KR101048148B1 - Method for preparing alpha arbutin using amylosucrase derived from Dinococcus thermos - Google Patents

Abstract

The present invention is Deinococcus The present invention relates to a method for producing alpha arbutin using amylosucrase derived from geothermalis ), and has a higher melanin synthesis inhibitory activity than arbutin by using sugar, which is a relatively inexpensive substrate than a substrate generally used in enzymatic synthesis. Alpha arbutin with low cytotoxic properties can be produced economically.

Deinococcus zeomeris, sugar, amylosucrase, alpha arbutin, hydroquinone, antioxidant

Description

Method for production of alpha-arbutin using amylosucrase from Deinococcus geothermalis}

The present invention relates to a method for producing alpha-albutin using an enzyme, and more specifically, Deinococcus It relates to a method for producing alpha arbutin using amylosucrase derived from geothermalis ).

Arbutin is a derivative in which glucopyranoside is beta-bound to hydroquinone, and has a melanin-inhibiting action, and is widely used as a therapeutic agent for skin diseases and a skin whitening substance.

On the other hand, as shown in the formula (1), alpha arbutin, a substance in which sugar and hydroquinone are connected by alpha glucosidic (α-glycosidic) bonds have been spotlighted as a new whitening raw material.

Alpha-arbutin, is a tyrosinase, while the inhibitory for (tyrosinase) river stable exhibit chemical properties, in vivo in a very good tyrosinase-inhibiting effect so show a lower value of IC _50, compared with arbutin 10 times Is showing.

Alpha arbutin is synthesized through an enzymatic sugar transfer reaction in which sugar is translocated to hytroquinone. The various sugar transfer products in which sugar molecules have been transduced have improved sweetness, increased solubility, proliferation of intestinal useful bacteria, and increased chemical stability. It is known that such functionality is added.

The sugar transition reaction of hydroquinone was carried out by Nishimura et al. ( Bciluus). subtilis ) X-23-derived alpha-amylase was the first success, and there has been a report on the synthesis of alpha arbutin using alpha-glucosidase and transglucosidase.

However, maltopentaose and maltose used as substrates are required to have a high cost, and there is an uneconomical problem such that various products are additionally generated in addition to the former products.

On the other hand, amylosucrase is an enzyme that hydrolyzes sugar into glucose and fructose by using sugar as a substrate, and it is known to produce alpha-1,4-linked water-insoluble glucan using the produced glucose. .

By the way, the study of amylosucrase is mainly biased to the production of water-insoluble glucan, the research to produce alpha arbutin using it has not been specifically known yet.

Accordingly, an object of the present invention is to develop and provide a method for preparing alpha arbutin by performing an enzymatic reaction by adding amylosucrase using hydroquinone and relatively inexpensive sugar as a substrate.

In order to achieve the above object, the present invention in the reaction solution containing a sugar and hydroquinone (hydroquinone) as a substrate Deinococcus thermalis ( Deinococcus It provides a method for producing alpha arbutin, characterized in that the enzyme reaction by adding amylosucrase derived from geothermalis ).

On the other hand, amylosucrase derived from Deinococcus geothermalis used in the present invention is Neiserria polysacchareria ( Neiserria) polysaccharea ) derived from amylosucrase, while the optimum pH is 6.0, the optimum pH is 7.0, since the hydroquinone used as the substrate can be oxidized at pH 7.0, in order to prevent oxidation, It is advisable to add additional agents. At this time, the antioxidant is not necessarily limited to a specific one, vitamin C may be used as an example.

On the other hand, Deinococcus Zeolites ( Deinococcus) of the present invention Amylosucrases derived from geothermalis ) can preferably be used in which the tag for purification is bound to the N-terminus or C-terminus.

Typically, the enzyme is obtained from the transformed transformed by introducing the gene encoding the enzyme, it must be purified to recover the enzyme. In the purification process, it is very important to increase the purity of the enzyme, and a method of tagging 5 'or 3' of the gene for purification is generally used. As a tag for purification, various ones can be selected in consideration of the pH characteristics of host proteins included in the strain used as a host. When E. coli is used as a host, cationic is considered when considering the pH of the main protein in E. coli. It may be efficient to use His-tag, an amino acid tag.

On the other hand, the activity of the enzyme is often changed by tagging, as confirmed by the present invention, Deinococcus thermalis of the present invention ( Deinococcus Geothermalis ) -derived amylosukrases showed no significant change in activity even with a his-tag.

Alpha arbutin production method of the present invention by using a relatively cheaper sugar as a substrate than the conventional substrate used in the enzymatic synthesis method, it is possible to economically produce alpha arbutin with a higher inhibition of melanin synthesis and less cytotoxic than arbutin. .

Hereinafter, the contents of the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited only to the following examples, and includes modifications of equivalent technical ideas.

Meanwhile, matters not specifically described in the present invention may be sufficiently achieved through conventional genetic engineering techniques and fermentation techniques, and thus detailed description thereof will be omitted.

Preparation Example 1 : heath -tag( His - tag)  bound Daynocaucus  Zeo Themalis ( Deinococcus geothermalis Derived recombination Amylosucrase  Produce

(One) Amylosukrases Encrypted Polynucleotides  Preparation of Vectors to Include

Deinococcus geothermalis) a polynucleotide (SEQ ID NO: coding for sucrase dehydratase derived as amyl 1) was used to-day furnace separate from the chromosome gene of Lactococcus fifth sseoreu Marlies (Deinococcus geothermalis), the method was as follows.

For cloning the amylosucrase gene, chromosomal DNA of Deinococcus geothermalis DSM 11300 was used as template DNA, and the forward primer pHCDGAS1 and reverse primer pHCDGAS2 were designed with the sequence shown in Table 1, Polymerase chain reaction (PCR) was performed by the method described in Table 2.

designation Gene sequence Remarks pHCDGAS1 5'- CATATG CTGAAAGACGTGCTCACT-3 ' Forward primer pHCDGAS2 5'- CTCGAG TGCTGGAGCCTCCCCGGCGGT-3 ' Reverse primer

denaturalization
(denaturation) Pre-denaturation 95/5 min Amplification
(amplification) Denaturation 95/1 min 30 times Annealing 60/1 min Extension 72/2 min Final extension
(final extension) Final extension 72/5 min

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 the pGEM-T Easy vector treated with restriction enzymes Nde I and Xho I to prepare pGEM-T-DGAS.

Subsequently, the pGEM-T-DGAS was treated with restriction enzymes Nde I and Xho I, and a fragment of about 1.9 kbp in length was separated by agarose electrophoresis, and the pHCEXHD expression vector (His tag included) was treated with the same restriction enzyme. PHCDGAS was prepared by ligation.

(2) Preparation of transformant

In order to mass produce amylosucrase, transformants transformed with the recombinant vector pHCDGAS were prepared.

50 μl of the E. coli MC1061 strain transformed into a transformant cell was dissolved on ice for about 30 minutes, and then 1 μl of the ligation solution to which the recombinant vector pHCDGAS to be transformed was added and mixed well. Next, after the transfer to the electric shock cuvette (cuvette), the electric shock with the electric shock (Hybaid CelljecT Uno, Hybaid Ltd, UK) was transformed.

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 cultivation, strains showing resistance were first selected by plating the plate on LB agar medium containing ampicillin (100 µg / ml).

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.

Thereafter, the plasmids were separated from the recovered cells through a plasmid separation kit and cleaved with Nde I and Xho I restriction enzymes to confirm that a polynucleotide encoding about 1.9 kb of amylosucrase was included. Strains were selected.

(3) His - tag ( heath -Tag) Combined  Recombination Amylosucrase  refine

The transformants selected above were incubated at 37 ° C. for 15 hours. After incubation, the cells were recovered by centrifugation at 4 ° C. and 4,000 rpm for 30 minutes. Lysis buffer [Lysis buffer] [50 mM NaH ₂ PO ₄ , pH 7.0, 300 mM NaCl, 10 mM imidazole, pH 7.0 ], 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 into Ni-NTA affinity chromatography to purify recombinant amylosucrase.

As shown in Fig. 1, is showed a molecular weight of about 75kDa, which is the molecular weight of the sucrase azepin with purified recombinant amyl match the theoretical value, this is sucrase enzyme in recombinant E.coli-amyl (E. coli) It was confirmed that it was successfully expressed in MC1061, it was confirmed that it is efficiently purified through the Ni-NTA affinity chromatography process.

Example  One; Preparation and Purification of Alpha Arbutin

Hydroquinone and sugar used for the preparation of alpha arbutin were purchased from Sigma Chemical Co .; St. Louis, Mo. USA. After mixing 25 g / L of hydroquinone and 50 g / L of sugar in Tris-Cl buffer (pH 7.0), 10 U / ml of the recombinant amylosucrase enzyme purified from Preparation Example 1 was added thereto, and 35 ° C. Reaction was carried out for 15 hours. The solution was boiled for 5 minutes after the reaction and filtered using Whatman filter paper.

Then, the reaction product was analyzed in the following experimental example.

Example  2; Preparation and Purification of Alpha Arbutin

Hydroquinone and sugar used for the preparation of alpha arbutin were purchased from Sigma Chemical Co .; St. Louis, Mo. USA. After mixing 25 g / L of hydroquinone and 50 g / L of sugar in Tris-Cl buffer (pH 7.0), 10 U / ml of the recombinant amylosucrase enzyme purified from Preparation Example 1 was added thereto, and 35 ° C. Reaction was carried out for 15 hours.

On the other hand, in order to prevent the oxidation of the hydroquinone substrate 4 g / L of ascorbic acid (vitamin C) was added, the solution was boiled for 5 minutes after the reaction, and filtered using Whatman filter.

Then, the reaction product was analyzed in the following experimental example.

Experimental Example  One; Thin layer chromatography  Analysis of the reaction product used

Thin layer chromatography was performed to analyze the reaction products obtained in the reactions of Examples 1 and 2.

The reaction products of Examples 1 and 2 were loaded in 1 μl each of Merck 60 F ₂₅₄ TLC plates (20 cm × 20 cm) and developed in ethyl acetate: 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.

As shown in FIG. 2, the sugars were transferred to the hydroquinone in the reaction product of Example 1, in which the ascorbic acid (vitamin C) was not added, and the reaction product of Example 2, in which the ascorbic acid (vitamin C) was added, to give alpha arbutin. It was confirmed that the generated. However, when the reaction was performed by adding ascorbic acid (vitamin C) to the reaction solution, it was confirmed that a greater amount of alpha arbutin was produced than otherwise.

Experimental Example  2; High performance liquid chromatography  Analysis of the reaction product used

The reaction product obtained in Example 2 was analyzed using high performance liquid chromatography (HPLC). The reaction product obtained in Example 2 was filtered through a 0.45 µm thin film filter paper and 25 µl was injected, and the flow rate was analyzed at 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 were as shown in Figure 3, it was confirmed that the alpha arbutin was prepared.

Experimental Example  3; ' Fast Atom Bombardment Mass Spectroscopy ' Molecular Weight Measurement of Alpha Arbutin

In order to confirm the molecular weight of the reaction product obtained in Example 2 FABMS (Fast atom bombardment mass spectroscopy: JEOL JMS 700, Japan) was used. As a result of the analysis, the reaction product obtained in Example 2 had a molecular weight of 272.1 as the molecular weight of the sample from the ionic peak at 271.1 [MH] ⁻ .

This shows that the reaction product is consistent with the molecular weight of alpha arbutin.

Experimental Example  4; ^One H and ¹³ C NMR Analysis of Alpha Arbutin Using

In order to confirm the structure of the reaction product obtained in Example 2 ¹ H and ¹³ C nuclear magnetic resonance analysis (NMR; Varian, USA) was performed by comparing with arbutin. The sample was used by dissolving the purely purified reaction product in Example 2 in heavy water (D ₂ O).

The results were shown in Table 3. COSY, HMQC and HMBC experiments showed that the reaction product was alpha arbutin. Table 3 below shows the structural analysis results using ¹ H and ¹³ C NMR for arbutin and alpha arbutin according to the present invention.

1 is an electrophoretic photograph showing purified recombinant amylosucrase.

Figure 2 shows the reaction product analysis results of the present invention using thin film chromatography.

Figure 3 shows the analysis of the reaction product of the present invention using HPLC.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Method for production of alpha-arbutin using amylosucrase from          Deinococcus geothermalis <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 1950 <212> DNA <213> Deinococcus geothermalis <220> <221> CDS (222) (1) .. (1950) <400> 1 atg ctg aaa gac gtg ctc act tct gaa ctg gcg gcg cag gta cga gac 48 Met Leu Lys Asp Val Leu Thr Ser Glu Leu Ala Ala Gln Val Arg Asp   1 5 10 15 gcc ttc gat gat gac cgt gac gcc gag acg ttc ctg ctg cgg ctg gaa 96 Ala Phe Asp Asp Asp Arg Asp Ala Glu Thr Phe Leu Leu Arg Leu Glu              20 25 30 cgc tac ggc gag gac ctc tgg gag agc ctg cgc gcg gtg tat ggc gac 144 Arg Tyr Gly Glu Asp Leu Trp Glu Ser Leu Arg Ala Val Tyr Gly Asp          35 40 45 cag gtg agg gcc ttg cca ggg cga ctg ctg gaa gtc atg ctc cac gcc 192 Gln Val Arg Ala Leu Pro Gly Arg Leu Leu Glu Val Met Leu His Ala      50 55 60 tat cac gcc cgc ccc gcg gag ctg cgg cgt ttg gac gag gcc cgg ctg 240 Tyr His Ala Arg Pro Ala Glu Leu Arg Arg Leu Asp Glu Ala Arg Leu  65 70 75 80 ctg cgg ccc gac tgg ctg caa cgt ccc gag atg gtg ggc tac gtc gcc 288 Leu Arg Pro Asp Trp Leu Gln Arg Pro Glu Met Val Gly Tyr Val Ala                  85 90 95 tac acc gac cgt ttt gcc gga acg ctg aag ggg gta gag gag cgc ttg 336 Tyr Thr Asp Arg Phe Ala Gly Thr Leu Lys Gly Val Glu Glu Arg Leu             100 105 110 gac tac ctg gag ggc ctg ggt gtg aag tac ctg cac ctg atg ccc ctt 384 Asp Tyr Leu Glu Gly Leu Gly Val Lys Tyr Leu His Leu Met Pro Leu         115 120 125 ctc agg ccg cgc gag ggc gaa aat gac ggt ggc tac gcg gtg cag gat 432 Leu Arg Pro Arg Glu Gly Glu Asn Asp Gly Gly Tyr Ala Val Gln Asp     130 135 140 tac cga gcg gtg cgt ccc gac ctg ggc acg atg gat gac ctc tcg gcc 480 Tyr Arg Ala Val Arg Pro Asp Leu Gly Thr Met Asp Asp Leu Ser Ala 145 150 155 160 ctc gcg cgg gcg ctg cgg ggc cgc ggc atc agc ctg gtg ctg gat ctc 528 Leu Ala Arg Ala Leu Arg Gly Arg Gly Ile Ser Leu Val Leu Asp Leu                 165 170 175 gtg ctg aac cac gtg gcg cgc gaa cat gcg tgg gcc cag aag gcg cgg 576 Val Leu Asn His Val Ala Arg Glu His Ala Trp Ala Gln Lys Ala Arg             180 185 190 gcg ggt gat ccc aag tac cgg gcc tac ttt cat ctc ttc ccc gac cgc 624 Ala Gly Asp Pro Lys Tyr Arg Ala Tyr Phe His Leu Phe Pro Asp Arg         195 200 205 agg ggg ccg gac gct ttt gaa gcc acc ctt cct gag atc ttt ccc gac 672 Arg Gly Pro Asp Ala Phe Glu Ala Thr Leu Pro Glu Ile Phe Pro Asp     210 215 220 ttc gcg ccg ggc aac ttc tcg tgg gac gag gag atc ggt gaa ggc gag 720 Phe Ala Pro Gly Asn Phe Ser Trp Asp Glu Glu Ile Gly Glu Gly Glu 225 230 235 240 ggg ggc tgg gtc tgg acc acc ttc aac agc tac cag tgg gac ctg aac 768 Gly Gly Trp Val Trp Thr Thr Phe Asn Ser Tyr Gln Trp Asp Leu Asn                 245 250 255 tgg gcc aac ccc gac gtg ttt ctg gag ttt gtg gac atc atc ctc tac 816 Trp Ala Asn Pro Asp Val Phe Leu Glu Phe Val Asp Ile Ile Leu Tyr             260 265 270 ctc gcc aac cgg ggc gtg gag gtg ttc cgg ctg gat gcg atc gcc ttc 864 Leu Ala Asn Arg Gly Val Glu Val Phe Arg Leu Asp Ala Ile Ala Phe         275 280 285 atc tgg aag cgg ctg gga acc gac tgc caa aac cag ccg gaa gtt cac 912 Ile Trp Lys Arg Leu Gly Thr Asp Cys Gln Asn Gln Pro Glu Val His     290 295 300 cac ctc acg cgg gcg ctg cgg gca gcc gcg cgc atc gtc gcg ccc gca 960 His Leu Thr Arg Ala Leu Arg Ala Ala Ala Arg Ile Val Ala Pro Ala 305 310 315 320 gtc gcc ttt aag gcc gag gcg atc gtg gcg ccc gcc gac ctg atc cac 1008 Val Ala Phe Lys Ala Glu Ala Ile Val Ala Pro Ala Asp Leu Ile His                 325 330 335 tac ctg ggc acc cgt gcg cac cac ggc aag gtg agc gac atg gcc tac 1056 Tyr Leu Gly Thr Arg Ala His His Gly Lys Val Ser Asp Met Ala Tyr             340 345 350 cac aac agc ctg atg gtg cag ctg tgg agt agc ctc gcc agc cgg aat 1104 His Asn Ser Leu Met Val Gln Leu Trp Ser Ser Leu Ala Ser Arg Asn         355 360 365 acc cgt ctc ttt gag gag gca ctg cgg gcg ttt ccc ccc aag ccc acg 1152 Thr Arg Leu Phe Glu Glu Ala Leu Arg Ala Phe Pro Pro Lys Pro Thr     370 375 380 agc acg acc tgg ggg ctg tac gtc cgc tgt cac gac gac atc ggc tgg 1200 Ser Thr Thr Trp Gly Leu Tyr Val Arg Cys His Asp Asp Ile Gly Trp 385 390 395 400 gcc atc agc gac gag gac gcg gcc cgg gcc gga ttg aac ggc gcg gcg 1248 Ala Ile Ser Asp Glu Asp Ala Ala Arg Ala Gly Leu Asn Gly Ala Ala                 405 410 415 cac cgg cac ttt ctc tcg gac ttc tac agc ggt cag ttt ccc ggc tcc 1296 His Arg His Phe Leu Ser Asp Phe Tyr Ser Gly Gln Phe Pro Gly Ser             420 425 430 ttt gcg cgg ggg ctg gtg ttt cag tac aac ccg gtg aac ggc gac cgg 1344 Phe Ala Arg Gly Leu Val Phe Gln Tyr Asn Pro Val Asn Gly Asp Arg         435 440 445 cgc atc agt ggc tcg gcg gcc agc ctc gct ggg ctg gag gca gcg ctg 1392 Arg Ile Ser Gly Ser Ala Ala Ser Leu Ala Gly Leu Glu Ala Ala Leu     450 455 460 gaa acc ggg gac ccg ggc cgc atc gag gac gcg gtg cgt cgc ctg ctg 1440 Glu Thr Gly Asp Pro Gly Arg Ile Glu Asp Ala Val Arg Arg Leu Leu 465 470 475 480 ctc ctc cac acg gtc att ctc ggc ttc ggc ggg gtg ccg ctg ctg tac 1488 Leu Leu His Thr Val Ile Leu Gly Phe Gly Gly Val Pro Leu Leu Tyr                 485 490 495 atg ggc gac gaa ctc gcc ctg ctg aat gac tac gcc ttc gag gac gtg 1536 Met Gly Asp Glu Leu Ala Leu Leu Asn Asp Tyr Ala Phe Glu Asp Val             500 505 510 ccc gaa cac gcg ccg gac aac cgc tgg gtg cat cgc ccg cag atg gat 1584 Pro Glu His Ala Pro Asp Asn Arg Trp Val His Arg Pro Gln Met Asp         515 520 525 tgg gcc ctc gcg gag cgg gtg cgg cag gag cct tcc tcg ccc gcc gga 1632 Trp Ala Leu Ala Glu Arg Val Arg Gln Glu Pro Ser Ser Pro Ala Gly     530 535 540 cgg gtg aac acg ggc ctg cgc cac ctc ctg cgg gtg cgc cgc gat acc 1680 Arg Val Asn Thr Gly Leu Arg His Leu Leu Arg Val Arg Arg Asp Thr 545 550 555 560 ccg cag ctg cac gcc agc atc gag agc cag gtg ctg ccc agc ccc gat 1728 Pro Gln Leu His Ala Ser Ile Glu Ser Gln Val Leu Pro Ser Pro Asp                 565 570 575 tcg cgt gcg ctt ctg ctg cgc cgc gac cat ccc ctc ggc ggg atg gtg 1776 Ser Arg Ala Leu Leu Leu Arg Arg Asp His Pro Leu Gly Gly Met Val             580 585 590 cag gtg tac aac ttc agc gag gag acg gtg atg ctg ccc agc cat gtt 1824 Gln Val Tyr Asn Phe Ser Glu Glu Thr Val Met Leu Pro Ser His Val         595 600 605 ctg cgg gac gtg ctg ggg gac cac gtc cag gac cgg ctg agc ggg agt 1872 Leu Arg Asp Val Leu Gly Asp His Val Gln Asp Arg Leu Ser Gly Ser     610 615 620 gcc ttt cgc cta gat cgg ccc acc gtt cgc ctg gag ggc tac cgg gca 1920 Ala Phe Arg Leu Asp Arg Pro Thr Val Arg Leu Glu Gly Tyr Arg Ala 625 630 635 640 ctg tgg ctg acc gcc ggg gag gct cca gca 1950 Leu Trp Leu Thr Ala Gly Glu Ala Pro Ala                 645 650 <210> 2 <211> 650 <212> PRT <213> Deinococcus geothermalis <400> 2 Met Leu Lys Asp Val Leu Thr Ser Glu Leu Ala Ala Gln Val Arg Asp   1 5 10 15 Ala Phe Asp Asp Asp Arg Asp Ala Glu Thr Phe Leu Leu Arg Leu Glu              20 25 30 Arg Tyr Gly Glu Asp Leu Trp Glu Ser Leu Arg Ala Val Tyr Gly Asp          35 40 45 Gln Val Arg Ala Leu Pro Gly Arg Leu Leu Glu Val Met Leu His Ala      50 55 60 Tyr His Ala Arg Pro Ala Glu Leu Arg Arg Leu Asp Glu Ala Arg Leu  65 70 75 80 Leu Arg Pro Asp Trp Leu Gln Arg Pro Glu Met Val Gly Tyr Val Ala                  85 90 95 Tyr Thr Asp Arg Phe Ala Gly Thr Leu Lys Gly Val Glu Glu Arg Leu             100 105 110 Asp Tyr Leu Glu Gly Leu Gly Val Lys Tyr Leu His Leu Met Pro Leu         115 120 125 Leu Arg Pro Arg Glu Gly Glu Asn Asp Gly Gly Tyr Ala Val Gln Asp     130 135 140 Tyr Arg Ala Val Arg Pro Asp Leu Gly Thr Met Asp Asp Leu Ser Ala 145 150 155 160 Leu Ala Arg Ala Leu Arg Gly Arg Gly Ile Ser Leu Val Leu Asp Leu                 165 170 175 Val Leu Asn His Val Ala Arg Glu His Ala Trp Ala Gln Lys Ala Arg             180 185 190 Ala Gly Asp Pro Lys Tyr Arg Ala Tyr Phe His Leu Phe Pro Asp Arg         195 200 205 Arg Gly Pro Asp Ala Phe Glu Ala Thr Leu Pro Glu Ile Phe Pro Asp     210 215 220 Phe Ala Pro Gly Asn Phe Ser Trp Asp Glu Glu Ile Gly Glu Gly Glu 225 230 235 240 Gly Gly Trp Val Trp Thr Thr Phe Asn Ser Tyr Gln Trp Asp Leu Asn                 245 250 255 Trp Ala Asn Pro Asp Val Phe Leu Glu Phe Val Asp Ile Ile Leu Tyr             260 265 270 Leu Ala Asn Arg Gly Val Glu Val Phe Arg Leu Asp Ala Ile Ala Phe         275 280 285 Ile Trp Lys Arg Leu Gly Thr Asp Cys Gln Asn Gln Pro Glu Val His     290 295 300 His Leu Thr Arg Ala Leu Arg Ala Ala Ala Arg Ile Val Ala Pro Ala 305 310 315 320 Val Ala Phe Lys Ala Glu Ala Ile Val Ala Pro Ala Asp Leu Ile His                 325 330 335 Tyr Leu Gly Thr Arg Ala His His Gly Lys Val Ser Asp Met Ala Tyr             340 345 350 His Asn Ser Leu Met Val Gln Leu Trp Ser Ser Leu Ala Ser Arg Asn         355 360 365 Thr Arg Leu Phe Glu Glu Ala Leu Arg Ala Phe Pro Pro Lys Pro Thr     370 375 380 Ser Thr Thr Trp Gly Leu Tyr Val Arg Cys His Asp Asp Ile Gly Trp 385 390 395 400 Ala Ile Ser Asp Glu Asp Ala Ala Arg Ala Gly Leu Asn Gly Ala Ala                 405 410 415 His Arg His Phe Leu Ser Asp Phe Tyr Ser Gly Gln Phe Pro Gly Ser             420 425 430 Phe Ala Arg Gly Leu Val Phe Gln Tyr Asn Pro Val Asn Gly Asp Arg         435 440 445 Arg Ile Ser Gly Ser Ala Ala Ser Leu Ala Gly Leu Glu Ala Ala Leu     450 455 460 Glu Thr Gly Asp Pro Gly Arg Ile Glu Asp Ala Val Arg Arg Leu Leu 465 470 475 480 Leu Leu His Thr Val Ile Leu Gly Phe Gly Gly Val Pro Leu Leu Tyr                 485 490 495 Met Gly Asp Glu Leu Ala Leu Leu Asn Asp Tyr Ala Phe Glu Asp Val             500 505 510 Pro Glu His Ala Pro Asp Asn Arg Trp Val His Arg Pro Gln Met Asp         515 520 525 Trp Ala Leu Ala Glu Arg Val Arg Gln Glu Pro Ser Ser Pro Ala Gly     530 535 540 Arg Val Asn Thr Gly Leu Arg His Leu Leu Arg Val Arg Arg Asp Thr 545 550 555 560 Pro Gln Leu His Ala Ser Ile Glu Ser Gln Val Leu Pro Ser Pro Asp                 565 570 575 Ser Arg Ala Leu Leu Leu Arg Arg Asp His Pro Leu Gly Gly Met Val             580 585 590 Gln Val Tyr Asn Phe Ser Glu Glu Thr Val Met Leu Pro Ser His Val         595 600 605 Leu Arg Asp Val Leu Gly Asp His Val Gln Asp Arg Leu Ser Gly Ser     610 615 620 Ala Phe Arg Leu Asp Arg Pro Thr Val Arg Leu Glu Gly Tyr Arg Ala 625 630 635 640 Leu Trp Leu Thr Ala Gly Glu Ala Pro Ala                 645 650  

Claims (5)

Deinococcus in a reaction solution containing sugar and hydroquinone as a substrate. Method for preparing alpha arbutin, characterized in that the enzyme reaction by adding amylosucrase derived from geothermalis ) The method of claim 1, To the reaction solution, Method for producing alpha arbutin, characterized in that additional antioxidant is added to prevent oxidation of hydroquinone The method of claim 2, The antioxidant, Method for producing alpha arbutin, characterized in that vitamin C The method of claim 1, Deinococcus amylosucrase derived from geothermalis ), Method for producing alpha arbutin, characterized in that the tag for purification is bound to the N- or C-terminus 5. The method of claim 4, The tag is, Method for producing alpha arbutin, characterized in that the (His-tag)

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