KR100941695B1 - Method for preparing of alpha-D-glucopyranosyl-1?4-arbutin by using amylosucrase from Deinococcus georthermails - Google Patents

Abstract

The present invention relates to a method for preparing alpha-D-glucopyranosyl- (1 → 4) -albutin using amylosucrase isolated from Deinococcus georthermails, which is a cosmetic whitening material. The high yield of alpha-D-glucopyranosyl- (1 → 4) -arbutin can be prepared by reacting with arbutin used as a.

Amylosucrase, Deinococcus georthermails, Arbutin, Arbutin Derivatives

Description

Method for preparing alpha-D-glucopyranosyl- (1 → 4) -albutin using amylosucrase isolated from Dienococcus geothermalis (Method for preparing of alpha-D-glucopyranosyl- (1 → 4)- arbutin by using amylosucrase from Deinococcus georthermails}

The present invention relates to a method for preparing alpha-D-glucopyranosyl- (1 → 4) -albutin using amylosucrase from a new Deinococcus georthermails.

The present invention relates to arbutin derivatives, more particularly sugar transfer arbutin, and a method for preparing the same, using amylosucrase derived from Deinococcus geothermalis , a new sugar transfer enzyme. Arbutin is a derivative in which glucopyranoside is combined with hydroquinone, which has less side effects when using hydroquinone and has melanin pigment inhibitory effect, which inhibits the increase of melanin pigmentation, thereby treating skin diseases and skin whitening. It is a natural compound used as a substance.

It is known that various sugar transition products in which sugar molecules have been translocated have added functions such as improved sweetness, increased solubility, proliferation of enterobacteriaceae, and increased chemical stability, and some arbutin sugar-transfer substances inhibit melanin pigments than general arbutin. This was known to be outstanding. The general arbutin sugar transfer reaction is known to produce a sugar transfer product by reacting CGTase and β-glucosidase, which are widely known as conventional sugar transfer enzymes. However, the substrate used at this time is a high cost required maltotriose and cellobiose, there is a non-economic problem, such as the production of a complex of several products instead of a single product of the former.

Amylosucrase hydrolyzes sugar into glucose and fructose, using sugar as a substrate. It is known to generate water-insoluble glucan with alpha-1,4-bond using the glucose produced at this time. Previous researchers have reported using amylosucrase only to produce water-insoluble glucans.

Thus, there is a need for new uses of amylosucrase and the development of methods for preparing single arbutin products using economic substrates in the preparation of arbutin saccharide products.

The present invention is to solve the requirements of the prior art as described above, the purpose of which is an enzyme of amylosucrase derived from Deinococcus geothermalis (amylosucrase), not a conventional sugar transfer enzyme It is to provide a single arbutin derivative is very low production cost but very excellent melanin synthesis inhibitory ability.

In addition, another object of the present invention is to add an amylosucrase derived from Deinococcus geothermails to the reaction solution containing sugar and arbutin as a substrate, characterized in that for enzymatic reaction It is to provide a method for producing alpha-D-glucopyranosyl- (1 → 4) -arbutin.

The object of the present invention can be achieved by a single arbutin or a derivative thereof using amylosucrase separated from the dienococcus geothermalis represented by the following formula (1).

(Formula 1)

delete

Another object of the present invention is to mix the recombinant amylosucrase enzyme 10 U / ㎖, arbutin 50g / L, sugar 40g / L in Wan Tris-Cl filling solution and then reacted at 30 ℃ for 12 hours Boiling the solution for 5 minutes to produce filtered single arbutin derivative; Isolating and purifying the single arbutin derivative; It can be achieved by a method for producing a single arbutin derivative using amylosucrase isolated from the Dienococcus geotheremis, characterized in that made.

In addition, another object of the present invention is to transfer glucose, which is a component of sugar, to arbutin by a sugar transfer reaction of amylosucrase, using amylosucrase isolated from the dienococcus geothermalis of formula 1 It can be achieved by a method for preparing a single arbutin derivative.

(Formula 1)

Amylosucrase derived from the Deinococcus geothermalis of the present invention exhibits a higher efficiency of the sugar transfer reaction than the existing sugar transfer enzymes, and the substrate is an expensive one used for the conventional sugar transfer reaction. By using sugar that is cheaper than the substrate to produce a single sugar product rather than a complex sugar product, the industrial effect can be very large. In addition, the single arbutin derivative made by the present invention was found to have higher melanin synthesis inhibitory activity and less cytotoxicity than general arbutin. Through this, it can be seen that the single arbutin derivative made by the present invention is superior to general arbutin. This would be of great industrial value.

Hereinafter, with reference to the accompanying drawings will be described in more detail the configuration and effects of the present invention through specific examples and comparative examples. However, the following examples are only intended to more clearly understand the present invention, and the present invention is not limited to the following examples.

Example 1 dienococcus geothermalis Deinococcus geothermails Preparation of Amylosucrase

1-1. Preparation of a vector comprising a polynucleotide encoding an enzyme

The polynucleotide encoding the amylosucrase derived from wild-type Dienococcus geothermalis, an enzyme of the present invention, was isolated from the chromosomal gene of Dienococcus geothermalis. That method is mentioned below.

The polynucleotide encoding the amylosucrase derived from the wild-type Dienococcus geothermalis uses the chromosomal DNA of Deinococcus geothermalis DSM 11300 as the template DNA, and the forward primer under the conditions shown in Table 1 below. Using DGAS1 (SEQ ID NO: 1) and reverse primer DGAS2 (SEQ ID NO: 2) as a primer, polymerase chain reaction (PCR) was obtained under the conditions shown in Table 2.

TABLE 1

designation Gene sequence SEQ ID NO: DGAS1 5'- GGATCC GATGCTGAAAGACGTGCTCACTTC-3 ' One DGAS2 5'- AAGCTT ATGCTGGAGCCTCCCCGGC-3 ' 2

TABLE 2

Denaturation Pre-denaturation 95 ℃ / 5 min Amplification Denaturation 95 ℃ / 1 min 35 cycles Annealing 60 ℃ / 1 min Extension 72 ℃ / 2 min Final extension Final extension 72 ℃ / 5 min

Table 3 below shows the results of structural analysis using ¹ H and ¹³ C NMR for a single arbutin derivative using amylosucrase isolated from the Dienococcus geotheremis according to the present invention.

Table 3

As a result of the PCR, a 1.9 kb PCR product including the whole vector was obtained. The obtained PCR product was conjugated with a pGEM-T Easy vector treated with restriction enzymes Bam HI and Eco RI to prepare pGEM-T-DGAS. The pGEM-T-DGAS was treated with restriction enzymes Bam HI and Eco RI, and a fragment of about 1.9 kbp length was separated by agarose electrophoresis, and treated with the same restriction enzyme with the pGEX-4T-1 expression vector. Ligation to prepare pGEX-DGAS. The N-terminus of the amino acid sequence encoding the amylosucrase contains glutathione-S-transferase and also contains an ampicillin resistance gene.

1-2. Preparation of Transformant

In order to mass-produce the amylosucrase, a transformant transformed with the recombinant vector pGEX-DGAS was prepared.

E. coli BL21 strains were inoculated in 5 ml of LB liquid medium and pre-incubated with stirring at 37 ° C. for 12 hours, and then inoculated again in 50 ml of LB liquid medium to measure the absorbance at 600 nm. Incubate until 0.6.

When the absorbance reached a reasonable level, it was left on ice for about 10 minutes. After removing the supernatant by centrifugation at 4 ° C. and 4,000 rpm for 20 minutes, 1 ml of cooled sterilized water was added to completely dissolve the cells. Then, 50 ml of sterile water was added again to remove the salt component and 5 at 6,000 rpm. The process of centrifugation and washing was repeated three times. The supernatant is completely removed by centrifugation at 6,000 rpm for 10 minutes to remove residual supernatant remaining in the cells. The cells were weighed, and 10% glycerol cooled to a volume of 1: 1 was added to dissolve pellets to prepare competent cells. 50 μl was transferred to a previously cooled microtube and stored at −70 ° C.

When performing transformation, 50 μl of stored transformed cells (competent cells) are dissolved in ice for about 30 minutes, and then 1 μl of the ligation solution to which the recombinant pGEM-DGAS to be transformed is added and mixed well. Electric shock was transferred to the cuvette (cuvette) and subjected to electrical shock with an electric shock (Hybaid CelljecT Uno, Hybaid Ltd, UK) to perform transformation.

To the reaction solution subjected to the transformation, 950 μl of LB liquid medium was added and incubated at 37 ° C. for about 40 minutes in an incubator. After incubation, the strain was plated on LB agar medium containing ampicillin (100 µg / ml), and the strains showing resistance were first selected.

The first selected strains were inoculated in 5 ml of LB liquid medium containing ampicillin, incubated at 37 ° C. for 12 hours, and centrifuged to obtain cells. Plasmids were isolated from the cells recovered by the plasmid separation kit and digested with Bam HI and Eco RI restriction enzymes to select one strain that identified a polynucleotide encoding about 1.9 kb of amylosucrase. .

1-3. Purification of Mutant Enzymes

The transgenic strains selected above were cultured at 37 ° C., and the absorbance was measured at 600 nm and cultured until the value became 0.4 to 0.6. IPTG (isopropyl β-D-1-thiogalactopyranoside) was added to the cultured bacteria so that the final concentration was 1 mM, followed by incubation at 25 ° C for 12 hours. After incubation, the cells were recovered by centrifugation at 4 ° C. and 4,000 rpm for 30 minutes, and phosphate buffered saline [140 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , and 1.8 mM KH ₂ PO ₄ , pH 7.3] followed by ultrasonic grinding. The pulverized pulverized liquid using the ultrasonic wave was centrifuged for 10 minutes under conditions of 4 ° C. and 12,000 rpm, and then a supernatant was obtained. The supernatant was injected and purified by Glutathione-Sepharose ^™ High Performance Affinity Chromatography, and glutathione-S-transfer adhered to the enzyme by thrombin treatment to remove glutathione-S-transferase attached to the purified N-terminus. Laze was removed. An electrophoretic photograph of the sample for each purification step is shown in FIG. 2. Line 1 represents a standard marker, number 2 is an unexpressed transformant, number 3 is a transformant expressed, number 4 is purified amylosucrase with glutathione S-transferase, and number 5 is pure purified amylose Rosucrase is shown.

As shown in FIG. 2, it was confirmed that the molecular weight of the enzyme exhibited a molecular weight of about 75 kDa consistent with the theoretical value as shown on the SDS-PAGE, and the amylosucrase recombinant mutase was successfully synthesized in E. coli BL21. It was expressed and confirmed to be efficiently purified through Glutathione-Sepharose ^™ High Performance affinity chromatography.

Example 2; Preparation and Purification of Arbutin Derivatives

Arbutin and sugar used in the experiment were Sigma Chemical Co. (St. Louis, Mo. USA). 10 U / ml of the purified recombinant amylosucrase enzyme was mixed with 50 g / L of arbutin and 40 g / L of sugar in Tris-Cl buffer (pH 8.0), followed by reaction at 30 ° C. for 12 hours. After the reaction, the solution was boiled for 5 minutes and filtered using Whatman filter paper. The reaction solution was passed through a gel permeation column (W-251) using high performance recycling liquid chromatography to separate and purify the reaction product.

Example 3; Analysis of reaction product using thin layer chromatography

Thin film chromatography was performed to analyze the reaction product obtained in the reaction of Example 2. After the completion of the reaction, each sample was loaded into Whatman K5F TLC plate (20 cm × 20 cm) by 1 μl and developed in n-butanol: acetic acid: distilled water = 3: 1: 1 developing solution. After development, the plate was well dried, soaked in a coloring solution in which 0.3% (w / v) N- (1-naphthyl) -ethylenediamine and 5% (v / v) sulfuric acid was dissolved in methanol, and then taken out in a 110 ° C oven. Color development for 10 minutes. The obtained result is shown in FIG. As can be seen from the second line, it was confirmed that a new substance was transferred to arbutin with sugar transfer.

Example 4; Analysis of Arbutin Derivatives Using High Performance Liquid Chromatography

The product obtained in Example 2 was analyzed using High Performance Liquid Chromatography (HPLC). The sample was analyzed by filtering the reaction product obtained in Example 2 with 0.45 탆 thin film filter and injecting 25 ㎕ with a flow rate of 1.0 ml / min. HPLC analysis was carried out using a Shimadzu LC-AD gradient pump, SPD-10A detector and LC-NH2 column (SUPELCOSIL ^TM ), and the solvent was A solution (acetonitrile) and B solution (water). The mixture was used as 1: 1. The results are shown in FIG. Peak 1 represents arbutin and peak 2 represents alpha-D-glucopyranosyl- (1 → 4) -arbutin.

Example 5; Molecular Weight Measurement of Alpha-D-Glucopyranosyl- (1 → 4) -Arbutin Using Fast Atom Bombardment Mass Spectroscopy

Fast atom bombardment mass spectroscopy: JEOL JMS 700, Japan (FABMS) was used to confirm the molecular weight of alpha-D-glucopyranosyl- (1 → 4) -albutin, the main transition of the present invention. The results are shown in FIG. In FIG. 5, alpha-D-glucopyranosyl- (1 → 4) -arbutin had a molecular weight of 433 from the ion peaks shown at 867 [2M-H] ⁻ and 433 [MH] ⁻ . It can be seen from this that the reaction product is consistent with the molecular weight of alpha-D-glucopyranosyl- (1 → 4) -arbutin.

Example 6; Structural analysis of alpha-D-glucopyranosyl- (1 → 4) -arbutin using ¹ H and ¹³ C NMR

¹ H and ¹³ C nuclear magnetic resonance analysis (NMR; Varian, USA) were performed to confirm the structure of alpha-D-glucopyranosyl- (1 → 4) -albutin, the main transition of the present invention. The sample in Example 2 was purified from the, alpha -D- glucopyranosyl was used by dissolving in heavy water a arbutin _{(D 2 O) - (1} → 4). The results are shown in Table 3. COSY, HMQC and HMBC experiments showed that the reaction product was alpha-D-glucopyranosyl- (1 → 4) -arbutin.

Example 7; Efficacy of alpha-D-glucopyranosyl- (1 → 4) -arbutin

7-1. Inhibitory Effect of Tyrosinase Extracted from Mushrooms

100 μl of 0.03% L-tyrosine, aqueous arbutin solution, and 50 μl of the obtained alpha-D-glucopyranosyl- (1 → 4) -arbutin at various concentrations were mixed in 640 μl of 67 mM phosphate buffer (pH 7.0), respectively. , 37 ° C for 10 minutes. 60 U mushroom extract tyrosinase (mushroom tyrosinase) is dissolved in 67 mM phosphate buffer (pH 7.0) 10 ㎕ is added to the reaction solution prepared first and reacted for 30 minutes at 37 ℃.

The mushroom extract tyrosinase activity was measured by absorbance at 475 nm and calculated using the following equation.

tyrosinase activity (%) = [(A-B) / (Cp-Cn)] X 100

In the above formula, A is the absorbance of the experimental group (67 mM phosphate buffer, L-tyrosine, arbutin or arbutin derivative, tyrosinase); B is the absorbance of the control group (67 mM phosphate buffer, water, arbutin or arbutin derivative, tyrosinase); Cp is the absorbance of the positive control (67mM phosphate buffer solution, L-tyrosine, tyrosinase); Cn is the absorbance of the negative control group (67mM phosphate buffer, water, tyrosinase). The results are shown in FIG. Alpha-D-glucopyranosyl- (1 → 4) -arbutin showed higher inhibitory activity of tyrosinase than normal arbutin. At 6 mM concentration, normal arbutin showed about 50% inhibition, alpha-D-glucose. Pyranosyl- (1 → 4) -arbutin showed about 60% inhibition.

7-2. Inhibition of melanin synthesis using melanoma cell line

B16F10 murine melanoma cells were purchased from Korea Cell Line Bank. B16F10 cell medium was a medium to which 10% fat bovine serum (FBS) (BioWhittaker) was added in Dulbecco's modified Eangle's medium (DMEM) (BioWhittaker, Walkersville, MD, USA). Culture conditions were incubated at 37 ℃. At this time, the incubator contained 5% CO ₂ in the incubator atmosphere.

B16F10 cells were cultured by releasing them in 2 ml DMEM medium to which 10% FBS was added to 60 mm cell culture dishes (Iwaki, Japan). After 48 hours, wash twice with 2 ml phosphate buffered saline and replace with 2 ml of the fresh medium mentioned above. And α-MSH is added so that the final concentration of 200 nM and arbutin or arbutin derivative to 1, 5, 10, 20 mM.

After 48 hours, the cells are washed twice with 2 ml phosphate buffered saline and the cells are collected using a scraper. The collected cell solution is centrifuged at 1,700xg for 5 minutes at 4 ° C and the supernatant is discarded. 200 μl of 1N NaOH added with 10% DMSO was added to the cell body, and reacted at 80 ° C. for 1 hour.

Melanin (melanin) was dissolved by stirring, the content of melanin was measured by absorbance of 405 nm using Synergy HT Multi-Detection Microplate Reader (Biotek Instruments, Ins., Winooski, VT, USA). The results are shown in FIG. Alpha-D-glucopyranosyl- (1 → 4) -arbutin was found to have higher melanogenesis inhibition than normal arbutin at concentrations above 5 mM. At 10 mM concentration, alpha-D-glucopyranosyl- (1 → 4) -arbutin showed about 80% inhibition, while normal arbutin showed about 60% inhibition.

7-3. Cytotoxicity Test

B16F10 cells were cultured in 96-well tissue culture plates (Nunc, USA) using 200 μl of DMEM with 10% FBS as a medium. After 48 hours, cells were washed twice with phosphate buffered saline and replaced with 200 μl of fresh medium. At this time, the final concentration of α-MSH is 200 nM and arbutin and arbutin derivatives are dissolved in phosphate buffered saline to add the final concentration of 1, 5, 10, 20 mM.

After 48 hours, cells are washed twice with phosphate buffered saline and replaced with fresh medium. At this time, 20 μl of 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl-tertazolium bromide (MTT) solution (5 mg / ml in PBS) was added to each culture solution, React for hours.

After removing the culture medium from the cell culture, the cells are dissolved in 100 µl of DMSO. The amount of formazan produced by conversion of MTT was measured with an absorbance of 570 nm using Synergy HT Multi-Detection Microplate Reader (Biotek Instruments, Ins, Winooski, VT, USA). The results are shown in FIG. Cell survival was about 79% in 20 mM normal arbutin and about 90% in alpha-D-glucopyranosyl- (1 → 4) -arbutin. This suggests that alpha-D-glucopyranosyl- (1 → 4) -arbutin has lower cytotoxicity than regular arbutin.

FIG. 1 is a chemical formula of alpha-D-glucopyranosyl- (1 → 4) -arbutin prepared using amylosucrase isolated from dienococcus geothermalis according to the present invention.

FIG. 2 is an electrophoretic photograph of a step-by-step sample of purification of a single arbutin derivative using amylosucrase isolated from dienococcus geothermalis according to the present invention.

Figure 3 shows the results of thin layer chromatography analysis of a single arbutin derivative using amylosucrase isolated from Dienococcus geothermalis according to the present invention.

Figure 4 shows the results of analysis using high performance liquid chromatography for a single arbutin derivative using amylosucrase isolated from Dienococcus geothermalis according to the present invention.

Figure 5 shows the results of molecular weight measurement using Fast Atom Bombardment Mass Spectroscopy for a single arbutin derivative using amylosucrase isolated from Dienococcus geothermalis according to the present invention.

Figure 6 shows the results of analysis of tyrosinase inhibitory effect extracted from mushrooms against a single arbutin derivative using amylosucrase isolated from the Dienococcus geothermalis according to the present invention.

Figure 7 shows the results of the analysis of the inhibition of malin synthesis using a melanoma cell line for a single arbutin derivative using amylosucrase isolated from Dienococcus geothermalis according to the present invention.

Figure 8 shows the cytotoxicity test results for a single arbutin derivative using amylosucrase isolated from Dienococcus geothermalis according to the present invention.

Claims (14)

delete Alpha-D-glucopyranosyl- characterized in that the reaction solution containing sugar and arbutin is added to amylosucrase derived from Deinococcus geothermails and subjected to enzymatic reaction. (1 → 4) -Method of arbutin delete delete delete The method of claim 2, The amylosucrase is, Method for producing alpha-D-glucopyranosyl- (1 → 4) -arbutin, characterized in that glutathione-S-transferase is contained at the N-terminus as a tag for purification. delete delete delete delete delete delete The method of claim 2, Enzyme reaction, Method for preparing alpha-D-glucopyranosyl- (1 → 4) -arbutin, characterized in that carried out for 12 hours at a temperature of 30 ℃ delete

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