KR102585963B1 - Method for producing p-nitrophenyl-maltooligosaccharides with sugar transfer reaction of cyclodextrin glucanotransferase - Google Patents

Abstract

The present invention relates to a method for producing p- nitrophenyl-maltooligosaccharides ( p NP maltooligosaccharides) using the glycotransferase reaction of cyclodextrin glucanotransferase, and more specifically, to cyclodextrin glue. Method for optimally producing p NP maltooligosaccharide from p -nitrophenol-α- _D -glucopyranoside ( α- p NPG) using the glycotransferase reaction of carnotransferase It's about. The present invention finds the optimal glycosyltransferase reaction of cyclodextrin glucanotransferase, thereby reducing by-products and producing p -nitrophenol-α- _D -glucopyranoside, α- p NPG . ), it is possible to provide a production method that maximizes the production of p NP maltooligosaccharides.

Description

Method for producing p-nitrophenyl-maltooligosaccharides with sugar transfer reaction of cyclodextrin glucanotransferase}

The present invention relates to a method for producing p- nitrophenyl-maltooligosaccharides ( p NP maltooligosaccharides) using the glycotransferase reaction of cyclodextrin glucanotransferase, and more specifically, to cyclodextrin glue. Method for optimally producing p NP maltooligosaccharide from p -nitrophenol-α- _D -glucopyranoside ( α- p NPG) using the glycotransferase reaction of carnotransferase It's about.

Unlike chemical glycotransfer reactions, which generally require cumbersome processes, enzymatic glycotransfer reactions are effective because they can expand the structural diversity of biomolecules and control physicochemical properties through the selective reaction of enzymes. To date, enzymatic transglycosylation reactions have been used in various research fields to increase the functionality, oxidation stability, or water solubility of biomolecules.

p- nitrophenyl-maltooligosaccharides ( p NP maltooligosaccharides) is a substrate that combines a p NP group with various maltooligosaccharides, and is converted to p -nitrophenol, a non-glycosidase, by glycosidase. The amount of p -nitrophenol released can be measured through the change in absorbance of the UV-vis spectrum. Because of the convenience of such measurements, p NP maltooligosaccharides are often used as indicators in enzyme kinetics studies, and various types of p NP maltooligosaccharides are required depending on the substrate specificity of the enzyme.

Previously, an enzymatic method using the glycotransfer reaction of amylase has been attempted to produce p NP maltooligosaccharides, but because the types of glycotransfer products that can be produced are limited, p NP with various carbohydrate chains has been used. There were difficulties in producing maltooligosaccharides. Recently, it was discovered using α- p NPG that CGTase causes a glycotransfer reaction with various regioselectivity depending on the configuration of the C-1 anomer of the sugar receptor, but p NP maltooligosaccharides ), follow-up research on the industrial production of is at a standstill, and there is a problem with the lack of research on the removal of by-products produced during the glycotransfer reaction. Therefore, it is necessary to develop a method that can effectively produce p NP maltooligosaccharides while reducing by-products using an enzymatic transglycosylation reaction.

Republic of Korea Patent Publication No. 10-2009-0059366 (publication date: 2009.06.11) relates to a heat-resistant amylase mutant enzyme and a method for producing maltooligosaccharide using the same, isolated from Pyrococcus furiosus. A method for producing chain maltooligosaccharides using heat-resistant amylase whose sequence has been changed using a special regiomutation method is described.

The present invention provides p NP from p -nitrophenol-α- _D -glucopyranoside (α- p NPG) by finding the optimal glycosyltransferase reaction of cyclodextrin glucanotransferase . The aim is to provide a method for optimally producing maltooligosaccharides.

The present invention uses p -nitrophenol-alpha- _D -glucopyranoside (α- p NPG) and alpha-glucoside (α-glucoside) as substrates to produce cyclodextrin glucanoside . A method for producing p- nitrophenyl-maltooligosaccharides is provided, which is characterized by an enzymatic reaction using transferase ( cyclodextrin glucanotransferas, CGTase).

Meanwhile, in the present invention, the method for producing p- nitrophenyl -maltooligosaccharides preferably uses cyclodextrin glucanotransferase (CGTase) and glucose oxidase during the enzymatic reaction. ) may be used.

Meanwhile, in the present invention, the p -nitrophenol-alpha-D-glucopyranoside ( p -nitrophenol-α- _D -glucopyranoside, α- p NPG) is preferably a sugar receptor, and alpha-glucoside (α- glucoside) may be a sugar donor.

Meanwhile, in the present invention, the alpha-glucoside may be, for example, one or more selected from maltose, β-CD, soluble starch, amylose, and amylopectin. .

Meanwhile, in the present invention, the enzymatic reaction is preferably carried out using 2 to 3% (w/v) of p -nitrophenol-alpha-D-glucopyranoside ( p -nitrophenol-α- _D -glucopyranoside, α- p NPG) and 0.2 to 1% (w/v) of alpha-glucoside.

Meanwhile, in the present invention, the enzyme reaction may be preferably performed at 50 to 70°C.

The present invention finds the optimal glycosyltransferase reaction of cyclodextrin glucanotransferase, thereby reducing by-products and producing p -nitrophenol-α- _D -glucopyranoside, α- p NPG . ) provides a production method that maximizes the production of p NP maltooligosaccharides.

Figure 1 is a schematic diagram showing the production of p NP maltooligosaccharides using cyclodextrin glucanotransferase (CGTase) and glucose oxidase.
Figure 2 is a photograph of the results of TLC analysis of the glycotransfer reaction according to various sugar donor substrates.
Figure 3 is a photograph of the results of TLC analysis of the glycotransfer reaction according to the concentration of the substrate.
Figure 4 is a photograph showing the results of TLC analysis of the effect of reducing the by-product of glucose oxidase (GOD).
Figure 5 shows the results of HPLC analysis of changes in p NP maltooligosaccharides production depending on the use of glucose oxidase (GOD).

The present invention uses p-nitrophenol-α-D-glucopyranoside (α-pNPG) and alpha-glucoside (α-glucoside) as substrates to produce cyclodextrin glucanotransfer. A method for producing p-nitrophenyl-maltooligosaccharides is provided, which is characterized by an enzymatic reaction using cyclodextrin glucanotransferas (CGTase). A production schematic diagram for this is shown in Figure 1. However, Figure 1 is a schematic diagram showing a preferred embodiment in which glucose oxidase is additionally used to prevent production of by-products.

In the present invention, p-nitrophenol-alpha-D-glucopyranoside (α-pNPG) is preferably a sugar receptor, and alpha-glucoside (α-glucoside) is preferably a sugar receptor. It is a party donor.

p-nitrophenol-alpha-D-glucopyranoside (α-pNPG) is a substance combining a p NP group and glucose, and is limited to those produced by specific methods. However, you can purchase and use a commercially available product, or use one manufactured directly through a chemical reaction.

Glucoside refers to a compound in which the hemiacetal hydroxyl group of glucose is ether bound to another compound. Depending on the binding method of glucose, it is divided into alpha (α) type and beta (β) type. In the present invention, alpha-glucoside ( α-glucoside) is used. The alpha-glucoside may be, for example, one or more selected from maltose, β-CD, soluble starch, amylose, and amylopectin.

In the present invention, p-nitrophenyl-maltooligosaccharides are produced by inducing a glycosyltransferase reaction using cyclodextrin glucanotransferase (CGTase) enzyme. Until recently, various enzymes such as cyclodextrin glucanotransferase (CGTase) and 4-alpha-glucotransferase (α-glucanotransferase, 4-α-GTase) were used to perform the enzymatic glycotransferase reaction. come. In the present invention, unlike 4-α-GTase, CGTase is an extracellular secreted enzyme and is produced from strains (GRAS) that are safe to eat, such as Bacillus sp., and is easy to recover after enzyme expression, so CGTase is used. By determining the optimal reaction conditions, it was possible to provide an effective method for producing p NP maltooligosaccharides.

Meanwhile, the method for producing p-nitrophenyl-maltooligosaccharides of the present invention preferably uses cyclodextrin glucanotransferase (CGTase) and glucose oxidase during the enzymatic reaction. It is recommended to use additionally. This is because cyclodextrin transferase produces maltooligosaccharides as by-products from glucose (G1) generated through a slight hydrolysis reaction, and the glucose oxidase reduces the production of these by-products.

Meanwhile, in the present invention, the enzymatic reaction is preferably carried out using 2 to 3% (w/v) of p-nitrophenol-alpha-D-glucopyranoside (α- It is recommended to react pNPG) with 0.2 to 1% (w/v) of alpha-glucoside. Additionally, in the present invention, the enzyme reaction is preferably performed at 50 to 70°C.

Meanwhile, according to the following experiment, p NP malto was obtained in a yield of about 68% by enzymatic reaction at 60°C using 3.12 U CGTase and 13.5 U GOD using 3% α- p NPG and 0.4% amylose as substrates. It was confirmed that oligosaccharides (maltooligosaccharides) were produced. Accordingly, in the present invention, by determining the optimal enzymatic reaction conditions using CGTase, an effective production method of p NP maltooligosaccharides was established, and its industrial applicability was confirmed.

Hereinafter, the contents of the present invention will be described in more detail through the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples, but also includes modifications of the technical idea equivalent thereto.

[Example 1: Using CGTase p [Determination of optimal transglycosylation reaction conditions for optimal production of NP maltooligosaccharides]

1) Purpose of experiment

In this example, we attempted to determine enzyme reaction conditions for optimal production of p NP maltooligosaccharides.

2) Analysis of transglycosylation reaction of CGTase according to various types of substrates (alpha-glucoside)

To determine the optimal sugar donor for the optimal production of p NP maltooligosaccharides, various alpha-glucosides such as maltose, β-CD, soluble starch, and amylose were used. This induced a glycotransfer reaction to α- p NPG.

Specifically, for 2% α- p NPG, 0.2% amylose, 0.5% maltose, 0.5% β-CD, and 0.5% soluble starch were used, respectively. The transglycosylation reaction was performed with 3.12 U of CGTase at pH 6.0 and 60°C for 6 hours. At this time, 1 U was defined as the amount of enzyme that hydrolyzes 5.26 mg of starch in 1 hour.

The glycotransfer reaction was analyzed using thin-layer chromatography (TLC). TLC was prepared by activating silica gel 60 TLC plates (Whatman, Maidstone, UK) at 110°C for 30 minutes, and then 1.2 ㎕ of the reaction was added. The completely dried TLC plate was developed in a developing solution (ethanol/butanol/water, 5:5:3, v/v/v). The TLC plate was washed with MeOH containing 0.3% (w/v) N-(1-naphthyl)-ethylene diamine and 5% (v/v) H ₂ SO ₄ After immersion in the solution, heat was applied at 110°C for about 10 minutes until the spots became clear.

The results of analysis of the glycotransferase reaction according to various sugar donor substrates using TLC were shown in Figure 2. In the sample using amylose as a sugar donor (lane 4), the p NP maltooligosaccharides production was the highest (the band appears darkest at α- p NPG2 to α- p NPG5) and the least by-products. (The bands from G1 to G7 appeared the faintest). Through this, it was confirmed that among several alpha-glucosides, amylose is the optimal sugar donor.

3) Analysis of glycotransferase reaction of CGTase according to substrate concentration

Since the glycotransfer reaction can only induce a glycotransfer reaction rather than hydrolysis when the concentration of the sugar receptor is sufficiently high, the glycotransfer reaction was performed by changing the concentration of α- p NPG, a sugar receptor.

In addition, to compare the production of p NP maltooligosaccharides according to the concentration of the sugar donor, a glycotransfer reaction was performed by varying the concentrations of amylose and amylopectin.

Glycotransfer was carried out by reacting 0.5 and 1% of amylopectin and 0.2 and 0.4% of amylose, respectively, as sugar donors, and 2 and 3% of α -p NPG, as sugar acceptors, with 3.12 U of CGTase at pH 6.0 and 60°C for 14 hours. The reaction was performed, and the glycotranslation reaction was analyzed using TLC as above.

The experimental results were shown in Figure 3. From Figure 3, the most suitable sample for the production of p NP maltooligosaccharides was shown to be lane 8 (0.4% amylose + 3% α- p NPG), with the most bands in α- p NPG2 to α- p NPG5. It appears dark, and the bands of G1 to G7, which are by-products, appear the faintest), and this condition was judged to be the most optimal among the samples.

[Example 2: Determination of glycotransfer reaction conditions for reducing by-products G1 to G7 and determination of conditions under the determined conditions p Quantitative analysis of NP maltooligosaccharides production]

1) Determination of transglycosylation reaction conditions to reduce by-products G1~G7

Under the optimal glycosyltransferase reaction conditions obtained through the results of Example 1, CGTase was accompanied by a slight hydrolysis reaction, and the glucose (G1) produced in this way acted as a sugar receptor in the glycosyltransferase reaction, producing G2 as a by-product. ~Produces G7 maltooligosaccharides. Therefore, we considered using glucose oxidase (GOD) to reduce G1, which is the cause of by-products. At this time, for an effective reaction between CGTase and GOD, the reactions were compared and analyzed at various temperatures.

Specifically, 0.4% amylose was reacted with 3% α- p NPG, and the reaction was performed by adding 3.12 U of CGTase and GOD at pH 6.0 at 50-70°C for 14 hours. The glycotransfer reaction was analyzed using TLC as in Example 1 above.

The experimental results were shown in Figure 4. As shown in Figure 4, GOD was confirmed to most effectively reduce by-products at 60°C. That is, in FIG. 4, the 60°C treated sample shows the greatest color change between the 'b' sample using only CGTase and the 'c' sample using both CGTase and GOD compared to samples under other temperature conditions (50°C, 70°C). It was found that the production of by-products G2~G4 was reduced the most by GOD treatment at a temperature of 60℃. Therefore, it was determined that glycosyltransferase reaction at 60°C using CGTase and GOD together was the optimal method for producing high purity p NP maltooligosaccharides.

2) At 60℃ with or without GOD used p Quantitative analysis of NP maltooligosaccharides production

Based on the above TLC analysis, we attempted to quantitatively analyze the production of p NP maltooligosaccharides at 60°C with or without GOD using HPLC. The enzyme reaction product was diluted 150 times with acetonitrile for HLPC analysis and then filtered using a 0.45 ㎛ filter (Advantec, Dublin, CA, USA). High-performance liquid chromatography (HPLC) analysis was performed using an UltiMate 3000 Standard LC System (Thermo Fisher Scientific, Waltham, CA) equipped with a polyamine II column (250 x 4.6 mml.D. 5 ㎛, 12 nm; YMC). MA, USA) was used. 20 μl of the analyte was injected and analyzed at 30°C and 300 nm using solvent A (deionized water) and solvent B (acetonitrile) at 1 mL/min. Solvent conditions for analysis were as follows; 0 min, 100% B; 0 ~ 9.5 min, 100 ~ 68.3% B; 9.5 ~ 17.0 min, 68.3 ~ 58.8% B; 17.0 ~ 22.0 min, 0% B.

The HPLC analysis results of p NP maltooligosaccharides production according to the presence or absence of GOD were shown in Table 1 and Figure 5.

Reaction condition Peak area (mAU*min) Total area
of products
(mAU*min) α- pNPG2 α- pNPG3 α- pNPG4 α- pNPG5 CGTase only 9.6±0.06 2.4±0.03 0.4±0.02 0.04±0.005 12.44 CGTase + GOD 14.9±0.5 4.1±0.07 0.9±0.09 0.2±0.09 20.1

When GOD was added, the production of p NP maltooligosaccharides increased by about 38%, and the length of maltooligosaccharides bound to the p NP group also increased. At this time, the yield of maltooligosaccharides was about 68%. From the above results, it was confirmed that when GOD was added, the production of p NP maltooligosaccharides was significantly increased.

In summary, in the present invention, it was confirmed that p NP maltooligosaccharides can be produced using CGTase, and the optimal production method of p NP maltooligosaccharides was established by finding the optimal transglycosylation reaction conditions. According to the above experimental results, p NP maltooligosaccharides were produced with a yield of about 68% through a reaction of 3.12 U CGTase and 13.5 U GOD at 60°C using 3% α- p NPG and 0.4% amylose. could be confirmed. As the optimal production method of p NP maltooligosaccharides has been established, the present invention is expected to have a very high possibility of being used as a mass production technique for p NP maltooligosaccharides, an artificial substrate for the analysis of newly developed enzymes. It is understandable.

Claims (6)

p -nitrophenol-alpha-D-glucopyranoside ( p -nitrophenol-α- _D -glucopyranoside, α- p NPG) uses a sugar acceptor, amylose, as a sugar donor, and is a cyclodextrin glucanotransfer. Through an enzymatic reaction using cyclodextrin glucanotransferas (CGTase) and glucose oxidase, high purity p -nitrophenyl -maltooligosaccharides are produced.
The p -nitrophenyl-maltooligosaccharides include p -nitrophenyl-α-D - maltoside (α -p NPG2), p -nitrophenyl-α-D-maltotrioside (α -p NPG3) ), p -nitrophenyl-α-D-maltotetraoside (α- p NPG4) and p -nitrophenyl-α-D-maltopentaoside (α- p NPG5),
The enzyme reaction is,
2~3% (w/v) of p -nitrophenol- α- _D -glucopyranoside (α- p NPG) and 0.2~0.4% (w/v) of A method for producing p - nitrophenyl-maltooligosaccharides using amylose and reacting at 60 to 70°C. delete delete delete delete delete