JP6321857B1 - Method for producing sugar carboxylic acid - Google Patents

Abstract

[PROBLEMS] To produce sugar carboxylic acid industrially by using a catalase preparation that rapidly decomposes hydrogen peroxide produced as a by-product in an oxidation reaction, and in a high yield and with a starch decomposition oxide or a transfer reaction oxide having a polymerization degree of 2 or more. To provide a way to produce. A method for producing a sugar carboxylic acid in which an aldehyde group on the reducing end side of a starch degradation product or transfer reaction product having a glucose residue at a reducing end of 2 or more is oxidized is obtained by hydrogen peroxide during saccharide oxidation. In the presence of a catalase preparation having a content ratio (B / A) of saccharification activity (B) to catalase activity (A) in the catalase preparation of 0.00002 or more and 0.005 or less. And a step of acting on the raw material substrate containing the starch decomposition product or the transfer reaction product. The catalase preparation is present in an amount such that its saccharification activity is 0.9 u / g or less with respect to the amount of reducing sugar (wt%) in the raw material substrate. [Selection figure] None

Description

  The present invention relates to a method for producing a sugar carboxylic acid in which an aldehyde group on the reducing end side of a starch degradation product or transfer reaction product having a glucose residue at a reducing end of 2 or more or a transfer reaction product is oxidized, a salt thereof, and a lactone thereof.

  Gluconic acid, which is one of the aldonic acids obtained by oxidizing the reducing end of glucose, is not only a monosaccharide but also has functionality such as bifidobacteria growth selectivity, as well as inorganic substances such as calcium. Since it has a characteristic of forming a stable salt with a cation, it is used as a mineral reinforcing agent. However, the solution stability is poor, and there is a drawback that it is precipitated when stored at a high concentration.

  As a material to compensate for these drawbacks, sugar carboxylic acids such as maltobionic acid in which glucose is bonded to the non-reducing terminal side of gluconic acid can be mentioned. Maltobionic acid, which is a sugar carboxylic acid, also forms a stable salt with an inorganic cation, but has good solubility and does not precipitate even when stored under high concentration conditions. In this way, it is expected that many functional substances can be obtained by oxidizing the reducing end of a saccharide higher than a disaccharide.

  Patent Documents 1 and 2 disclose methods using microorganisms such as Acinetobacter genus, Burkholderia genus, Gluconobacter genus and Acetobacter genus as a technique for oxidizing maltose, lactose, cellobiose and the like having a polymerization degree of 2. ing. In addition, as an enzymatic method for oxidizing starch degradation products having a polymerization degree of 4 or more, a method using a saccharide oxidase preparation derived from a microorganism belonging to the genus Microdochium or a saccharide oxidase preparation derived from a microorganism belonging to the genus Acremonium is known. It has been.

JP 2001-245657 A JP 2007-028917 A Japanese Patent No. 4417550 Japanese Patent No. 3310008 International Publication WO2014 / 042237 Pamphlet Japanese Patent No. 3910213 Special Table 2000-502904

  The saccharide oxidases of Patent Documents 3 to 5 generate hydrogen peroxide as a by-product by a reaction that oxidizes saccharides. Hydrogen peroxide has the power to denature proteins, such as being used as a bactericidal or bleaching agent, and hydrogen peroxide produced as a by-product during oxidization of carbohydrates denatures and deactivates carbohydrate oxidase. For this reason, in order to oxidize starch degradation products and transfer reaction products having a polymerization degree of 2 or more industrially and efficiently using carbohydrate oxidase, it is necessary to rapidly decompose hydrogen peroxide.

  In the glucose oxidase preparations of Patent Documents 6 and 7, hydrogen peroxide is generated in the process of oxidizing glucose to gluconic acid. It is described that a catalase preparation is used as a production technique for rapidly decomposing hydrogen peroxide produced as a by-product.

  Even when starch degradation products and transfer products with a polymerization degree of 2 or more are oxidized, adding a catalase preparation together with a carbohydrate oxidase can quickly decompose hydrogen peroxide produced as a by-product during carbohydrate oxidation. I can do it. However, although the cause is unknown, an oxide having a degree of polymerization of 2 or more cannot be stably produced in a high yield.

  The present invention has been made in view of the above circumstances, and uses a catalase preparation that quickly decomposes hydrogen peroxide produced as a by-product in an oxidation reaction, and has high yield and has a degree of polymerization of 2 or more. It is an object of the present invention to provide a method for industrially producing a sugar carboxylic acid using a product or a transfer reaction oxide.

  The inventors of the present invention are that a starch degradation product or transfer reaction product having a polymerization degree of 2 or more as a raw material is hydrolyzed by a starch degrading enzyme such as α-glucosidase or glucoamylase, which is a contaminating enzyme contained in a catalase preparation. Has been found to be the cause of destabilizing the production of sugar oxides having a degree of polymerization of 2 or more. In contrast to the production of gluconic acid by an enzymatic method, the raw material glucose is a monosaccharide, so that it is not necessary to consider hydrolysis by a contaminating enzyme in the catalase preparation. Based on this discovery, a method of stably obtaining a starch degradation product having a polymerization degree of 2 or more or an oxide of its transfer reaction product by using a calatase preparation prepared so that the amylolytic enzyme, which is a contaminating enzyme, is less than required. As a result, the present invention has been completed. More specifically, the present invention provides the following.

(1) A method for producing a sugar carboxylic acid in which an aldehyde group on the reducing end side of a starch degradation product or transfer reaction product having a glucose residue at a reducing end of 2 or more is oxidized,
Carbohydrate oxidase that produces hydrogen peroxide as a by-product during oxidization of saccharides has a saccharification activity (B) content ratio (B / A) of 0.00002 to 0.005 in catalase preparation to catalase activity (A). In the presence of a catalase preparation, which comprises the step of acting on a raw material substrate containing the starch degradation product or transfer reaction product,
The catalase preparation is a method in which the saccharification activity is present in an amount of 0.9 u / g or less with respect to the amount of reducing sugar (wt%) in the raw material substrate.

(2) A method for producing a sugar carboxylic acid in which an aldehyde group on the reducing end side of a starch degradation product or transfer reaction product having a glucose residue at the reducing end of 2 or more is oxidized,
A saccharide oxidase agent that produces hydrogen peroxide as a by-product during oxidization of saccharides has a saccharification activity (B) content ratio (B / A) to catalase activity (A) in the catalase preparation of 0.005 or less and saccharification Including the step of acting on a raw material substrate containing the starch degradation product or transfer reaction product in the presence of a catalase preparation having an activity (B) of 0.1 u / ml or more,
The catalase preparation is a method in which the saccharification activity is present in an amount of 0.9 u / g or less with respect to the amount of reducing sugar (wt%) in the raw material substrate.

  (3) The method according to (1) or (2), wherein the amount of the carbohydrate oxidase agent added is 1 u / g or more and 30 u / g or less with respect to the amount of reducing sugar (wt%) in the raw material substrate.

  (4) The catalase preparation is present in an amount such that the catalase activity is 40 u / g or more and 1000 u / g or less with respect to the amount of reducing sugar (wt%) in the raw material substrate (1) to (3) the method of.

  (5) The catalase preparation is present in any amount (1) to (4), wherein the saccharification activity is 0.00008 u / g or more with respect to the amount of reducing sugar (wt%) in the raw material substrate. Method.

  (6) The production method according to any one of (1) to (5), wherein the concentration of the starch degradation product or transfer reaction product in the action step is 10% (w / w) or more.

  According to the present invention, sugar carboxylic acids that are useful as materials for solubilizing mineral components and the like in foods, medicines, and industrial fields can be produced with high yield.

  Hereinafter, specific embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments, and may be implemented with appropriate modifications within the scope of the object of the present invention. Can do. In addition, although description may be abbreviate | omitted suitably about the location where description overlaps, the summary of invention is not limited.

In one embodiment of the present invention, a saccharide oxidase agent that produces hydrogen peroxide as a by-product during oxidization of a saccharide has a content ratio (B / A) of saccharification activity (B) to catalase activity (A) in the catalase preparation. In the presence of a catalase preparation of 0.00002 or more and 0.005 or less, comprising a step of acting on a raw material substrate containing the starch degradation product or transfer reaction product,
The catalase preparation is a method in which the saccharification activity is present in an amount of 0.9 u / g or less with respect to the amount of reducing sugar (wt%) in the raw material substrate.

Another embodiment of the present invention is a method for producing a sugar carboxylic acid in which an aldehyde group on the reducing end side of a starch degradation product or transfer reaction product having a degree of polymerization of 2 or more having a glucose residue at the reducing end is oxidized,
A saccharide oxidase agent that produces hydrogen peroxide as a by-product during oxidization of saccharides has a saccharification activity (B) content ratio (B / A) to catalase activity (A) in the catalase preparation of 0.005 or less and saccharification Including the step of acting on a raw material substrate containing the starch degradation product or transfer reaction product in the presence of a catalase preparation having an activity (B) of 0.1 u / ml or more,
The catalase preparation is a method in which the saccharification activity is present in an amount of 0.9 u / g or less with respect to the amount of reducing sugar (wt%) in the raw material substrate.

  For example, when maltose having a polymerization degree of 2, which is a starch degradation product having a glucose residue at the reducing end, is used as a raw material, maltobionic acid can be obtained by acting a sugar oxidase agent that produces hydrogen peroxide as a by-product during sugar oxidation. Hydrogen peroxide is produced, but the by-produced hydrogen peroxide denatures and deactivates the oxidase. For this reason, in order to produce maltobionic acid in a high yield, it is necessary to add a catalase preparation that rapidly decomposes hydrogen peroxide.

(Catalase preparation)
Examples of the catalase preparation referred to in the present invention include catalase preparations derived from microorganisms such as the genus Aspergillus and Micrococcus, and specific examples include a catalase preparation derived from Aspergillus nigr or Micrococcus lysodeikticus. It also includes selecting and using a commercially available glucose oxidase preparation having catalase activity as a side activity.

  In the catalase preparation of the present invention, when there are many contaminating enzymes having saccharification activity such as glucoamylase and α-glucosidase, the degree of polymerization is 2 or more having a glucose residue at the reducing end as a raw material for sugar carboxylic acid. The starch degradation product or transfer reaction product, and the sugar carboxylic acid obtained by oxidizing these raw materials are decomposed, making it impossible to produce a sugar carboxylic acid having a stable quality.

  Therefore, in the present invention, in order to stably produce a sugar carboxylic acid such as maltobionic acid at a high yield, the content ratio (B / A) of saccharification activity to catalase activity (A) in the catalase preparation is 0.005 or less. Some catalase preparations are used. Preferably, B / A is 0.0045 or less, 0.003 or less, 0.002 or less, 0.0015 or less, 0.001 or less, 0.0005 or less, or 0.0004 or less.

  The catalase preparation used in the present invention does not consist only of a highly purified catalase enzyme like a reagent from the viewpoint of industrially producing a sugar carboxylic acid, but rather contains a contaminating enzyme within an acceptable range. Specifically, B / A is preferably 0.00002 or more, and specifically may be 0.0001 or more, 0.0002 or more, 0.0003 or more, or 0.0004 or more. Even if the catalase preparation has saccharification activity at such a ratio, the main reaction of saccharide oxidation proceeds more rapidly than the saccharification reaction, so that the yield is unlikely to decrease.

  In the present invention, as shown in Comparative Example 5 of Test Example 4, even if the content ratio (B / A) of saccharification activity (B) to catalase activity (A) in the catalase preparation is 0.005 or less, If a large amount of catalase preparation is added to the raw sugar, the contaminating enzyme in the catalase preparation hydrolyzes the raw material substrate, and the composition of the sugar carboxylic acid such as maltobionic acid may not be obtained. . Therefore, the saccharification activity in the catalase preparation is 0.9 u / g or less (preferably 0.8 u / g or less, 0.7 u / g or less) with respect to the amount of reducing sugar (wt% per solid content) in the raw material substrate. , 0.65 u / g or less), it is necessary to make the catalase preparation act.

  The catalase activity (A) in the catalase preparation is preferably 5000 u / ml or more, specifically 10000 u / ml or more, 15000 u / ml or more, 20000 u / ml or more, 22500 u / ml or more. When it has high catalase activity, even if saccharification activity is high to some extent, it is easy to keep the influence on the yield small.

  The catalase activity (A) in the catalase preparation is preferably 500000 u / ml or less, and specifically may be 2500000 u / ml, 150,000 u / ml, 100000 u / ml or less, 75000 u / ml or less. Since the catalase preparation used in the present invention has a low saccharification activity, even if it does not have an excessive catalase activity, the influence on the yield can be kept small.

  The saccharification activity (B) in the catalase preparation is preferably 250 u / ml or less, specifically, 100 u / ml or less, 50 u / ml or less, 30 u / ml or less, or 25 u / ml or less.

  On the other hand, the saccharification activity (B) in the catalase preparation may be within an acceptable range, and is preferably 0.1 u / ml or more, specifically 0.5 u / ml or more, 1.0 u / ml. As described above, it may be 1.5 u / ml or more and 2.0 u / ml or more. Even if this level of saccharification activity is present, the main reaction of carbohydrate oxidation proceeds more rapidly than the saccharification reaction, so that it is difficult to lead to a decrease in yield.

  The saccharification activity in the catalase preparation may be within an acceptable range. Specifically, it is 0.00008 u / g or more with respect to the amount of reducing sugar (wt% per solid content) in the raw material substrate, preferably 0.8. It may be 0005 u / g or more, 0.001 u / g or more, 0.0015 u / g or more. Even if this level of saccharification activity is present, the main reaction of carbohydrate oxidation proceeds more rapidly than the saccharification reaction, so that it is difficult to lead to a decrease in yield.

  Further, in the production of sugar carboxylic acids such as maltobionic acid, the catalase preparation is preferably present at 40 u / g or more and 1000 u / g or less with respect to the amount of reducing sugar (per solid content) in the raw material substrate, more preferably , 60 u / g or more and 500 u / g or less. In the present invention, since the saccharification activity in the catalase preparation is kept low, even if a sufficient amount of the catalase preparation is used to suppress the degradation of the carbohydrate oxidase by hydrogen peroxide, the yield is unlikely to decrease. In addition, degradation of starch degradation products and transfer reaction products having a polymerization degree of 2 or more, which is a raw material due to saccharification activity, is suppressed, and it is difficult to cause a decrease in yield even if a saccharide oxidation reaction is performed over a certain period of time. Catalase activity is not required.

The catalase activity in the catalase preparation of the present invention is measured as follows.
Follow the method of titrating the residual hydrogen peroxide after the enzyme reaction with sodium thiosulfate (supervised by Michio Kosaki “Enzyme Use Handbook”, Jinjinshokan 1985 edition, p404-410). That is, 5 ml of a substrate solution obtained by diluting commercially available 30% by weight hydrogen peroxide 800-fold with 50 mM phosphate buffer (pH 7.0) is placed in a container and placed in a constant temperature water bath at 30 ° C. for 15 minutes to obtain a constant temperature. Add 1 ml of the sample enzyme solution kept at 30 ° C., and after 5 minutes, add 2 ml of 0.5N sulfuric acid and shake well to stop the enzyme action. To this was added 1 ml of 10 wt% potassium iodide solution, 1 drop of 1% ammonium molybdate and 5 drops of 0.5% starch reagent as an indicator, and while stirring this solution, 0.005N sodium thiosulfate solution (for determination) In the blank, 1 ml of water is added instead of the sample, the amount of hydrogen peroxide decomposed by the catalase action is calculated by subtracting the value of the sample from the blank value, and the catalase activity of the sample enzyme solution from the standard curve Ask for. In addition, 1U has shown the activity which decomposes | disassembles 1 micromol hydrogen peroxide in 1 minute.
Catalase activity (U / ml) = A × n
n: Dilution ratio A: Obtain the value A on the x-axis of y = (T ₀ −T _S ) × 24.18 / T ₀ × 2.5 × f from the standard curve graph. f: 0.005N sodium thiosulfate factor T _0: blank titration value (ml)
T _S : Titration value of sample (ml)
24.18 / T ₀ : Correction value for change in activity measurement due to initial substrate concentration 2.5: 1 ml of 0.005N sodium thiosulfate solution corresponds to 2.5 μmol of hydrogen peroxide

  The saccharification activity defined in the present invention is a force that hydrolyzes the starch degradation product by glucoamylase activity and α-glucosidase activity to release glucose, and the saccharification activity of the present invention is the substrate 4-nitrophenyl β-maltoside. From (G2-β-PNP), the activity of releasing 1 μmol of PNP per minute can be defined as 1U.

  The saccharification activity in the catalase preparation is obtained by reacting the catalase preparation with 4-nitrophenyl β-maltoside to produce 4-nitrophenyl β-glucoside, which is decomposed by β-glucosidase to produce 4-nitrophenol, It is measured by quantifying 4-nitrophenol. Specifically, the saccharification activity in the catalase preparation is measured using a saccharification power measurement kit or a saccharification power fractionation determination kit manufactured by Kikkoman Corporation.

(Measurement of saccharification activity using a saccharification test kit manufactured by Kikkoman)
When using a kit for measuring saccharification power manufactured by Kikkoman, 0.5 ml of a substrate solution containing 4-nitrophenyl β-maltoside is mixed with 0.5 ml of an enzyme solution containing β-glucosidase and preliminarily added at 37 ° C. for 5 minutes. After performing the temperature, 0.1 ml of a measurement sample is added, mixed, and reacted at 37 ° C. for 10 minutes. To stop the reaction, 2 ml of enzyme stop solution containing sodium carbonate is added and mixed. The saccharification power is measured by quantifying the liquid after the reaction at a wavelength of 400 nm, and the activity is calculated from the following formula.
Saccharification activity (U / ml) = (Es−Eb) × 0.171 × n
Es: Absorbance of measurement sample Eb: Absorbance of blank n: Dilution ratio of enzyme solution

(Sugar oxidase preparation)
The saccharide oxidase preparation referred to in the present invention means a substance that oxidizes a saccharide having a glucose residue at the reducing end and having a polymerization degree of 2 or more to generate hydrogen peroxide as a by-product. Examples include saccharide oxidase preparations derived from microorganisms belonging to the genus Microdocium, saccharide oxidase preparations derived from microorganisms belonging to the genus Acremonium, and specifically, saccharide oxidases derived from Acremonium chrysogenum and the like.

  In producing a sugar carboxylic acid such as maltobionic acid, the sugar oxidase is preferably from 1 u / g to 30 u / g, more preferably from 2 u / g to 20 u / g, based on the amount of reducing sugar (wt%) in the raw material substrate. Act at g or less. In the present invention, since the decomposition of hydrogen peroxide by the catalase preparation is sufficiently performed, the carbohydrate oxidation reaction can be performed at a sufficient rate regardless of the increase of hydrogen peroxide produced as a by-product. In addition, degradation of starch degradation products and transfer reaction products having a polymerization degree of 2 or more, which is a raw material due to saccharification activity, is suppressed, and it is difficult to cause a decrease in yield even if a saccharide oxidation reaction is performed over a certain period of time. Does not require the amount of carbohydrate oxidase.

The enzyme activity of the carbohydrate oxidase of the present invention is measured as follows.
0.1 ml monopotassium phosphate-sodium hydroxide buffer solution (pH 7.0) containing 0.15% (w / v) phenol and 0.15% (w / v) Triton X-100, 10% maltose Mix 0.5 ml of hydrate solution, 0.5 ml of 25 U / ml peroxidase solution, and 0.1 ml of 0.4% (w / v) 4-aminoantipyrine solution, and incubate at 37 ° C. for 10 minutes. .1 ml was added to start the reaction. The carbohydrate oxidation activity was measured by measuring the increase in absorbance at a wavelength of 500 nm as the enzyme reaction progressed. For Bunrank, 0.1M phosphate buffer (pH 7.0) was used, and the amount of enzyme required to oxidize 1 μmol of maltose monohydrate per minute was defined as 1 unit. Calculate activity.
Maltose oxidation activity (U / ml)
= {(A5-A2)-(Ab5-Ab2)} * 2.218 * n
A2, A5: Absorbance after 2 minutes and 5 minutes after starting the reaction (sample)
Ab2, Ab5: Absorbance 2 minutes and 5 minutes after the start of the reaction (blank)
n: dilution rate of enzyme solution

(Raw sugar)
The carbohydrate used as a raw material in the present invention is a starch degradation product or transfer reaction product having a glucose residue at the reducing end and having a polymerization degree of 2 or more, and maltose, isomaltose, maltotriose, isomaltotriose, maltotetraose. , Maltohexaose, panose, malto-oligosaccharide, isomalto-oligosaccharide, starch syrup, powder cake, dextrin, branched dextrin, isomaltodextrin and the like.

  The concentration of the raw sugar during the production of the sugar carboxylic acid is preferably 10 to 50 (wt)%, more preferably 20 to 40 (wt)% in consideration of the concentration in the purification step. In the present specification, “(wt)%” means the content (mass) of the target component, and here, the content of the carbohydrate in the liquid.

(Reaction temperature and reaction pH)
The reaction temperature in the reaction step between the carbohydrate oxidase and catalase is preferably, for example, about 20 to 60 ° C, and more preferably 25 to 40 ° C.

  The reaction pH is preferably about 4 to 10 and more preferably in the range of pH 5 to 8. Moreover, since reaction pH falls in the process in which sugar carboxylic acid produces | generates, it is necessary to add and adjust a neutralizing agent to a reaction liquid. Examples of the neutralizing agent include sodium hydroxide, potassium hydroxide, calcium carbonate, magnesium carbonate, sodium carbonate, sodium hydrogen carbonate, calcium oxide and the like.

  Moreover, since oxygen is required in the oxidation reaction of the present invention, it is preferable to maintain the amount of dissolved oxygen in the reaction solution by aeration and stirring of air or oxygen.

(Sugar carboxylic acid)
The sugar carboxylic acid prepared by using the method of the present invention is a starch decomposition product having a polymerization degree of 2 or more, preferably 4 or more, or an aldehyde group on the reducing end side of the transfer reaction product is oxidized. There is no particular limitation. The degree of polymerization of the starch decomposition product or transfer reaction product may be, for example, 2 to 100, preferably 4 to 100 or the like. More specifically, the sugar carboxylic acid is more specifically maltodextrin oxide, powdered oxide, starch oxalate, maltohexanoic acid, maltotetraonic acid, maltotriionic acid, maltobionic acid, isomalt. Examples thereof include dextrin oxide, panose oxide, isomaltorionic acid, isomaltionic acid, nigerovionic acid, and cordibionic acid. Among these, the sugar carboxylic acid may be a free acid, a lactone, or a salt thereof.

  Although it does not specifically limit as salt of sugar carboxylic acid, Calcium salt, magnesium salt, potassium salt, sodium salt, zinc salt, iron salt, copper salt etc. are mentioned.

  The oxidation reaction to the sugar carboxylic acid can be confirmed from the decrease in the amount of reducing sugar, and for example, a colorimetric determination method by the Nelson-Somogi method can be used.

  Moreover, it can also confirm by analyzing raw material saccharide | sugar and sugar carboxylic acid by HPLC. For example, after an oxidation reaction using maltose as a raw material, elution: 35 ° C., 1.0 ml / min, sodium hydroxide concentration: 100 mM, acetic acid by HPAED-PAD method (pulsed amperometry detector, CarboPac PA1 column) Sodium concentration: Maltose and maltobionic acid can be quantified by measurement under conditions of 0 min-0 mM, 2 min-0 mM, and 20 min-20 mM.

  The sugar carboxylic acid prepared using the method of the present invention can be used for foods and drinks, cosmetics, pharmaceuticals, chemical products and the like.

(Test Example 1) Effect of catalase preparation A on maltobionic acid production 9 g of maltose (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in distilled water in an Erlenmeyer flask with a baffle to make 30 g (30 wt%), and then calcium carbonate (Wako Pure) 1.5 g of sugar oxidase preparation (sugar oxidase activity 300 u / ml) from Acremonium chrysogenum (36 u, 4 u / g substrate), catalase preparation A (catalase activity 75060 u / ml) derived from the genus Aspergillus, Saccharification activity 24.8u / ml, Saccharification activity / catalase activity ratio = 0.00033) Add 0-4500U (0-500u / g substrate), shake reaction at 30 ° C, 200rpm and effect on production efficiency of maltobionic acid Evaluated.

  The amount of maltobionic acid in the reaction composition was determined by HPAED-PAD method (pulsed amperometry detector, CarboPac PA1 column), elution: 35 ° C., 1.0 ml / min, sodium hydroxide concentration: 100 mM, sodium acetate concentration: 0. Analysis was performed under conditions of min-0 mM, 2 min-0 mM, and 20 min-20 mM.

  As a result of the evaluation, when no catalase was added in Comparative Example 1, the conversion rate to maltobionic acid remained at 25%. On the other hand, in Examples 1 to 6 using a predetermined catalase preparation in an appropriate amount, 90% or more could be converted to maltobionic acid.

(Test Example 2) Effect on Catalyst Formulation A on Dextrin Oxide Production After 9 g of dextrin (trade name NSD700, manufactured by Sanei Saccharification Co., Ltd.) was dissolved in distilled water in an Erlenmeyer flask with baffle to make 30 g (30 wt%), 0.75 g of calcium carbonate (manufactured by Wako Pure Chemical Industries), 60 μl (18 u, 2 u / g substrate) of saccharide oxidase preparation (carbohydrate oxidative activity 300 u / ml) derived from Acremonium chrysogenum, and catalase preparation A (catalase activity derived from Aspergillus genus) 75060 u / ml, saccharification activity 24.8 u / ml, saccharification activity / catalase activity ratio = 0.00033) 0-9000 U (0-300 u / g substrate) was added, and the dextrin oxidation efficiency was carried out at 30 ° C. and 200 rpm. The impact on was evaluated. For the oxidation rate, the amount of reducing sugar in the reaction solution was quantified by the Nelson-Somogi method, and the conversion rate was calculated by the following formula.
(Reducing sugar amount before reaction start-reducing sugar amount in reaction solution) / Reducing sugar amount before reaction start × 100 = oxidation rate (%)

  As a result of the evaluation, when no catalase of Comparative Example 2 was added, the carbohydrate oxidase was denatured and inactivated by the amount of hydrogen peroxide produced as a by-product, so that the oxidation rate remained at 34%. In Examples 7 to 11 using a predetermined catalase preparation in an appropriate amount, 90% or more could be oxidized.

(Test Example 3) Effect of catalase preparation B on dextrin oxide production In a conical flask with a baffle, 9 g of dextrin (trade name NSD700, manufactured by Sanei Saccharification Co., Ltd.) was dissolved in distilled water to make 30 g (30 wt%). 0.75 g of calcium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.), sugar oxidase preparation derived from Acremonium chrysogenum (carbohydrate oxidizing activity 300 u / ml) 60 μl (18 u, 2 u / g substrate), and catalase preparation B (catalase activity derived from Aspergillus genus) 27400 u / ml, saccharification activity 244 u / ml, saccharification activity / catalase activity ratio = 0.0089) were added in the amounts shown in Table 3, and the shaking reaction was performed at 30 ° C. and 200 rpm to evaluate the effect on dextrin oxidation efficiency. For the oxidation rate, the amount of reducing sugar in the reaction solution was quantified by the Nelson-Somogi method, and the conversion rate was calculated by the following formula.
(Reducing sugar amount before reaction start-reducing sugar amount in reaction solution) / Reducing sugar amount before reaction start × 100 = oxidation rate (%)

  Further, the sugar composition of the reaction product was determined by HPAED-PAD method (pulsed amperometry detector, CarboPac PA1 column), elution: 35 ° C., 1.0 ml / min, sodium hydroxide concentration: 100 mM, sodium acetate concentration: The analysis was performed under the conditions of 0 min-0 mM, 5 min-0 mM, 55 min-40 mM.

  As a result of the evaluation, even if the saccharification activity is 0.9 u / g or less per reducing sugar of the raw material substrate, Comparative Example 3 in which the saccharification activity / catalase activity ratio is 0.005 or more has an oxidation rate of 80% or less and is in the composition The amount of gluconic acid obtained by oxidation of glucose produced by hydrolysis of 9 is about 9%, which indicates that the dextrin as a raw material is affected by hydrolysis.

  Further, in Comparative Example 4 in which the saccharification activity is 0.9 u / g or more per reducing sugar of the raw material substrate and the saccharification activity / catalase activity ratio is 0.005 or more, the oxidation rate is 70% or less, and It can be seen that the amount of gluconic acid in which glucose produced by hydrolysis is oxidized is about 16%, and the dextrin as a raw material is affected by hydrolysis.

(Test Example 4) Effect of catalase preparation C on dextrin oxide production In a conical flask with a baffle, 9 g of dextrin (trade name NSD700, manufactured by Sanei Saccharification Co., Ltd.) was dissolved in distilled water to make 30 g (30 wt%). Calcium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.) 0.75 g, Acrenium chrysogenum-derived carbohydrate oxidase preparation (carbohydrate oxidation activity 300 u / ml), Aspergillus-derived catalase preparation C (catalase activity 52000 u / ml, saccharification activity 64 u / ml) The ratio of saccharification activity / catalase activity = 0.0012) was added in the amount shown in Table 4, and a shaking reaction was performed at 30 ° C and 200 rpm to evaluate the effect on dextrin oxidation efficiency. For the oxidation rate, the amount of reducing sugar in the reaction solution was quantified by the Nelson-Somogi method, and the conversion rate was calculated by the following formula.
(Reducing sugar amount before reaction start-reducing sugar amount in reaction solution) / Reducing sugar amount before reaction start × 100 = oxidation rate (%)

  Further, the sugar composition of the reaction product was determined by HPAED-PAD method (pulsed amperometry detector, CarboPac PA1 column), elution: 35 ° C., 1.0 ml / min, sodium hydroxide concentration: 100 mM, sodium acetate concentration: The analysis was performed under the conditions of 0 min-0 mM, 5 min-0 mM, 55 min-40 mM.

  As a result of the evaluation, even when the ratio of saccharification activity / catalase activity = 0.005 or less, Comparative Example 5 in which the saccharification activity is 0.9 u / g or more per reducing sugar of the raw material substrate has an oxidation rate of 70% or less and a composition The amount of gluconic acid obtained by oxidizing glucose produced by hydrolysis in the medium is about 19%, which indicates that the dextrin as a raw material is affected by hydrolysis. On the other hand, in Example 12, in which the saccharification activity is 0.9 u / g or less per reducing sugar of the raw material substrate, the oxidation rate is 90% or more and the amount of gluconic acid in the composition is 7% or less, and the molecular weight is greatly reduced. There was no oxidation reaction.

(Test Example 5) Influence of catalase preparation C on maltobionic acid production To a conical flask with a baffle, 30 g of 30 (wt)% hymaltose solution, 0.75 g of calcium carbonate (manufactured by Wako Pure Chemical Industries), saccharide oxidation from Acremonium chrysogenum Table 5 shows enzyme preparation (sugar oxidation activity 300 u / ml) and catalase preparation C derived from the genus Aspergillus (catalase activity 52000 u / ml, saccharification activity 64 u / ml, saccharification activity / catalase activity ratio = 0.0012) And the shaking reaction was carried out at 30 ° C. and 200 rpm to evaluate the influence on the production efficiency of maltobionic acid.

  The amount of maltobionic acid in the reaction composition was determined by HPAED-PAD method (pulsed amperometry detector, CarboPac PA1 column), elution: 35 ° C., 1.0 ml / min, sodium hydroxide concentration: 100 mM, sodium acetate concentration: 0. Analysis was performed under conditions of min-0 mM, 2 min-0 mM, and 20 min-20 mM.

  As a result of evaluation, Comparative Example 6 in which the saccharification activity is 0.9 u / g or more per reducing sugar of the raw material substrate even when the saccharification activity / catalase activity ratio is 0.005 or less is that maltose of the raw material is hydrolyzed, While the conversion amount to maltobionic acid was 80% or less, the saccharification activity was 0.9 u / g or less per reducing sugar of the raw material substrate, and in Example 13, there was almost no influence of hydrolysis by contaminating enzymes. 98% or more was converted to maltobionic acid.

(Test Example 6) Influence on dextrin oxide production by catalase preparation D In an Erlenmeyer flask with baffle, 9 g of dextrin (trade name NSD700, manufactured by Sanei Saccharification Co., Ltd.) was dissolved in distilled water to make 30 g (30 wt%). 0.75 g of calcium carbonate (manufactured by Wako Pure Chemical Industries), sugar oxidase preparation from Acrenium chrysogenum (sugar oxidation activity 300 u / ml) 60 μl (18 u, 2 u / g substrate), and catalase preparation D (catalase activity from Aspergillus genus) 54400 u / ml, saccharification activity 242 u / ml, saccharification activity / catalase activity ratio = 0.0044) were added in the amounts shown in Table 6, and the shaking reaction was performed at 30 ° C. and 200 rpm to evaluate the effect on dextrin oxidation efficiency. For the oxidation rate, the amount of reducing sugar in the reaction solution was quantified by the Nelson-Somogi method, and the conversion rate was calculated by the following formula.
(Reducing sugar amount before reaction start-reducing sugar amount in reaction solution) / Reducing sugar amount before reaction start × 100 = oxidation rate (%)

  Further, the sugar composition of the reaction product was determined by HPAED-PAD method (pulsed amperometry detector, CarboPac PA1 column), elution: 35 ° C., 1.0 ml / min, sodium hydroxide concentration: 100 mM, sodium acetate concentration: The analysis was performed under the conditions of 0 min-0 mM, 5 min-0 mM, and 40 min-40 mM.

  As a result of the evaluation, even when the saccharification activity / catalase activity ratio is 0.005 or less, the saccharification activity is 0.9 u / g or more per reducing sugar of the raw material substrate. In Comparative Example 7, the oxidation rate is 70% or less, and The amount of gluconic acid obtained by oxidizing glucose produced by hydrolysis in the composition is 26%, which indicates that the dextrin as a raw material is affected by hydrolysis. On the other hand, in Example 14 where the saccharification activity is 0.9 u / g or less per reducing sugar of the raw material substrate, the oxidation rate is 90% or more, and the amount of gluconic acid in the composition is 8% or less, which greatly reduces the molecular weight. There was no oxidation reaction.

(Test Example 7)
(Example 15)
In addition to maltose 70.3 wt%, glucose maltose (Bx.75%, containing 1.2 wt% glucose, 15.0 wt% maltotriose and 13.5 wt% malto-oligosaccharide (degree of polymerization 4) or higher) After adding 1320 g of distilled water to 880 g of Sanei Saccharification Co., Ltd. and dissolving to 30% wt, 86 g of calcium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.), sugar oxidase preparation derived from Acremonium chrysogenum (carbohydrate oxidative activity 300 u / ml) ) 4.4 ml (1320u, 2u / g substrate) and catalase preparation A derived from the genus Aspergillus (catalase activity 75060u / ml, saccharification activity 24.8u / ml, saccharification activity / catalase activity ratio = 0.00033) 0.66m (149500 U, 70 u / g substrate) 00rpm, it was subjected to aeration-agitation in the air ventilation 2L / minute. Twelve hours after the start of the reaction, 1.1 ml (330 u, 0.5 u / g substrate) of a carbohydrate oxidase agent and 0.22 ml of a catalase preparation (16500 u, 25 u / g substrate) were additionally added to carry out an oxidation reaction.

For the transition of the oxidation reaction, the amount of reducing sugar in the reaction solution was quantified by the Nelson-Somogi method, and the conversion rate was calculated by the following formula.
(Reducing sugar amount before reaction start-reducing sugar amount in reaction solution) / Reducing sugar amount before reaction start × 100 = oxidation rate (%)

  Further, the sugar composition of the reaction product was determined by HPAED-PAD method (pulsed amperometry detector, CarboPac PA1 column), elution: 35 ° C., 1.0 ml / min, sodium hydroxide concentration: 100 mM, sodium acetate concentration: The analysis was performed under the conditions of 0 min-0 mM, 2 min-0 mM, and 20 min-20 mM.

  Table 7 shows the saccharification activity / catalase activity ratio = 0.005 or less (0.00033) and the saccharification activity is 0.9 u / g or less (0.066) per reducing sugar of the raw material substrate. The oxidation rate at the time from 45 hours to 45 hours is shown. As shown in Table 7, after 45 hours of reaction, the product was 100% oxidized. The product at this time contained calcium 4.1 wt%, maltobionic acid 67.4 wt%, gluconic acid 1.2 wt%, maltotrione It is composed of 14.4 wt% acid and 12.9 wt% malto-oligosaccharide acid (polymerization degree 4) or more, and it was confirmed that there is no influence of hydrolysis or the like due to saccharification activity.

Claims (7)

A method for producing a sugar carboxylic acid in which an aldehyde group on the reducing end side of a starch degradation product or transfer reaction product having a glucose residue at the reducing end of 2 or more is oxidized,
Carbohydrate oxidase that produces hydrogen peroxide as a by-product during oxidization of saccharides has a saccharification activity (B) content ratio (B / A) of 0.00002 to 0.005 in catalase preparation to catalase activity (A). In the presence of a catalase preparation, which comprises the step of acting on a raw material substrate containing the starch degradation product or transfer reaction product,
The catalase formulation, method of its saccharification activity is present in an amount at most 0.9 U / g for the amount of reducing sugars the raw material substrate. A method for producing a sugar carboxylic acid in which an aldehyde group on the reducing end side of a starch degradation product or transfer reaction product having a glucose residue at the reducing end of 2 or more is oxidized,
A saccharide oxidase agent that produces hydrogen peroxide as a by-product during oxidization of saccharides has a saccharification activity (B) content ratio (B / A) to catalase activity (A) in the catalase preparation of 0.005 or less and saccharification Including the step of acting on a raw material substrate containing the starch degradation product or transfer reaction product in the presence of a catalase preparation having an activity (B) of 0.1 u / ml or more,
The catalase formulation, method of its saccharification activity is present in an amount at most 0.9 U / g for the amount of reducing sugars the raw material substrate.   The method according to claim 1 or 2, wherein the sugar carboxylic acid excludes maltobionic acid. The amount of the saccharide oxidase agent, the method of claims 1 to 3, wherein any one for the reducing sugar amount of the raw material in the substrate is less than 1u / g or more 30u / g. Catalase preparations, method of claims 1 4, wherein any one of the catalase activity are present in an amount for the amount of reducing sugar is not more than 40u / g or more 1000 u / g of the raw material substrate. The catalase formulation, method according to claims 1 5 or its saccharification activity is present in an amount at 0.00008u / g or more for the amount of reducing sugars the raw material substrate. The manufacturing method according to any one of claims 1 to 6, wherein a concentration of the starch decomposition product or transfer reaction product in the action step is 10% (w / w) or more.