KR101717015B1 - A method for measuring agar-hydrolazing enzymes - Google Patents

Abstract

The present invention relates to a method for measuring activities of agarase. According to the present invention, instead of a prior art which involves the measurement of activities of enzymes from reducing sugar after completion of a reaction between a substrate and an enzyme, the method for measuring the activities of the agarase enables the measurement of the activities of agarase based on the degree of decomposed agarose. Additionally, real-time activity measurement is also possible even with low contents of enzymes without getting affected by pH or salt concentration.

Description

[0001] The present invention relates to a method for measuring the activity of an agar hydrolyzing enzyme,

The present invention relates to a method for measuring the activity of an agar hydrolyzing enzyme.

The world is spurring the development of future energy sources to replace fossil fuels such as petroleum and coal due to depletion of underground resources, and bio-energy is expected to account for about 30% of future renewable energy. Currently, the global biofuels market is shifting from first grain biomass of sugarcane, corn and starch to biomass of second generation. However, the first-generation biomes have been abandoned due to ethical problems caused by the possibility of food use and saturation of the cultivation area. In the second generation woody system, lignin and hemicellulose, which are degradable materials, surround the cellulose, This is a difficult disadvantage. Therefore, the development of bioethanol using algae is emerging as a future energy source.

In order to produce bio-alcohols using seaweed, a saccharification process is required. The glycosylation process involves chemical hydrolysis through acid treatment and enzymatic method using seaweed hydrolase. Currently, the chemical hydrolysis method through acid treatment is applied for the bio-energy production process, but it is difficult to selectively decompose algae into the desired low-molecular-active type in the case of acid treatment, and the process hindrance to the neutralization process again, Enzymatic hydrolysis methods are attracting attention because of the problem of contamination.

Among green algae, red algae, and brown algae, which are capable of producing bio-alcohols, red algae are very important ecologically and economically. For example, Coralline red algae strengthens the marine coral reef area, and some red algae such as Gelidium amansii are used as a raw material of agar and are mainly used as a favorite food. Especially, in Korea, the production of bio-alcohol by the enzymatic decomposition method of Ugokgasari is attracting attention because the production of Ugokgasari is much higher than that of other countries.

Accordingly, the present invention has raised the problems of the conventional method for measuring agar hydrolytic enzymes that hydrolyze agar, and discussed a new method for measuring the activity.

Agarase, an agar hydrolyzing enzyme, exists in an alpha form and a beta form. The α-agarase hydrolyzes the (α1 → 3) bond and the β-agarase belongs to the glycoside hydrolase family which hydrolyzes the (β1 → 4) bond. On the basis of amino acid sequence homology, Agarase is a GH-16, GH-50, GH-86, GH-118, GH-NC (not classified) protein on the carbohydrate activation enzyme database ( http://www.cazy.org ) And the α-agarase corresponds only to the GH-96 group, and the domain of the GH-16 group β-agarase has been studied extensively. The GH-16 enzyme module is linked to the signal peptide and the CBM-6 module is located at the C-terminus. GH-50 activity of six kinds of α-agarase belonging to the GH-50 group The 275 amino acid sequence of the module is well conserved and is located at the C-terminal, unlike the agarase of the GH-16 group. A homology study confirms that the central active residues (Glu, Asp) are well conserved, although the sequence of each agarase shows significant differences. The carbohydrate-binding module (CBM) breaks down the crystalline structure of polysaccharides and helps the active site. The CBM6 module, which does not exhibit activity, recognizes the first repetition site of disaccharide in the enzyme of GH-16 group and acts to hydrolyze agarose by binding agarose to the active site of βg-agarase.

The active site of the agarase consists of two Glu residues and one Asp residue inside the GH-16 activity module. The step of hydrolyzing the agarose by the agar hydrolyzing enzyme proceeds in two steps in total. The first step is the glycosylation, which is the process by which the agarose binds to the agarase. This process is accomplished by the interaction of two Glu residues as acids and bases, where one Glu residue is responsible for the hydrogen bonding of the hydrogen atom of the carboxylic acid to the oxygen atom forming the 1,4-bond of the sugar, and the other Glu residue It serves to electrostatically bind by providing electrons to the first carbon of the partially positively charged D-galactose. Through this process, the weakened 1,4-bond is cleaved to form an aglycone, and the remaining portion is attached to the Glu residue of the enzyme. The second step is the hydrolytic degradation process. The hydroxyl group formed by the negative Glu residue dislodging the hydrogen atom of the water molecule attacks the first carbon of the sugar bound to the enzyme and terminates the bond with the enzyme, thereby dropping the sugar from the enzyme. The Asp residue located between two Glu residues plays a very important role in maintaining the polarity of the two Glu residues.

Most carbohydrase activity measures, including agar hydrolytic enzymes, are based on the analysis of reducing sugars. The hydrolysis products of agar are reducing sugars with aldehyde groups. Therefore, the activity of agarase can be measured by measuring the amount of reducing sugar as a product. At present, the Nelson-somogyi (NS) method using copper and non-molybdate reagents [Non-Patent Documents 1 and 2] and the use of 3,5-dinitrosalicylic acid (DNS ) Method is mainly used.

Although the DNS method is known to be 10 times less accurate than the NS method and does not provide stoichiometric data by measuring the amount of reducing sugar higher than the actual number of hemiacetal reducing sugars, Has been recommended as a method for measuring activity against carboxymethylcellulose. The DNS method is a method in which the yellow color of 3,5-dinitrosalicylic acid is reduced to 3-amino, 5-nitrosalicylic acid by reducing sugar to turn into reddish orange color. The NS method is a method of measuring the blue color that is produced by reducing the non-molybdate salt of copper ion reduced by the reducing sugar.

The NS method published by N. Nelson in 1944 and Somogyi in 1952, and the DNS method published by GL Miller in 1959 have been used for 70 years without improvement, and the NS method known to be more than 10 times more accurate than the NS method There are also various problems in Edo. The reagents necessary for the preparation of the reagents of the NS method include copper sulfate, sodium sulfate, sodium carbonate, sodium bicarbonate, Rochelle salt, molybdic acid, arsenic acid, and sulfuric acid. It contains up to eight species including harmful species. To prepare the reagent using molybdic acid, a reaction time of at least 48 to 72 hours is required. For the activity measurement, the reaction time of the enzyme and the substrate is 30 minutes, the heating is 10 minutes for the reduction reaction of copper, Min, the reaction time with the non-molybdate reagent is 10 minutes, and the time required for measuring the absorbance is about 1 hour. If there are many samples to be measured, the time taken for the active measurement increases exponentially. Thus, the NS method is a time-consuming and labor-intensive method.

There are other limitations to the method of quantifying reducing sugars such as the DNS method and the NS method. For example, the product produced by Agarase, an agarase, differs depending on the enzyme such as NA2 (neoagarobiose), NA4, NA6, NA8, and the like. In addition, there are differences in the types of polysaccharides that each enzyme can recognize. That is, the number of reducing sugars that can be produced by decomposing agar with different enzymes at the same time depends on the kind of enzyme. Therefore, it is not reasonable to compare enzymes with different substrate binding sites and products with the same activity measurement method.

The agar hydrolytic enzymes that have been published so far have not been able to use the DNS method and the NS method, which have the above problems, and define the unit by a different method, making comparative analysis of the enzyme impossible. For example, agar hydrolytic enzymes sold by Sigma are determined by the NS method, and the amount of enzyme required to produce 1.0 μg of reducing sugar at pH 6.0 and 43 ° C is defined as 1 unit. On the other hand, the agar hydrolytic enzyme provided by Thermo scientific defines a unit as the amount of enzyme that can be completely dissolved when 100 μl of 1% low melting point agarose is reacted at 42 ° C. for 30 minutes.

The present invention proposes a new activity measurement method and a unit calculation method for solving the problems of the existing activity measurement method described above.

1. N. Nelson (1944), " A photometric adaptation of the Somogyimethod for the determination of glucose ", J. Biol. Chem., 153, 2, 375-380 2. M. Somogyi (1952), "Notes on sugar determination", J. Biol. Chem., 195, 19-23

It is an object of the present invention to provide an enzyme activity unit according to a method for measuring the activity of agar hydrolyzing enzyme in real time through the degree of degradation of agarose and a method for measuring the activity of agar hydrolyzing enzyme.

As means for solving the above problems,

Adding a sample containing an agar hydrolyzing enzyme to 1.5% agarose solution and reacting at 43 캜 for 5 minutes;

Adding the Rule solution after the reaction;

Measuring absorbance at 460 nm of the solution to which the Rule solution is added; And

And measuring the amount of agar hydrolyzing enzyme by substituting the absorbance value into the following general formula (1): < EMI ID =

[Formula 1]

1 unit (enzyme activity unit) = Amount of agarase required to react with 1.5% agarose solution at 43 ° C for 5 minutes to reduce the absorbance by 10%.

The method of measuring the activity of an agarase according to the present invention is a method of measuring the activity of an agarase by measuring the degree of degradation of the agarose instead of the conventional technique of measuring the activity of the enzyme from the reducing sugar produced after the substrate- Can be measured, is not influenced by the pH or salt concentration, and can measure its activity in real time even with a low content of enzyme.

Figure 1 shows the results of amplified AgaB34 electrophoresis.
FIG. 2 shows the results of electrophoresis of DNA fragments extracted from pET26b-AgaB34 and restriction enzyme-treated fragments (M: marker 1: fragment of AgaB34 gene (1,362 bp); fragment of pET26b (+) (5,230 bp); 3: designed pET26b-AgaB34 (6,592 bp); 4: truncated pET26b-AgaB34 (5,230, 1,362 bp).
FIG. 3 shows the result of measuring the OD value at 600 nm in order to examine conditions under which the AgaB34 protein is most induced.
FIG. 4 shows the activity of agarase according to the concentration of IPTG (0.2, 0.4, 0.6, 0.8, 1.0).
FIG. 5 shows the results of electrophoresis of the expression level of AgaB34 cultured with 0.4 mM concentration of IPTG when the OD value at 600 nm was 0.4.
FIG. 6 shows electrophoresis results of dialysis of ammonium sulfate of purified AgaB34 protein using the ammonium sulfate precipitation method at a final degree of saturation of 10% to 80%.
FIG. 7 shows the results of measuring the activity of AgaB34 against an ammonium sulfate precipitation solution having a final degree of saturation of 10% to 80%.
FIG. 8 shows the electrophoresis results of the purified AgaB34 protein (1: fractions obtained in the washing step, 2: purified AgaB34).
Figure 9 illustrates the overall purification process of the AgaB34 protein.
10 is a graph showing the change of absorbance according to the content of potassium iodide.
11 shows the color change of the solution according to the content of the agarase.

The present invention relates to a method for measuring the activity of an agar hydrolyzing enzyme.

Specifically, the present invention provides

Adding a sample containing an agar hydrolyzing enzyme to an agarose solution to react;

Adding the Rule solution after the reaction;

Measuring the absorbance of the solution to which the Rule solution is added; And

And measuring the amount of agar hydrolyzing enzyme by substituting the absorbance value into the following general formula (1): < EMI ID =

[Formula 1]

1 unit (enzyme activity unit) = Amount of agarase required to react with 1.5% agarose solution at 43 ° C for 5 minutes to reduce the absorbance by 10%.

Conventional methods for measuring enzyme activity have been problematic in that a long reaction time is required because the amount of reducing sugar is measured and thus the reaction between the substrate and the enzyme must be completed.

The method of measuring the activity of the agarase according to the present invention is characterized in that the activity of the agarase is measured through the degree of degradation of the agarose instead of the conventional technique of measuring the activity of the enzyme from the reducing sugar after the end of the substrate- . Specifically, enzyme activity can be measured in real time by changing the color of the solution from orange to pale yellow by binding of agarose cut with agarose hydrolyzing enzyme to iodine (I) of rugol solution. In addition, the method of measuring the activity of an agar hydrolyzing enzyme according to the present invention is not influenced by the pH or salt concentration, and its activity can be measured with a low content of the enzyme.

In the present invention, a method for measuring the activity of an agarase comprises the step of adding a sample containing an agarose hydrolyzing enzyme to a 1.5% agarose solution and reacting at 43 캜 for 5 minutes.

The solvent of the agarose solution may be distilled water. The agarose in the agarose solution is contained at 1.5% (w / v). The agarose solution can be obtained by adding agarose to distilled water at 90 ° C to 110 ° C or heating distilled water to which agarose has been added at a temperature of 90 ° C to 110 ° C.

The agarose solution is subjected to a step of adding a sample containing agar hydrolyzing enzyme and lowering the temperature to 43 캜 for 5 minutes.

In one embodiment of the present invention, the agar hydrolyzing enzyme is selected from the group consisting of α-agarase, β-agarase, β-porphyranase, α- Neoagarobiose hydrolase, beta-agarobiose hydrolase, endo-beta-galactosidases, endo-beta-1,3-glucan, Endo-β-1,3-glucanases, laminarinases, endo-β-1,3-1,4-glucanases, lichenases, But are not limited to, κ-carrageenases or xyloglucanases.

The reaction solution is subjected to a step of adding Lugol solution.

In the present invention, Rugol solution means a solution in which iodine (I) and potassium iodide (KI) are present in an aqueous solution state. The Rulal solution may be prepared by incorporating iodine (I) and potassium iodide (KI) in a weight ratio of 1: 3 to 1:10, such as 1: 5 to 1: 7, preferably 1: 6 . The solvent of the Rule solution may be distilled water. Since iodine (I) and potassium iodide (KI) are contained in distilled water in the weight ratio range in the Rugol solution, iodine (I) can be completely dissolved in the solvent so that the Rugol solution can be maintained in stability without being significantly affected by temperature have. In the following examples, in order to establish the conditions under which the stability of the Rule solution was maintained, the Rule solution was prepared at 4 ° C, 22 ° C, and 37 ° C, and the absorbance changes with time were observed. As a result, It was confirmed that the absorbance value was maintained. In addition, the stability and absorbance of the rye solution according to the concentration of potassium iodide (KI) were measured, and it was confirmed that the ryeol solution maintained the stability and the absorbance value in the weight ratio range of the iodine (I) and potassium iodide (KI) .

In one embodiment, the solution to which the Rule solution is added may further comprise heating at 60 占 폚 or higher for 5 to 20 minutes. When the solution to which the Rugol solution is added is heated to the above-mentioned temperature range, a large amount of iodine (I) can be bound between the irregularly cleaved agarose gaps due to the activity of the agarase, so that the agarase activity is inhibited, The error of the activity value can be reduced when measuring the activity of the degrading enzyme. In addition, the structure of the agarose is loosened so that the agarose cut by the agar hydrolyzing enzyme forms a complex with the iodine (I) of the solution of the agar to measure the activity of the agar hydrolytic enzyme.

The solution to which the Rule solution is added is subjected to a step of measuring the absorbance at 460 nm. It is preferable to measure absorbance at 460 nm at which the absorbance of cyan green, which is a complementary color, can exhibit the maximum value because the solution binds with iodine (I) .

Finally, in the method of measuring the activity of the agar hydrolyzing enzyme of the present invention, the absorbance value measured in the above step is substituted into the following general formula 1 to measure the amount of agar hydrolyzing enzyme:

[Formula 1]

1 unit (enzyme activity unit) = Amount of agarase required to react with 1.5% agarose solution at 43 ° C for 5 minutes to reduce the absorbance by 10%.

In the following examples, agarose hydrolyzed by agar hydrolyzing enzyme according to the present invention measures iodine (I) of the agarose solution and the solution of iodine (I) to change the color of the solution from orange to pale yellow , And the activity of the agarase was confirmed by measuring the absorbance at 460 nm together with the change in color. Thus, the enzyme activity unit of the agarase was defined as shown in the general formula (1).

Hereinafter, the present invention will be described in more detail with reference to the following Examples. However, the scope of the present invention is not limited by the following Examples.

Manufacturing example . Recombination Of AgaB34  refine

1) Agarase Of AgaB34  Gene synthesis

Oligonucleotide primers were synthesized to include Xho I and Nde I at both ends of AgaB34 in order to insert into glutathione S-transferase (GST) -free pET26b (+) affecting the structure of agarase enzyme AgaB34. Are shown in Table 1. One primer pair designed was synthesized with pUC57 by Cosmo Genetech (Seoul, Korea). PCR was carried out by adding 20 pmole of primer, 10 μM dNTP, 5 μl of 10 × e-Taq buffer and 1.25 U of e-Taq DNA polymerase (Solgent, Daejeon) synthesized to the synthesized pUC57-AgaB34 plasmid. 0.8% agarose gel electrophoresis (FIG. 1) and purified using a PCR purification kit. The PCR reaction conditions are listed in Table 2.

Sequence pET26b (+) AgaB34
forward 5'-GGAATTC CATATG AAAGGTTTTACGAAACAT-3 '
Nde I AgaB34
reverse 5'-CCG CTCGAG AGCTTCATGCCATTCCAG-3 '
Xho I

Segment Temperature Time Cycle First denaturation 95 ℃ 2 min 1 cycle Second denaturation 95 ℃ 20 sec 25 cycles Annealing 60 ° C 40 sec Extension 72 ℃ 3 min 36 sec Additional extension 72 ℃ 5 min 1 cycle

As shown in the results of Fig. 1, the AgaB34 gene was confirmed by confirming the band of 1,362 bp.

2) AgaB34  Preparation of Recombinant Expression Vector for Expression

The amplified AgaB34 gene was inserted into the T-vector using the T-Blunt PCR cloning kit (Solgent, Deajeon, Korea) to facilitate restriction enzyme treatment by recombining pST-AgaB34. The pET26b (+) vector and pST-AgaB34 were digested with restriction enzymes Nde I and Xho I at 37 ° C for 3 hours to obtain a double restriction enzyme-treated AgaB34 DNA fragment. The size of the AgaB34 DNA fragment thus obtained was confirmed by DNA agarose electrophoresis and pET26b (+) was also digested with the same enzyme for 3 hours to insert the AgaB34 DNA fragment into the pET26b (+) vector, -cloning site: MCS), a vector portion in which a DNA fragment between Nde I and Xho I was cleaved was obtained. The AgaB34 gene, which was digested with Nde I and Xho I, and the pET26b (+) gene from which the multiple cloning site was excised were separated and purified by gel extraction kit. The purified AgaB34 gene and pET26b (+) vector were reacted at 16 ° C for 16 hours using T4 DNA ligase (Elpis Biotech, Korea). To produce a large amount of the cleaved pET26-AgaB34 gene, a sample reacted by the hanahan method was transformed into TOP10 (Hanahan D., 1991). Transformation proceeded as follows.

10 μl of pET26b (+) and AgaB34 ligation solution was carefully added to TOP10 competent cells, and heat shocked at 42 ° C for 1 minute and 30 seconds. The ligation solution distributed around the competent cells penetrated into the cells Respectively. After the heat shock, the cells were stabilized on ice for 3 minutes. Then, 400 μl of LB liquid medium, which had been previously heated to 37 ° C., was added to 100 μl competent cells and shaking incubation was performed at 37 ° C. for 45 minutes. Transformed samples were isolated using antibiotic selection. 3 ml of transformed TOP10 competent cells were cultured in LB-agar solid medium containing 30 μg / ml kanamycin for 16 hours using kanamycin antibiotic characteristics of pET26b (+). Selectively grown colonies were cultured in a small volume in a liquid medium and cultured at 37 ° C for 6 hours. DNA was extracted with DNA mini-prep kit. The extracted DNA was digested with Nde I and Xho I at 37 ° C for 1 hour and confirmed by DNA agarose electrophoresis.

As a result of the electrophoresis of FIG. 2, the size of the 1,362 bp AgarB34 DNA fragment was confirmed and the size of the fragment treated with the pET26b (+) restriction enzyme of 5,320 bp was confirmed.

3) Using E. coli AgaB34  Expression of protein

2), 10 μl of the DNA was transformed into an expression strain BL21 (DE3) star in the same manner as in 2). The transformed BL21 (DE3) star was also cultured for 16 hours in LB solid medium containing kanamycin. The transformed colony was again cultured in LB liquid medium for 6 hours. The E. coli transformed with pET26b-AgaB34 / BL21 (DE3) star was cultured at 1% and the protein inducer IPTG was added at a final concentration of 0.6 mM according to the OD ₆₀₀ value of the culture medium. The induced conditions were selected through Fig. In order to compare the expression levels according to IPTG concentration, IPTG was added to final concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 to compare the activity (FIG. 4).

As a result, it was confirmed that pET26b-AgaB34 was cultured at 37 ° C for 9 hours when the absorbance of OD ₆₀₀ was 0.4 (about 6 hours) and IPTG was added to a final concentration of 0.4 mM 5). Therefore, the OD value at 600 nm was measured with a UV / VIS spectrophotometer. When the concentration reached 0.4, the IPTG inducer was added to a final concentration of 0.4 mM, and the AgaB34 was cultured for 9 hours.

4) Recombination Of AgaB34  refine

3), pET26-AgaB34 was harvested at 4 ° C and 8,000 g for 15 minutes using a freezing centrifuge, and pET26b (+) pel B, and the cells were discarded by autoclave, and the supernatant was collected and stored in a 4 ° C chamber until the purification experiment. AgaB34 was purified using the ammonium sulfate precipitation method as the primary purification method. Specifically, in order to precipitate and concentrate all the proteins contained in the supernatant, a small amount of ammonium sulfate was added in a 4 ° C chamber so that the final saturation was 10% (56 g / L). After the final degree of saturation reached 10%, after 6 hours, it was precipitated with a freezing centrifuge at 4 ° C, 3,000 g for 30 minutes. The precipitate was discarded and the supernatant was taken separately and 183 g / L ammonium sulfate was added to achieve a final saturation of 40%. And then sedimented at 4 ° C and 3,000 g for 30 minutes using a freezing centrifuge. After centrifugation, the supernatant was discarded and the precipitate was homogenized to 10 ml of 20 mM Tris-HCl buffer A (pH 8.0). Protein electrophoresis, activity measurement and dialysis were performed three times for 8 hours in 20 mM Tris-HCl (pH 8.0) to remove ammonium sulfate which interfered with quantitative measurement. The results of electrophoresis analysis of the dialyzed solution are shown in FIG. The results of measuring the activity of each of the precipitating solutions are shown in FIG. The results of the electrophoresis analysis (FIG. 6) and the activity measurement results (FIG. 7) and the supernatant were saturated with ammonium sulfate by 40% to obtain an enzyme solution containing the target protein.

The enzyme solution obtained using the ammonium sulfate precipitation method was purified using DEAE Sepharose CL-6B, an anion exchange resin. The theoretical pI value of AgaB34 is 6.79, which can be dissolved in a buffer solution at pH 8.0 and purified with anion exchange resin. Thus, the dialyzed sample was passed through a DEAE Sepharose CL-6B column, and a washing buffer, in which NaCl was dissolved to 0.3 M in a buffer solution A, was poured into a column to remove unnecessary proteins attached to the bead . In order to purify the agarase attached to the beads, a fraction was obtained by eluting with an elution buffer in which NaCl was dissolved to 0.4 M in the buffer solution A. The amount of protein was determined by measuring the OD value at 280 nm with UV / VIS for each fraction, and the enzyme activity was measured for each fraction to obtain fractions having agarase activity (lane 1 in FIG. 8). The obtained agarase was eluted with NaCl buffer solution and again dialyzed 3 times for 8 hours in 50 mM potassium phosphate buffer solution (pH 6.0). Purified proteins were confirmed by electrophoresis (lane 2 in FIG. 8). All experiments were carried out at 4 ° C to protect beads from agarase activity for enzyme stability. The results of purification through ammonium sulfate precipitation and anion exchange resin are shown in Table 3, and electrophoretic analysis results of the purified proteins are shown in FIG.

The overall purification method is shown in Fig.

step Total protein (mg / ml) Enzyme activity (Enzyme activity, units ^* ) Specific
Activity, units / mg) Fold purification (Fold purification) Recovery (%,%) Crude extract 150,000 1,400,000 9 One 100 Ammonium sulfate precipitation ((NH _{4) 2} SO ₄
Precipitation) 553 500,000 940 104 59 DEAE-sepharose
column 1.3 30,000 27,200 3,000 2.5

Units: Amount of enzyme producing 1 μg of D-galctose per minute

As can be seen in Table 3, about 1.3 mg of purified agarase per 1 L of culture was obtained. As can be seen from FIG. 8, lane 1 showed no band around 53 kDa and showed almost no agarase, and lane 2 showed that AgaB34 protein was purified through a single band of 53 kDa.

Example  One. Ulgol ( Lugol ) Confirm the stability of the solution

1X Rugol solution was stable at 4 ℃, 22 ℃, and 37 ℃, and the absorbance changes with time (Table 4). As a result, the lowest standard deviation was observed for all Rogol reagents manufactured at 4 ° C, but the value was still not stable. The reagents prepared at all temperatures had absorbances ranging from 10 minutes to 1 hour and 30 minutes Respectively.

Time (min, min) Absorbance ( 460 nm ) 4 ℃ 22 ℃ 37 ℃ 0 0.066 0.088 0.173 10 0.161 0.104 0.184 20 0.128 0.094 0.190 30 0.139 0.102 0.207 40 0.136 0.115 0.198 50 0.141 0.111 0.216 60 0.140 0.113 0.209 90 0.144 0.130 0.224 120 0.154 0.133 0.251 Standard Deviation 0.006 0.011 0.017

Iodine (I) and potassium iodide (KI) were dissolved in the same 30 ml of distilled water to dissolve 2X and 3X of 1X, respectively, in order to examine the effect of Rugol concentration on the absorbance. It was prepared at 37 ℃ considering the reaction conditions, and the change of absorbance with time was also observed (Table 5).

Time (min, min) Absorbance ( 460 nm ) 1X Ulgol 2X Ulgol 3X Ulgol 0 0.248 0.560 0.895 30 0.247 0.556 0.897 45 0.250 0.552 0.870 60 0.248 0.560 0.881 75 0.214 0.563 0.917 90 0.219 0.561 0.909 105 0.235 0.524 0.874 120 0.220 0.582 0.934 135 0.257 0.582 0.937 150 0.254 0.570 0.920 165 0.251 0.568 0.910 180 0.258 0.563 0.865 Standard Deviation 0.015 0.015 0.024

Finally, in order to measure the stability and absorbance of the Rule solution according to the concentration of potassium iodide (KI), the amount of potassium iodide at room temperature was increased from 0.2 g to 0.6 g per 0.1 g, (Fig. 10).

In Table 4, 5 and 10, when 0.1 g of iodine and 0.6 g of potassium iodide are dissolved in 30 ml of distilled water at room temperature to prepare a solution of ryeol, the iodine (I) is completely dissolved, .

Comparative Example  1. Nelson- Somogyi  Measurement of enzyme activity by method

The pET26-AgaB34 / BL21star (DE3) strain was cultured by 1 L and the supernatant obtained after collecting was defined as crude extracts. The crude enzyme solution was purified by ammonium sulfate precipitation and anion exchange resin, and the enzyme was quantified by Nelson-Somogyi method. 1.5% (w / v) agarose was used as the substrate. 200 μl of the substrate solution and 100 μl of the crude enzyme solution or fraction were reacted at 43 ° C for 30 minutes and 50 μl of the Nelson-Somogyi copper reagent (16 mM Copper Sulfate, 1.3 M Sodium Sulfate, Sodium Carbonate, 190 mM Sodium Bicarbonate, and 43 mM Sodium Potassium Tartrate Solution) were added and the mixture was boiled in boiling water for 10 minutes. To the solution cooled to room temperature, 50 μl of Ars-Mol reagent (40 mM molybdic acid, 19 mM arsenic acid and 756 mM sulfuric acid solution) was added, and 0.6 ml of distilled water was added to remove the bubbles . The absorbance was measured at 540 nm after centrifugation at 14,000 rpm for 5 minutes to remove agarose and impurities. D-galactose was dissolved in PBS buffer solution (pH 6.0) as a standard solution so as to be 0.000%, 0.002%, 0.004%, 0.006%, 0.008% and 0.010%, and enzyme activity was measured by Nelson-Somogyi method. . The activity of agarase was defined as 1 unit of the amount of D-galactose-producing enzyme per minute.

Example  2. Enzyme activity measurement

50 μl of the crude enzyme solution and the purified enzyme were reacted with 1.0 ml of 1.5% (w / v) agarose at 43 ° C. for 5 minutes, 100 μl of Rulol solution was added to the whole reaction solution, Respectively. The absorbance of the solution after 10 minutes was measured at 460 nm.

Recombination Of AgaB34 Crude enzyme solution (μl) Absorbance ( 460 nm ) Enzyme Activity Unit Nelson- Somogyi
Unit by method 0 0.809 0 0 10 0.737 0.9 0.0003 30 0.432 4.7 0.0010 50 0.211 7.4 0.0016

As a result, as shown in Table 6, it can be seen that the absorbance decreases as the amount of the enzyme increases. Visually, it can be seen that the color decreases as the amount of the enzyme increases as shown in FIG. Based on the results of this absorbance change, the enzyme activity units of agar hydrolytic enzymes can be defined as follows.

1 Unit: Amount of enzyme required to react with 1.5% agarose solution at 43 ° C for 5 minutes to reduce the absorbance by 10%

Claims (5)

Adding a sample containing agar hydrolyzing enzyme to 1.5% (w / v) agarose solution and reacting at 43 ° C for 5 minutes;
Adding the Rule solution after the reaction;
Measuring absorbance at 460 nm of the solution to which the Rule solution is added; And
Measuring the amount of agar hydrolyzing enzyme by substituting the absorbance value into the following general formula 1:
[Formula 1]
1 unit (Enzyme activity unit) = Amount of agarase required to react with 1.5% (w / v) agarose solution at 43 ° C for 5 minutes to reduce the absorbance by 10%.
The method according to claim 1,
A method for measuring the activity of an agarase comprising iodine (I) and potassium iodide (KI) in a weight ratio of 1: 3 to 1:10.
3. The method of claim 2,
Wherein the weight ratio of iodine (I) to potassium iodide (KI) is 1: 5 to 1: 7.
The method according to claim 1,
A method for measuring the activity of agar hydrolyzing enzyme which is distilled water.
The method according to claim 1,
And heating the solution to which the rugol solution has been added at 60 캜 or higher for 5 to 20 minutes before the absorbance measurement step.

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