JP2001197898A - Method of producing trehalose-containing saccharide solution, product therefrom and application thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of producing a trehalose-containing saccharide solution, by using an enzyme at a high temperature to obtain the product solution industrially advantageously and economically, to provide the trehalose- containing saccharide solution by utilizing the same method, to provide a dried product therefrom, and to provide an application thereof. SOLUTION: This method of producing the trehalose-containing saccharide solution comprises acting a maltose-forming enzyme, a heat-resistant maltose phosphorylase, and a heat-resistant trehalose phosphorylase on a liquidized starch solution at the same time, or comprises acting the maltose-forming enzyme on the liquidized starch solution to conduct maltose-forming reaction and further acting the heat-resistant maltose phosphorylase and the heat-resistant trehalose phosphorylase while conducting the maltose-forming reaction. Thus the method gives the trehalose-containing saccharide solution, and further gives the dried product therefrom. Food and drink containing the solution, or containing the dried product, is prepared through the method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing a trehalose-containing sugar solution using maltose-forming enzyme, thermostable maltose phosphorylase and thermostable trehalose phosphorylase, the sugar solution or a dried product thereof, and a food or drink containing the same.

[0002]

2. Description of the Related Art One of the methods for producing trehalose by an enzymatic method is maltose-phosphorylase (Maltose-ortholase).
ophosphate β-D-glucosyltransferase) and trehalose-orthophosphate β-D-gl
ucosyltransferase) and maltose simultaneously in the presence of inorganic phosphate (Patent No. 1513517)
No.). The present applicant has filed an application for thermostable maltose phosphorylase and trehalose phosphorylase which are heat stable, and discloses that an enzymatic reaction at a high temperature, which is industrially advantageous, is possible (Japanese Patent Application Laid-Open No. 8-131166). JP-A-9-37780).

[0003] A method has also been reported for producing a saccharified solution containing a high concentration of trehalose by simultaneously reacting β-amylase, a debranching enzyme, maltose phosphorylase and trehalose phosphorylase on a starch liquefied solution (M. Yos).
hida et al., Starch, 49 , 21 (1997)). This method is suitable for actual production from the viewpoint of shortening the reaction time and controlling the process. On the other hand, after liquefying starch with α-amylase or the like, β-
When amylase is acted on, high-molecular β-limit dextrin (branched dextrin) is produced together with maltose. Branched dextrins have characteristics such as being difficult to age and having low hygroscopicity, and can be used as food materials.
(JP-B-52-46290).

[0004]

In the saccharification industry, the reaction temperature is generally raised to prevent decay of sugar. Furthermore, in the process of producing high molecular weight dextrin, aging of components may cause product quality (white turbidity), decrease in yield, trouble in the filtration process, etc. To prevent this, production at high temperature (enzyme) Reaction) is required. In addition, increasing the reaction temperature is advantageous in that the enzyme reaction rate is increased and the reaction time can be shortened, and the cost is also advantageous. An object of the present invention is to provide a method for producing a trehalose-containing sugar solution under industrially advantageous and economical high-temperature conditions using an enzyme, and a trehalose-containing sugar solution or a trehalose-containing sugar solution obtained by the method. It is intended to provide a dried product and its use.

[0005]

Means for Solving the Problems The present inventors have studied the production of a trehalose-containing sugar solution by allowing a maltogenic enzyme such as β-amylase, a thermostable maltose phosphorylase and a thermostable trehalose phosphorylase to act on a starch liquefied solution. In which, when a maltose-forming enzyme is allowed to act to produce a substantially maximum amount of maltose, then these three enzymes are simultaneously acted on, as compared to the case where thermostable maltose phosphorylase and thermostable trehalose phosphorylase are acted on, or After the maltose synthase is acted on, if maltose-forming maltose phosphorylase and thermostable trehalose phosphorylase are allowed to act during the maltose-forming reaction, these enzymes can be made to act at a higher temperature. Aging of high molecular weight dextrin Technique, found that it is possible to carry out the enzymatic reaction at a high trehalose yield from and in a shorter time, and completed the present invention.

According to the present invention, a maltose-forming enzyme, a thermostable maltose phosphorylase and a thermostable trehalose phosphorylase are simultaneously allowed to act on a liquefied starch solution.
The present invention relates to a method for producing trehalose, wherein a maltose-forming enzyme is caused to act on a liquefied starch solution to carry out a maltose-forming reaction, and heat-resistant maltose phosphorylase and heat-resistant trehalose phosphorylase are allowed to act during the progress of the maltose formation reaction. The heat-resistant maltose phosphorylase is preferably obtained from a wild-type microorganism having the enzyme-producing ability or produced by a recombinant microorganism into which a heat-resistant maltose phosphorylase gene derived from the microorganism has been introduced. The thermostable trehalose phosphorylase is preferably obtained from a wild-type microorganism having the enzyme-producing ability or produced by a recombinant microorganism into which a thermostable trehalose phosphorylase gene derived from the microorganism has been introduced. Sex trehalose phosphorylase. The present invention also relates to a trehalose-containing sugar solution produced by the above method or a concentrate or a dried product obtained by evaporating water from the trehalose-containing sugar solution. The present invention further relates to a food or beverage containing such a trehalose-containing sugar solution or a concentrate or a dried product obtained by evaporating water therefrom.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION In the present specification,% for sugar concentration and sugar composition means mass / mass%. In the present specification, hereinafter, maltose phosphorylase is referred to as MPa.
se, trehalose phosphorylase is abbreviated as TPase. The starch liquefied liquid used in the present invention is not particularly limited as long as it is a liquefied liquid obtained by liquefying starch (enzyme (α-amylase)) or acid. Sources of starch include, for example, barley, wheat, corn, tapioca, potato, sweet potato and the like. Examples of the α-amylase used in the enzyme liquefaction include those of bacterial, mold, plant, and animal origin, including bacteria of the genus Bacillus, such as Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus stearo. Α-Amylase derived from Thermophilus is preferred in terms of heat resistance. In the case of acid liquefaction, examples of the acid used include oxalic acid and hydrochloric acid.
During liquefaction, if the starch milk concentration is too high, the viscosity at the time of gelatinization becomes large, causing a problem on the handling surface.
It is preferably performed at 0%. Also, the pH of starch milk during liquefaction
In the case of enzyme liquefaction, any pH may be used as long as the α-amylase to be used acts. Generally, the pH is 6.0 to 7.5. In the case of acid liquefaction, there is no particular limitation.
Perform at ~ 3.5.

The liquefaction temperature may be any temperature at which the starch does not age, and in the case of enzyme liquefaction, it is generally 60 ° C. to 120 ° C.
95 ° C., preferably using an α-amylase from Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus stearothermophilus.
The reaction is performed at an elevated temperature of 0 ° C. The reaction may be carried out until the desired degree of liquefaction (DE value) is reached. In the present invention, the starch liquefied liquid is usually from DE1 to 20, preferably from DE5 to DE1.
Five. B. licheniformis, B. amyloliquefaciens, B. stearothermophilus α-amylase of 95 ° C. to 11
When the reaction is carried out at a high temperature of 0 ° C., the reaction time is generally 30 minutes to 4 hours, preferably 30 minutes to 2 hours. In the case of acid liquefaction, liquefaction is generally performed at 80 ° C. to 160 ° C. for 5 minutes to 5 minutes.
Time, preferably 10 minutes to 1 hour, to obtain the same DE value as in the case of enzyme liquefaction.

In the present invention, β-amylase is usually used as the maltogenic enzyme, but maltogenic α-amylase can be used together with or instead of β-amylase. β-amylase is an enzyme that produces β-maltose,
Amylases whose main product is α-maltose are called maltogenic α-amylases. The β-amylase used in the present invention is not particularly limited, and examples thereof include those of plant origin such as sweet potato, barley malt, wheat, soybean, radish, or Bacillus megaterium, Bacillus cereus, Pseudomonas, and Strepto. Microbial sources such as Myces. Among them, those derived from soybean and barley malt are preferred in terms of heat resistance.

The origin of the maltogenic α-amylase used in the present invention is not particularly limited.
Maltogenic-α-amylase produced by actinomycetes (eg, Streptomyces hygroscopicus SF-1084, Streptomyces precox NA-273, Streptomyces tosensis), maltogenic-α of Bacillus stearothermophilus -Maltogenic α-amylase produced by expressing an amylase gene in Bacillus subtilis (Bacillus subtilis) [Patent No. 26
No. 96537, commercially available products (maltogenase,
Novo Co., Ltd.)], and the above-mentioned maltogenic α-amylase obtained by genetic recombination is preferred in terms of heat resistance and maltose-generating ability.

[0011] Heat resistant MPas used in the present invention
e, heat-resistant TPase refers to a heat-resistant TPase
Mase and TPase which maintain 80% or more of untreated activity after treatment at a temperature of 5 ° C., preferably 50 ° C., and more preferably 60 ° C. for 15 minutes. Sex TPase is not particularly limited. Examples of such heat-resistant Mase include Bacillus bacteria such as Bacillus species R.
K-1 strain (FERM BP-5592), Bacillus species MK-1 strain (FERM BP-5593)
(EP0757098A3) and Plesiomonas S
H-35 strain (FERM BP-5144) (EP070)
7062A1) and the like, commercially available heat-resistant MPase (eg, Mase sold by Oriental Yeast Co., Ltd.), and the like.
Particularly, heat-resistant Mase produced by Bacillus sp. RK-1 strain is preferable from the viewpoint of heat resistance. Thermostable Mase from culture solution of Bacillus species RK-1 strain
The collection of the enzyme is described in JP-A-9-37780, and the enzyme can be collected in substantially the same manner when other microorganisms are used. Heat-resistant TPa used in the present invention
Examples of se include Bacillus bacteria such as Bacillus
Stearothermophilus SK-1 strain (FERM BP-
5594) (EP0757098A3) and heat-resistant TPases produced by Plesiomonas strain SH-35 (FERM BP-5144), and the like, but heat-resistant TPases produced by Bacillus bacteria, particularly Bacillus stearothermophilus SK-1 strain, are exemplified. TPase is preferred from the viewpoint of heat resistance. For the collection of heat-resistant TPase from a culture of Bacillus stearothermophilus SK-1 strain, see Japanese Patent Laid-Open No.
The enzyme can be collected in substantially the same manner when other microorganisms are used.

Heat-resistant MPas used in the present invention
e, thermostable TPases may also be derived from wild-type microorganisms that produce them.
Heat-resistant MPase or heat-resistant TPase produced by a recombinant microorganism into which a Pase gene has been introduced may be used. In the above description, “derived from” is intended to include not only the structural gene of the enzyme of the wild-type microorganism but also the structural gene of the enzyme of the recombinant microorganism obtained using the structural gene. Examples of the microorganism serving as a donor of such a thermostable MPase gene include the above-described Bacillus species RK-1 strain and Bacillus sp.
The strain MK-1 and the strain Plesiomonas SH-35 are exemplified, and the strain Bacillus sp. RK-1 is preferred from the viewpoint of heat resistance. In addition, such heat-resistant TPa
Examples of the microorganism serving as a donor of the se gene include the above-mentioned Bacillus stearothermophilus SK-1 strain and Plesiomonas SH-35 strain, and Bacillus stearothermophilus SK-1 strain is heat-resistant. It is preferable from the viewpoint of properties.

Methods for obtaining chromosomal DNA from a donor strain include, for example, the method of Marmer [Marmur, J., J. Mol. Bi.
ol., 3 , 208 (1961)] and the method of Saito and Miura [Saito, H.
andMiura, K., Biochem. Biophys. Acta. 72 , 619 (196
3)] etc., but other similar methods can also be used. By cutting the chromosomal DNA thus obtained with a restriction enzyme, a DNA fragment containing a thermostable MPase gene or a thermostable TPase gene can be prepared, but the type of restriction enzyme used is one that does not disrupt the gene. Anything can be used. Alternatively, a thermostable Mase gene or a thermostable TP
As a method for obtaining the ase gene, a PCR method can also be used.

For the purpose of enhancing the expression efficiency of the thermostable MPase gene or the thermostable TPase gene, the promoter of the gene can be replaced with another promoter known to have strong promoter activity. Among them, when a Bacillus genus is expressed as a host, an α-amylase promoter derived from Bacillus amyloliquefaciens is preferable from the viewpoint of expression efficiency. The thus-prepared fragment of the gene is cloned to prepare a recombinant DNA molecule (expression vector). The vector used at this time is capable of autonomous replication in a host strain, and the integrated fragment is stably used. Anything that can be held can be used.
This vector preferably has a selection marker (for example, a drug resistance gene such as an ampicillin resistance gene or a kanamycin / neomycin resistance gene) to confirm introduction into a host.

As an example of a vector used to construct an expression vector, when Bacillus is used as a host, pUB110 (Pro. Natl. Acad. Sc.
USA, 75 , 1423 (1978)), pC194 or derivatives thereof. When E. coli is used as a host, pBR322 (Gene, 2 , 95 (1977)) or pUC1
9 (Messing, Methods, Enzymol., 101 , 20 (1983)), p.
CR2.1, and a shuttle vector pHY300PLK that can be replicated in Escherichia coli and Bacillus subtilis (Jpn. J. Genet., 6
0 , 235 (1985)). The above recombinant DNA
The technology is, for example, Molecular Cloning A Laboratory Man
ual (Cold Spring Harbor Laboratory, 1989).

The obtained expression vector is then introduced into a host microorganism. The host microorganism is not particularly limited. For example, in the case of Escherichia coli, DH5α strain, HB10
1 strain, C600 strain, JM109 strain, INVαF ′ strain and the like. In the case of Bacillus genus, Bacillus subtilis 168 strain, BD170 strain, ISW1214 strain and the like can be mentioned. As a host, a microorganism of the genus Bacillus is preferred because it has been used for fermentation production and has no pathogenicity. Among them, the Bacillus subtilis 168 strain has been elucidated genetically, and is one of the certified host-vector systems in the Guidelines for Recombinant DNA Experiments of the Science and Technology Agency and is regarded as a particularly safe host. Furthermore, since the entire nucleotide sequence of the chromosome has already been determined (Nature 390 , 249 (1997)), it is suitable as a safe host.

The method of introducing the expression vector into the host microorganism is not limited. For example, methods such as transduction, cell fusion, electroporation and the like can be mentioned. For example, in the case of an E. coli host, the method of Mandel and Higa (J. Mol. Biol., 53 , 159 (19
70), Hanahan's method (J. Mol. Biol., 166 , 111 (197
8)) etc. can be used. After the introduction, a recombinant microorganism is obtained with a selection marker, and the obtained recombinant microorganism is cultured to obtain a heat-resistant Mase or a heat-resistant TPas.
Since e is accumulated, it may be collected by a conventional method.

In the present invention, the maltose-forming enzyme is allowed to act on the starch liquefied solution at the same time, or the maltose-forming enzyme is allowed to act on the starch liquefied solution, and the maltose-forming reaction proceeds. Inside, heat-resistant Mase and heat-resistant TPas
e. When these enzymes are used in this way, maltose-forming enzyme is allowed to act on the liquefied starch solution to produce maltose to the maximum, and then heat-resistant MPa
These enzymes can be acted on at higher temperatures as compared to the case where se and thermostable TPase are acted on, so that the aging of high-molecular-weight dextrins is prevented, and the trehalose yield is shortened in a shorter time. Can carry out an enzymatic reaction.

The above heat-resistant Mase and heat-resistant TP
The time of the action of the ase is the same as that of the maltogenic enzyme or the maltose-generating reaction by the maltogenic enzyme may be in progress, but preferably within 12 hours after the maltogenic enzyme is actuated, more preferably within 4 hours. The reaction time is even more preferably within 1 hour, and most preferably, simultaneously with the maltogenic enzyme in view of the working efficiency and the shortening of the reaction time. In addition, heat-resistant MP
The ase and the heat-resistant TPase usually act at the same time, but they may act at different times. In addition, "acting" an enzyme on a starch liquefied liquid means adding, mixing, etc.
It shall mean contacting the starch liquefied liquid with the enzyme.

There are no particular restrictions on the amounts of the three enzymes, maltogenic enzyme, heat-resistant MPase and heat-resistant TPase.
It is appropriate to use 0.1 to 100 units, preferably 5 to 50 units per g. The use ratio of each enzyme is not particularly limited, but is preferably 1:20 to 20: 1, more preferably 1: 5 to 5: 1 for any two of the three enzymes in terms of unit ratio. , 1: 2 to 2: 1
Is even more preferred.

The units of each enzyme are as follows: maltose-forming enzyme; β-amylase;
The amount of enzyme required to generate a reducing power equivalent to 1 mg of glucose in 10 minutes under the condition of 0 ° C. was defined as 1 unit, and for maltogenic α-amylase, pH 5.0, 37
The amount of the enzyme that hydrolyzes 1 μmole maltotriose per minute under the condition of ° C. was defined as one unit.

The activity of the heat-resistant MPase and the heat-resistant TPase was measured by the following method. (Mase activity measurement) 0.4 ml of crude enzyme solution, 0.5
0.06 ml of M phosphate citrate buffer (pH 6.0),
0.6 ml of 2 g / dl maltose and 0.14 of distilled water
of the mixture, and a maltose decomposition reaction was performed at 60 ° C. for 15 minutes. After the reaction, the reaction was stopped by boiling for 10 minutes, 0.02 ml of the reaction stop solution was collected, 3 ml of glucose test reagent glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.) was added, and the mixture was reacted at room temperature for 20 minutes. After that, the absorbance at 505 nm was measured to determine the amount of glucose in the reaction solution. The amount of enzyme capable of phosphorylating 1 μmol of maltose per minute was determined from the amount of generated glucose, and this was defined as one unit. (Measurement of TPase activity) The amount of glucose in the reaction solution was quantified in the same manner as in MPase, except that the substrate was changed to 2 g / dl trehalose, and the amount of an enzyme capable of phosphorylating 1 μmol of trehalose per minute was determined. This was defined as one unit.

The reaction temperature in the method for producing a trehalose-containing sugar solution of the present invention is not particularly limited as long as the enzyme reaction proceeds. However, considering the propagation of various bacteria, the efficiency of the reaction and the aging of dextrin as described above. It is preferable to carry out the reaction at a temperature as high as possible within a range where the deactivation of the enzyme is small. For example,
When a maltose synthase is first allowed to act, and then heat-stable MPase and heat-stable TPase are allowed to act after a certain period of time, the first maltose formation reaction is not particularly limited. Considering this, the reaction can be preferably performed at a temperature of 60 to 70 ° C. However, in actual production, it is difficult to adjust the temperature in a short time, so it is preferable to perform the reaction at the same temperature as the subsequent trehalose generation reaction.

[0024] Regardless of whether the trehalose-forming reaction is carried out simultaneously with or at the same time as the maltose-forming reaction, it is generally appropriate to carry out the reaction at 45 to 70 ° C. However, since the heat resistance varies depending on the type of the heat-resistant MPase and the heat-resistant TPase to be used, it is preferable to determine the reaction temperature in consideration of the heat resistance of each enzyme in actual production. Specifically, Bacillus sp. RK-1 and heat-resistant TPase
When used in combination with those obtained from Bacillus stearothermophilus SK-1 strain, it is preferably 60 to 70 ° C., more preferably 65 ° C. in consideration of trehalose conversion efficiency and dextrin aging. It is near. In addition, Bacillus sp. R
Heat-resistant Mase and heat-resistant TP produced by a recombinant microorganism into which a heat-resistant MPase gene derived from strain K-1 has been introduced.
Bacillus stearothermophilus SK-
When used in combination with a heat-resistant TPase produced by a recombinant microorganism into which a heat-resistant MPase gene derived from one strain has been introduced, the temperature is preferably 55 to 65 ° C, more preferably around 60 ° C. Furthermore, a commercially available heat-resistant MPa
When using se, the temperature is preferably 45 to 60 ° C, more preferably around 55 ° C.

The pH condition in the production of trehalose of the present invention is not particularly limited, but usually, the pH of the liquefied liquid is preferably adjusted to 4.5 to 6.5 using a pH adjuster.
More preferably, if it is adjusted to 5.5 to 6.5, the later p
The H adjustment need not be performed. Oxalic acid, slaked lime, phosphates such as disodium phosphate,
Dipotassium phosphate or the like can be used. Regarding the reaction time of the trehalose production reaction in the present invention,
Although there is no particular limitation, regardless of whether the three enzymes act simultaneously or the two enzymes used in the trehalose production reaction are applied later, usually about 24 to 72 hours after the two enzymes used in the trehalose production reaction act. Appropriate.

A sugar solution containing trehalose at a high concentration can be obtained by the above complex enzyme reaction. This sugar solution or a concentrate or dried product obtained by evaporating water from this sugar solution is also one embodiment of the present invention. Preferably, this sugar solution is appropriately purified by a conventional method (enzyme inactivation, filtration, decolorization, deodorization, deionization, etc.), and the concentrate or dried product is also prepared from such a purified sugar solution. . Powder is preferred as the dried product. The sugar solution obtained by the complex enzyme reaction contains dextrin and has low hygroscopicity, so that it can be easily pulverized by spray drying or the like.

The sugar solution, concentrate or dried product of the present invention has low reducibility and is stable because trehalose is non-reducing. For this reason, even when mixed and processed with other materials, especially amino acids, oligopeptides, proteins, etc., the degree of browning and off-flavor generation is low, and the mixed other materials are hardly damaged. In addition, trehalose is known to have an anti-aging effect of starch and an effect of suppressing protein denaturation, and as a preservative for storing chilled food for a long time, as well as a food material to be frozen or dried or It can be used advantageously for the purpose of preventing protein denaturation in beverages. Also,
Since the sugar solution or the concentrate or the dried product of the present invention is in good harmony with various substances having other tastes such as acidity, salty taste, astringency, umami, bitterness, and has high acid resistance and heat resistance, it is generally used. Can be advantageously used for sweetening and improving the taste of foods and drinks.
Furthermore, the sugar solution, concentrate or dried product of the present invention can be used as a bulking agent because it contains dextrin. Further, it can be used after being molded into various shapes such as granules, spheres and tablets.

As described above, the sugar solution, concentrate or dried product of the present invention may be used as a sweetener, a bulking agent, a taste improver, a quality improver, a stabilizer, an excipient, etc.
It can be advantageously used for feeds, cosmetics, pharmaceuticals, etc. Examples of foods and drinks include various seasonings (for example, amino acids, peptides, soy sauce, powdered soy sauce, miso, powdered miso, moromi, hishio, sprinkle, mayonnaise, dressing, vinegar, three cups vinegar, powdered sushi vinegar, Chinese ingredients, Ten soup, noodle soup, sauce,
Ketchup, grilled meat sauce, curry roux, stew ingredients, soup ingredients, dash ingredients, etc.), various Japanese sweets (for example,
Senbei, hail, okoshi, mochi, manju, seaweed, bean jam, sheep gong, water sheep gong, nishikidama, jelly, castella,
Candy, etc.), various Western confectionery (for example, bread, biscuit, crackers, cookies, pie, pudding, butter cream,
Custard cream, cream puff, waffle, sponge cake, donut, chocolate, chewing gum, caramel, candy, etc.), syrups (eg, fruit syrup pickled, ice honey, etc.), pastes (eg, flower paste, fruit paste, spread, etc.) ), Jam, marmalade, pickles (for example, Fukugami pickles, betta pickles, senmai pickles, lucky pickles, etc.), livestock meat products (for example, ham, sausage, etc.), fish meat products (for example, kamaboko, bamboo wheels, etc.), noodles (for example, Chinese noodles) , Udon, etc.), various delicacies, tsukudani, alcoholic drinks, coffee, cocoa, juice, carbonated drinks, stamina drinks, lactic acid drinks, lactic acid drinks, instant foods and drinks (eg, instant juice, instant coffee, etc.), tempura fries Such as Clothing materials for the lower thereof. In addition, it can be used for raising livestock, poultry, other honeybees, silkworms, fish, and other breeding animals for the purpose of improving the palatability of feed and feed.

The content of the sugar solution or the concentrate or the dried product of the present invention in foods, feeds, feeds, feeds, cosmetics, pharmaceuticals, etc. is not particularly limited. Minutes), generally 0.1% ~
99.5% is appropriate, and preferably about 0.2% to 30%.

[0030]

Next, the present invention will be described more specifically with reference to examples. Example 1 Bacillus sp. Heat-resistant MPas prepared from RK-1 strain
e and production of trehalose-containing sugar solution using heat-resistant TPase prepared from Bacillus stearothermophilus SK-1 strain 1-1 Bacillus sp. Mase of RK-1 strain and TPa of Bacillus stearothermophilus SK-1 strain
Preparation of SE <Preparation of crude MPase> 100 ml of a medium (pH 7.0) containing 1 g / dl of yeast extract, 2 g / dl of polypeptone, and 1 g / dl of maltose was placed in a 500 ml pleated Erlenmeyer flask, and autoclaved at 121 ° C. for 20 minutes. Bacillus sp. RK-1 strain (FER
MBP-5592) and inoculated with one platinum loop.
The culture obtained for a period of time was used as a seed culture solution. About 3 L of a medium having the same composition as that of the seed culture was put into a 5 L fermenter and sterilized. After adjusting the temperature to 55 ° C., the seed culture solution was 2 v / v%.
Was inoculated and cultured with aeration and agitation for 18 hours while maintaining the temperature at 55 ° C. and the pH at 6.0 to 7.0. After completion of the culture, the culture was centrifuged to remove the cells, ammonium sulfate was dissolved in the supernatant to a saturation of 40 to 60%, and the resulting protein precipitate was collected by centrifugation. (PH 6.0)
, Dialyzed against the same buffer, concentrated,
20 ml of a crude enzyme solution having a Pase activity of about 300 units / ml was obtained.

<Preparation of crude TPase enzyme>
12 g / dl, polypeptone 0.4 g / dl, NaCl
Medium (pH 7.0) 100 containing 0.2 g / dl
into a 500 ml pleated Erlenmeyer flask and add 121 ml.
Bacillus stearothermophilus SK-1 strain (FERM BP)
-5594) was inoculated with one platinum loop and cultured at 55 ° C for 16 hours to obtain a seed culture solution. In a 5 L fermenter, yeast extract 1 g / dl, polypeptone 2 g / dl,
Approximately 3 L of a medium containing 1 g / dl of trehalose was added thereto and sterilized. After adjusting the temperature to 55 ° C., 2 v / v% of a seed culture was inoculated, and 4% while maintaining the temperature at 55 ° C. and pH 6.0 to 7.0.
The cells were cultured with aeration and stirring for 0 hours. After completion of the culture, the culture was centrifuged to remove the cells, ammonium sulfate was dissolved in the supernatant to 80% saturation, and the precipitated protein was collected by centrifugation. This was dissolved in a 10 mM acetate buffer (pH 6.0), dialyzed against the same buffer, and concentrated, followed by TPas.
20 ml of a crude enzyme solution having an e activity of about 220 units / ml was obtained.

1-2 Production of Trehalose-Containing Sugar Solution Corn starch was allowed to react with α-amylase (Termamyl, Novo) to react until DE10, and then the α-amylase was acid-inactivated to prepare a starch liquefied solution. This was adjusted to a solid concentration of 33%, and Na ₂ HPO ₄ was added to a final concentration of 400 ppm, and then the pH was adjusted to 6. Β-amylase (sold by Nagase & Co., Ltd./β-amylase), Mase and TPa prepared in Example 1-1
Then, 5 units, 10 units, and 10 units of the crude enzyme solution per 1 g of the solid content in the liquefied starch solution were added and reacted at 70 ° C. for 24 hours. Sample a part of the sugar solution before and after the reaction,
After filtration, the total saccharide was measured by the phenol-sulfuric acid method, and the recovery was 99.9%. After heating the reaction solution to deactivate the enzyme, the solution was decolorized with activated carbon according to a conventional method, desalted and purified with an ion exchange resin, and further concentrated to obtain a trehalose-containing sugar solution having a water content of about 50%. . This sugar solution contains about 23% of trehalose, about 2% of glucose, about 32% of maltose, about 9% of maltotriose, and about 34% of branched dextrin having a degree of polymerization of 4 or more per solid content.
Included. This was spray-dried by a conventional method to obtain a powdered trehalose-containing sugar solution. When the sugar composition of this powder product was examined, it was the same as before spray drying.

Examples 2 and 3 Same as Example 1 except that the reaction temperature was 65 ° C. and the reaction time was 36 hours (Example 2) or the reaction temperature was 60 ° C. and the reaction time was 48 hours (Example 3). Enzyme reaction, total sugar measurement before and after the reaction, desalting / purification, concentration, water content about 50%
Trehalose-containing sugar solution was obtained. The total sugar recovery after the reaction was 99.5% in Example 2 and 98.3 in Example 3.
%Met. The sugar solution of Example 2 contained about 37% of trehalose, about 2% of glucose, about 18% of maltose, about 9% of maltotriose, and about 34% of branched dextrin having a degree of polymerization of 4 or more per solid content. The sugar solution of Example 3 contains about 36% of trehalose, about 2% of glucose, about 19% of maltose, about 19% of maltotriose, and about 9% of maltotriose, and about 4% or more of branched dextrin per degree of solids. 34%
Included.

COMPARATIVE EXAMPLE 1 Na ₂ HPO ₄ was added to a starch liquefied liquid (DE10) having a solid content of 33% to a final concentration of 400 ppm, and then a pH of 6 was obtained.
Was adjusted. To this, β-amylase was added in an amount of 5 units per g of solids in the liquefied starch solution, and reacted at 70 ° C. for 24 hours. Then, the crude enzyme solution of MPase and the crude enzyme solution of TPase prepared in Example 1 were each added in an amount of 10 units per g of solids in the liquefied starch solution, and reacted at 70 ° C. for 24 hours. A part of the sugar solution before and after the reaction was partially sampled, and after filtration, the total sugar was measured by the phenol-sulfuric acid method. As a result, the recovery of the total sugar after the reaction was 99.8%. After heating the reaction solution to deactivate the enzyme, the solution was decolorized with activated carbon according to a conventional method, desalted and purified with an ion exchange resin, and further concentrated to obtain a trehalose-containing sugar solution having a water content of about 50%. . This sugar solution contains about 18% of trehalose, about 2% of glucose, about 37% of maltose, about 9% of maltotriose, and about 3% of branched dextrin having a degree of polymerization of 4 or more per solid content.
It contained 4%.

Comparative Examples 2 and 3 Na ₂ HPO ₄ was added to a starch liquefied liquid (DE10) having a solid content of 33% to a final concentration of 400 ppm, and the pH was adjusted to 6 or less.
Was adjusted. To this, β-amylase was added at 5 units per g of solids in the liquefied starch solution, and reacted at 65 ° C. for 36 hours (Comparative Example 2) or at 60 ° C. for 48 hours (Comparative Example 3).
Then, the crude enzyme solution of MPase and the crude enzyme solution of TPase prepared in Example 1 were each added in an amount of 10 units per g of solids in the liquefied starch solution, and the mixture was added at 65 ° C. for 36 hours (Comparative Example 2).
Alternatively, the reaction was carried out at 60 ° C. for 48 hours (Comparative Example 3). Then, the whole sugar measurement before and after the reaction, desalting / purification,
Concentration was performed to obtain a trehalose-containing sugar solution having a water content of about 50%. The total sugar recovery after the reaction was 99.6% and 9%, respectively.
In the case of Comparative Example 2, the obtained sugar solution contained about 29% of trehalose, about 2% of glucose,
It contains about 26% maltose, about 9% maltotriose, and about 34% branched dextrin having a degree of polymerization of 4 or more.
In the case of Comparative Example 3, about 33% trehalose per solid content,
It contained about 2% glucose, about 22% maltose, about 9% maltotriose, and about 34% branched dextrin with a degree of polymerization of 4 or higher.

Example 4 Mase and TPas prepared from a recombinant Bacillus subtilis strain
Production of trehalose-containing sugar solution using e. Preparation of 4-1 Mase expression vector Bacillus amyloliquefaciens IFO15535 was cultured, and chromosomal DNA was recovered by a conventional method. Gene, 1
5 , 43 (1981), two types of primers having the sequences of SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing were synthesized, and (α-amylase promoter was modified by PCR). DNA fragment contained)
Was synthesized. The end of the obtained fragment was phosphorylated using T4 DNA kinase, and the end was blunted using S1 nuclease. Then, digest with Xbal, 3.
The mixture was subjected to 0 g / dl agarose electrophoresis to recover a 0.25 kb DNA fragment containing the α-amylase promoter region.

In addition, Bacillus sp. RK-1 strain (FE
RM BP-5592) from the chromosome of
No. 62683, SEQ ID NO: 3
A fragment containing the MPase structural gene was amplified by PCR using two types of primers shown in SEQ ID NO: 4 and SEQ ID NO: 4. The obtained 2.3 kb fragment was ligated with T4 DNA
After phosphorylation of the end using a kinase, the end was blunted using S1 nuclease. Then, it was digested with EcoR1 and subjected to 0.8 g / dl agarose electrophoresis.
A 2.3 kb DNA fragment containing the Pase structural gene was recovered. On the other hand, plasmid pUB110 (SIGMA
Was digested with the restriction enzyme Xbal-EcoR1, and 0.25 containing the α-amylase promoter region obtained previously.
The kb DNA fragment and the 2.3 kb DNA fragment containing the MPase structural gene were ligated. As a result, an MPase expression vector pUB-P-MP (FIG. 1) was obtained.

4-2 Preparation of TPase Expression Vector Bacillus stearothermophilus SK-1 strain (FER
MBP-5594) from the chromosome of JP-A-10-32
A fragment containing a TPase structural gene was amplified by PCR using two types of primers shown in SEQ ID NO: 5 and SEQ ID NO: 6, based on the description in JP-A-7887. The resulting 3.1 kb fragment was phosphorylated at the ends using T4 DNA kinase, and then blunt-ended using S1 nuclease. Then, it cuts with EcoR1 and 0.8g
/ Dl agarose electrophoresis to recover a 3.1 kb DNA fragment containing the TPase structural gene. On the other hand,
Plasmid pUB110 was replaced with the restriction enzyme Xbal-EcoR.
After digestion with 1, the TPase expression vector pUB-P-TP (FIG. 2) was obtained in the same manner as in 4-1.

4-3 Mase Expression Vector and T
Transformation of Pase expression vector into Bacillus subtilis (preparation of recombinant B. subtilis strain) Using the MPase expression vector and TPase expression vector obtained in 4-1 and 4-2, in a conventional manner,
Bacillus subtilis 168 strain was transformed and the recombinant B. subtilis strain B. subtilis / pUB-P-MP and B. subtilis / pUB-P-MP subtilis / pUB-P-TP was obtained.

4-4 Recombinant MP from Recombinant Bacillus subtilis
Preparation of recombinant Bacillus subtilis strain B. subtilis
/ PUB-P-MP and B.I. subtilis / pU
Each of BP-TP was added to 100 ml of L medium (1 g / g).
dl tryptone, 0.5 g / dl yeast extract, 0.5 g
/ DlNaCl, pH 7.2, 10 µg / ml kanamycin) and cultured at 37 ° C overnight. 1 ml of culture solution
And harvested by centrifugation, 0.85 g / dlN
Suspension in an aCl aqueous solution was repeated, and the cells were washed. next,
The bacteria suspended in 0.85 g / dl NaCl aqueous solution
100 ml of 2 × Y in a pleated Erlenmeyer flask
T medium (1.6 g / dl tryptone, 1.0 g / dl yeast extract, 0.5 g / dl NaCl, pH 7.2, 10
μg / ml kanamycin) and cultured for 20 hours using a rotary shaker. After completion of the culture, the culture solution was subjected to ultrasonic treatment to destroy the cells, and cell residues were removed by centrifugation to prepare a crude enzyme. When the activity of the crude enzyme was measured, Mase was about 240,000 units and TPase was about 300,000 per liter of culture solution.
Units.

4-5 Preparation of Trehalose-Containing Sugar Solution α-Amylase was allowed to act on corn starch to give DE10
After the reaction, the α-amylase was inactivated by acid to prepare a starch liquefied solution. This was adjusted to a solid concentration of 33%,
After adding Na ₂ HPO ₄ to a final concentration of 400 ppm, the pH was adjusted to 6. The β-amylase (sold by Nagase & Co., Ltd./β-amylase) and the recombinant enzyme solution of the recombinant MPase and the crude enzyme solution of the TPase prepared in 4-4 were each added in 5 units per g of solids in the liquefied starch solution. , 10
Units and 10 units were added and reacted at 65 ° C. for 36 hours. A part of the sugar solution before and after the reaction was sampled, and after filtration, the total sugar was measured by the phenol sulfuric acid method. As a result, the recovery was 99.5%. After heating the reaction solution to deactivate the enzyme, the solution is decolorized with activated carbon, desalted and purified with an ion exchange resin, and further concentrated to a water content of about 50 according to a conventional method.
% Trehalose-containing sugar solution was obtained. The sugar solution contained about 33% of trehalose, about 2% of glucose, about 22% of maltose, about 9% of maltotriose, and about 34% of branched dextrin having a degree of polymerization of 4 or more per solid content. This was spray-dried by a conventional method to obtain a powdered trehalose-containing sugar solution. When the sugar composition of this powder product was examined, it was the same as before spray drying.

Examples 5 and 6 The reaction temperature was 60 ° C. and the reaction time was 48 hours (Example 5).
Alternatively, the enzyme reaction, measurement of total sugars before and after the reaction, desalting / purification, and concentration were performed in the same manner as in 4-5 of Example 4 except that the reaction temperature was 55 ° C. and the reaction time was 60 hours (Example 6). Do
A trehalose-containing sugar solution having a water content of about 50% was obtained. The total sugar recovery after the reaction was 98.3% in Example 5 and 93.1% in Example 6. The sugar solution of Example 5 contained about 36% of trehalose and about 2% of glucose per solid.
%, Maltose about 19%, maltotriose about 9
%, About 34% of branched dextrin having a degree of polymerization of 4 or more. The sugar solution of Example 6 contains about 37% of trehalose, about 2% of glucose, about 18% of maltose,
It contained about 9% maltotriose and about 34% branched dextrin with a degree of polymerization of 4 or higher.

Comparative Example 4 Na ₂ HPO ₄ was added to a starch liquefied liquid (DE10) having a solid content of 33% to a final concentration of 400 ppm, and the pH was adjusted to 6
Was adjusted. To this, β-amylase was added in an amount of 5 units per g of the solid content in the liquefied starch solution, and reacted at 65 ° C. for 36 hours. Next, the recombinant Mpa prepared in 4-4 of Example 4 was used.
The se crude enzyme solution and the recombinant TPase crude enzyme solution were added at 10 units per g of solids in the liquefied starch solution, and reacted at 65 ° C. for 36 hours. A part of the sugar solution before and after the reaction was sampled and filtered, and then the total sugar was measured by the phenol-sulfuric acid method. The total sugar recovery was 99.6%. After heating the reaction solution to deactivate the enzyme, the solution was decolorized with activated carbon, desalted and purified using an ion exchange resin, and concentrated to obtain a trehalose-containing sugar solution having a water content of about 50% according to a conventional method. This sugar solution contains about 25% trehalose per solid,
It contained about 2% glucose, about 30% maltose, about 9% maltotriose, and about 34% branched dextrin with a degree of polymerization of 4 or higher.

Comparative Examples 5 and 6 Na ₂ HPO ₄ was added to a starch liquefied liquid (DE10) having a solid content of 33% to a final concentration of 400 ppm, and the pH was adjusted to 6
Was adjusted. To this, β-amylase was added in an amount of 5 units per g of solids in the liquefied starch solution, and reacted at 60 ° C. for 48 hours (Comparative Example 5) or at 55 ° C. for 60 hours (Comparative Example 6). Next, the recombinant Mpa prepared in 4-4 of Example 4 was used.
The crude enzyme solution and the crude enzyme solution of the recombinant TPase were added in an amount of 10 units per g of solids in the liquefied starch solution.
For 48 hours (Comparative Example 5) or at 55 ° C. for 60 hours (Comparative Example 6). Thereafter, in the same manner as in Comparative Example 3, total saccharide measurement before and after the reaction, desalting / purification, and concentration were performed to obtain a trehalose-containing sugar solution having a water content of about 50%. The recovery rates of the total sugars after the reaction were 98.2% and 93.3%, respectively, and the obtained sugar solution contained about 3% trehalose per solid in Comparative Example 5.
It contains about 3%, about 2% glucose, about 22% maltose, about 9% maltotriose, and about 34% branched dextrin having a degree of polymerization of 4 or more. In Comparative Example 6, about 35% trehalose per solid is contained. %, Glucose about 2%, maltose about 20%, maltotriose about 9%, polymerization degree 4
It contained about 34% of the above branched dextrin.

Example 7 Production of a Trehalose-Containing Sugar Solution Using Commercially Available Mpase and TPase Prepared from Bacillus stearothermophilus SK-1 Strain Corn α-starch was reacted with α-amylase to give DE10
After the reaction, the α-amylase was inactivated by acid to prepare a starch liquefied solution. This was adjusted to a solid concentration of 33%,
After adding Na ₂ HPO ₄ to a final concentration of 400 ppm, the pH was adjusted to 6. Β-amylase (sold by Nagase & Co., Ltd./β-amylase), MPase (sold by Oriental Yeast Co., Ltd./maltose phosphorylase derived from bacteria) and Bacillus stearothermophilus prepared in 1-1 of Example 1 5 units, 10 units, and 10 units of TPase crude enzyme solution of SK-1 strain were added per g of solids in the liquefied starch solution, and reacted at 60 ° C. for 48 hours. A part of the sugar solution before and after the reaction was partially sampled, and after filtration, the total sugar was measured by the phenol-sulfuric acid method.
3%. After heating the reaction solution to deactivate the enzyme, the solution is decolorized with activated carbon according to a conventional method, desalted and purified with an ion exchange resin, and further concentrated to about 50% water.
Trehalose-containing sugar solution was obtained. The sugar solution contained about 23% of trehalose, about 2% of glucose, about 32% of maltose, about 9% of maltotriose, and about 34% of branched dextrin having a degree of polymerization of 4 or more per solid content. This was spray-dried by a conventional method to obtain a powdered trehalose-containing sugar solution. When the sugar composition of this powder product was examined, it was the same as before spray drying.

Examples 8 and 9 With the same combination of enzymes and the amount of enzyme added as in Example 7,
The reaction was performed at the reaction temperature and reaction time shown in Table 1 below. As a result, the yield and sugar composition were as shown in Table 1.

COMPARATIVE EXAMPLE 7 Na ₂ HPO ₄ was added to a starch liquefied liquid (DE10) having a solid content of 33% to a final concentration of 400 ppm, and then a pH of 6 was obtained.
Was adjusted. To this, β-amylase was added in an amount of 5 units per g of solids in the liquefied starch solution, and reacted at 60 ° C. for 48 hours. Then, Mase (sold by Oriental Yeast Co., Ltd./maltose phosphorylase) and 1-1 of Example 1
Bacillus stearothermophilus SK-1 prepared in
The crude TPase enzyme solution of the strain was added in an amount of 10 units per g of solids in the liquefied starch solution, and reacted at 60 ° C. for 48 hours. A part of the sugar solution before and after the reaction was sampled and filtered, and then the total sugar was measured by the phenol-sulfuric acid method. As a result, the total sugar recovery was 98.2%. After heating the reaction solution to deactivate the enzyme, decolorize with activated carbon according to a conventional method,
The trehalose-containing sugar solution having a water content of about 50% was obtained by desalting and purifying with an ion exchange resin and further concentrating. This sugar solution is
About 18% trehalose, about 2% glucose, about 37% maltose, about 9% maltotriose per solid
%, About 34% of branched dextrin having a degree of polymerization of 4 or more.

Comparative Examples 8 and 9 Trehalose-containing sugar solutions were obtained by changing only the reaction temperature and the reaction time according to the type, amount and timing of addition of the same enzymes as in Comparative Example 7. The recovery rate and sugar composition were measured in the same manner as in Comparative Example 7, and the results are shown in Table 1.

Table 1 summarizes the conditions for producing the trehalose-containing sugar solutions of the above Examples and Comparative Examples and the sugar compositions of the obtained sugar solutions.

[0050]

[Table 1]

From Table 1, it can be seen that commercially available β-amylase, Bacillus sp. Examples 1, 2, and 3 using MPase prepared from strain RK-1 and TPase prepared from Bacillus stearothermophilus SK-1
(Simultaneous addition of 3 enzymes) and Comparative Examples 1, 2, 3 (1 enzyme and 2
70 ° C., 65 ° C., 60 ° C.
It can be seen that the examples are far superior to the comparative examples in terms of the yield of trehalose and the reaction time at any of ° C. A similar tendency is observed when using Mase and TPase prepared from a recombinant B. subtilis strain in addition to commercially available β-amylase, and using TPase prepared from commercially available Mase and Bacillus stearothermophilus SK-1. Can also be seen. This is Mase, T
It is considered that the heat resistance of the Pase increases under the conditions in the present invention. In addition, the sugar recovery rate tended to depend on the reaction temperature regardless of the origin of the enzyme. This is because if the reaction temperature is low, the unsaccharified high-molecular-weight dextrin cannot be completely dissolved in the reaction solution, so that it is not subject to decomposition by an enzyme and precipitates.

Example 7 Bread 100 parts by mass of flour, 2 parts by mass of yeast, 0.12 parts by mass of yeast food, 5 parts by mass of trehalose-containing sugar liquid powder (the product of the present invention), 2 parts by mass of salt, 5 parts by mass of oil and fat, water Bread dough is prepared by kneading 62 parts by mass according to a conventional method.
Aged for a minute and baked. The obtained bread had better appearance, flavor, and texture as compared to the control without the product of the present invention, and was particularly excellent in softness and retention of elasticity.

Example 8 Fried food A croquette ingredient pre-molded by mixing ground baron potato, minced onion, ground meat, salt, pepper and water was mixed with a trehalose-containing sugar liquid powder (product of the present invention).
Was added to a batter added to a concentration of 20% by mass, bread crumbs were added, and the mixture was fried in oil at 175 ° C. for 3 minutes. This was frozen and stored at −25 ° C. for 2 weeks, and then thawed by heating in a microwave oven. This product had better appearance, flavor, and texture as compared to the control without the product of the present invention, and particularly had a crisp and light texture.

Example 9 Noodle noodles prepared by a conventional method using 100 parts by weight of udon flour (medium flour), 5 parts by weight of trehalose-containing sugar liquid powder (the present invention), 2 parts by weight of sodium chloride and 34 parts by weight of water. Make the dough, corner 10
A 2.8 mm-thick noodle string was formed from the incisor No. 2 and boiled for 10 minutes. This was stored in a chilled state at 8 ° C. and returned to the boil for 2 minutes after one day. As a result, the product had a smoother texture than the control without the product of the present invention. Met.

Example 10 100 parts by mass of Chinese noodle flour (strong flour), 5 parts by mass of trehalose-containing sugar solution powder (the present invention), 1 part by mass of salt, 1 part by mass of starch, 2 parts by mass of alcohol, 32 parts by mass of water Was made into noodle dough by a conventional method, and a noodle string having a thickness of 1.35 mm was formed with incisors having a corner of No. 22 and packaged with a polyethylene film. This was stored in a chilled state at 8 ° C., and after one day, the odor was evaluated. As a result, the odor of alcohol was suppressed as compared with the control without the product of the present invention. When this product was boiled for 3 minutes and the taste was evaluated, no difference from the control was found.

[0056]

As shown in the production method of the present invention, the maltose-forming enzyme, the heat-resistant MPase and the heat-resistant TPase are simultaneously allowed to act on the liquefied starch solution or the maltose-forming enzyme is reacted with the maltose-forming enzyme from the liquefied starch solution during the reaction. When trehalose is generated by the action of a thermophilic MPase and a heat-resistant TPase, a maltose-forming enzyme is applied to a liquefied starch solution to generate a substantial amount of maltose, and then the trehalose is applied by the action of the heat-resistant MPase and the heat-resistant TPase. Trehalose can be produced at a higher temperature and at a higher yield in a shorter time than in the case of producing, and consequently, bacterial contamination can be reduced, aging in the production process can be suppressed, and enzyme cost can be reduced. In addition, the trehalose-containing sugar solution obtained by the conventional production method using phosphorylase (a production method in which MPase, TPase, β-amylase, and a debranching enzyme simultaneously act on a liquefied starch solution) has high hygroscopicity, and therefore, the syrup product is used However, since the trehalose-containing sugar solution provided by the present invention contains dextrin and has low hygroscopicity, it can be easily pulverized by spray drying or the like. By using a powdered product, it can be used for a premix product such as a hot cake mix or a pan mix.

[0057]

[Sequence List] SEQUENCE LISTING <110> Showa Sangyo Co., Ltd. <120> Method for producing trehalose-containing sugar solution, and products and applications <130> SHO1101 <160> 6 <210> 1 <211> 29 <212> DNA <213 > Bacillus amyloliquefaciens <400> 1 cgctctagag ccccgcacat acgaaaaga <210> 2 <211> 26 <212> DNA <213> Bacillus amyloliquefaciens <400> 2 gtttcctctc cctctcattt tcttat <210> 3 <211> 20 <212> DNA <213> Bacill sp. <400> 3 atgtattaca acaggttgtt <210> 4 <211> 29 <212> DNA <213> Bacillus sp. <400> 4 cgcgaattct cacacatact ccttcgtat <210> 5 <211> 10 <212> DNA <213> Bacillus stearothermophilus <400> 5 atgtcttggt caattagctc <210> 6 <211> 29 <212> DNA <213> Bacillus stearothermophilus <400> 6 aaagaattct cgtcgcgctt ccaccgatt

[Brief description of the drawings]

FIG. 1 shows a step of obtaining an MPase expression vector pUB-P-MP.

FIG. 2 shows a step of obtaining a TPase expression vector pUB-P-TP.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (Reference) C12N 1/19 C12N 1/19 4B064 1/21 1/21 4B065 5/10 9/10 9/10 5/00 A15 / 09 ZNA 15/00 ZNAA (72) Inventor Shinsuke Miyoshi 1-16 Sakura, Tsukuba, Ibaraki Prefecture Showa Sangyo Co., Ltd. Research Institute Biotechnology Research Center F-term (reference) 4B024 AA05 BA10 CA03 DA07 EA04 FA02 GA11 4B032 DB01 DK12 DL01 4B036 LF13 LH10 4B046 LA02 LA05 LC01 LG13 4B050 CC03 DD02 LL05 4B064 AF03 CA02 CA19 CA21 CB30 CC24 DA10 4B065 AA15Y AA18Y AA19X AB01 AC14 BA02 CA29 CA41

Claims (10)

[Claims] 1. A maltose-forming reaction is carried out by simultaneously reacting a maltose-forming enzyme, a thermostable maltose phosphorylase and a thermostable trehalose phosphorylase on a liquefied starch solution or by reacting a maltose-forming enzyme on a liquefied starch solution. A process for producing trehalose, wherein a thermostable maltose phosphorylase and a thermostable trehalose phosphorylase are allowed to act during the progress. 2. After reacting maltose synthase, 12
The method according to claim 1, wherein the reaction temperature is 50 to 70C after the heat-resistant maltose phosphorylase and the heat-resistant trehalose phosphorylase are allowed to act on the heat-resistant maltose phosphorylase and the heat-resistant trehalose phosphorylase within the time. 3. A heat-resistant maltose phosphorylase which is obtained from a wild-type microorganism having the enzyme-producing ability, or which is produced by a recombinant microorganism into which a heat-resistant maltose phosphorylase gene derived from the microorganism is introduced. Wherein the thermostable trehalose phosphorylase is obtained from a wild-type microorganism having the enzyme-producing ability, or a thermostable trehalose phosphorylase produced by a recombinant microorganism into which a thermostable trehalose phosphorylase gene derived from the microorganism is introduced. The method according to claim 1. 4. The microorganism having a thermostable maltose phosphorylase producing ability and the microorganism having a thermostable trehalose phosphorylase producing ability belong to the genus Bacillus.
The manufacturing method as described. 5. A wild-type microorganism having a thermostable maltose phosphorylase-producing ability is obtained from Bacillus sp. RK-1 (FE
RM BP-5592) and a wild-type microorganism having the ability to produce thermostable trehalose holase, Bacillus stearothermophilus SK-1 (FERM BP-559).
The method according to claim 3 or 4, wherein the method is (4). 6. A recombinant microorganism into which a thermostable maltose phosphorylase gene has been introduced and a recombinant microorganism into which a thermostable trehalose phosphorylase gene has been introduced.
5. The method according to claim 3, wherein the method belongs to Sacillus. 7. The method according to claim 7, wherein the thermostable maltose phosphorylase is Bacillus sp. RK-1 (FERM BP-5592), wherein the thermostable trehalose phosphorylase is Bacillus stearothermophilus SK-1 (FE
RM BP-5594), wherein the trehalose-forming enzyme reaction is carried out at a temperature of 60 ° C to 70 ° C. 8. The thermostable maltose phosphorylase of Bacillus sp. A heat-resistant maltose phosphorylase produced by a recombinant microorganism belonging to Bacillus subtilis into which a heat-resistant maltose phosphorylase gene derived from RK-1 (FERM BP-5592) has been introduced, wherein the heat-resistant trehalose phosphorylase is Bacillus stearothermophilus. SK-1 (FERM BP-5594) is a thermostable trehalose phosphorylase produced by a recombinant microorganism belonging to Bacillus subtilis into which a thermostable trehalose phosphorylase gene derived from SK-1 (FERM BP-5594) has been introduced. The method according to claim 1 or 2, wherein the method is performed. 9. A trehalose-containing sugar solution produced by the method according to any one of claims 1 to 8, or a concentrate or a dried product obtained by evaporating water therefrom. 10. A food or drink comprising the trehalose-containing sugar solution according to claim 9 or a concentrate or a dried product obtained by evaporating water therefrom.