KR101252928B1 - Immobilized enzyme using pH-sensitive polymer and preparation method of linear alpha-1,4-glucans using the same - Google Patents

Abstract

The present invention relates to an immobilized enzyme immobilized on a pH-sensitive polymer carrier and a method for producing a linear α- (1,4) -glucan using the immobilized enzyme, and more specifically, a pH-sensitive polymer carrier such as Eudragit L100. An immobilization enzyme obtained by immobilizing amylosucrase (NpAS) derived from Neisseria polysaccharea, and a method for producing linear α- (1,4) -glucan using the immobilized enzyme.
Since the immobilized enzyme according to the present invention maintains the enzyme activity while improving the thermal stability of the enzyme itself, it is possible to biosynthesize linear α- (1,4) -glucan stably and economically using the immobilized enzyme.

Description

Immobilized enzyme using pH-sensitive polymer and preparation method of linear alpha-1,4-glucans using the same}

The present invention relates to an immobilized enzyme immobilized on a pH-sensitive polymer carrier and a method for producing linear alpha-1,4-glucan using the immobilized enzyme.

Amylosucrase (NpAS) obtained from Neisseria polysaccharea is a unique glucan sucrase obtained from family 13 of glycoside hydrolase, which is linear α- (1,4)-from sucrose while freeing fructose in the reaction medium. It is known as an enzyme that promotes the synthesis of glucan. The linear α- (1,4) -glucan thus synthesized can be usefully used as food resistant, starch, UV filter and emission control substrate in food, cosmetics and pharmaceuticals, respectively.

Unlike other enzymes involved in the synthesis of α- (1,4) -glucan, NpAS is an glucosyl donor because it does not require expensive nucleotide-activated sugars such as ADP-glucose or UDP-glucose. It has been regarded as a very attractive biocatalyst for industrial synthesis and structural modification of saccharides.

However, one of the major drawbacks to overcome for industrial applications of NpAS is poor stability. The half-life of NpAS is only 21 hours at 30 ° C. and is reported to be completely inactivated after 48 hours (FEBS Letters 471 (2-3): 219-223, 2000; J. Bacteriol. 181 (2): 375-381 , 1999).

In order to remedy this problem, membrane-immobilized NpAS is known in the art (Biotechnology Progress 18 (5): 964-968, 2002). Heterogeneous photoinitiated graft polymerization of glycidyl methacrylate produced epoxy groups for covalent enzyme immobilization on the microfiltration membrane. The membrane-immobilized NpAS thus obtained showed low enzymatic activity compared to unimmobilized NpAS. In addition, linear α- (1,4) -glucans tend to have strong chain-chain associations resulting in precipitation, thus making it difficult to separate linear α- (1,4) -glucan products from membrane-immobilized NpAS. there is a problem.

Therefore, there is a need to develop a process capable of improving the stability of NpAS and synthesizing linear α- (1,4) -glucan economically.

Thus, the present inventors synthesized immobilized NpAS by covalently or non-covalently binding NpAS to a pH-dependent autoprecipitator such as EL100, and the immobilized NpAS thus obtained is particularly useful for linear α- (1,4) -glucan biosynthesis. The present invention has been completed by focusing on the stable and economical biosynthesis of linear α- (1,4) -glucan as stability and reusability are improved.

It is an object of the present invention to provide an immobilized enzyme and a method for preparing the same, which can improve the stability and reusability of NpAS useful for biosynthesis of linear α- (1,4) -glucan.

In addition, another object of the present invention to provide a synthetic method for synthesizing linear α- (1,4) -glucan stably and economically using the immobilized enzyme.

In order to achieve the above object, the present invention provides an immobilized enzyme, characterized in that the amylosucrase is immobilized on a pH sensitive polymer carrier.

The amylosucrase is derived from any one of the microorganisms selected from the group consisting of the microorganisms Neisseria polysaccharea , Deinococcus geothermalis and Alteromonas macleodii , in particular may be derived from Neisseria polysaccharea , the microorganisms are disclosed in US Pat. Can be separated according to.

The microorganism Neisseria polysaccharea is cultivated under conditions that allow the microorganism to grow to produce the enzyme amylosucrase, and is generally isolated and incubated at a temperature of about 37 ° C. and pH 7.0 to 7.2. The incubation time is not limited as long as the microorganism can sufficiently grow, but may be incubated for 24 to 48 hours, for example. After the microbial culture is completed, cells can be collected from the culture by a common solid-liquid phase separation method. For example, cells may be obtained by centrifugation at 4 ° C. to remove the supernatant.

In particular, the amylosucrase gene derived from the microorganism Neisseria polysaccharea can be obtained by expressing a recombinant protein in a host cell, for example, E. coli, Bacillus, lactic acid bacteria, fungi.

Cellular enzyme sources can use coenzymes extracted from cells according to common methods. For example, coenzymes can be obtained by extracting enzymes from cells by grinding using ultrasonic or homogenation and treating the extracts by centrifugation or membrane filtration. The coenzyme can be used without further treatment or can be purified and purified in a universal manner. For example, the crude enzyme extract can be filtered to remove insoluble matter and the filtrate is passed through a nickel-nitrilotriacetic acid (Ni-NTA) affinity column to purify recombinant His ₆ -tagged amylosucrase, 4 The amylosucrase can be eluted using Amicon Ultra Centrifugal Filter Devices at ° C to obtain an electrophoretic homogeneous enzyme.

Purifying enzymes may be used directly or immobilized on the carrier material by universal methods. For example, methods such as bonding to ion exchangers, covalent bonding and adsorption to resins and membranes, and the like can be used. Through this immobilization, the enzyme can be easily recovered and used several times as a synthetic catalyst. Since the purification process of enzymes is generally time-consuming and expensive, fixing and reusing enzymes can contribute to significant cost savings.

The activity of the purified amylosucrase according to the present invention can be measured as follows. That is, 100 μl of enzyme solution is added to 900 μl of 50 mM Tris-HCl buffer (pH 7.0) containing 0.1 M sucrose as a substrate, and the mixture is reacted at 35 ° C. for 30 minutes, and the reaction mixture is 100 ° C. Heat the enzyme at 10 minutes to stop the enzymatic reaction. The amount of fructose liberated into the reaction mixture is analyzed using the dinitrosalicylic acid (DNS) method using fructose as a standard. One unit of amylosucrase activity is defined as the amount of enzyme that promotes production of 1 μmol fructose per minute under assay conditions.

The pH-sensitive polymer carrier is a pH-dependent autoprecipitator that automatically precipitates according to the pH change, methacrylic acid-methyl methacrylate copolymers (eudragit L100 and S100), methacrylic acid-methylacrylate copolymer (MPM -05) and methacrylic acid-methylacrylate-methylmethacrylate copolymer (MPM-06).

According to one embodiment of the present invention, Eudragit L100 (EL100) may be used as the pH-dependent autoprecipitation agent, and the EL100 is a copolymer of methacrylic acid and methyl methacrylate, which is nontoxic, inexpensive, and commercially available. Do. EL100 is widely used as a carrier for enzyme immobilization due to its unique pH-dependent autoprecipitation properties. The enzyme bound to EL100, either covalently or non-covalently, is used in soluble form during the enzymatic reaction and can be recovered in insoluble form after enzymatic reaction by lowering the pH of the reaction mixture. Linear α- (1,4) -glucan produced by the enzymatic reaction of NpAS is recovered as a semi-crystalline precipitated form. Thus, pH dependent autoprecipitation agents are very useful for this bioconversion process.

In particular, the pH dependent autoprecipitation agent may be soluble at a pH of 4.0-9.0, but insoluble at a pH of 2.5-3.3.

In addition, the present invention comprises the steps of adjusting the pH of the solution in which the pH-sensitive polymer carrier is dissolved to 5.0-10.0 (first step); Adding and stirring amylosucrase to the polymer solution (second step); Adjusting the pH of the reactants to 3.0-4.3 (step 3); Centrifuging the reactant to separate the precipitate (fourth step); And it provides a method for producing an immobilized enzyme comprising the step of washing the precipitate (step 5).

In the first step, the pH of the polymer solution in which the pH-sensitive polymer carrier, for example, EL100 is dissolved in a solvent such as Tris solution is adjusted to 5.0-10.0, but more preferably 6.0-8.0. If the pH of the polymer solution is out of the above range, a problem may occur in the chemical structure stability of the polymer polymer.

In addition, the second step may be stirred by adding 22 to 176 parts by weight of amylosucrase to 100 parts by weight of the polymer solution, more preferably 44 to 88 parts by weight. If amylosucrase is out of the content range, it may cause a problem of deterioration of the efficiency of immobilization reaction between the carrier and the enzyme.

In addition, the third step adjusts the pH of the reactants to 3.0 to 4.3, but more preferably may be 3.8 to 4.3. If the pH of the reactant is out of the above range, problems such as irreversible inactivation of the immobilized enzyme and a decrease in recovery rate of the polymer carrier may occur.

In addition, the fourth step is centrifuged for 10-20 minutes at 3,000-5,000 g to separate the precipitate, the fifth step is to wash the precipitate 2-3 times with dilute HCl solution (pH 4.0-4.3) and the like.

Detailed description of the amylosucrase and pH-sensitive polymer carrier used in the method for preparing the immobilized enzyme according to the present invention is the same as described above.

The present invention also provides a method for producing linear α-1,4-glucan using immobilized enzyme in which amylosucrase is immobilized on a pH sensitive polymer carrier.

More specifically, the present invention comprises the steps of using sucrose as a substrate, and adding the immobilized enzyme to perform an enzymatic reaction; Centrifuging the enzyme reactant to separate the precipitate; And it provides a method for producing a linear α-1,4-glucan comprising the step of washing and freeze-drying the precipitate.

The enzyme reaction is preferably carried out for 12-120 hours at a temperature of 30-40 ℃ and pH of 6.0-8.5. If the enzyme reaction is out of the above conditions, it may cause problems such as lowering enzyme stability, lowering glucan production yield, lowering efficiency of immobilized enzyme reuse.

The centrifugation is performed at 5,000-7,000 g for 10-20 minutes to separate the precipitate, the precipitate is washed 5-10 times with purified water and freeze-dried.

In particular, the concentration of sucrose as a substrate for the production of insoluble linear α-1,4-glucan is preferably 0.1 to 1.0 M, it may cause a problem that effective insoluble glucan production is difficult to fall outside the above range.

In addition, the present invention comprises the steps of using the sucrose as a substrate, and adding an immobilized enzyme immobilized amylosucrase on a pH-sensitive polymer carrier to perform an enzyme reaction; Adjusting the pH of the supernatant obtained after centrifuging the enzyme reactant to 3.8-4.0; And it provides a method for recovering the immobilized enzyme comprising the step of washing the precipitate obtained after centrifuging the enzyme reactant.

According to the present invention, immobilized NpAS obtained by binding NpAS to a pH-dependent autoprecipitator such as EL100 maintains enzyme activity while improving the thermal stability of the enzyme itself, so that it is stable and economically linear α- (1, 4) -glucan can be biosynthesized.

Figure 1 shows the sedimentation behavior of the immobilized NpAS with pH changes,
2 shows the effect of temperature on the enzyme activity of immobilized NpAS and unimmobilized NpAS,
3 shows the effect of pH on the enzyme activity of immobilized NpAS (B) and unimmobilized NpAS (A),
4 shows the thermal stability of immobilized NpAS and unimmobilized NpAS at 40 ° C.,
5 shows high performance size exclusion chromatography of linear α- (1,4) -glucan produced by immobilized NpAS and unimmobilized NpAS, respectively.

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

Example 1 NpAS Enzyme Preparation and Purification

1. Recombination E. coli  Stock Culture Preparation of BL21

Through a number of PCR using two primers based on the nucleotide sequence as sucrase Neisseria polysaccharea amyl obtain a gene corresponding to NpAS from Neisseria polysaccharea ATCC 43768. That is, the PCR reaction was carried out using N. polysaccharea genomic DNA as a template and two oligonucleotides (SEQ ID NO: 1: NPAS1 5'- GGA TCC GAT GTT GAC CCC CAC GCA GCA A-3 '; and SEQ ID NO: 2: NPAS2 5'- GGC AAG CTT CAG GCG ATT TCG AGC CAC AT-3 ').

The resulting PCR product was cloned in a T-easy vector (Promega, Madison, Wis., USA) and confirmed by DNA sequencing any mistakes in the PCR reaction. After cutting the insert using Bam HI and Hin dIII, the obtained fragment was inserted into a pRSET-B vector treated with the same enzyme to obtain an NpAS expression vector pRSET-NpAS. E. coli BL21 was transformed with the pRSET-NpAS for efficient gene expression.

2. Enzyme Preparation

The liquid nutrient culture medium consisting of 1% (w / v) bactotriptone, 0.5% (w / v) yeast extract, 0.5% (w / v) sodium chloride, and water was adjusted to pH 7.0. 500 ml of culture medium was placed in a 1000 ml Erlenmeyer flask sterilized at 120 ° C. for 15 minutes, then cooled and inoculated with a stock culture of recombinant E. coli BL21 prepared before, followed by 37 ° C. in the presence of 0.01% (w / v) ampicillin. Incubated at. When the optical density of the cells reached about 0.6, 1 ml of 0.1 M isopropyl-β-D-thiocalactopyranoside was added to the culture to induce gene expression. Cells were harvested by incubating at 16 ° C. for 12 hours and then centrifuging at 5,500 × g for 20 minutes at 4 ° C.

3. Enzyme Purification

The previously prepared culture was completely resuspended in 50 ml of 50 mM Tris-HCl buffer (pH 7.0), and then the cells were disrupted using an Sonic Dismembrator 550, Fisher Scientific Co. Model D100. The resulting mixture was centrifuged at 11,000 × g for 20 minutes at 4 ° C. to remove cellular debris. The resulting supernatant was filtered through a 0.45 μm syringe filter and then 5 ml of the filtrate was passed through a nickel-nitrilotriacetic acid (Ni-NTA) affinity column to purify recombinant His ₆ -tagged amylosucrase. Ni-NTA affinity column was first washed with 20 ml wash buffer (50 mM Tris, 300 mM sodium chloride, 20 mM imidazole, pH 7.0) before eluting His ₆ -tagged amylosucrase, followed by 5 ml of elution buffer (50 mM The amylosucrase protein was eluted with Tris, 300 mM sodium chloride, 250 mM imidazole, pH 7.0) and concentrated using Amicon Ultra Centrifugal Filter Devices (Millipore Corporation, Billerica, USA) at 4 ° C. Electrophoresis yielded enzymes with a single protein band. In this case, the protein content of the obtained enzyme was analyzed by the Lori method using bovine serum albumin as a standard, it was found to be 2.20 mg / ml, it was confirmed that the molecular weight is 76 kDa through SDS-PAGE analysis.

Example 2 Preparation of Immobilized NpAS

EL100 with an average molecular weight of 1.35 × 10 ⁵ Da was added to R ㆆ hm Pharma Co. Purchased from (Darmstadt, Germany). Immobilization of NpAS was performed according to some variations of the known methods (Enzyme and Microbial Technology 27 (9): 672-679, 2000). 50 mg of EL100 was completely dissolved in 25 mL of 100 mM Tris solution. The pH of the solution was then adjusted to 7.0 by addition of 1M HCl and the total volume was adjusted to 50 mL with distilled water. 2 mL of NpAS (2.53 U / mL) was added to 5 mL of this polymer solution. After stirring for 1 hour at room temperature, the pH of the solution was adjusted to 4.3 by the dropwise addition of 0.1 M HCl. The precipitate obtained by centrifugation at 3,000 g for 10 minutes was recovered as fixed NpAS. After washing twice with dilute HCl (pH 4.3) and re-dissolving in 50 mM Tris-HCl buffer (pH 8.0), the obtained immobilized NpAS was used in subsequent experiments.

Example 3 Activity Analysis of Immobilized NpAS

Enzyme analysis was performed for 30 min at 35 ° C. in 50 mM Tris-HCl buffer (pH 7.0) using 0.1 M sucrose as substrate. One unit of NpAS activity (U) corresponds to the amount of enzyme that promotes the release of 1 μmol reducing sugar per minute under assay conditions. The amount of reducing sugar liberated in this reaction was determined using fructose as a standard via the dinitrosalicylic acid (DNS) method (Journal of Biological Chemistry 108: 51-54, 1935). Protein concentration was measured by Lowry method (J Biol Chem 193: 265-275, 1951) using bovine serum albumin as a standard.

Protein and activity recovery yields of immobilized NpAS were 95.7% and 86.7%, respectively, and about 91% of immobilized NpAS showed enzymatic activity. Immobilized NpAS also showed high specific activity of 1.04 U / mg protein.

Example 4 Sedimentation Behavior of Immobilized NpAS

The sedimentation behavior of EL100 and immobilized NpAS was evaluated by measuring the turbidity of each suspension at 470 nm as a function of pH (Journal of Biotechnology 42 (1): 75-84, 1995).

As a result, as shown in FIG. 1, the EL100 solution having a pH of 4.2 or higher did not show any absorbance at 470 nm, and was completely dissolved. However, as the pH decreased, the polymer began to precipitate out of solution and the relative turbidity increased. Below pH 3.3, the EL100 dispersion showed maximum turbidity and the polymer did not dissolve at all. The dissolution-insoluble transition curve of immobilized NpAS shifted significantly at higher pH, immobilized NpAS completely dissolved above pH 5.4 and not below pH 4.4.

From these results, the significant shift in the dissolution-insoluble transition curve was due to high absorbance and reached 0.84 mg protein / mg polymer. However, this settling pH shift to higher levels was useful for recovering NpAS activity because it reduces the chance that NpAS will be irreversibly denatured by pH dropping.

Example 5 Examination of Temperature and pH Dependence of Immobilized NpAS

The optimum temperature and optimal pH for the enzymatic reaction of immobilized NpAS were examined as follows. That is, the optimum reaction temperature was determined by measuring the enzyme activity at a temperature of 20 to 55 ℃. In addition, the pH dependence of unimmobilized NpAS and immobilized NpAS was determined in various buffer systems including pH 4.0 to 10.0 (50 mM sodium acetate buffer, 50 mM sodium phosphate buffer, 50 mM Tris-HCl buffer, and 50 mM glycine-NaOH buffer). Was evaluated by measuring enzymatic activity.

Unimmobilized NpAS and immobilized NpAS showed similar dependence profiles on temperature and pH. That is, as shown in Figure 2 both showed the best activity at 42.5 ℃. And, with respect to pH dependence, both showed the best activity at pH 8.5 of 50 mM Tris-HCl buffer as shown in FIG. Enzyme activity was significantly decreased when the pH of Tris-HCl buffer decreased from 8.5 to 7.0, showing only about 60% of enzyme activity at pH 7.0. Meanwhile, according to the known documents, the buffer condition for the NpAS reaction was 50 mM Tris-HCl buffer (pH 7.0).

Example 6 Evaluation of Thermal Stability of Immobilized NpAS

The thermal stability of the immobilized NpAS was evaluated by heat-treating the unimmobilized NpAS and the immobilized NpAS in a 40 ° C. water bath for 100 hours. The heat-treated enzymes were collected and enzyme activity was measured at predetermined time intervals.

As shown in Figure 4, the immobilized NpAS significantly increased thermal stability compared to the non-immobilized NpAS. After 96 hours of incubation, approximately 76% of the enzyme activity remained in the immobilized NpAS, while the unimmobilized NpAS was completely inactivated. By plotting the natural log of residual enzyme activity with the change of incubation time, the first inactivation reaction rate of unimmobilized NpAS was confirmed, and the inactivation rate phase ( ki ) was 2.74 × 10 ⁻² h ⁻¹ . Regarding immobilized NpAS, a multifaceted inactivation pattern was observed. The first inactivation reaction rate was applied by dividing the time into three steps, and the inactivation rate constants were calculated, respectively. During the first stage of culture (0-6 hours), immobilized NpAS quickly lost 8% of the catalytic activity with a relatively high ki of 1.36 × 10 ⁻² h ⁻¹ , which was about half of the unimmobilized enzyme. Corresponding. However, during the second stage of culture (6-24 hours), ki significantly decreased to 0.4 × 10 ⁻² h ⁻¹ . After 24 hours, the rate of inactivation further slowed down, and ki in the third stage of culture (6-24 hours) was about 5% of the unimmobilized enzyme. Indicative of the nonuniform sensitivity of immobilized NpAS to temperature from a gradual decrease in ki . In the non-immobilized NpAS, a large amount of precipitate was observed after 3 hours of incubation, whereas in the immobilized NpAS, no precipitate was observed even after 100 hours of incubation.

The binding of NpAS to the EL100 polymer effectively prevented protein aggregation, thus stabilizing the active form of the enzyme. Unimmobilized NpAS showed poor thermal stability at 40 ° C., although the best enzymatic activity at 42.5 ° C. Therefore, in order to minimize the effect of enzyme inactivation on the properties of the product, the enzymatic reaction for the production of linear α- (1,4) -glucan was carried out at 35 ° C., not at the optimum temperature of 42.5 ° C.

Example 7 Repeated Production of Linear α- (1,4) -Glucan by Immobilized NpAS

To examine the reusability of immobilized NpAS, repeated production of α- (1,4) -glucan was performed as follows. Repeat production of linear α- (1,4) -glucan with NpAS immobilized at 35 ° C. in 50 mM Tris-HCl buffer (pH 7.0) using 0.1 M sucrose as substrate and with 400 U / L enzymatic activity It was. After 18 hours of enzyme reaction, the reaction mixture was centrifuged at 5,000 g for 10 minutes. The supernatant was carefully collected and the immobilized NpAS was reused, and the precipitate was washed five times with distilled water, then lyophilized and weighed. Reuse of immobilized NpAS was performed as follows: pH of the collected supernatant was adjusted to 4.0 by addition of 0.1 M HCl, and the precipitate was collected as immobilized NpAS by centrifugation at 3,000 g for 10 minutes. The precipitate was washed twice with dilute HCl (pH 4.0) and then redissolved in 50 mM Tris-HCl buffer (pH 8.0). The stability index was defined as the recovery yield ratio of activity for the protein of immobilized NpAS during the regeneration reaction.

As a result, Table 1 summarizes protein and activity recovery yields after each reaction cycle. Immobilized NpAS showed good stability during repeated enzymatic reactions. Although only about 71-75% of the enzyme protein was recovered after each reaction cycle, most of them retained their enzyme activity. Therefore, the stability index, defined as the ratio of enzyme activity recovery yield to protein recovery yield, was observed to be high, from 0.93 to 0.96.

Significant loss (25-29%) of protein after each reaction cycle contained enzymes from linear α- (1,4) -glucan precipitates, as well as the release of enzyme proteins from EL100 polymers with pH changes by centrifugation. This may be due in part to incomplete separation of the supernatant. In addition, α- (1,4) -glucan aggregates contain small particles with a diameter of less than 5 μm and exhibit loose pore structure, which not only adsorbs to the surface but also traps into immobilized NpAS to some extent protein recovery. Yield can be reduced.

enzyme Enzyme Activity Recovery Yield (%) Protein recovery yield (%) Stability indicator Immobilized NpAS 86.7 ± 0.4 ^b 95.7 ± 1.3 0.91 Immobilized NpAS
(1 time reuse) 70.5 ± 1.2 74.4 ± 1.3 0.95 Immobilized NpAS
(2 reuse) 66.9 ± 1.5 72.2 ± 0.8 0.93 Immobilized NpAS
(3 reuses) 68.8 ± 0.2 71.3 ± 0.3 0.96 Immobilized NpAS
(4 reuse) 69.4 ± 0.8 72.6 ± 0.9 0.96

From Table 2, the yield of linear α- (1,4) -glucan produced repeatedly by immobilized NpAS was nearly constant, and there was no difference compared to the product produced by unimmobilized NpAS.

enzyme Yield (%) HPSEC Peak Molecular Weight (Mw _p , kDa) DP _peak Unimmobilized NpAS 26.4 ± 0.4 34.6 ± 0.7 214 ± 4  NpAS 26.6 ± 0.6 36.3 ± 0.6 224 ± 3 Immobilized NpAS
(1 time reuse) 26.1 ± 0.3 33.7 ± 0.8 208 ± 5 Immobilized NpAS
(2 reuse) 25.7 ± 0.4 29.9 ± 0.2 185 ± 1 Immobilized NpAS
(3 reuses) 25.2 ± 0.5 28.8 ± 0.3 178 ± 2

Example 8 Molecular Weight Distribution of Linear α- (1,4) -Glucan Using HPSEC

The molecular weight distribution was analyzed via HPSEC to characterize the linear α- (1,4) -glucan product produced by immobilized NpAS.

Lyophilized linear α- (1,4) -glucan (50 mg) was evenly moistened with 0.5 mL of distilled water, followed by addition of 4.5 mL of dimethylsulfoxide (DMSO). This suspension was mechanically stirred with heating on a water bath for 1 hour and then further stirred at room temperature for 12 hours. 1 mL of this dispersion was reacted with 6 mL of ethanol to precipitate linear α- (1,4) -glucan. After carefully removing the supernatant by centrifugation at 7,000 g for 10 minutes, the linear α- (1,4) -glucan pellets were redissolved in boiling water (10 mL) and further stirred for 30 minutes on a boiling water bath. The hot sample solution was filtered through a syringe filter (0.45 m), followed by a high performance size exclusion chromatography (HPSEC) system using Shodex OHpak SB-804 and SB-802.5 columns (Showa Denko, Tokyo, Japan). , Dionex, USA) to measure the molecular weight.

At this time, the final concentration of the injected sample filtrate was 1.0 mg / mL, the injection volume was 50 μL. The column was kept at 50 ° C. while the temperature of the RI detector was fixed at 30 ° C. Elution was performed using water for HPLC (JT Baker, Phillipsburg, NJ, USA) at a flow rate of 1.0 mL / min. Glucose, Maltotriose, Maltopentaose, Maltoheptaose and P5 ( M _w = 5.8 × 10 ³ ), P10 ( M _w = 11.8 × 10 ³ ), P20 ( M _w = 22.8 × 10 ³ ), P50 ( M Standard using a series of pullulan standards (Showa Denko, Tokyo, Japan), including _w = 47.3 × 10 ³ ), P100 ( M _w = 11.2 × 10 ⁴ ), and P200 ( M _w = 21.2 × 10 ⁴ ) A curve was obtained.

As shown in FIG. 5, both linear α- (1,4) -glucan products produced from immobilized NpAS and unimmobilized NpAS showed similar HPSEC profiles, and the related DP (polymerization degree) in molecular weight and peak elution time are shown in Table 2. Indicated. Based on the standard curve, the linear α- (1,4) -glucan product produced by unimmobilized NpAS exhibits a broad molecular weight distribution of 980 to 235,000 Da, which corresponds to a chain length distribution of DP 6 to DP 1,451. Corresponds. In immobilized NpAS, the first use produced linear α- (1,4) -glucan products with the same properties as unimmobilized NpAS. Interestingly, as well as a slight increase in the shoulder peak of 14.73 minutes (corresponding to DP 61), a slight decrease in peak DP was observed as the reuse time increased.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Industry-Academia Cooperation Group Of Sejong University <120> Immobilized enzyme using pH-sensitive polymer and preparation          method of linear alpha-1,4-glucans using the same <130> DP-2010-0649 <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> NPAS1 <400> 1 ggatccgatg ttgaccccca cgcagcaa 28 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> NPAS2 <400> 2 ggcaagcttc aggcgatttc gagccacat 29

Claims (8)

Amylosucrase was transformed into methacrylic acid-methyl methacrylate copolymers (eudragit L100 and S100), methacrylic acid-methylacrylate copolymers (MPM-05) and methacrylic acid-methylacrylate-methylmetha An enzyme complex immobilized with amylosucrase, which is immobilized on any one of pH sensitive polymer carriers selected from the group consisting of acrylate copolymers (MPM-06). The enzyme complex of claim 1, wherein the amylosucrase is derived from any one microorganism selected from the group consisting of microorganisms Neisseria polysaccharea , Deinococcus geothermalis, and Alteromonas macleodii . delete A method for producing linear α-1,4-glucan using an enzyme complex immobilized with amylosucrase according to claim 1 or 2. The method of claim 4,
Using sucrose as a substrate and adding an enzyme complex immobilized with amylosucrase to perform an enzymatic reaction;
Centrifuging the enzyme reactant to separate the precipitate; And
Washing and freeze-drying the precipitate
Method for producing a linear α-1,4-glucan comprising a. The method of claim 5, wherein the enzymatic reaction is performed at a temperature of 30-40 ° C. and a pH of 6.0-8.5 for 12-120 hours. The method of claim 5, wherein the concentration of sucrose for preparing insoluble linear α-1,4-glucan is 0.1-1.0 M. Carrying out an enzymatic reaction using sucrose as a substrate and adding an enzyme complex immobilized with amylosucrase according to claim 1;
Adjusting the pH of the supernatant obtained after centrifuging the enzyme reactant to 3.8-4.0; And
Centrifuging the enzyme reactant to wash the precipitate obtained
Method for recovering the enzyme complex immobilized amylosucrase comprising a.

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