KR100746666B1 - A method purifying fructosyl transferase form Penicillium citrinum KCCM 11663 - Google Patents

Abstract

The present invention is penicillium citrinum ( Penicillium) citrinum ) A method for isolating and purifying fructosyl transferase from KCCM 11663 strains, and in particular, sugar among various enzymes produced by penicillium citrinum KCCM 11663 cultured in a medium composition containing sucrose. The present invention relates to a method for the separation and purification of a new fructosyl transferase to make neoprelacto oligosaccharides. In general, fructo oligosaccharides and neoprelacto oligosaccharides were produced together, but as a result of reacting with sucrose using the purified fructosyl transferase of the present invention, fructo oligosaccharides are not produced and only neoprelacto oligosaccharides can be selectively produced. It is easy to use as an additive in many food and pharmaceutical industries.

Penicillium citrinum KCCM 11663, fructosyl transferase, sucrose, neofloto oligosaccharide

Description

A method purifying fructosyl transferase form Penicillium citrinum KCCM 11663}

1 is a diagram showing the difference in fructosyl transferase activity according to the concentration of ammonium sulfate,

2 is a diagram showing the difference in the fructosyl transferase activity of the eluted protein according to the concentration of the salt added by extracting the protein precipitate by DEAE-sepharose chromatography,

3 is a diagram showing the difference in the fructosyl transferase activity of the eluted protein according to the concentration of the salt added by extracting the protein precipitate by carboxymethyl-sepharose chromatography,

4 is a diagram showing the fructosyl transferase activity of the eluted protein after Sepadex G 75 chromatography.

The present invention is penicillium citrinum ( penicillium citrinum ) relates to a method for the isolation and purification of fructosyl transferase from KCCM 11663 strain.

Fructo oligosaccharides are β- (2,1) -binding of 1 to 3 molecules of fructose to sucrose, and thus 1-kestose, nystose and fructosyl varnish. Oligosaccharide composed of toss (fructosyl nystose), widely distributed in asparagus, onions, honey, etc. Since plant-derived fructo oligosaccharides are greatly influenced by seasonal and storage factors in their production, they are currently produced through microbial processes. As a method for producing fructo oligosaccharides, oreovacsidium flulans using enzymatic methods using fructosyl transferase or β-fructofuranosidase and microbial cells themselves. ( Aureobasidium pullulans ), Aspergillus niger or Fusarium sp. strains, etc. There is a cell immobilization method that directly reacts with the sucrose solution.

Neoprelacto oligosaccharide is a structure in which 1-3 molecules of fructose (fructose) are bound to sucrose and β- (2,6), which is different from the existing fructo oligosaccharide structure, and neokeestose (Neokestose, 6G-). It is composed of β-fructofuranosyl-sucrose, Neonystose (6G-β-fructofuranosyl-kestose), and Neofructosyl nystose (6G-β-fructofuranosyl-nystose). Japanese Patent No. 10-165192 discloses a penny room Solarium sheet rinum (Penicillium citrinum ) FERM-15944 fungus was used to produce a mixture of neoprelacto oligosaccharides along with existing fructo oligosaccharides. The neoprelacto oligosaccharides have excellent moisturizing effect, excellent sweetness, low calorie, anti-carious activity, and enteric bacteria. Because it has a function of proliferation and intestinal local immunity, it has been exemplified that there are various applications such as sweeteners, functional foods, feeds, medicines, and plant protection agents. In addition, Korean Patent No. 10-2002-0004445 identified soil-derived penicillium citrinum KCTC 18080P, and reacted the cultured cells with a high concentration of sucrose solution to produce fructo oligosaccharides and neoprelacto oligosaccharides together.

In order to be used as an additive in many food and pharmaceutical industries in the future, neoprelacto oligosaccharides must be produced as pure substances, not mixtures with fructo oligosaccharides. Existing production methods require a new process to separate the produced fructo oligosaccharides and neoprelacto oligosaccharides, so it is necessary to develop a process capable of producing only neoprelacto oligosaccharides.

Accordingly, the inventors of the present invention continued to produce only neoprelacto oligosaccharides, and found an enzyme that selectively produces only neoprelacto oligosaccharides from the penicillium citrinum KCCM 11663 strain, and developed a method for separating and purifying them. The present invention has been completed.

An object of the present invention is penicillium citrium citrinum ) to provide a separate purification method of fructosyl transferase from KCCM 11663 strain.

In order to achieve the above object, the present invention provides a method for isolation and purification of fructosyl transferase from penicillium citrinum KCCM 11663 strain. Specific purification method is as follows.

(1) a first step of culturing the penicillium citrinum KCCM 11663 by activating shaking culture for 15-35 ° C. and 100-300 rpm for 60-90 hours in a medium containing sucrose as a single carbon source;

(2) a second step of mass production of fructosyl transferase by shaking culture for 15-35 ° C. and 80-120 hours at 400-700 rpm in a medium having the same composition as the seed culture of the first step;

(3) a third step of centrifuging the fermentation broth of step 2 to remove the cells and precipitating the remaining filtrate with 50-60% ammonium sulfate to concentrate the protein;

(4) a fourth step of performing the first chromatography of the concentrated protein in the third step through a DEAE-sepharose column and eluting the protein with a salt of 0.05-0.2M;

(5) performing a second chromatography on the protein eluted in the fourth step through a carboxymethyl-sepharose column and eluting the protein with a salt of 0.05-0.2 M;

(6) penicillium citrine, comprising a sixth step of purifying fructosyl transferase by performing third-order chromatography on a Sephadex G75 column of the eluted protein. A method for purifying fructosyl transferase from KCCM 11663 is provided.

The fructosyl transferase is characterized by selectively producing only neoprelacto oligosaccharides such as neokestos or neonistos from sucrose.

Also provided is a purified fructosyl transferase obtained by the above purification method and having the following characteristics (a) to (e).

(a) Optimum Temperature 50 ℃

(b) optimum pH 5.0

(c) stable at a temperature of 20-50 ° C. and pH 5.0-7.0

(d) isoelectric point pi 5.0 to 6.0

(e) SDS-PAGE measurement molecular weight 80-90 kDa.

Provided is a method of producing neoketos or neoflicto oligosaccharides of neoketos or neonitos in sucrose using fructosyl transferase having the above characteristics.

Hereinafter, the present invention will be described in detail.

The fructose transferase of the present invention can be purified by the following method.

Penicillium citrinum KCCM 11663 with sucrose as a single carbon source, 15-35 in a medium composed of NaNO ₃ , K ₂ HPO ₄ , yeast extract, MgSO ₄ · 7H ₂ O, KCl or FeSO ₄ · 7H ₂ O A first step of culturing by activating by incubating shaking culture at 60 ° C., preferably 25-30 ° C., at 100-300 rpm, preferably 180-230 rpm, for 60-90 hours, preferably 70-80 hours;

Shake at 15-35 ° C., preferably 25-30 ° C., 400-700 rpm, preferably 450-550 rpm, 80-120 hours, preferably 90-100 hours, in a medium of the same composition as the first stage seed culture Culturing the second step of mass production of fructosyl transferase;

Centrifuging the fermentation broth of the second step to remove the cells and depositing the remaining filtrate with 50-60% ammonium sulfate, preferably 55% ammonium sulfate to concentrate the protein;

Fourth step of eluting the protein with a salt of 0.05-0.2M, preferably 0.1M after performing the first chromatography on the concentrated protein in the third step through a column of DEAE-sepharose (DEAE-sepharose) step;

(5) A second step of eluting the protein eluted in the fourth step through a carboxymethyl-sepharose column and eluting the protein with a salt of 0.05-0.2M, preferably 0.1M. Step 5;

(6) penicillium citrine, comprising a sixth step of purifying fructosyl transferase by performing third-order chromatography on a Sephadex G75 column of the eluted protein. A method for purifying fructosyl transferase from KCCM 11663 is provided.

The fructosyl transferase is characterized by selectively producing only neoprelacto oligosaccharides such as neokestos of formula (1) or neonisuto of formula (2) from sucrose.

Therefore, the purified fructosyl translase obtained by the above purification method has an optimum activity at an optimum temperature of 50 ° C. and an optimum pH of 5.0, while maintaining stability at a temperature of 20 to 50 ° C. and a pH of 5.0 to 7.0. , Isoelectric point is pi 5.0 to 6.0, characterized in that the SDS-PAGE measurement molecular weight is 80-90 kDa.

In another aspect, the present invention provides a method for producing neoketos or neo-fructose oligosaccharides of neokestos or neo-nistos at several cross using fructosyl transferase having the above characteristics. 40-70%, preferably 50-60% sucrose solution and the purified fructosyl transferase were reacted for 2-10 hours, preferably 5-7 hours, and neokes except fructo oligosaccharides in sucrose Only neoflacto oligosaccharides of toss and neonisuto can be produced purely.

Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples. The following examples and experimental examples are only specific examples of the present invention, and do not limit the scope of the present invention.

Example  One. Penicillium Citrinum KCCM  11663 Cultivation

For seed culture of penicillium citrinum KCCM 11663, a medium having a composition as shown in Table 1 employing sucrose as a single carbon source is used. The strain was cultured for 72 hours at 28 ° C., 200 rpm by adding 50 ml of medium in a 250 ml flask.

Badge composition ingredient Composition (g / L) Sucrose 126.5 NaNO ₃ 2 K ₂ HPO ₄ 5 Yeast extract 29 MgSO _{4 7} H ₂ O One KCl One FeSO ₄ 7H ₂ O 0.02

Experimental Example  One. Fructosyl Transferase  Separation and Purification

1-1. Penicillium Citrinum KCCM  Cultivation of 11663

The main culture of the penicillium citrinum KCCM 11663 cultured in Example 1 is a medium having the same composition as the culture medium of the species and cultured in a 2.5 L fermenter (Kobiotech, Korea) at 28 ° C., 500 rpm, and 1.5 vm for 96 hours. Incubation resulted in mass production of fructosyl transferase.

1-2. Fructosyl Transferase  concentration

Fructo was recovered by centrifuging the culture solution of the penicillium citrinum KCCM 11663 cultured in Experimental Example 1-1, removing the cells and precipitating by adding 0-70% ammonium sulfate to the protein in the filtrate. The thickening effect of the seal transferase was observed.

As a result, as shown in Figure 1, when the precipitate was added to 55% ammonium sulfate it was confirmed that the concentration of fructosyl transferase is the most excellent. Therefore, the culture of the cultured penicillium citrinum KCCM 11663 was recovered and centrifuged to remove the cells and precipitated by adding 55% ammonium sulfate to the protein in the filtrate.

1-3. Diethylaminoethyl - Sepharose ( DEAE - sepharose ) Chromatography

The concentrated protein precipitate in Experimental Example 1-2 was recovered by centrifugation and subjected to primary chromatography through a column filled with diethylaminoethyl-sepharose.

Then, the protein was eluted at different salt ion concentration gradients (0-0.3 M NaCl) to measure the protein concentration and the activity of fructosyl transferase.

As a result, the activity of fructosyl transferase was measured in the section eluted at a concentration of 0.1M as shown in FIG.

Therefore, the fraction in which the activity of fructosyl transferase was measured was recovered and concentrated again by ultrafiltration in order to perform secondary chromatography.

1-4. Carboxymethyl - Sepharose (CM- sepharose ) Chromatography

The concentrated fraction in Experimental Example 1-3 was flowed into a column filled with carboxymethyl-sepharose to perform secondary chromatography. Then, the protein was eluted through various salt ion concentration gradients (0-0.3M NaCl) to measure the protein concentration and the activity of fructosyl transferase.

As a result, the activity of fructosyl transferase was measured in the section eluted at a concentration of 0.1M as shown in FIG.

Therefore, the fraction in which the activity of fructosyl transferase was measured was recovered and concentrated again by ultrafiltration.

1-5. Sephadex G75 ( Sephadex  G 75) Chromatography

The fraction recovered in Experimental Example 1-4 was run on a column filled with Sephatex G75 to carry out tertiary chromatography.

As a result, as shown in Fig. 4, most of the impure proteins were removed by primary and secondary chromatography, and the fructosyl transferase was finally purified by the tertiary chromatography.

The fructosyl transferase of the present invention purified by the above method was analyzed by SDS polyacrylamide gel electrophoresis, the molecular weight was in the range of about 80-90 kDa, showed optimum activity at pH 5.0, 50 ℃, isoelectric point Was in the range of about pI 5.0-6.0 and thermal and pH stability experiments showed that the enzyme was stable at pH 5.0-7.0 and very stable at temperatures ranging from 20-50 ° C.

In addition, as shown in Table 2, fructosyl transferase selectively producing only neoprelacto oligosaccharides through the entire separation and purification process was purified about 8-fold from 2 L of the culture medium of the penicillium citrinum KCCM 11663 strain, and 10.5%. Was recovered and showed an inactivation of 1.655 U / mg in the final purification step.

Purification of fructosyl transferase Purification stage Volume (ml) Overall potency (U) Inactive (U / mg) Protein amount (mg) % Recovery Purified drainage Culture filtrate 2000 100.5 0.219 459.3 100 - Ammonium Sulfate Precipitation 200 71.8 0.569 126.2 71.4 2.60 DEAE-sepharose 200 49.2 0.898 54.81 48.9 4.10 CM-sepharose 150 23.2 0.987 23.51 23.1 4.51 Sephadex G75 80 10.59 1.655 6.41 10.5 7.56

Experimental Example  2. Refined Fructosyl Transferase  Used Neoprelacto  Production of Oligosaccharides

Production rate after 6 hours reaction with 60% sucrose solution was measured using the purified fructosyl transferase through the separation and purification process of Experimental Example 1.

As a result, when reacted with the concentrated culture solution as shown in Table 3, the fructo oligosaccharides and neoprelacto oligosaccharides were produced together but as a result of the reaction using the purified fructosyl transferase was not produced fructo oligosaccharides neo Kestos was selectively produced at about 11.2% solids, and neonithos was produced at about 9.34% solids, and it was confirmed that the neoprelacto oligosaccharide content was more than about 20% of the total solids.

Production of Neoprelacto Oligosaccharides Using Purified Fructosil Transferase Sugars (%, output for total solids) Culture filtrate concentrate Fructosyl transferase Fructose, glucose 30.17 37.27 Sucrose 35.46 42.15 1-kestose 10.23 - Nystose 9.56 - Neokestose 8.34 11.24 Neonystose 6.24 9.34

As described above, the present invention is the isolation of a new fructosyl transferase that makes the sugar neoprelacto oligosaccharides among the enzymes produced by the penicillium citrinum KCCM 11663 cultured in the medium composition containing sucrose (sucrose) It relates to a purification method. Neoprelacto oligosaccharides have excellent functions of moisturizing, sweetness, low calorie, anti-caries, intestinal bacterial growth and intestinal local immunity. It is possible to apply various fields such as accelerators. In general, fructo oligosaccharides and neoprelacto oligosaccharides are produced together, and their yield is low, and there is a technical difficulty of repurifying the fructo oligosaccharides when necessary. There was a restriction on the use of the bay. However, as a result of reacting sucrose with the purified fructosyl transferase of the present invention, only fructo oligosaccharides can be selectively produced without producing fructo oligosaccharides, which is a considerable process cost when used as an additive in various food and pharmaceutical industries. It is a useful invention for the industry using neoprelacto oligosaccharides because it is possible to reduce the yield and high yield production.

Claims (4)

delete delete delete SDS-PAGE Determination from Penicillium citrinum KCCM 11663 Produces neoketos or neonitosose neoprelacto oligosaccharides in sucrose using fructosyl transferase having a molecular weight of 80-90 kDa. How to.

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