New Method for Measuring Enzyme Activity

Abstract

A method for selecting dextransucrase having transglycosylase activity on a glucose acceptor from among various enzymes, particularly various dextransucrases, and a method for measuring its activity are provided.

Description

Technical Field

[0001] The present disclosure relates to a method for selecting a dextransucrase having transglycosylation activity towards a glucose acceptor among enzymes, particularly various dextransucrases, and a method for measuring its activity. Background Art

[0002] Dextransucrase releases glucose from sugar and, at the same time, catalyzes the polymerization reaction of polymers of glucose, thereby producing polysaccharides such as dextran or oligosaccharides as polymer materials. In addition, when substances such as stevia (steviol glycoside), polyphenols, etc. are present in the substrate, dextransucrase releases glucose from sugar because of its broad substrate specificity, and then shows transglycosylation activity capable of transferring glucose to stevia, polyphenols, etc. In this regard, transglycosylated stevia, polyphenols, etc. may have improved industrial values such as improved solubility, improved taste, etc.

[0003] The above activities of dextransucrase are generally measured by determining reducing sugar (DNS), and this method analyzes how much sugar is hydrolyzed into fructose and glucose by measuring free fructose. However, the above method can only measure the hydrolysis degree of dextransucrase on sugar and has the problem that it does not reflect transglycosylation activity. In other words, when using DNS to select an improved dextransucrase, there is a high possibility of only selecting a dextransucrase having excellent sugar hydrolysis or dextran polymerization activity, and a dextransucrase having excellent transglycosylation activity is not selected.

[0004] However, even though there are the above problems, there is no method other than DNS for measuring dextransucrase activity, and there is still a need to develop a new method for measuring dextransucrase activity. Summary of the Invention

[0006] Technical Problem

[0007] The present inventors have developed a high-speed selection method for rapidly and efficiently selecting or improving a dextransucrase having transglycosylation activity towards a glucose acceptor by measuring the difference between the amount of free glucose after a sugar hydrolysis reaction using dextransucrase and the amount of free glucose after a sugar hydrolysis reaction and a transglycosylation reaction, thus completing the present disclosure.

[0008] Technical Solution

[0009] The present disclosure provides a method for selecting a dextransucrase having transglycosylation activity towards a glucose acceptor, the method comprising the steps of: (i) determining the amount of free glucose by reacting a sugar with the dextransucrase; (ii) determining the amount of free glucose by reacting a mixture of the sugar and the glucose acceptor with the dextransucrase; and (iii) comparing the amount of free glucose in (i) with the amount of free glucose in (ii).

[0010] Advantageous effects

[0011] The method of the present disclosure can be used to analyze the activity of a dextransucrase having transglycosylation activity towards a glucose acceptor by measuring the difference between the amount of free glucose after the sugar hydrolysis reaction by the dextransucrase and the amount of free glucose after the sugar hydrolysis reaction and the transglycosylation reaction, thereby quickly and efficiently selecting or improving an enzyme having excellent dextransucrase activity and having transglycosylation activity towards a glucose acceptor. Brief description of the drawings

[0013] Figure 1 Showing the amount of free glucose after the sugar hydrolysis reaction, the amount of free glucose after the sugar hydrolysis reaction and the transglycosylation reaction, and the difference between the two, as measured relative to a control enzyme;

[0014] Figure 2 Showing the amount of free glucose after the sugar hydrolysis reaction, the amount of free glucose after the sugar hydrolysis reaction and the transglycosylation reaction, and the difference between the two, as measured relative to an experimental enzyme; and

[0015] Figure 3 Showing the transglycosylation pattern according to the concentrations of the control and experimental enzymes.

[0016] Description of the invention

[0017] The present disclosure will be described in detail below. At the same time, each description and embodiment disclosed in the present disclosure can also be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in the present disclosure fall within the scope of the present disclosure. Further, the scope of application of the present disclosure is not limited by the following specific description.

[0018] One aspect of the present disclosure provides a method for selecting a dextransucrase having transglycosylation activity towards a glucose acceptor, the method comprising the steps of: (i) determining the amount of free glucose by reacting a sugar with the dextransucrase; (ii) determining the amount of free glucose by reacting a mixture of the sugar and the glucose acceptor with the dextransucrase; and (iii) comparing the amount of free glucose in (i) with the amount of free glucose in (ii).

[0019] Existing reducing sugar assays (DNS) effectively confirm the degree of hydrolysis of sugars by dextransucrase, but have limitations in measuring transglycosylation activity. Specifically, there are limitations in directly confirming whether a glucose acceptor has been glycosylated, and thus, the present disclosure is characterized by the development of a method for indirectly confirming the glycosylation of a glucose acceptor by comparing the amount of free glucose.

[0020] As used herein, the term "dextransucrase", which is an enzyme secreted from microorganisms, refers to an enzyme that produces polysaccharides such as dextran or oligosaccharides as polymer materials by releasing glucose from sugars and, at the same time, catalyzing the polymerization reaction of polymers of glucose, or an enzyme that exhibits transglycosylation activity capable of releasing glucose from sugars and then transferring the glucose to a glucose acceptor such as stevia (steviol glycoside), polyphenols, etc. when the glucose acceptor is present in the substrate.

[0021] As used herein, the term "dextransucrase having transglycosylation activity towards a glucose acceptor" refers to a dextransucrase among the above dextransucrases that exhibits transglycosylation activity capable of releasing glucose from sugars and then transferring the glucose to a glucose acceptor such as stevia (steviol glycoside), polyphenols, etc. when the glucose acceptor is present in the substrate. In other words, as used herein, "dextransucrase having transglycosylation activity towards a glucose acceptor" can be a dextransucrase having sugar hydrolysis activity and transglycosylation activity towards a glucose acceptor.

[0022] A "glucose acceptor" is a target substance to which glucose is transferred, and in the present disclosure, the glucose acceptor can be a natural product excluding glucose, specifically stevia or polyphenols and more specifically stevia. However, the glucose acceptor is not limited as long as it is a natural product to which glucose is transferred.

[0023] As used herein, the term "stevia" refers to a sweet compound present in the leaves of Stevia rebaudiana, which is a plant of the Asteraceae family and is native to South America. Stevia is often used as a sugar substitute as a sweetener because it is not metabolized in the human body and does not induce a blood glucose response. Stevia is also referred to as steviol glycoside.

[0024] For the method of the present disclosure, step (i) can be determining the amount of free glucose by reacting a sugar with dextransucrase.

[0025] The dextransucrase reaction can be carried out by adding various concentrations of dextransucrase, but is not limited thereto.

[0026] Specifically, in step (i), sugar is used as a substrate, and dextransucrase solution is mixed and allowed to react, and then the amount of free glucose produced by hydrolysis can be measured according to the reaction time.

[0027] As a method for measuring the amount of free glucose, any method known in the art can be used without limitation. For example, methods such as glucose oxidase (GOD) - peroxidase (POD) assay, reducing sugar assay (DNS), liquid chromatography (LC) using a refractive index detector (RID), etc. can be used. In the present disclosure, the method for measuring the amount of free glucose can be the GOD-POD assay.

[0028] For the present disclosure, step (ii) can be to determine the amount of free glucose by reacting a mixture of sugar and a glucose acceptor as a substrate with dextransucrase.

[0029] The dextransucrase can be reacted by adding dextransucrase at the same concentration as in step (i), but it is not limited thereto.

[0030] Specifically, in step (ii), a mixture of sugar and a glucose acceptor is mixed with dextransucrase solution and allowed to react, and then the amount of free glucose produced by hydrolysis can be measured according to the reaction time.

[0031] The method for measuring the amount of free glucose is the same as described above.

[0032] In the present disclosure, steps (i) and (ii) can be carried out sequentially or simultaneously, but it is not limited thereto.

[0033] For the method of the present disclosure, step (iii) can be to compare the amount of free glucose in step (i) with the amount of free glucose in step (ii).

[0034] Specifically, such a comparison can be made by subtracting the amount of free glucose in step (ii) from the amount of free glucose in step (i).

[0035] In the present disclosure, the selection can be to select a dextransucrase having transglycosylation activity towards a glucose acceptor among dextransucrases, but it is not limited thereto.

[0036] Specifically, for the method of the present disclosure, in step (iii), the amount of free glucose in step (i) is compared with the amount of free glucose in step (ii), and as a result, if the amount of free glucose in step (ii) decreases compared to the amount of free glucose in step (i), then the dextransucrase is determined to be a dextransucrase having transglycosylation activity towards a glucose acceptor, and thus this dextransucrase can be selected as a dextransucrase having transglycosylation activity towards a glucose acceptor.

[0037] Further, with respect to the method of the present disclosure, in step (iii), the amount of free glucose in step (i) is compared with the amount of free glucose in step (ii), and as a result, when the amount of free glucose in step (i) is similar to the amount of free glucose in step (ii), it can be determined that the dextransucrase is a dextransucrase having no transglycosylation activity towards a glucose acceptor.

[0038] In the present disclosure, the selection may be to select a dextransucrase having transglycosylation activity towards a glucose acceptor, but is not limited thereto. The selection may also include measuring its activity, but is not limited thereto.

[0039] Specifically, with respect to the method of the present disclosure, in step (iii), the amount of free glucose in step (i) is compared with the amount of free glucose in step (ii), and as a result, if the amount of free glucose in step (ii) decreases compared to the amount of free glucose in step (i), then it can be determined that the dextransucrase is a dextransucrase having transglycosylation activity towards a glucose acceptor. Therefore, it is possible to select a dextransucrase having transglycosylation activity towards a glucose acceptor.

[0040] Further, with respect to the method of the present disclosure, in step (iii), the amount of free glucose in step (i) is compared with the amount of free glucose in step (ii), and as a result, if the amount of free glucose in step (ii) decreases compared to the amount of free glucose in step (i), and the level of the decrease in amount increases with an increase in the dextransucrase concentration or is higher than those levels of other dextransucrases, then the transglycosylation activity of the dextransucrase can be measured by means of the level of the decrease in amount.

[0041] That is, when the level of the decrease in the amount of free glucose in step (ii) is higher than the amount of free glucose in step (i), it is determined that the transglycosylation activity of the dextransucrase is high, and thus the transglycosylation activities of different dextransucrases can be compared and / or measured.

[0042] The hydrolysis reaction in step (i) and the hydrolysis reaction and transglycosylation reaction in step (ii) can be carried out within 1 hour, but are not limited thereto. However, depending on the enzyme, when the hydrolysis reaction and transglycosylation reaction continue even after 1 hour, these reactions can be carried out within a specific time limit.

[0043] In the present disclosure, dextransucrase having high saccharohydrate hydrolysis activity and low transglycosylation activity, and simultaneously having high polysaccharide polymerization activity releases glucose (a) after saccharohydrate hydrolysis, increasing the amount of free glucose. However, when a glucose acceptor (stevioside, polyphenol) (b) is added, the glucose remains similar to (a) in the reactor because the amount of glucose used for transglycosylation is very small, and thus the value of (a)-(b) approaches 0 ( Figure 3 , left side). However, dextransucrase having high saccharohydrate hydrolysis activity and high transglycosylation activity, and simultaneously having low polysaccharide polymerization activity releases glucose (a) after saccharohydrate hydrolysis, increasing the amount of free glucose, and when a glucose acceptor (stevioside, polyphenol) (b) is added, the free glucose is transferred to the glucose acceptor, and thus the value of (a)-(b) can be presented as the amount of transglycosylated glucose. In this regard, it was observed that as the transglycosylation activity against the glucose acceptor (stevioside, polyphenol) increased, the value of (a)–(b) also increased ( Figure 3 , right side).

[0044] Therefore, as described above, by examining the difference in the amount of free glucose after (a) saccharohydrate hydrolysis and (b) polysaccharide polymerization or transglycosylation reaction of dextransucrase, enzymes having dextransucrase activity can be distinguished and selected.

[0045] Specific embodiments of the invention

[0046] The present disclosure will be described in more detail below with reference to the examples. However, these examples are for illustrative purposes only, and it is obvious to those skilled in the art that the scope of the present disclosure is not intended to be limited by these examples.

[0047] Example 1: Evaluation of glycosyltransferase activity using the difference between the amount of free glucose after sugar hydrolysis and the amount of free glucose after sugar hydrolysis and transglycosylation Example 2: Difference between the amount of free glucose after sugar hydrolysis caused by the control enzyme and the amount of free glucose after sugar hydrolysis and transglycosylation

[0048] Enzyme solutions having different dextransucrase activities (0.5 U / mL, 1.0 U / mL, 2.0 U / mL, 3.0 U / mL, based on the reducing sugar assay (DNS)) were each added to a 200 mM sugar solution and hydrolyzed, and then the amount of free glucose was first measured. Then, the enzyme solutions having different dextransucrase activities were separately added to a sugar solution of the same concentration (200 mM) containing 200 mM stevioside, and hydrolysis and transglycosylation were carried out, and then the amount of free glucose was measured. Assuming that the difference between the two measured amounts is the amount of pure transglycosylated glucose, it was examined whether the transglycosylation activity of the enzyme could be evaluated.

[0049] Specifically, a separate sugar substrate and each control or experimental dextransucrase enzyme solution (0.5 U / mL to 3.0 U / mL) were mixed and allowed to react, and then, according to each reaction time, the amount of free glucose produced by hydrolysis was first measured by the GOD (glucose oxidase)–POD (peroxidase) assay (1). Further, a sugar and stevia substrate mixture and each control or experimental dextransucrase enzyme solution (0.5 U / mL to 3.0 U / mL) were mixed and allowed to react, and the amount of free glucose produced by hydrolysis and transglycosylation was measured by the GOD-POD assay according to each reaction time (2). Thereafter, the measured value of (2) was subtracted from the measured value of (1), and it was checked whether the amount of transglycosylated glucose had a pattern dependent on the enzyme concentration. At this time, dextransucrase derived from Leuconostoc mesenteroides ATCC 13146, which has sugar hydrolysis activity, low transglycosylation activity, and high polysaccharide polymerization activity, was used as the negative control group, and dextransucrase derived from Lactobacillus mali DSM20444, which has sugar hydrolysis activity, high transglycosylation activity, and low polysaccharide polymerization activity, was used as the experimental group.

[0050] Figure 1 Example 3: Difference between the amount of free glucose after sugar hydrolysis caused by the experimental enzyme and the amount of free glucose after sugar hydrolysis and transglycosylation

[0051] After the sugar hydrolysis reaction using dextransucrase derived from Leuconostoc mesenteroides ATCC 13146, the amount of free glucose was first measured by the GOD (glucose oxidase)–POD (peroxidase) assay (1). After the hydrolysis / transglycosylation reaction using the sugar and stevia substrate mixture, the amount of free glucose in the reaction mixture was then measured (2). The value of (1)-(2) was expected to be the amount of glucose used in the transglycosylation reaction, and the values of (1) and (2) were similar to each other, indicating that the value of (1)-(2) was close to 0 (Table 1).

[0052] Specifically, the control dextransucrase, which has sugar hydrolysis activity, low transglycosylation activity, and high polysaccharide polymerization activity, did not show a difference between the amount of free glucose after sugar hydrolysis and the amount of free glucose after sugar hydrolysis and transglycosylation (Table 1), indicating no pattern according to activity and reaction time (Table 2 and Figure 2 ). The free glucose produced by sugar hydrolysis was immediately used as a substrate for the polysaccharide polymerization reaction, and thus the amount of free glucose detected in the reaction mixture was low.

[0053] [Table 1]

[0054]

[0055]

[0056] [Table 2]

[0057]

[0058] Example 4: Evaluation of the reproducibility of glycosyltransferase activity using the difference between the amount of free glucose after sugar hydrolysis and the amount of free glucose after sugar hydrolysis and transglycosylation Figure 3

[0059] After the saccharide hydrolysis reaction using dextransucrase derived from Lactobacillus mali DSM20444, the amount of free glucose was first measured by the GOD (glucose oxidase)–POD (peroxidase) assay (1). After the hydrolysis / transglycosylation reaction using a saccharide and stevioside substrate mixture, the amount of free glucose in the reaction mixture was subsequently measured (2). The value of (1)–(2) was expected to be the amount of glucose used in the transglycosylation reaction, and it was observed that as the transglycosylation activity increased, the value of (1)–(2) increased (Table 3).

[0060] Specifically, dextransucrases having high saccharide hydrolysis activity and high transglycosylation activity, and at the same time having low polysaccharide polymerization activity, showed a clear pattern according to each enzyme activity for a 1-hour reaction time (Table 4 and ​ ).

[0061] [Table 3]

[0062]

[0063]

[0064] [Table 4]

[0065]

[0066] ​ ​

[0067] To evaluate the reproducibility of Example 1, experiments were conducted by varying the stevioside concentration and enzyme concentration. Enzyme solutions having different dextransucrase activities (0.5 U / mL, 1.0 U / mL, 3.0 U / mL, 5.0 U / mL, based on the reducing sugar assay (DNS)) were each added to a 200 mM saccharide solution and a hydrolysis reaction was carried out, and then the amount of free glucose was first measured. Enzyme solutions having different dextransucrase activities were separately added to a saccharide solution of the same concentration (200 mM) containing 100 mM stevioside, and a hydrolysis reaction and a transglycosylation reaction were carried out, and then the amount of free glucose was measured. Assuming that the difference between the two measured amounts was the amount of pure transglycosylated glucose, it was checked whether the transglycosylation activity of the enzyme could be evaluated.

[0068] Specifically, a separate sugar substrate and each control or experimental dextransucrase enzyme solution (0.5 U / mL to 5.0 U / mL) were mixed and allowed to react, and subsequently, according to each reaction time, the amount of free glucose produced by hydrolysis was first measured by the GOD (glucose oxidase)–POD (peroxidase) assay (1). Further, a sugar and stevioside substrate mixture and each control or experimental dextransucrase enzyme solution (0.5 U / mL to 3 U / mL) were mixed and allowed to react, and according to each reaction time, the amount of free glucose produced by hydrolysis and transglycosylation reactions was measured by the GOD-POD assay (2). Thereafter, the measured value of (2) was subtracted from the measured value of (1), and it was checked whether the amount of transglycosylated glucose had a pattern dependent on the enzyme concentration (Tables 5 and 6).

[0069] [Table 5]

[0070]

[0071] [Table 6]

[0072]

[0073] As a result, a transglycosylation pattern was observed within 1 hour after the start of the reaction according to the enzyme concentration, which was the initial stage of the reaction, and it was confirmed that it was possible to select a dextransucrase having transglycosylation activity towards a glucose acceptor ( ​ ).

[0074] Based on the above description, those skilled in the art will understand that the present disclosure can be implemented in different specific forms without changing its technical spirit or essential characteristics. In this regard, it should be understood that the above embodiments are not restrictive but illustrative in all aspects. The scope of the present disclosure is defined by the appended claims rather than the preceding description, and all changes and modifications falling within the scope of the claims or equivalents of such scope are therefore intended to be encompassed by the claims.

Claims

1. A method for selecting a dextransucrase having transglycosylation activity towards a glucose acceptor, the method comprising the steps of: (i) determining the amount of free glucose by reacting a sugar with a dextransucrase; (ii) determining the amount of free glucose by reacting a mixture of a sugar and a glucose acceptor with a dextransucrase; and (iii) comparing the amount of free glucose in (i) with the amount of free glucose in (ii), wherein the glucose acceptor in step (ii) is stevioside.

2. The method according to claim 1, wherein steps (i) and (ii) are carried out sequentially or simultaneously.

3. The method according to claim 1, wherein as a result of the comparison, when the amount of free glucose in (ii) is lower compared to the amount of free glucose in (i), the dextransucrase is determined to be a dextransucrase having transglycosylation activity towards a glucose acceptor.

4. The method according to claim 1, wherein the selection is a selection of a dextransucrase having transglycosylation activity towards a glucose acceptor among various dextransucrases.

5. The method according to claim 1, wherein the selection is to select a dextransucrase having transglycosylation activity towards a glucose acceptor or to measure its activity.

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