer was collected. The precipitate was rinsed with cold 70% (v / v) ethanol and vacuum dried to obtain DNA. The obtained DNA was dissolved in SSC buffer (pH 7.1) so as to have a concentration of about 1 mg / ml, and frozen at −80 ° C.

<Preparation of transformant and recombinant DNA>
Take 1 ml of the DNA solution obtained by the method of Example 2, add about 20 units of restriction enzyme Hind III, incubate at 37 ° C. for 30 minutes to partially decompose the DNA, and then sucrose density gradient ultracentrifugation. A DNA fragment having a length of about 2,000 to 5,000 base pairs was collected. Separately, after the plasmid vector “Bluescript II SK (+)” manufactured by Stratagene Cloning Systems was completely cleaved with the restriction enzyme Hind III by a conventional method, 0.3 μg of the cleaved plasmid vector was obtained in advance. About 3 μg of the DNA fragment was ligated using the “DNA ligation kit” manufactured by Takara Shuzo according to the attached instructions, and the resulting recombinant DNA was subjected to stratagene cloning by the ordinary competent cell method. -A gene library was prepared by transforming 100 µl of E. coli "Epicurian Coli XL1-Blue" manufactured by Systems.

The thus obtained transformant as a gene library was prepared by a conventional method, and 10 g / l tryptone, 5 g / l yeast extract, 5 g / l sodium chloride, 75 mg / l ampicillin sodium salt and 50 mg / l 5 -Inoculated on an agar plate medium (pH 7.0) containing bromo-4-chloro-3-indolyl-β-galactoside, cultured at 37 ° C for 18 hours, and then about 4,000 white colonies formed on the medium. The pieces were fixed on an Amersham nylon membrane “Hybond-N +”. Separately, the oligonucleotide of the base sequence represented by 5′-TTYGAYGAARAAYAYATGCC-3 ′ based on the first to seventh amino acid sequences in the amino acid sequence shown in SEQ ID NO: 7 of the Sequence Listing, which was clarified by the method of Experiment 4 Was synthesized by isotope labeling using [γ- ³² P] ATP and T4 polynucleotide kinase according to a conventional method to obtain a synthetic DNA as a probe. Among the colonies fixed on the nylon membrane obtained earlier, a colony showing a remarkable association with the probe was selected by applying a normal colony hybridization method, and the transformant was named “TKP1”. .

  This transformant TKP1 was inoculated in an L-broth medium (pH 7.0) containing 100 μg / ml ampicillin sodium salt according to a conventional method, and cultured with shaking at 37 ° C. for 24 hours. After completion of the culture, the cells were collected from the culture by centrifugation, and the recombinant DNA was extracted by the usual alkali-SDS method. When the base sequence of this recombinant DNA was analyzed by a normal dideoxy method, the recombinant DNA was derived from Thermoanaerobium brokki (ATCC 35047) and has a chain length of 3956 base pairs in SEQ ID NO: 9 of the base sequence shown in FIG. As shown in FIG. 5, in this recombinant DNA, the DNA was ligated downstream of the recognition site by the restriction enzyme Hind III. On the other hand, the amino acid sequence deduced from this base sequence is as shown in SEQ ID NO: 9, and this amino acid sequence and the N-terminal amino acid sequence of the present invention Combibiose phosphorylase confirmed by the method of Experiment 4 and When comparing the amino acid sequences shown in SEQ ID NOs: 1 to 3 and SEQ ID NOs: 6 to 8 in the Sequence Listing, which are the middle part amino acid sequences, the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3 in the Sequence Listing are respectively It completely matched with the first to fifth, 590th to 594th and 357th to 361rd amino acid sequences in the amino acid sequence shown in SEQ ID NO: 9. In addition, the amino acid sequences shown in SEQ ID NOs: 6, 7, and 8 in the sequence listing are the 1st to 10th, 590th to 599th, and 357th to 366th amino acids in the amino acid sequence shown in SEQ ID NO: 9 in the sequence listing, respectively. Completely matched the amino acid sequence. The above is the fact that the cordobiose phosphorylase of the present invention comprises the amino acid sequence shown in SEQ ID NO: 4 in the Sequence Listing, and the enzyme in Thermoanaerobium brokki (ATCC 35047) is SEQ ID NO: in the Sequence Listing. It shows that it is encoded by DNA of the base sequence shown in FIG. The recombinant DNA prepared as described above and confirmed in the base sequence was named “pTKP1”.

<Production of cordobiose phosphorylase by the transformant>
100 ml of an aqueous solution containing 16 g / l polypeptone, 10 g / l yeast extract and 5 g / l sodium chloride was placed in a 500 ml Erlenmeyer flask, treated in an autoclave at 121 ° C. for 15 minutes, cooled and aseptically adjusted to pH 7.0. Thereafter, 10 mg of ampicillin sodium salt was aseptically added to prepare a liquid medium. This liquid medium was inoculated with the transformant TKP1 obtained by the method of Example 3 and cultured at 37 ° C. for about 20 hours with aeration and stirring as a seed culture solution. Next, 7 l of a medium having the same composition as that used for seed culture was prepared in a 10 l fermenter according to the seed culture, inoculated with 70 ml of the seed culture, and cultured with aeration and stirring for about 20 hours. According to a conventional method, the culture is centrifuged to recover the cells, suspended in 10 mM phosphate buffer (pH 7.0), sonicated to disrupt the cells, and further centrifuged to insoluble. Things were removed and the supernatant was collected. The supernatant was dialyzed against 10 mM phosphate buffer, and then the Codybiose phosphorylase activity in the dialysate was measured. As a result, about 500 units of the enzyme were produced per liter of culture.

  As a first control, Escherichia coli XL1-Blue strain was cultured under the same conditions as in the case of the above-described transformant except that no ampicillin was added to the medium. Was collected and dialyzed. As a second control, Thermoanaerobium brokkii (ATCC 35047) was used in the same manner as in the case of the transformant except that it did not contain ampicillin. The culture was allowed to stand at 0 ° C., and the supernatant of the crushed cell was collected from the culture and dialyzed as in the case of the transformant described above. No enzyme activity was found in the first control dialysate. The enzyme activity was observed in the second control dialysate, which in this case was about 100 units per liter of culture, which was clearly lower than that of transformant TKP1.

  The dialyzate obtained by the method of Example 4 was further purified by column chromatography using DEAE-Toyopearl 650 gel and Ultrogel AcA44 according to the method shown in Experiment 2, and the purified enzyme was further tested. Analysis was performed according to the method shown in FIG. Molecular weight 83,000 ± 5,000 dalton by SDS-polyacrylamide gel electrophoresis, molecular weight 500,000 ± 30,000 dalton by gel filtration, isoelectric point 4.4 ± by polyacrylamide gel electrophoresis 0.5, optimum temperature of cordobiose phosphorylase activity around 65 ° C., optimum pH around 5.5, temperature stability up to around 65 ° C., pH stability about 5.5 to 10.0, Experiment 1 It was substantially the same as the physicochemical properties of the enzyme prepared by the method shown in 2 or 2. The above results indicate that the cordobiose phosphorylase of the present invention can be produced satisfactorily by recombinant DNA technology, and the productivity of the enzyme is also significantly improved.

<Enzyme solution>
The transformant TKP1 obtained by the method shown in Example 3 was cultured by the method shown in Example 4. The bacterial cells obtained by centrifuging the culture broth were subjected to ultrasonic disruption, and the Codybiose phosphorylase activity of the supernatant of the disrupted solution was measured. When this activity was converted per 1 ml of the culture solution, it was about 0.5 units. The supernatant of the crushed liquid is concentrated with an ultrafiltration membrane, and the concentrated liquid is dialyzed to obtain an enzyme solution having about 10 units of cordobiose phosphorylase activity per ml with respect to the total activity of the original culture solution. % Yield.

<Corgebiose-containing sugar solution>
In a 25 mM dipotassium phosphate-citrate buffer solution (pH 6.0) containing 5% maltose, 5 units of commercially available maltose phosphorylase derived from bacteria is used per 1 g of maltose, and Combibiose phosphorylase prepared by the method of Example 1 is used in 1 g of maltose. 40 units per unit were added, and the reaction was carried out at 30 ° C. for 72 hours. The reaction solution was heated at 100 ° C. for 30 minutes to inactivate the enzyme, then cooled, decolorized and filtered with activated carbon according to a conventional method, purified by desalting with H-type and OH-type ion exchange resins, By concentrating, a syrup-like cordierbiose-containing sugar solution having a concentration of about 75% was obtained at a yield of about 95% per raw material solid.

  This product contains about 30% of Codybiose per solid and also contains Codybiosyl glucose, has good taste sweetness, moderate viscosity, moisturizing, sweetener, taste improver, Widely used as stabilizers, bifidobacteria growth promoters, mineral absorption promoters, etc., can be advantageously used in various compositions such as foods, drinks, cosmetics, pharmaceuticals, and molded products.

<Corgebiose high content powder>
A sugar solution containing about 30% cordierbiose per solid obtained by reaction and purification by the method of Example 6 was used as a raw material to adjust the solid concentration to about 20%. To this, 5 units of glucoamylase per 1 g of solid were added to pH 4 5. The reaction was carried out at 40 ° C. for 16 hours to decompose the remaining maltose. The reaction was stopped by heating at 100 ° C. for 30 minutes, and then concentrated to a concentration of about 40%. In order to increase the content of cordobiose in the sugar solution, an alkali metal type strongly acidic cation exchange resin (manufactured by Tokyo Organic Chemical Industry Co., Ltd., trade name “XT-1016”, Na type, cross-linking degree 4%) is used. Four stainless steel columns with a jacket of 3 cm and a length of 1 m were filled with water suspension and connected in series so that the total length of the resin layer was about 4 m. While maintaining the temperature in the column at 40 ° C, the sugar solution is added to the resin at 5 v / v%, and 40 ° C warm water is flowed at a flow rate of SV0.15 for fractionation, and a fraction containing high Cozybiose is collected. did. Further, purification, concentration, vacuum drying, and pulverization gave a powder having a high content of cordobiose with a yield of about 20% per raw material solid.

  This product contains approximately 90% cordierbiose per solid, has good sweetness and moderate moisture retention, sweetener, taste improver, stabilizer, bifidobacteria growth promoter, mineral Widely used as an absorption accelerator and the like, it can be advantageously used in various compositions such as foods and drinks, cosmetics, pharmaceuticals and molded products.

<Glucosyl sorbose-containing sugar solution>
An aqueous solution containing 5% each of β-D-glucose-1-phosphate and L-sorbose was adjusted to pH 5.5, and cordierbiose phosphorylase prepared by the method of Example 1 was added to β-D-glucose-1-phosphate. It added so that it might become 10 units per g of acid, and it was made to react at 60 degreeC for 72 hours. The reaction solution was heated at 90 ° C. for 30 minutes to inactivate the enzyme, then cooled, decolorized and filtered with activated carbon according to a conventional method, purified by desalting with H-type and OH-type ion exchange resins, By concentrating, a syrup-like glucosyl sorbose-containing sugar solution having a concentration of about 75% was obtained with a yield of about 95% per raw material solid.

  This product contains about 30% glucosyl sorbose per solid, has good taste sweetness, moderate viscosity, and moisture retention, sweetener, taste improver, stabilizer, bifidobacteria growth promoter As a mineral absorption promoter, it can be used advantageously in various compositions such as foods, drinks, cosmetics, pharmaceuticals, and molded products.

<High sugar content glucosyl sorbose>
An aqueous solution containing 5% of β-D-glucose-1-phosphate and 10% of L-sorbose was adjusted to pH 6.0, and cordierbiose phosphorylase prepared by the method of Example 1 was added to β-D-glucose- It added so that it might become 30 units per 1g of phosphoric acid, and it was made to react at 60 degreeC for 72 hours. After the reaction, the reaction mixture was cooled, commercial baker's yeast was added so that the wet mass per solid substance was 5%, and the reaction solution was controlled at pH 5 to 6 using 1N sodium hydroxide solution for 6 hours at 27 ° C. Reacted. The reaction solution was heated at 90 ° C. for 30 minutes to stop the reaction, then cooled, decolorized and filtered with activated carbon according to a conventional method, purified by desalting with H-type and OH-type ion exchange resins, and further concentrated. As a result, a syrup-like glucosyl sorbose-containing sugar solution having a concentration of about 75% was obtained at a yield of about 65% per solid material.

  This product contains about 40% glucosyl sorbose per solid, has good taste sweetness, moderate viscosity, and moisture retention, sweetener, taste improver, stabilizer, bifidobacteria growth promoter As a mineral absorption promoter, it can be used advantageously in various compositions such as foods, drinks, cosmetics, pharmaceuticals, and molded products.

<Glucosyl sorbose high content powder>
Using a sugar liquid containing about 40% glucosyl sorbose per solid obtained by reaction and purification by the method of Example 9, this was adjusted to a solid concentration of about 45%. In order to increase the glucosyl sorbose content in the sugar solution, an alkali metal type strongly acidic cation exchange resin (manufactured by Tokyo Organic Chemical Industry Co., Ltd., trade name “XT-1016”, Na type, crosslinking degree 4%) is used. Four jacketed stainless steel columns having an inner diameter of 3 cm and a length of 1 m were filled with water suspension and connected in series so that the total length of the resin layer was about 4 m. While maintaining the temperature in the column at 40 ° C., the sugar solution is added to the resin at 5 v / v%, and 40 ° C. warm water is flowed at a flow rate of SV0.15 to perform fractionation, and a fraction containing high glucosyl sorbose is obtained. Collected. Furthermore, purification, concentration, vacuum drying, and pulverization gave a powder containing high glucosyl sorbose in a yield of about 25% per raw material solid.

  This product contains about 90% glucosyl sorbose per solid, has good taste sweetness, moderate moisture retention, sweetener, taste improver, stabilizer, bifidobacterial growth promoter, As a mineral absorption accelerator, it can be widely used in various compositions such as foods, cosmetics, pharmaceuticals and molded products.

<Kojibiosyl fructoside-containing sugar solution>
An aqueous solution containing 10% cordobiose and 10% sucrose was prepared, and to this was added cordozyme phosphorylase prepared by the method of Example 1 to 40 units per gram of cordobiose, in the presence of 5 mM sodium dihydrogen phosphate. The reaction was carried out at pH 5.0 and 60 ° C. for 72 hours. The reaction solution was heated at 90 ° C. for 30 minutes to inactivate the enzyme, then cooled, decolorized and filtered with activated carbon according to a conventional method, purified by desalting with H-type and OH-type ion exchange resins, By concentrating, a syrup-like cordobiosyl fructoside-containing sugar solution having a concentration of about 75% was obtained with a yield of about 95% per raw material solid.

  This product contains about 55% Kojibiosyl fructoside per solid, has good sweetness, moderate viscosity, and moisturizing properties, and various compositions such as food and drink, cosmetics, pharmaceuticals, and molded products It can be used to advantage.

<Cody biosyl fructoside high content powder>
The sugar solution containing about 55% of cordobiosyl fructoside per solid which was reacted and purified by the method of Example 11 was used as a raw material, and this was adjusted to a solid concentration of about 45%. In order to increase the content of Kojibiosyl fructoside in this molasses, an alkaline earth metal type strongly acidic cation exchange resin (Dow Chemical Co., trade name “Dowex 50W × 4”, Ca type) was used. Then, column chromatography was carried out according to the method of Example 10, and a fraction containing Kojibiosyl fructoside was collected. Furthermore, purification, concentration, vacuum drying, and pulverization yielded a powder having a high content of cordobiosyl fructoside in a yield of about 40% per raw material solid.

  This product contains about 95% Kojibiosyl fructoside per solid, has good sweetness and moderate moisturizing properties, and is used in various compositions such as food and drink, cosmetics, pharmaceuticals, and molded products. It can be used advantageously.

<Kojibiosyl glucose-containing sugar solution>
An aqueous solution containing 10% β-D-glucose-1 phosphate and 20% maltose is adjusted to pH 5.0, and cordierbiose phosphorylase prepared by the method of Example 1 is adjusted to 40 units per gram of maltose. And reacted at 60 ° C. for 72 hours. The reaction solution was heated at 90 ° C. for 30 minutes to inactivate the enzyme, then cooled, decolorized and filtered with activated carbon according to a conventional method, purified by desalting with H-type and OH-type ion exchange resins, By concentrating, a syrup-like cordierbiosylglucose-containing sugar solution having a concentration of about 75% was obtained with a yield of about 95% per raw material solid.

  This product contains about 45% kojibiosylglucose per solid, has good sweetness, moderate viscosity, and moisturizing properties, and various compositions such as foods, cosmetics, pharmaceuticals, and molded products Can be advantageously used.

<Corgebiosylglucoside-rich sugar solution>
An aqueous solution containing 5% β-D-glucose-1-phosphate and 10% trehalose was adjusted to pH 5.0, and cordobiose phosphorylase prepared by the method of Example 1 was added to β-D-glucose-1-phosphate. It added so that it might become 20 units per gram of acid, and it was made to react at 60 degreeC for 72 hours. Sodium hydroxide was added to the reaction solution and heated at 100 ° C. while maintaining an alkalinity of pH 10 or higher, then cooled, decolorized with activated carbon and filtered according to conventional methods, and desalted with H-type and OH-type ion exchange resins. Purification and further concentration yielded a syrup-like cordierbiosylglucoside-rich sugar solution having a concentration of about 75% in a yield of about 60% per raw material solid.

  This product contains about 95% kojibiosyl glucoside per solid, has good sweetness, moderate viscosity, and moisturizing properties, and various compositions such as foods, cosmetics, pharmaceuticals, and molded products. Can be advantageously used.

<Corgebiose-containing sugar solution>
In a 25 mM dipotassium phosphate-citrate buffer solution (pH 6.0) containing 5% maltose, 5 units of commercially available maltose phosphorylase derived from bacteria and 1 g of maltose prepared by the method of Example 5 were used. 40 units per unit were added, and the reaction was carried out at 30 ° C. for 72 hours. The reaction solution was heated at 100 ° C. for 30 minutes to inactivate the enzyme, then cooled, decolorized and filtered with activated carbon according to a conventional method, purified by desalting with H-type and OH-type ion exchange resins, By concentrating, a syrup-like cordierbiose-containing sugar solution having a concentration of about 75% was obtained at a yield of about 95% per raw material solid.

  This product contains about 30% of Codybiose per solid and also contains Codybiosyl glucose, has good taste sweetness, moderate viscosity, moisturizing, sweetener, taste improver, Widely used as stabilizers, bifidobacteria growth promoters, mineral absorption promoters, etc., can be advantageously used in various compositions such as foods, drinks, cosmetics, pharmaceuticals, and molded products.

<Corgebiosylglucoside-rich sugar solution>
An aqueous solution containing 5% β-D-glucose-1-phosphate and 10% trehalose was adjusted to pH 5.0, and cordobiose phosphorylase prepared by the method of Example 5 was added to β-D-glucose-1-phosphate. It added so that it might become 20 units per gram of acid, and it was made to react at 60 degreeC for 72 hours. Sodium hydroxide was added to the reaction solution and heated at 100 ° C. while maintaining an alkalinity of pH 10 or higher, then cooled, decolorized with activated carbon and filtered according to conventional methods, and desalted with H-type and OH-type ion exchange resins. Purification and further concentration yielded a syrup-like cordierbiosylglucoside-rich sugar solution having a concentration of about 75% in a yield of about 60% per raw material solid.

  This product contains about 95% kojibiosyl glucoside per solid, has good sweetness, moderate viscosity, and moisturizing properties, and various compositions such as foods, cosmetics, pharmaceuticals, and molded products. Can be advantageously used.

<Sweetener>
To 1 part by mass of the high glucosyl sorbose powder obtained by the method of Example 10, 0.05 part by mass of α-glycosyl stevioside (manufactured by Toyo Seika Co., Ltd., trade name α-G sweet) is added and mixed uniformly. A powder sweetener was produced. The product is refined and has about twice the sweetness of sugar, and calories are about half that of sugar per degree of sweetness. Therefore, this sweetener is suitable as a low-calorie sweetener for seasoning low-calorie foods and drinks for people who restrict intake of calories, such as obese and diabetics. In addition, this sweetener is suitable for seasoning foods and the like that suppress dental caries because it produces less acid by caries-inducing bacteria and produces less insoluble glucan.

<Hard candy>
80 parts by weight of reduced maltose starch syrup (water content 25%) is added to 30 parts by weight of the glucosyl sorbose-containing sugar solution obtained by the method of Example 8, mixed and dissolved, and concentrated under reduced pressure until the water content is less than 2%. Then, 1 part by mass of citric acid and an appropriate amount of lemon flavor and colorant were mixed, and then molded according to a conventional method to produce a hard candy. This product is a crisp hard candy that has an elegant sweetness, low hygroscopicity, and does not cause dripping.

<Chewing gum>
3 parts by weight of glucose and 2 parts by weight of a gum base heated and melted to a degree of softening are added to and mixed with 4 parts by weight of the cordieroyl fructoside-rich powder obtained by the method of Example 12, and an appropriate amount of peppermint flavor is added. After mixing, chewing gum was manufactured by kneading and forming with a roll according to a conventional method. This product is excellent in both texture and flavor.

<Chocolate>
With respect to 15 parts by mass of the Codybiose-rich powder obtained by the method of Example 7, 40 parts by mass of cocoa paste, 10 parts by mass of cocoa butter, 10 parts by mass of sucrose and 15 parts by mass of whole milk powder are mixed and passed through a refiner. did. After reducing the particle size, the mixture was placed in a conche, 0.5 parts by mass of lecithin was added, and the mixture was kneaded at 50 ° C. for two days and nights. Subsequently, it poured into the molding machine according to the conventional method, and it solidified by shaping | molding and manufactured chocolate. This product has no fear of fat bloom or sugar bloom and has good melting and flavor when placed on the tongue.

<Custard cream>
500 parts by mass of corn starch, 500 parts by mass of maltose and 5 parts by mass of sodium chloride are added to 400 parts by mass of the high glucosyl sorbose powder obtained by the method of Example 10, and mixed thoroughly through a sieve, and further 1,400 parts by mass of chicken egg Add 5,000 parts by weight of boiled milk gradually, and continue to stir over boiling. When the corn starch is completely gelatinized and the whole becomes translucent, stop the heat, A custard cream was prepared by cooling and adding a small amount of vanilla flavor. This product is smooth and glossy and is not too sweet and delicious.

<Uiro>
90 parts by mass of Kojibiosyl fructoside-containing sugar solution obtained by the method of Example 11 was added and kneaded by adding 90 parts by mass of rice flour, 20 parts by mass of corn starch, 20 parts by mass of sugar, 1 part by mass of powdered green tea powder and water. After that, this was put in a container and steamed for 60 minutes to produce a matcha uiro. This product had good shine, good mouthfeel and good flavor. Moreover, the aging of starch was suppressed and it was stable for a long time.

<Betara pickles>
3 parts by mass of maltose, 0.05 parts by mass of licorice preparation, 0.008 parts by mass of malic acid, 0.07 parts by mass of sodium glutamate, based on 1 part by mass of the cordobiosyl glucose-containing sugar solution obtained by the method of Example 13. After uniformly mixing 0.03 parts by mass of potassium sorbate and 0.2 parts by mass of pullulan, a pickle was prepared. In accordance with a conventional method, 30 kg of radish was submerged with salt and then soaked with sugar. The soup was then soaked in a seasoning solution prepared with 4 kg of the base of the pickles. This product had good color and aroma, moderate sweetness, and crispness.

<Lactic acid bacteria beverage>
With respect to 130 parts by mass of the sugar solution containing Kojibiose obtained by the method of Example 6, 175 parts by mass of skim milk powder and 50 parts by mass of powder containing lactosucrose (trademark “Hayashibara Shoji Co., Ltd., registered trademark“ milk oligo) ” Dissolve in 1,150 parts by mass, sterilize at 65 ° C. for 30 minutes, cool to 40 ° C., inoculate 30 parts by mass of a starter of lactic acid bacteria, and incubate at 37 ° C. for 8 hours. A lactic acid bacteria beverage was obtained. This product is a lactic acid bacteria beverage with good flavor. In addition, this product contains oligosaccharides and not only stably retains lactic acid bacteria, but also has a bifidobacteria growth promoting action.

<Synthetic sake>
6518 parts by mass of a 30 v / v% alcohol aqueous solution (when the temperature is 15 ° C.), 600 parts by mass of a 70% aqueous glucose solution, 50 parts by mass of the sugar solution containing cordobiose obtained by the method of Example 6, 11.1 parts by mass of succinic acid, 75 % Lactic acid aqueous solution 3.66 parts by mass, sodium glutamate 2.3 parts by mass, glycine 1.2 parts by mass, alanine 1.2 parts by mass, sodium succinate 2.22 parts by mass, sodium chloride 1.6 parts by mass, natural salt 1 .4 parts by mass and 2500 parts by mass of water were mixed to obtain a raw liquor having an alcohol content of about 20 v / v%. This was diluted with water to obtain a product having an alcohol content of 15 to 16 v / v%. This product has a mellow sweetness and refined flavor and is delicious.

<Emulsion>
With respect to 3.5 parts by mass of the glucosyl sorbose-rich sugar solution obtained by the method of Example 9, 0.5 part by mass of polyoxyethylene behenyl ether, 1 part by mass of polyoxyethylene sorbitol tetraoleate, lipophilic monostearic acid Add 1 part by weight of glycerin, 0.5 part by weight of behenyl alcohol, 1 part by weight of avocado oil, 1 part by weight of α-glycosyl rutin, vitamin E and preservatives. 5 parts by mass of glycol, 0.1 part by mass of carboxyvinyl polymer and 85.3 parts by mass of purified water were added and emulsified with a homogenizer to produce an emulsion. This product is excellent in moisture retention and can be advantageously used as a sunscreen, whitening agent, and the like.

<Skin cream>
For 4 parts by mass of cordierbiosyl fructoside-containing powder obtained by the method of Experiment 12, 2 parts by mass of polyoxyethylene glycol monostearate, 5 parts by mass of glyceryl monostearate, 2 parts by mass of α-glycosyl rutin In addition, 1 part by mass of liquid paraffin, 10 parts by mass of glyceryl trioctanoate and an appropriate amount of preservative were added and dissolved by heating according to a conventional method. To this, 5 parts by mass of 1,3-butylene glycol and 66 parts by mass of purified water were further added. The mixture was emulsified with a homogenizer, and an appropriate amount of a fragrance was added and mixed by stirring to produce a skin cream. This product is a cream with good elongation and can be advantageously used as a sunscreen, skin cleansing agent, fair skin agent and the like.

<Toothpaste>
With respect to 15 parts by mass of a high-concentration cordierbiosylglucoside sugar solution obtained by the method of Example 14, dicalcium phosphate part by mass, sodium lauryl sulfate 1.5 parts by mass, glycerin 25 parts by mass, polyoxyethylene sorbitan laurate 0. Toothpaste was obtained by mixing with 5 parts by mass, 0.02 parts by mass of saccharin, 0.05 parts by mass of preservative and 13 parts by mass of water. This product has good gloss and detergency and is suitable as a toothpaste.

<Tube nutrients>
80 parts by mass of Codybiose-containing powder obtained by the method of Example 7, 190 parts by mass of dried egg yolk, 209 parts by mass of skim milk powder, 4.4 parts by mass of sodium chloride, 1.85 parts by mass of potassium chloride, 4 parts by mass of magnesium sulfate A blend consisting of 0.01 parts by weight of thiamine, 0.1 parts by weight of sodium ascorbate, 0.6 parts by weight of vitamin E acetate and 0.04 parts by weight of nicotinamide was prepared. Each 25 g of this blend was filled into a laminated aluminum parcel and heat sealed to obtain a product. This product is used by dissolving one bag in about 150 to 300 ml of water to give a nutritional supplement and administering it to the nasal cavity, esophagus, stomach, etc. by the tube method.

<Tablets>
After adding 50 parts by mass of aspirin, 10 parts by mass of maltose, and 4 parts by mass of corn starch to 4 parts by mass of the glucosyl sorbose high content powder obtained by the method of Experiment 8, the mixture is uniformly mixed and then 1 Tableting was performed with a tablet of 680 mg, a tablet thickness of 5.25 mm, and a hardness of 8 kg ± 1 kg. This product is an easy-to-drink tablet with moderate sweetness.

<Strawberry jam>
150 parts by weight of raw strawberries, 60 parts by weight of sucrose, 20 parts by weight of maltose, 40 parts by weight of a sugar solution containing Kojibiose obtained by the method of Example 15, 5 parts by weight of pectin and 1 part by weight of citric acid were boiled in a pan and bottled. Got the product. This product is a jam with good flavor and color.

<Sweetened condensed milk>
In 100 parts by weight of raw milk, 3 parts by weight of the sugar solution containing Kojibiosyl glucoside and 1 part by weight of sucrose obtained by the method of Example 16 are dissolved, sterilized by heating with a plate heater, and then concentrated to a concentration of about 70%. The product was obtained by canning under aseptic conditions. This product has a mild sweet taste and good flavor and can be advantageously used for seasoning infant foods, fruits, coffee, cocoa, tea and the like.

  As described above, the present invention is based on the discovery of a phosphorylase capable of producing cordobiose that has not been known so far, that is, cordobiose phosphorylase. The cordobiose phosphorylase of the present invention has a high temperature in the optimum temperature and temperature stability, a wide pH range in the pH stability, and the optimum pH is within the pH range, and further enzyme production from the producing microorganism The amount is also high. Therefore, if β-D-glucose-1-phosphate is used as a sugar donor and the enzyme of the present invention is allowed to act in the presence of various sugars, Cojibiose, which has been conventionally known but extremely difficult to obtain, Various glucosyl transfer saccharides such as glucosyl sorbose and the transfer sugar-containing saccharides can be produced in large quantities and at low cost.

  The glucosyl transfer sugar and the transfer sugar-containing sugar produced in this way are refined sweeteners, taste improvers, quality improvers, body imparting agents, viscosity modifiers, moisturizers, illumination imparting agents, etc. Furthermore, since it is a great feature that it can be widely applied to various compositions such as foods and drinks, cosmetics, pharmaceuticals, and molded products as nutritional supplements, the present invention is not limited to the food, cosmetics, and pharmaceutical fields, Contributing to the agricultural, aquatic and livestock industries and the chemical industry is an invention that has great significance.

It is a figure which shows the influence of the temperature which acts on the enzyme activity of the cordobiose phosphorylase of this invention. It is a figure which shows the influence of pH which acts on the enzyme activity of the cordobiose phosphorylase of this invention. It is a figure which shows the influence of the temperature which acts on the stability of the cordobiose phosphorylase of this invention. It is a figure which shows the influence of pH which acts on stability of the cordobiose phosphorylase of this invention. It is a figure which shows the restriction enzyme map of the recombinant DNA of this invention. The arrow in the figure indicates the DNA encoding the cordobiose phosphorylase of the present invention.

Claims (5)

Cozybiose is decomposed in the presence of inorganic phosphoric acid and / or its salt to produce D-glucose and β-D-glucose-1 phosphate and / or its salt, and β-D-glucose-1 phosphate and / or its salt Alternatively, it has the action of producing cordobiose and inorganic phosphoric acid and / or a salt thereof from a salt thereof and D-glucose, and β-D-glucose-1 phosphate and / or a salt thereof as a sugar donor. Combibiose phosphorylase derived from a microorganism belonging to the genus Thermoanaerobium having an action of catalyzing the transfer of a glucosyl group to the saccharide of β-D-glucose-1-phosphate and / or a salt thereof and other saccharides And a method for producing a glucosyl transfer sugar-containing carbohydrate that is allowed to act under conditions of a substrate concentration of 5% by mass or more and a pH of 4.0 to 9.0. β-D-glucose-1 phosphate and / or salt thereof causes cordierbiose to act on cordobiose in the presence of inorganic phosphate and / or salt thereof, or presence of inorganic phosphate and / or salt thereof or the action of maltose phosphorylase to maltose under, or inorganic phosphate and / or glucosyl-transferred saccharide containing saccharide according to claim 1, wherein those generated by the action of trehalose phosphorylase trehalose in the presence of a salt thereof Production method.   The method for producing a glucosyltransferase-containing saccharide according to claim 1 or 2, wherein the other saccharide is a saccharide selected from D-glucose, L-sorbose, maltose, cordobiose, trehalose and sucrose.   The glucosyl transfer according to any one of claims 1 to 3, comprising a method selected from yeast fermentation and column chromatography as means for removing contaminating saccharides other than glucosyl transfer saccharide produced and / or remaining in the reaction solution. A method for producing a sugar-containing carbohydrate. Cozybiose is decomposed in the presence of inorganic phosphoric acid and / or its salt to produce D-glucose and β-D-glucose-1 phosphate and / or its salt, and β-D-glucose-1 phosphate and / or its salt Alternatively, it has the action of producing cordobiose and inorganic phosphoric acid and / or a salt thereof from a salt thereof and D-glucose, and β-D-glucose-1 phosphate and / or a salt thereof as a sugar donor. Combibiose phosphorylase derived from a microorganism belonging to the genus Thermoanaerobium, which has an action of catalyzing the transfer of a glucosyl group to the saccharides of maltose together with maltose phosphorylase in the presence of inorganic phosphate and / or its salt, and the substrate concentration Trehalose is allowed to act under conditions of 5% by mass or more and pH 4.0 to 9.0, or trehalose is added to trehalose in the presence of inorganic phosphoric acid and / or a salt thereof. With over scan phosphorylase, substrate concentration 5% by mass or more, kojibiose manufacturing method which comprises causing to act under the conditions of pH4.0 to 9.0.

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