KR102049573B1 - Method for preparing fructooligosaccharides - Google Patents

Abstract

The present invention relates to a method for producing fructooligosaccharide in high yield by treating an enzyme having high activity on a reaction substrate, and a fructooligosaccharide produced by the method.

Description

Method for producing fructooligosaccharides {Method for preparing fructooligosaccharides}

The present invention relates to a method for producing fructooligosaccharide in high yield by treating an enzyme having high activity, and to a fructooligosaccharide produced thereby.

Fructoligosaccharide is a natural oligosaccharide of 3 or more saccharides, and is a mixture of saccharides in which 1 to 3 fructose are bonded to the fructose residues of sugar, and its constituents are 1-Kestose (GF2), Nystose (GF3). , 1-F Fructofuranosyl nystose. Fructoligosaccharide is contained in vegetables, mushrooms, fruits such as bananas, onions, asparagus, burdock, garlic, honey and chicory root, and is found in Agave vera curz (agave genus) and pork potatoes. Fructooligosaccharide has a sweetness of 60% of sugar, and it dissolves well in water, showing a refreshing sweetness similar to sugar, and has been used as a low-calorie food for a long time because it is difficult to digest and absorb in our body. In addition, since it contains more than 33% dietary fiber, when consumed by our body, it helps to inhibit the growth of intestinal bacteria, bifitus, and the growth of harmful bacteria in the intestine in the lower intestine, and helps to facilitate bowel movements. In addition, fructooligosaccharide has the function of increasing the solubility of calcium in the large intestine and promoting absorption by ingestion.

From the 1980s, fructooligosaccharides began to be produced using enzymes obtained from microorganisms. Β-Fructofuranosidase, an enzyme that produces fructo-oligosaccharide from sugar, is an enzyme that produces fructooligosaccharides produced by microorganisms. Toss is drawn and finally 1-F fructofuranosyl nystus is produced. This enzyme can be isolated from bacteria or fungi. Reported microorganisms include Aspergillus niger, Aureobasidium pullulans, Athrobacter sp. Penicillium frequentens and others.

An object of the present invention is to provide a method for producing fructooligosaccharide in high yield by treating an enzyme having high activity on a reaction substrate.

Another object of the present invention is to provide a fructooligosaccharide produced by treating an enzyme having a high activity on a reaction substrate.

Another object of the present invention is to provide an Aspergillus Niger strain having high activity per unit protein of an enzyme derived from the strain derived from the wild type.

A novel Aspergillus Niger mutant (non-GMO) derived from the wild type capable of producing a fructo-oligosaccharide converting enzyme having an excellent ability to convert sugar into fructooligosaccharide in excellent yield was isolated and identified, and their activity was investigated. The composition of the medium for optimization was confirmed. In addition, the present invention was completed by confirming the optimum pH condition or reaction time of the cell reaction having excellent conversion ability from sugar to fructooligosaccharide using the cells, and establishing conditions for efficiently mass-producing fructooligosaccharide.

Hereinafter, the present invention will be described in more detail.

An example of the present invention is to produce fructooligosaccharide in a high yield by treating at least one selected from the group consisting of an enzyme with high activity, a bacterial cell of a strain producing the enzyme, a culture, a lysate, and a supernatant of a lysate. A method and a fructooligosaccharide produced by the method are provided.

According to the production invention of the present invention, fructooligosaccharides can be prepared in high yield, and the fructooligosaccharides are 1-kesotse (GF2), nystose (GF3), and 1-F-fructose. It may contain at least one selected from the group consisting of furanosyl nystose (1-F-Fructofuranosyl nystose, GF4).

In a preferred embodiment, when the fructooligosaccharide of the present invention is reacted with a fructooligosaccharide converting enzyme at a temperature of 60°C for 30 hours, 1-kestose is 10 to 60% by weight based on 100% by weight of the total solid content of fructooligosaccharide. %, preferably 15 to 45% by weight, more preferably 28 to 35% by weight.

In a preferred embodiment, when the fructooligosaccharide of the present invention is reacted with a fructooligosaccharide converting enzyme for 30 hours at a temperature of 60°C, 0 to 50% by weight or nytose based on 100% by weight of the total solid content of fructooligosaccharide It may include 0.01 to 50% by weight, preferably 0 to 35% by weight or 0.01 to 35% by weight.

In a preferred embodiment, when the fructooligosaccharide of the present invention is reacted with a fructooligosaccharide converting enzyme at a temperature of 60° C. for 30 hours, 1-F-fructofuranosyl based on 100% by weight of the total solid content of fructooligosaccharide Nistose may be included in an amount of 0 to 15% by weight or 0.01 to 15% by weight, preferably 0 to 10% by weight or 0.01 to 10% by weight, more preferably 0 to 5% by weight or 0.01 to 5% by weight. .

When the fructooligosaccharide converted by the fructooligosaccharide converting enzyme of the present invention is prepared by reacting with sugar for 30 hours at a temperature of 60°C, the values measured in Area% as measured by HPLC are 1-Kestose and Nistose And 1-F-fructofuranosyl nystos (1-kestose: the total weight of nystos and 1-F-fructofuranosyl nystos) in a content ratio of 0.5 to 2.0, preferably 0.8 to 1.5 weight. It may be characterized in that it is produced to be %.

In the production method of the present invention, sugar may be used as the reaction substrate, and at least one selected from the group consisting of fructose and glucose is additionally generated by using sugar as the reaction substrate, and the reaction product is fructooligosaccharide, fructose, glucose and sugar. It may be one or more selected from the group consisting of.

In a preferred embodiment, when the fructooligosaccharide converting enzyme of the present invention is reacted for 30 hours at a temperature of 60° C. using sugar as a reaction substrate, the fructooligosaccharide is based on 100% by weight of the total solid content of the reaction substrate. 50 to 70% by weight, preferably 55 to 65% by weight, more preferably 60 to 63% by weight can be produced.

In one embodiment, the step of reacting or treating the strain with a substrate may be performed by culturing the lysate of the strain, the supernatant of the lysate, or an enzyme derived from the cell in a culture medium containing sugar. In another embodiment, the step of reacting with the substrate is a step of contacting the strain (cell, a culture of the strain, a lysate of the strain and/or a supernatant of the lysate) with a substrate, e.g., mixing the strain with a substrate. Alternatively, it may be carried out by contacting the substrate with the carrier on which the strain is immobilized. By reacting the strain with the substrate as described above, a substrate, for example, sugar can be converted into fructooligosaccharide to produce fructooligosaccharide.

In the fructooligosaccharide production method, for efficient fructooligosaccharide production, the concentration of sugar used as a substrate may be 30 to 90, preferably 40 to 80% (w/v) based on the total reactant. The sugar may be used in the form of a buffer solution or a solution dissolved in water (eg, distilled water).

The enzyme used in the production method of the present invention may be a fructooligosaccharide producing enzyme, for example, beta-fructofuranosidase, and may be produced from Aspergillus Niger strain.

The Aspergillus strain is preferably Aspergillus niger (Aspergillus niger) strain, has a strong starch saccharification power, and has the ability to produce a large amount of citric acid, gluconic acid, etc. from sugar, which is used in the organic acid fermentation industry. It is known in the art that the strain of the present invention is a random mutation, for example, NTG (N-methyl-N-nitroso-guanidin) or UV irradiation with wild type Aspergillus Niger isolated from fermented food. It may be a mutant strain (non-GMO) that has undergone a first or more mutation.

Preferably, the strain of the present invention may be a strain of accession number KCTC13140BP ( Aspergillus niger SYG-Neo1) deposited with KCTC as of 2016.10.28.

The term used in the specification of the present invention, strain activity is the strain activity calculated by the following equation (1), the amount of 1-kestose produced for 1 minute per unit volume of the reaction solution including the supernatant and the substrate of the strain culture (mM ) Means the activity of the strain measured by the amount of enzyme capable of producing.

[Equation 1]

For example, the Aspergillus Niger mutant strain used in the production method of the present invention is a wild-type Aspergillus Niger strain subjected to random mutations at least two times, and the strain activity calculated by Equation 1 above is 150 to It may be 600 U/ml, preferably 200 to 400 U/ml. The strain activity is characterized in that the relative strain activity is 150 to 800%, preferably 300 to 650%, based on 100% of the wild-type Aspergillus Niger strain activity.

In the case of genetically modified strains, that is, GMOs, consumers are very reluctant to purchase when used in food, and there is a disadvantage that may be harmful in terms of environmental ecology due to genetic manipulation. In addition, in the case of using an enzyme of a genetically modified microorganism (GMO), there is an advantage in that it is possible to produce fructooligosaccharide with high efficiency, but there is a disadvantage that a lot of cost and time are consumed in verifying safety for human consumption. On the other hand, in the case of using a safety-proven natural strain (Non-GMO strain with edible experience), the ability to convert high-produced fructooligosaccharides by improving the natural strain (Non-GMO strain with edible experience) in order to save the above troubles. It is important to construct a mutation with

The fermented food may be, for example, one or more selected from the group consisting of miso, cheonggukjang, nuruk, red pepper paste, natto, and meju, but is not limited thereto.

The strain of the genus Aspergillus of the present invention is characterized in that it can produce a fructooligosaccharide converting enzyme capable of converting sugar to fructooligosaccharide at a particularly high titer in high yield. The strain may include a nucleic acid sequence encoding a fructofuranosidase enzyme and/or a regulatory sequence that is operably linked to an upstream thereof to regulate the expression of the fructofuranosidase enzyme in the strain. have. Preferably, the strain of the present invention may have the 18S rRNA sequence of SEQ ID NO: 5.

The enzyme having a high fructooligosaccharide conversion ability of the present invention may be at least one selected from the group consisting of the bacterial body of the strain, the culture of the strain, the lysate of the strain, and the supernatant of the lysate. Preferably, the enzyme may be a fructofuranosidase enzyme, preferably a beta-fructofuranosidase enzyme, contained in the supernatant of the lysate of the strain. For example, the beta-fructofuracinodase enzyme of the present invention may have the gene sequence of SEQ ID NO: 7.

The excellent conversion ability of the enzyme prepared by the strain of the present invention allows the production of fructooligosaccharides with excellent conversion ability compared to the conventionally most widely used Meiji strains, as well as a shorter time required for the production of the same amount of fructooligosaccharides. Accordingly, the Aspergillus Niger strain of the present invention or the enzyme produced therefrom is not only a useful substitute for fructooligosaccharide, particularly a conventional strain that can be usefully applied to mass production thereof, but also improves the yield of fructooligosaccharide production. I can.

The enzyme activity used in the specification of the present invention is the fructooligosaccharide conversion activity, and the unit protein calculated by dividing the strain activity calculated by the formula of Equation 2 below by the weight of the protein solid in the reaction solution including the supernatant and the substrate of the strain culture. Sugar is active.

[Equation 2]

For example, the enzyme used in the production method of the present invention has an enzyme activity per unit protein of 300 to 1500 U/mg, preferably 300 to 1400 U/mg, more preferably 300 to 500 U/mg or 1200 to 1400. It can be U/mg. The strain activity is characterized in that the relative enzyme activity is 150 to 1000%, preferably 600 to 1000% or 250 to 400% high, based on 100% of the enzyme activity generated from the wild-type Aspergillus Niger strain.

When preparing fructooligosaccharide with the strain of the present invention, when 1 kg of sugar as a reaction substrate reacts for 30 hours at a concentration of 60 (w/v)%, 0.005 to 0.050 g, preferably 0.007 to 0.03 g, more preferably It may be characterized in that 0.009 to 0.02g of the enzyme reacts.

The enzyme of the present invention has excellent storage stability, and when the enzyme activity measured by Equation 2 is 100% at the time of initial storage, when stored for 7 months at a temperature of 4, 25 or 37°C, 60 to 100%, preferably It was confirmed that the activity was maintained in the range of 70 to 100%, more preferably 75 to 95%, so that long-term storage is very excellent. For example, when stored for 7 months at a temperature of 4° C., it may be maintained at 90 to 100%, preferably 90 to 95%.

The published standards for export to Japan are for the composition of fructooligosaccharides: monosaccharides (fructose + glucose) 33% or less, sugar 10% ± 1.5, 1-Kestose (GF2) 26% ± 3, Nystose (GF3) 26% ± 3, 1-F-Fructofuranosyl nystose (GF4) 5% ± 3, the total fructooligosaccharide content must be 57% or more, but the domestic standard is a product if the fructooligosaccharide is 55% or more regardless of the sugar composition. The fructooligosaccharide produced from the strain of the present invention may satisfy all of the above specifications.

The temperature at which the fructooligosaccharide converting enzyme of the present invention is reacted with the substrate is 40 to 80°C, preferably 50 to 70°C, and more preferably 55 to 65°C, and the enzyme activity is excellent. In addition, the enzyme exhibits excellent enzyme activity when reacted at a temperature of 60° C. in the range of pH 4 to 8, preferably pH 5 to 7, more preferably pH 5.5 to 6.5.

The method for producing fructooligosaccharide of the present invention may further include a step of aeration during culturing for production of a converting enzyme, and may be performed at 0.1 to 5 vvm, preferably 0.5 to 3 vvm. Due to the aeration, the conversion efficiency of the strain to fructooligosaccharide can be further increased.

The fructooligosaccharide converting enzyme of the present invention may be characterized in that it is reacted with the substrate under conditions of 3 to 60 hours, preferably 12 to 48 hours.

Another example of the present invention provides a medium composition for culturing a strain of the genus Aspergillus having a property of converting a reaction substrate into fructooligosaccharide or a composition for producing fructooligosaccharide comprising the strain.

The matters related to the Aspergillus genus strain or the fructooligosaccharide converting enzyme may be equally applied to the Aspergillus genus strain culture medium composition or the composition for producing fructooligosaccharide.

The strain applied to the medium composition or production composition of the present invention has excellent fructooligosaccharide conversion ability, and the fructooligosaccharide conversion ability is an enzyme that the Aspergillus Niger strain converts sugar into fructooligosaccharide, It is preferably obtained by producing a beta-fructofuranosidase enzyme, and the composition may include a medium composition in which fructo-oligosaccharide converting enzyme activity is optimized.

Accordingly, the medium composition of the present invention may have a C/N ratio of 1:3 to 1:10, preferably in a weight ratio of 1:3 to 1:7.

As another example of the present invention, as a wild type-derived Aspergillus Niger strain, a strain having high activity per unit protein of an enzyme produced from the strain or a fructooligosaccharide converting enzyme produced by the strain is provided. do.

The matters regarding the method for producing fructooligosaccharide may be equally applied to the strain and enzyme.

As an example of the present invention, there is provided a functional food containing fructooligosaccharide produced by treating an enzyme having a high activity on a reaction substrate.

The matters regarding the method for producing fructooligosaccharide may be equally applied to the functional food.

The Aspergillus Niger strain of the present invention can produce fructooligosaccharides having an activity of converting fructooligosaccharides from sugar in a high yield even on a large scale, and can produce fructooligosaccharides in a high yield compared to strains that are widely used in the past. It is expected to be widely used in the health food and pharmaceutical industries related to functional sugars, as it can not only manufacture, but also produce enzymes with high storage stability.

1 is a contour diagram analysis result of Aspergillus Niger strain of the present invention.
2 is a result of analysis by the reaction optimization tool of Aspergillus Niger strain of the present invention.
3 is a result of comparing the activity change as a result of culturing the parent strain Aspergillus Niger and strains 1 and 2 of the present invention in a 5L fermentation plant.
4 shows the results of SDS-PAGE of the enzyme protein for each purification step.

The present invention will be described in more detail with reference to the following exemplary examples, but the scope of protection of the present invention is not intended to be limited to the following examples.

Example 1: Isolation of food-derived microorganisms

1.1 Culture and isolation of strains in the sample

To search for microorganisms that produce fructo-oligosaccharide converting enzymes, domestic doenjang, cheonggukjang, yeast, red pepper paste, natto and meju (purchased at department stores, marts and traditional markets: Lotte Department Store in Daegu, Chilseong Market in Daegu, E-mart in Dunsan-dong, Daejeon, Daedeok, Daejeon) Lotte Mart) samples were collected from representative fermented foods. 1 g of the collected food sample was suspended in 10 mL of 0.85% NaCl, and 100 ul of the suspension was spread on Wallerstein Laboratory Nutrient Agar (WLNA), Potato Dextrose Agar (PDA), Inulin Agar (IA) medium, and cultured at 28° C. for 3 days. Among the colonies grown in solid medium, those of different shapes and sizes were sorted, separated, and cultured with shaking at a temperature of 30°C for 5 days in the production medium. The composition of each medium is shown in Table 1 below.

Medium ingredient WLNA(g/L) PDA(g/L) IA(g/L) Production medium Yeast extract 4 - One 35 Potato starch - 4 - - Inulin - - 20 - Bactocasitone 5 - - - Sucrose - - - 150 Dextrose 50 20 - - K ₂ HPO ₄ One - One - KCl 0.125 - - - MgSO ₄ -7H ₂ O 0.25 - 0.5 - FeCl ₃ 0.0025 - - - MnSO _{4 -4H 2} O 0.0025 - - - NaNO ₃ - - 3 - Bromocresol green 0.022 - - - Agar 20 20 20 - CMC* - - - 5 pH pH6 - pH5 pH6.5

* CMC: carboxymethylcellulose sodium salt

The cultured cells were centrifuged to take the supernatant, and the supernatant was used as a crude enzyme. Crude enzyme was reacted for 24 hours at 40°C using 100g/L sugar as a substrate, and the reaction was analyzed by TLC (thin layer chromatography). TLC plate was developed three times using TLC silica gel 60 F254 and 85% acetonitrile as an electric solvent, and immersed in a mixed solution of methanol: sulfuric acid: N-(1-Naphthyl_ethylene diamine dihydrochloride = 95: 4.7: 0.3 After performing heat treatment, color development was observed, 10 strains in which sugar was converted to fructooligosaccharide among the 467 strains were screened through TLC results.

The 467 strains screened in the solid medium were 48 from yeast sample, 68 from Cheonggukjang sample, 13 from red pepper paste sample, 138 from soybean paste sample, 19 from Meju powder sample, 46 from Meju sample, and natto sample. The distribution was found to be 4 in the sample, 8 in the makgeolli sample, and 123 in the other.

1.2 Selection of parent strain

The 10 strains selected in Example 1.1 were inoculated in a liquid medium containing 15% sugar, 3.5% yeast extract and 0.5% carboxymethylcellulose-CMC (pH6.5) at a temperature of 30°C. It was cultured with shaking for 5 days. The culture medium was centrifuged to separate the supernatant from the cells, and then only the supernatant was taken and used as a Crude enzyme. Crude enzyme was reacted for 30 minutes at a temperature of 40° C. using 100 g/L sugar as a substrate, and the activity of the fructooligosaccharide-generating enzyme was compared through HPLC RI analysis. Agilent HPLC was used, and specific analysis conditions are as follows.

[HPLC R1 analysis conditions]

Column: NH2-P 50 4E (shodex) column

Mobile phase solvent: 70% acetonitrile

Analysis temperature: 30℃

Flow rate: 1ml/min

Injection volume: 10l

Detector: RI detector

Identification was also carried out for 10 isolated strains for which the activity was compared. PCR was performed to identify the strain, and after PCR was performed using universal primers NL1 and NL4, nucleotide sequence analysis was performed. After nucleotide sequence analysis, the strain was identified by referring to the NCBI database.

Table 2 shows the results identified by analyzing the strain. The identified strains were Pichia farinose, Yarrowia lipolytica, Millerozyma farinose, Aspergillus oryzae, and Aspergillus Niger, and the nucleotide sequences of their 18S ribosomal RNA are sequences of SEQ ID NOs: 1 to 5. Among them, Aspergillus Niger, which has the best activity, was selected as the final excellent isolated strain, and a later experiment was conducted on this.

Identified strain name 18S rRNA sequence number Type of food Count Strain activity (U/mL) pichia farinose SEQ ID NO: 1 Miso One 3.3 Yarrowia liplytica SEQ ID NO: 2 Cheonggukjang One 1.71 Milerozyma farinose SEQ ID NO: 3 Cheonggukjang One 2.47 Aspergillus oryzae SEQ ID NO: 4 Meju 6 20-25 Aspergillus niger SEQ ID NO: 5 Meju One 30 sum 10

Example 2. Selection of high-producing primary mutant strains of fructooligosaccharide

2.1 Selection of primary mutant strains

The parent strain isolated in Example 1 was plated on Potato dextrose Agar (PDA) solid material, and then incubated for 3 days at a temperature of 28°C. Only the spores generated in the solid medium were collected to prepare and store glycerol stock. To use the prepared Glycerol stock for mutation, glycerol was removed and washed three times. Mutation was performed using the washed spores, and the mutant was used with NTG (N-methyl-N-nitroso-guanidin) and UV 254nm. 1 ml of an NTG solution dissolved in 2 mg/ml was mixed with the washed spores and left at room temperature for 10 minutes. After removing the NTG solution through the centrifugation process, the washing process was performed three times. After adding 1 ml of sterilized water to the washed spores, they were suspended, diluted appropriately, and spread on a solid medium. The solid medium composition is shown in Table 3 below.

Medium ingredients g/L Glycerol 30 2-Deoxyglucose 1, 5 MgSO ₄ 7H ₂ O One Sodium glutamate 2 KCl 0.5 K ₂ HPO ₄ One FeSO ₄ 7H ₂ O 0.01 Agar 20 pH pH 6

Using 2-deoxyglucose as a maker, it was spread on a solid medium containing 2-deoxyglucose and irradiated at UV 254 nm for 1 minute. Colonies selected through mutation were cultured in liquid (production medium in Table 1) to measure activity. 100g/L of sugar was reacted at a reaction temperature of 40℃ for 30 minutes at pH (40 mM McIlvain buffer, pH5), then treated with boiling water for 10 minutes to inactivate the enzyme to stop the reaction, and then activate the converted value through HPLC analysis. Was measured. At this time, the primary mutant strain having excellent conversion activity was selected.

2.2 Optimal C/N ratio setting

An experiment was conducted to set the optimal C/N ratio using the first mutant strain selected in Example 2.1. Using the experimental design method, after incubation for 5 days in the range of 150 to 350 g/L for sugar and 30 to 60 g/L for yeast extract, the activity results were analyzed using Minitab's response optimization tool. The medium composition was used by removing CMC from the existing production medium and adding 100 mM MES.

The results of the contour diagram analysis are shown in FIG. 1, and when the contour diagram is confirmed, it was confirmed that the activity tends to increase as the concentration of sugar and yeast extract increases. In addition, the results of comparing the activity of each medium condition are shown in Table 4, and the results of analyzing the reaction optimization tool are shown in FIG. 2. Sugar was 250 g/L and yeast extract showed maximum activity when it was 53 g/L (CMC 100 mM). Therefore, the medium C/N ratio capable of increasing the activity of the fructooligosaccharide strain was confirmed to be 4.7. The strain activity was calculated by the following equation.

[Equation 1]

Strain (test area) Sugar (g/L) Yeast extract (g/L) C/N ratio Strain activity (U/ml) Primary mutant stock (Daejo-gu) 150 35 4.3 81 Primary variant stock (optimal condition) 250 53 4.7 140

Example 3. Fructooligosaccharide Selection of high-producing secondary mutant strains

In addition to using the first mutant strain of Example 2.1 as a parent strain, after culturing in the same manner as in Example 2.1, a total of 1522 through mutations using NTG (N-methyl-N-nitroso-guanidin) and UV 254 nm as mutants. The fructooligosaccharide conversion activity of the mutant strains of dogs was tested, and the secondary mutant strain having the most activity was selected.

The strain was deposited with KCTC on October 28, 2016 and was given the accession number KCTC13140BP.

Example 4. Evaluation of activity of strains producing high fructooligosaccharides

Colonies selected through mutations in Examples 2 and 3 were measured for activity in liquid culture (production medium in Table 1). When measuring activity, if more than GF3 is produced, it is difficult to quantify GF2, so the enzyme must be diluted and used so that only GF2 is produced. 100 g/L of sugar was reacted at a reaction temperature of 40°C for 30 minutes in pH 5 (40 mM McIlvain buffer), and then treated with boiling water for 10 minutes to inactivate the enzyme to stop the reaction. The reaction solution in which the enzyme was deactivated was diluted twice to perform HPLC analysis, and the activity was measured using the converted value. .

The strain activity was expressed by Equation 1 below, and the enzyme activity was expressed by the strain activity per unit protein derived by Equation 2 below.

[Equation 1]

[Equation 2]

The strain activity measurement results and the enzyme activity measurement results expressed as strain activity per unit protein are shown in Table 5 below.

Strain Strain activity (U/ml) Protein (mg/ml) Enzyme activity (U/mg) Parent strain 35 15 47 1st variant 81 25 65 2nd variant 221 43 409

Example 5. Cultivation of strains by fermentation tank

The second mutant strain of Example 3 was cultured in a 5L fermenter with a medium composition having a C/N ratio of 4.7. The culture conditions were maintained at a pH of 5.5 to 6.5 during cultivation using ammonia water, aerated at 1 vvm, and incubated for 144 hours at a temperature of 30° C. and rpm 300 to 650. The seed culture was inoculated to be 3 to 5% of the fermentation tank culture. Sampling during culture was performed to analyze the activity and protein pattern.

After culturing the secondary mutant strain in a 5L fermenter, a supernatant was obtained, and the fructooligosaccharide conversion activity and strain activity over time were calculated in the same manner as in Example 4 and shown in Fig. 3, and the fructooligosaccharide conversion activity and The protein concentration in the culture supernatant is shown in Table 6 below. As can be seen in Table 6, it was confirmed that the fructooligosaccharide-producing enzyme was produced in the highest yield by confirming that the production activity of the fructooligosaccharide converting enzyme was high and the protein content was high in the secondary mutant strain. As a comparative example, fructosylfuranosidase (Meiji) from Meiji was used, and the mixture was mixed with the substrate in the same manner as described above, and the reaction was carried out for comparison.

Strain Strain activity (U/ml) Protein (mg/ml) Activity per unit protein (U/mg) Parent strain 150 30 400 1st variant 315 50 504 2nd variant 455 70 1300 Comparative example - 740

From the above results, when the reactant sugar is reacted for 30 hours at a concentration of 1 kg of 60 (w/v)% during the production reaction of fructooligosaccharide, the secondary mutant is 0.012 g of enzyme, and the comparative strain is 0.008 g of enzyme. Was found to be added.

Example 6. Isolation and purification of fructooligosaccharide-producing enzyme

In Example 5, in order to obtain enzymes from the culture broth of mutant strain 2 cultured in a 5L fermenter, micro filteration (MF) and ultra filteration (UF) were first performed. First, centrifugation was performed to remove the cells, and the supernatant obtained after the centrifugation was filtered under reduced pressure using 10 μm filter paper, and the filtrate passed through the filter was recovered. The filtrate from which the cells were removed was passed through a 30 KDa semipermeable membrane to recover a material larger than the semipermeable membrane size including the fructooligosaccharide producing enzyme, and dialysis was performed with a 20 mM sodium phosphate buffer (pH 7.2) solution to adjust the pH of the solution. The buffer was exchanged until it reached 6.8 ~ 7.2. The UF treatment solution was centrifuged to recover the supernatant, and FPLC purification was performed. Q Sepharose FF was used as the resin, 20 mM Sodium phosphate (pH7.2) was used as buffer A, and a mixture of buffer A and 1 M NaCl (pH7.2) was used as buffer B. After filling the resin, buffer A of about 2 to 3 times the volume of the resin was flowed at a flow rate of 10 ml/min to sufficiently stabilize. When the resin was sufficiently stabilized, the supernatant treated with impurities after UF treatment was loaded at a flow rate of 10 ml/min, the eluate was detected with UV 260 nm, and the eluted fraction was recovered and the enzyme activity was measured. When purifying the fructooligosaccharide-producing enzyme from the culture supernatant, it was confirmed that the low molecular weight except for the target protein was removed step by step and showed a pattern almost similar to the enzyme band of Meiji Co., which is currently used commercially.

In addition, the entire sequence (complete cds) of beta-fructofuranosidase, an enzyme that produces fructooligosaccharides of the fructo-oligosaccharide-producing enzyme, was analyzed to show the sequence of SEQ ID NO: 7 (second variant), and the parent strain As a result of comparing the sequence of (wild type) (SEQ ID NO: 6) with the sequence of the secondary mutant, it was confirmed that 1376 bp in the gene sequence of the parent strain was changed from G to C.

Example 7. Enzyme long-term storage stability evaluation

In order to evaluate the long-term storage stability of the fructooligosaccharide-producing enzyme isolated in Example 6, the enzyme activity was measured at 7 months at temperatures of 4, 25, and 37°C. Enzyme activity was calculated in the same manner as in Example 4.

As a result, it was confirmed that the activity was maintained at 92% at a temperature of 4°C, so that long-term storage stability was very excellent, and the storage stability was very excellent by maintaining the activity at 80% and 79%, respectively, even at a temperature of 25°C or 37°C. Was confirmed.

Example 8. Production of fructooligosaccharide using fructooligosaccharide production enzyme

Using the fructooligosaccharide producing enzyme isolated in Example 6, a reaction with sugar was carried out to confirm whether the production of fructooligosaccharide was in accordance with the standard. The published standards for export to Japan are for the composition of fructooligosaccharides: monosaccharides (fructose + glucose) 33% or less, sugar 10% ± 1.5, 1-Kestose (GF2) 26% ± 3, Nystose (GF3) 26% ± 3, 1-F-Fructofuranosyl nystose (GF4) 5% ± 3, the total fructooligosaccharide content must be 57% or more, but the domestic standard is a product if the fructooligosaccharide is 55% or more regardless of the sugar composition. In order to check whether the sugar composition meets the standards, 1.5 Kg of sugar and 1 Kg of distilled water were mixed to prepare a substrate having a final concentration of 60%.

When necessary, the pH of the substrate was adjusted by adding HCl or NaOH so that the pH was about 6.2 to 6.5. However, there is no pH adjustment during the reaction. Purified enzyme was added to the prepared substrate, and the reaction was carried out at a temperature of 60°C. The initial reaction was stirred at 200 rpm for 1 hour to mix the enzyme and the substrate, after which the reaction was allowed to stand still. The reaction product was analyzed by HPLC RI, and the production rate of each sugar composition was evaluated using the Area% value. As a comparative example, fructosylfuranosidase (Meiji) from Meiji was used, and the mixture was mixed with the substrate in the same manner as described above, and the reaction was carried out for comparison.

In the case of using the fructooligosaccharide-producing enzyme isolated in Example 6, when the reaction was performed for 15 to 30 hours, the fructooligosaccharide was 55% or more. In particular, when the reaction was performed for 22 hours, the fructooligosaccharide content was 61.39%. It was confirmed that it was high. The sugar composition generated during the reaction for 30 hours is shown in Table 7 below together with the comparative example results.

Kinds Fru Glu Suc GF2 GF3 GF4 FOS GF2/(GF3+GF4) Example 7 1.28% 26.88% 10.83% 31.66% 26.04% 3.31% 61.01% 1.1 Comparative example 2.35% 27.80% 10.74% 25.86% 26.72
% 6.53% 59.11% 0.78

As can be seen in Table 7 above, fructooligosaccharides can be produced at a higher level than the comparative example strains that are currently most actively used commercially, and specifically, among fructooligosaccharides, the production rate of GF2, that is, 1-kestose, is higher. In addition, it was confirmed that it conforms to Japanese export standards.

Example 9. Production of fructooligosaccharide according to reaction conditions

9.1 Evaluation of activity according to pH conditions

When converting sugar to fructooligosaccharide using the fructooligosaccharide-producing enzyme isolated in Example 6, enzyme activity according to changing the pH to pH 6, pH 7, pH 8 and pH 8.5 at a reaction temperature of 60°C Was measured and examined after 0, 1, 2, 3, and 4 hours. The enzyme activity was calculated in the same manner as in Example 4, and the value converted to a relative activity value by setting the value at the time of reaction to 100% is shown in Table 8 below.

pH Before reaction 1 hours 2 hours 3 hours 4 hours 6 100% 37.76% 22.28% 4.18% 3.62% 7 100% 18.54% 3.87% 0.64% 0.49% 8 100% 5.35% 0.61% 0.37% 0.27% 8.5 100% 1.34% 0.36% 0.15% 0.12%

As described above, it was confirmed that the enzyme activity is the best in the condition of pH 6 and that the activity can be maintained for a long time.

9.2 Activity evaluation according to temperature conditions

In the case of converting sugar to fructooligosaccharide using the fructooligosaccharide producing enzyme isolated in Example 6, the enzyme activity was shown to be the most excellent at a reaction temperature of 60, 70, 80 and 90°C under the condition of pH 6 The resulting enzyme activity was measured and examined after 0, 1, 2, 3, and 4 hours. The enzyme activity was calculated in the same manner as in Example 4, and the value converted to a relative activity value by setting the value at the time of reaction to 100% is shown in Table 9 below.

Temperature Before reaction 1 hours 2 hours 3 hours 4 hours 60℃ 100 37.76 22.28 4.18 3.62 70℃ 100 0.26 0.04 0.01 0.00 80℃ 100 0.01 0.00 0.00 0.00 90℃ 100 0.00 0.00 0.00 0.00

As described above, when the temperature was higher than 70°C, most of the enzymes were deactivated at the same time as the reaction started, and it was confirmed that excellent enzyme activity could be maintained for a long time, particularly at a reaction temperature of 60°C.

Name of donated institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC13140BP

Consignment Date: 20161028

<110> SAMYANG CORPORATION <120> Method for preparing fructooligosaccharides <130> DPP20184552KR <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 646 <212> DNA <213> 18s rRNA of pichia farinose <400> 1 gaagattggn gtattcttct aggtatcttg ccagcgctta attgcgcggc tggtgctatt 60 agaagtccat aagttcttac acacaggtgt tttttttgtt tgtgaaaaaa atttactttg 120 gtctggaact agaaatagtt ttgggccaga gggcaactta acttcaattt atattgaatt 180 gtttttaaat ttatttgtca aattattgat attaatcaaa aatcttcaaa actttcaaca 240 acggatctct tggttctcgc atcgatgaag aacgcagcga aatgcgataa gtaatatgaa 300 ttgcagattt tcgtgaatca tcgaatcttt gaacgcacat tgcgcccttt ggtattccaa 360 agggcatgcc tgtttgagcg tcatttctct ctcaaaccgc aaggtttggt gttgagcaat 420 atagatattt cggtatctat ttgcttgaaa tggattggca tgagtattta cagtagataa 480 atgccgtttg actcttcaat gtattaggtc taaccaactc gttgaaacag ttagcggtag 540 tatctgtgta aaagaggctc ggccttacaa caatctacaa agtttgacct caaatcaggt 600 aggaataccc gctgaactta agcatatcaa aagcccggag gaaang 646 <210> 2 <211> 354 <212> DNA <213> 18s rRNA of Yarrowia liplytica <400> 2 ggtttccgta ggtgaacctg cggaaggatc attattgatt ttatctattt ctgtggattt 60 ctggtwtatt acagcgtcat tttatctcaa ttataactat caacaacgga tctcttggct 120 ctcacatcga tgaagaacgc agcgaaccgc gatatttatt gtgacttgca gatgtgaatc 180 atcaatcttt gaacgcacat tgcgcggtat ggcattccgt accgcacgga tggaggagcg 240 tgttcgctct gggatcgcat tgctttcttg aaatggattt tttaaactct caattattac 300 gtcatttcac ctccttcatc cgagattacc cgctgaactt aagcatatca ataa 354 <210> 3 <211> 813 <212> DNA <213> 18s rRNA of Milerozyma farinose <400> 3 atgggaggga aaagaagcca aaactctgat aaagacggca gaaggaaacg acataaggtt 60 tcggggttca tagatcctaa tacaagcgga atatacgcta catgtaatag agggaaagaa 120 aaccaatgcc gcaatgaatt gataaatttc ttcagtgaaa aagcagaaga gtattatggt 180 gatcttgatc tagaaagcga caaggaagat aaacaggaat tgactataga agagcagatt 240 gctgcagagg taggtaacct caaggacaag ggaaagaata aaaaagaaac tttcaagcct 300 attgacctag gatgtgaatg cttgattttc ttcaagactc gaaaacccgt tcagcctgcc 360 gaatttgtgc aaaggatatg ccaggagtgc catgacagca aaagaaagac aactaggtac 420 acgcagaagt tgacgccgat ctccttttcg gtctctccgt ccatcgagga gttgaaaaaa 480 ttggccaaga tagtgcttgg gccccatttt cataaggaag aaggacaaga gccacataaa 540 tttgcgatca atgttaccag acgtaacttt aacaccttgc caaagagcga cattataaaa 600 acggtagctg aatgcgtggg cagggagcac ggccattcag ttgacttaaa ggcattcgat 660 aaattaatac tagttgagtg ctataaaagc aatataggca tgagtgtagt tgaaaattat 720 aaccaactag aacggttcaa cttgcagcaa atctttgaca agaaccaaga gggagccgaa 780 gtcgaagcca agtccgattc taacgtagct tag 813 <210> 4 <211> 544 <212> DNA <213> 18s rRNA of Aspergillus oryzae <400> 4 ggggacctgc ggaaggatca ttaccgagtg tagggttcct agcgagccca acctcccacc 60 cgtgtttact gtaccttagt tgcttcggcg ggcccgccat tcatggccgc cgggggctct 120 cagccccggg cccgcgcccg ccggagacac cacgaactct gtctgatcta gtgaagtctg 180 agttgattgt atcgcaatca gttaaaactt tcaacaatgg atctcttggt tccggcatcg 240 atgaagaacg cagcgaaatg cgataactag tgtgaattgc agaattccgt gaatcatcga 300 gtctttgaac gcacattgcg ccccctggta ttccgggggg catgcctgtc cgagcgtcat 360 tgctgcccat caagcacggc ttgtgtgttg ggtcgtcgtc ccctctccgg gggggacggg 420 ccccaaaggc agcggcggca ccgcgtccga tcctcgagcg tatggggctt tgtcacccgc 480 tctgtaggcc cggccggcgc ttgccgaacg caaatcaatc tttccaggtg acctcgatca 540 gaga 544 <210> 5 <211> 571 <212> DNA <213> 18s rRNA of Aspergillus niger <400> 5 ggtttccgta ggtgaacctg cggaaggatc attaccgagt gctgggtcct tcggggccca 60 acctcccacc cgtgcttacc gtaccctgtt gcttcggcgg gcccgccttc gggcggcctg 120 gggcctgccc ccgggaccgc gcccgccgga gaccccaatg gaacactgtc tgaaagcgtg 180 cagtctgagt cgattgatac caatcagtca aaactttcaa caatggatct cttggttccg 240 gcatcgatga agaacgcagc gaaatgcgat aactaatgtg aattgcagaa ttcagtgaat 300 catcgagtct ttgaacgcac attgcgcccc ctggtattcc ggggggcatg cctgtccgag 360 cgtcatttct cccctccagc cccgctggtt gttgggccgc gcccccccgg gggcgggcct 420 cgagagaaac ggcggcaccg tccggtcctc gagcgtatgg ggctctgtca cccgctctat 480 gggcccggcc ggggcttgcc tcgaccccca atcttctcag attgacctcg gatcaggtag 540 ggatacccgc tgaacttaag catatcaata a 571 <210> 6 <211> 1965 <212> DNA <213> beta-fructofuranosidase from Aspergillus niger wild type <400> 6 atgaagctca ccactaccac cctggcgctc gccaccggcg cagcagcagc agaagcctca 60 taccacctgg acaccacggc cccgccgccg accaacctca gcaccctccc caacaacacc 120 ctcttccacg tgtggcggcc gcgcgcgcac atcctgcccg ccgagggcca gatcggcgac 180 ccctgcgcgc actacaccga cccatccacc ggcctcttcc acgtggggtt cctgcacgac 240 ggggacggca tcgcgggcgc caccacggcc aacctggcca cctacaccga tacctccgat 300 aacgggagct tcctgatcca gccgggcggg aagaacgacc ccgtcgccgt gttcgacggc 360 gccgtcatcc ccgtcggcgt caacaacacc cccaccttac tctacacctc cgtctccttc 420 ctgcccatcc actggtccat cccctacacc cgcggcagcg agacgcagtc gttggccgtc 480 gcgcgcgacg gcggccgccg cttcgacaag ctcgaccagg gccccgtcat cgccgaccac 540 cccttcgccg tcgacgtcac cgccttccgc gatccgtttg tcttccgcag tgccaagttg 600 gatgtgctgc tgtcgttgga tgaggaggtg gcgcggaatg agacggccgt gcagcaggcc 660 gtcgatggct ggaccgagaa gaacgccccc tggtatgtcg cggtctctgg cggggtgcac 720 ggcgtcgggc ccgcgcagtt cctctaccgc cagaacggcg ggaacgcttc cgagttccag 780 tactgggagt acctcgggga gtggtggcag gaggcgacca actccagctg gggcgacgag 840 ggcacctggg ccgggcgctg ggggttcaac ttcgagacgg ggaatgtgct cttcctcacc 900 gaggagggcc atgaccccca gacgggcgag gtgttcgtca ccctcggcac ggaggggtct 960 ggcctgccaa tcgtgccgca ggtctccagt atccacgata tgctgtgggc ggcgggtgag 1020 gtcggggtgg gcagtgagca ggagggtgcc aaggtcgagt tctccccctc catggccggg 1080 tttctggact gggggttcag cgcctacgct gcggcgggca aggtgctgcc ggccagctcg 1140 gcggtgtcga agaccagcgg cgtggaggtg gatcggtatg tctcgttcgt ctggttgacg 1200 ggcgaccagt acgagcaggc ggacgggttc cccacggccc agcaggggtg gacggggtcg 1260 ctgctgctgc cgcgcgagct gaaggtgcag acggtggaga acgtcgtcga caacgagctg 1320 gtgcgcgagg agggcgtgtc gtgggtggtg ggggagtcgg acaaccagac ggccaggctg 1380 cgcacgctgg ggatcacgat cgcccgggag accaaggcgg ccctgctggc caacggctcg 1440 gtgaccgcgg aggaggaccg cacgctgcag acggcggccg tcgtgccgtt cgcgcaatcg 1500 ccgagctcca agttcttcgt gctgacggcc cagctggagt tccccgcgag cgcgcgctcg 1560 tccccgctcc agtccgggtt cgaaatcctg gcgtcggagc tggagcgcac ggccatctac 1620 taccagttca gcaacgagtc gctggtcgtc gaccgcagcc agactagtgc ggcggcgccc 1680 acgaaccccg ggctggatag ctttactgag tccggcaagt tgcggttgtt cgacgtgatc 1740 gagaacggcc aggagcaggt cgagacgttg gatctcactg tcgtcgtgga taacgcggtt 1800 gtcgaggtgt atgccaacgg gcgctttgcg ttgagcacct gggcgagatc gtggtacgac 1860 aactccaccc agatccgctt cttccacaac ggcgagggcg aggtgcagtt caggaatgtc 1920 tccgtgtcgg aggggctcta taacgcctgg ccggagagaa attga 1965 <210> 7 <211> 1965 <212> DNA <213> beta-fructofuranosidase from Aspergillus niger 2nd mutant strain <400> 7 atgaagctca ccactaccac cctggcgctc gccaccggcg cagcagcagc agaagcctca 60 taccacctgg acaccacggc cccgccgccg accaacctca gcaccctccc caacaacacc 120 ctcttccacg tgtggcggcc gcgcgcgcac atcctgcccg ccgagggcca gatcggcgac 180 ccctgcgcgc actacaccga cccatccacc ggcctcttcc acgtggggtt cctgcacgac 240 ggggacggca tcgcgggcgc caccacggcc aacctggcca cctacaccga tacctccgat 300 aacgggagct tcctgatcca gccgggcggg aagaacgacc ccgtcgccgt gttcgacggc 360 gccgtcatcc ccgtcggcgt caacaacacc cccaccttac tctacacctc cgtctccttc 420 ctgcccatcc actggtccat cccctacacc cgcggcagcg agacgcagtc gttggccgtc 480 gcgcgcgacg gcggccgccg cttcgacaag ctcgaccagg gccccgtcat cgccgaccac 540 cccttcgccg tcgacgtcac cgccttccgc gatccgtttg tcttccgcag tgccaagttg 600 gatgtgctgc tgtcgttgga tgaggaggtg gcgcggaatg agacggccgt gcagcaggcc 660 gtcgatggct ggaccgagaa gaacgccccc tggtatgtcg cggtctctgg cggggtgcac 720 ggcgtcgggc ccgcgcagtt cctctaccgc cagaacggcg ggaacgcttc cgagttccag 780 tactgggagt acctcgggga gtggtggcag gaggcgacca actccagctg gggcgacgag 840 ggcacctggg ccgggcgctg ggggttcaac ttcgagacgg ggaatgtgct cttcctcacc 900 gaggagggcc atgaccccca gacgggcgag gtgttcgtca ccctcggcac ggaggggtct 960 ggcctgccaa tcgtgccgca ggtctccagt atccacgata tgctgtgggc ggcgggtgag 1020 gtcggggtgg gcagtgagca ggagggtgcc aaggtcgagt tctccccctc catggccggg 1080 tttctggact gggggttcag cgcctacgct gcggcgggca aggtgctgcc ggccagctcg 1140 gcggtgtcga agaccagcgg cgtggaggtg gatcggtatg tctcgttcgt ctggttgacg 1200 ggcgaccagt acgagcaggc ggacgggttc cccacggccc agcaggggtg gacggggtcg 1260 ctgctgctgc cgcgcgagct gaaggtgcag acggtggaga acgtcgtcga caacgagctg 1320 gtgcgcgagg agggcgtgtc gtgggtggtg ggggagtcgg acaaccagac ggccacgctg 1380 cgcacgctgg ggatcacgat cgcccgggag accaaggcgg ccctgctggc caacggctcg 1440 gtgaccgcgg aggaggaccg cacgctgcag acggcggccg tcgtgccgtt cgcgcaatcg 1500 ccgagctcca agttcttcgt gctgacggcc cagctggagt tccccgcgag cgcgcgctcg 1560 tccccgctcc agtccgggtt cgaaatcctg gcgtcggagc tggagcgcac ggccatctac 1620 taccagttca gcaacgagtc gctggtcgtc gaccgcagcc agactagtgc ggcggcgccc 1680 acgaaccccg ggctggatag ctttactgag tccggcaagt tgcggttgtt cgacgtgatc 1740 gagaacggcc aggagcaggt cgagacgttg gatctcactg tcgtcgtgga taacgcggtt 1800 gtcgaggtgt atgccaacgg gcgctttgcg ttgagcacct gggcgagatc gtggtacgac 1860 aactccaccc agatccgctt cttccacaac ggcgagggcg aggtgcagtt caggaatgtc 1920 tccgtgtcgg aggggctcta taacgcctgg ccggagagaa attga 1965

Claims (11)

A fructooligosaccharide converting enzyme consisting of an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 7, 1 selected from the group consisting of cells, cultures, lysates, and supernatants of lysates of Aspergillus niger producing the enzymes A method for producing fructooligosaccharide, which comprises reacting at least species with sugar as a reaction substrate to obtain a reaction product solution containing fructooligosaccharide.
Reacting with sugar as the reaction substrate to obtain a reaction product solution containing fructooligosaccharide, is carried out at a pH range of 5.5 to 6.5 and a temperature range of 55 to 65 ℃,
The fructooligosaccharide converting enzyme has a storage stability of 60 to 100% relative enzyme activity based on 100% enzyme activity at the time of initial storage at storage conditions for 7 months at a temperature of 4 ℃,
The activity of the fructooligosaccharide converting enzyme is relative to the enzyme activity of 150 to 1000% based on 100% of the enzyme activity produced in the wild type Aspergillus Niger strain,
The reaction product solution, based on 100% by weight of the saccharide solids content of 28-35% by weight of 1-Kestose, 0.01-35% by weight of Nittos and 0.01-5% by weight of 1-F-fractopuranosyl It includes a nystos,
How to produce fructooligosaccharides. The method of claim 1, wherein the fructooligosaccharide is 50 to 70% by weight of the fructooligosaccharide is produced based on 100% by weight of the total solid sugar of the reactor. The weight ratio of 1-kestos (1-kestos / nistos and 1-F-fractofuranosyl to the total weight of the nystose and 1-F-fructofuranosyl nistos according to claim 1). The total weight of the nistos) is 0.5 to 2.0, the method for producing fructooligosaccharide. The method of claim 1, wherein the strain is a mutant strain that improves the wild-type Aspergillus niger strain. The method of claim 1, wherein the enzyme has a relative enzymatic activity of 60 to 100% based on 100% of the enzyme activity at the time of storage for 7 months at a temperature of 4 ° C., the method for producing fructooligosaccharide. The method of claim 1, wherein the enzyme has a fructooligosaccharide conversion activity of 300 to 1,500 U / mg. A fructooligosaccharide converting enzyme, consisting of an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 7,
The fructooligosaccharide converting enzyme has a storage stability of 60 to 100% relative enzyme activity based on 100% of enzyme activity at the time of storage for 7 months at a temperature of 4 ° C., and reacts with sugar as a substrate at 5.5 PH range of 6.5 to 55 and temperature range of 55 to 65 ℃,
The activity of the fructooligosaccharide converting enzyme is a fructooligosaccharide converting enzyme having a relative enzyme activity of 150 to 1000% based on 100% of the enzyme activity produced in the wild-type Aspergillus niger strain. The fructooligosaccharide converting enzyme of claim 7, wherein the enzyme has a fructooligosaccharide converting activity of 300 to 1,500 U / mg. The fructooligosaccharide converting enzyme of claim 7, wherein the enzyme has a relative enzyme activity of 75 to 95% based on 100% of the enzyme activity at the time of storage for 7 months at a temperature of 4 ° C. 8. The enzyme of claim 7, wherein the enzyme is fructooligosaccharide comprising 1-kestos, nystose, and 1-F-fructofuranosylnisto, and nystose and 1-F-fractofuranosyls To produce fructooligosaccharides having a weight ratio of 1-kestos to the total weight of tosses (total weight of 1-kestos / nistos and 1-F-fructofuranosyl nithose) of 0.5 to 2.0, Fructooligosaccharide Converting Enzyme. The fructooligosaccharide according to claim 7, wherein the activity of the enzyme is 600-1000% or 250-400% relative enzyme activity based on 100% of the enzyme activity produced in the wild-type Aspergillus niger strain. Conversion enzymes.

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