KR100800506B1 - Novel cyclodextrin glycosyltransferase specific for intermolecular transglycosylation of L-ascorbic acid-alpha-glycoside from Bacillus sp BL-12 and its application - Google Patents

Abstract

The present invention is a cyclodextrin glycosyltransferase derived from Bacillus sp. BL-12 having the intermolecular sugar transfer specificity of L-ascorbic acid, a natural antioxidant vitamin C. And relates to the production of L- ascorbic acid-α-glycoside (L-ascorbic acid-α-glycoside), which is a stable sugar transition derivative using the same. In the present invention, alkalophilic soil microbial Bacillus sp. Biel-12 strain that secretes and produces a sugar-transfer cyclodextrin glycosyltransferase, cyclodextrin glycosyltransferase enzyme, the base sequence of the gene encoding the enzyme, and using this enzyme Claims are made regarding the preparation of the intermolecular sugar transfer derivative L-ascorbic acid-α-glycoside.

     Bacillus sp. Biel-12 strain, cyclodextrin glycosyltransferase enzyme, gene sequence of the enzyme, L-ascorbic acid, intermolecular sugar transition, L-ascorbic acid-α-glycoside preparation

Description

Novel cyclodextrin glycosyltransferase specific for intermolecular transglycosylation of L-ascorbic acid-alpha-glycoside from Bacillus sp BL-12 and its application}

1 (A) is an electron micrograph of a novel soil microbial alkalophilic Bacillus sp. Biel-12 secreting the intermolecular translocation specific cyclodextrin glycosyltransferase of L-ascorbic acid, Figure 1 (B) is a classification of the above strain For identification, phylogenetic diagram prepared by analyzing 16S ribosomal DNA sequence and comparing with other microorganisms

Figure 2 is a graph showing the change in culture pattern and enzyme activity of the novel soil microbial Bacillus sp. Biel-12 of the present invention

3 is a result of verifying the molecular weight of the enzyme by protein electrophoresis by separating and purifying the molecular weight-specific cyclodextrin glycosyltransferase between L-ascorbic acid secreted and produced by Bacillus sp. Biel-12 of the present invention.

Figure 4 is a graph examining the enzyme reaction characteristics of the cyclodextrin glycosyltransferase isolated and purified in the present invention in terms of the appropriate reaction pH and temperature

Figure 5 shows the preparation of the sugar-transfer derivative L- ascorbic acid-α-glycoside using the cyclodextrin glycosyltransase of the present invention, the mixing ratio of the enzyme addition amount and the sugar donor and sugar receptor is L- ascorbic acid-α-glyco Graph showing side conversion rate

Figure 6 is a graph showing the change in sugar transfer rate during the preparation of L-ascorbic acid-α-glycoside using swelling starch as a sugar donor cyclodextrin glycosyltransferase of the present invention.

Cyclodextrin glycosyltransferase, a sugar transfer enzyme, is an intramolecular sugar transfer agent that synthesizes cyclodextrin directly from starches such as starch, dextrin, amylose, and amylopectin. Multifunctional enzyme that catalyzes the intramolecular transglycosylation reaction, the intermolecular transglycosylation reaction that transfers sugar molecules to specific sugar receptors, and the hydrolysis reaction that hydrolyzes cyclodextrins and starches. It is a widely used enzyme (Okada, S., Kitahata, S., Shiosaka, M., Bunya, H., Kubota, M., Sakai, S., Tsujisaka, Y., Denpun Kagaku (1991), 38: 211-215).

Cyclodextrin glycosyltransferase has a high molecular weight in the range of 35,000-88,000 daltons depending on the type of production strain, and generally has high activity at a temperature of 40-55 ° C. and a pH of 5.5-6.5. On the other hand, if number of the alkaline Bacillus (Bacillus) spp cyclodextrin glycosyl transferase of the resulting raised in some cases having a high activity at pH 9 and above. Also known are heat-resistant enzymes produced by Thermoanaerobacter sp., A superthermophilic microorganism (Okada, S., Kitahata, S., Shiosaka, M., Bunya, H., Kubota, M., Sakai). , S., Tsujisaka, Y., Denpun Kagaku (1991), 38: 211-215; Mori., S., Hirose, S., Oya, T., Kitahata, S., Oyo Toshitsu Kagaku (1994), 41 : 245-253).

One of the enzyme reactions catalyzed by the cyclodextrin glycosyltransferase is the intermolecular sugar transfer reaction, which is a terminal sugar molecule in a sugar donor such as starch, cyclodextrin, or maltooligosaccharide, which is a sugar or disaccharide, inositol It is an enzyme reaction that transfers sugar alcohols such as and various glycosides such as stevioside. As described above, various sugar transition products in which sugar molecules have been transferred are known to have added functionality such as improved sweetness, increased solubility, intestinal useful microbial growth effect, and increased chemical stability. In order to utilize the sugar transfer function of the cyclodextrin glycosyltransferase in various functional carbohydrate materials and new sugars, research is being actively conducted. (Miyake, T., Hijiya, H., US patent (1985), No. 4537763; Miyake, T., Hijiya, H., US patent (1986), No.4621137; Yoneyama., M., Iritani, S , Miyake., T., US patent (1996), No. 5565435).

L-ascorbic acid is the chemical name of natural vitamin C. It has many physiological functions including strong antioxidant activity, collagen synthesis promotion, and skin whitening. Therefore, it is widely used as an essential nutrient, vitamin C enhancer, food acidulant, reducing agent, antioxidant, bleach and stabilizer, and in medicine, it is used as prevention and treatment of susceptible disease such as viral disease, bacterial disease and malignant tumor. . In addition, in the cosmetic field has an excellent effect as a skin cleanser, skin whitening agent is used as a whitening cosmetic material, skin reducing agent, ultraviolet absorber and melanin production inhibitor. However, in the presence of oxygen, heat, etc., there is a drawback that it is easily oxidized and decomposed to easily lose physiological activity.

It was designed to improve the stability of L-ascorbic acid by using a sugar transfer enzyme such as alpha-amylase, cyclodextrin glucanotransferase, etc. at the carbon position 2 using L-ascorbic acid as a sugar receptor. L-ascorbic acid-α-glycoside, a sugar transition derivative made by transferring a molecule of glucose in the sugar donor. It is known to be highly stable against light, heat, oxygen and the like and maintain good physiological activity in vivo. L-ascorbic acid-α-glycoside was produced and commercialized by various sugar donors such as liquefied starch, dextrin, and cyclodextrin in Hayashibara, Japan, and its production method is Japanese Patent Publication No. 127,072 / 89 or Korean Patent No. 1001624950000.

However, for the domestic industrial production of L-ascorbic acid-α-glycoside, it is urgently needed to independently secure new cyclodextrin glycosyltransferase with high specificity for L-ascorbic acid, a sugar receptor. There is a desire to develop a process for the industrial production of acid-α-glycosides.

The technical problem of the present invention is to search for novel alkalophilic microorganisms that secrete high-potency cyclodextrin glycosyltransferase to L-ascorbic acid, and to investigate the physiological and biochemical characteristics of the detected strain and the nucleotide sequence of 16S ribosomal gene. (16S ribosome DNA sequence) is performed to classify and identify strains by performing phylogenetic analysis. It also determines the isolation, nucleotide sequence and amino acid sequence of the gene encoding the cyclodextrin glycosyltransferase secreted and produced by the isolate. In addition, the enzymatic properties of the produced alkaline microorganisms were analyzed from the viewpoint of L-ascorbic acid to L-ascorbic acid-α-glycoside in terms of sugar transition, and this was L-ascorbic acid-α-glyco, which is a heat and oxygen-stable glycoside It is used to produce the side industrially.

In order to achieve the above object, in the present invention, a novel call numbered Biel-12 by searching for alkalescent microorganisms that secrete cyclodextrin glycosyltransferase with a high glycemic potential to L-ascorbic acid from various soils in Korea. Alkaline microorganisms were isolated. Morphological, Physiological, and Biochemical Properties of Selected New Alkaloid Microbial Biel-12 and Bacillus sp. BL-12 by Analyzing the Sequence of 16S Ribosome DNA Gene Classified and identified as The strain was deposited with the patent strain on January 23, 2006 with the accession number KACC 91214P to the Korea Agricultural Microbiological Conservation Center (KACC) in accordance with the Budapest Treaty (initial deposit date).

New novel alkaline microbial bacillus biel-12 secreted and produced extracellular cyclodextrin glycosyltransferase and produced a gene encoding the cyclodextrin glycosyltransferase produced by this strain. Genes were isolated from genomic DNA and their base and amino acid sequences were analyzed.

As a result of examining the glycosides of the cyclodextrin glycosyltransferase produced by the novel alkalophilic microbial Biel-12 against various sugar receptors, the glycosides to L-ascorbic acid were excellent and L-ascorbic acid-α- It was confirmed that the glycosides suitable for glycoside preparation were enzymes.

The following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Screening and Isolation of Cyclodextrin Glycosyltransferase Production Novel Alkalophilic Microorganism Ti-1

The alkalescent soil microbial Biel-12, which secretes a large amount of cyclodextrin glycosyltransferase with high specificity of L-ascorbic acid intermolecular translocation from the soil, was newly selected. As a result of observing the form of the newly selected strain with a scanning electron microscope, as shown in Figure 1 (A) showed a long rod shape of 0.2 ~ 0.4 × 1.5 ~ 2.3 ㎛ size. The morphology, biochemical and physiological characteristics of the new isolates are shown in Table 1 according to the method presented in Bergey's Manual of Systematic Bacteriology .

TABLE 1 Morphology, Biochemistry, and Physiological Characteristics of Alkaline Soil Microbial Biel-12 Secreting Cyclodextrin Glycosyltransferase

Investigation Item result Investigation Item result Gram staining + Dihydroxiacetone + Form rod Urease ― Size 0.2 ~ 0.4㎛ × 1.5 ~ 2.3㎛ Degradation of tyrosine ― Motility motile Voges-Prokauer test ― Spore size 0.9 ~ 1.1㎛ Acid production by glucose + Spores form Ellipsoidal Indole formation ― Anaerobic growth ― H2S formation + Catalase + NaCl 5%, 10%, more than 15% + / ― / ― Hydrolysis of casein, gelatin, agar, agarose, amylose, carragenan, dextrin, and starch + Utilization of citrate, cellobiose, galactose, glucose, lactose, maltose, raffinose, ribose, and sucrose. +

L-ascorbic acid intermolecular translocation specificity New soil microbial Biel-12 secreting cyclodextrin glycosyltransferase showed high growth rate at 30-40 ° C, motility, gram positive, catalase, dehydration It was positive for dihydroxyacetone and negative for urease. Casein, gelatin, resolution to starch, formation of indole and hydrogen sulfide (H2S), and glucose, galactose, maltose, mannose and mannitol Various parties were available. Thus, the strain Biel-12 was found to belong to the genus Bacillus ( bacillus ).

In order to more clearly classify and identify new strains, 16S ribosomal DNA fragments were amplified from chromosomal dienes using polymerase amplification (Polymerase chain reaction), and then the base sequences of the amplified 16S ribosomal DNA fragments were determined. The determined nucleotide sequence was analyzed by NJ neighbor joining program and subjected to phylogenetic analysis of 16S ribosomal DNA, and the results are shown in FIG. 1 (B).

As shown in Figure 1 (B) it was confirmed that the novel alkalescent soil microbial Biel-12 is a microorganism of a new species belonging to the genus Bacillus ( bacillus ). The new strain that secretes the L-ascorbic acid intermolecular specificity cyclodextrin glycosyltransferase was named Bacillus sp. BL-12, which was deposited with the Korean Agricultural Microbial Conservation Center under accession number KACC 91214P. Deposited January 23, 2006 (initial deposit date).

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Example 2 Secretion Production and Separation Purification of Cyclodextrin Glycosyltransferase Using Novel Alkalophilic Soil Microbial Bacillus SP Biel-12

        Bacillus sp. Biel-12 selected in the present invention was selected from Horicoshi medium (1% soluble starch, 0.5% yeast extract, 0.5% peptone, 0.5% sodium chloride, 0.1% monobasic phosphate, 0.02% magnesium sulfate, 1% sodium carbonate, pH 9.5) was incubated for 48 hours shaking at 37 ℃. Figure 2 shows the change in cell mass and secreted cyclodextrin glycosyltransferase enzyme activity according to the incubation time, the enzyme activity increased rapidly up to 48 hours cultivation and reached the highest value of 980 units per liter of culture medium and then stopped I'm in.

        The crude enzyme solution was concentrated by ultrafiltration (limit molecular weight 50,000 Daltons) and dialyzed five times with 10 mM Tris-HCl buffer (pH 7.0) to hydrophobic phenylsepharose Ciel-4 ratio chromatography. The enzyme was adsorbed on (Phenyl sepharose CL-4B chromatography) and washed with 1M Trismaleic acid-NaOH buffer (pH 6.5), and then the adsorbed enzyme was 25% (w / v) ethylene glycol. eluted with (ethylene glycol). The eluted enzyme solution was concentrated by ultrafiltration with 10 mM Tris-hydrochloric acid buffer (pH 7.0), and the protein amount and enzyme activity were measured. The concentrated solution was separated again by Sephadex G-75 gel permeation chromatography (Sephadex-75 gel permeation chromatography) and purified by protein electrophoresis.

           The molecular weight of the purified cyclodextrin glycosyltransferase was measured by SD-polyacrylamide gel electrophoresis, and the molecular weight was about 70,000 daltons as shown in FIG. 3, and most of the cyclodextrin glycosyls reported so far. The molecular weight of the transferase was in the range of 64,000-75,000 Daltons.

Example 3 Enzymatic Properties of L-ascorbic Acid Molecular Transfer Specificity Cyclodextrin Glycosyltransferase

        (1) optimum reaction pH and temperature

Cyclodextrin glycosyl in 20 mM tris-hydrochloric acid (pH 6.0) buffer containing calcium chloride was mixed with cyclodextrin glycosyltransferase and soluble starch of the same activity for 30 minutes at 30 ° C to 90 ° C at 10 ° C intervals. The optimum reaction temperature of the transferase was observed and showed the highest activity at 50 ° C. as in FIG. 4 (A).

Transition specific cyclodextrin glycosyltransfer by adding 10% (w / v) soluble starch and 0.3 unit / milliliter of cyclodextrin glycosyltransferase to various pH buffers for 30 minutes at 50 ° C. As a result of measuring the activity by the addition of the raise was as shown in Figure 4 (B), showed the highest reactivity at pH 9, it was confirmed that the alkaline enzyme.

(2) substrate specificity for various sugar receptors

Table 2 shows the results of examining the sugar transfer reaction yield of the purified intermolecular sugar specificity cyclodextrin glycosyltransferase using soluble starch as the sugar donor and various glycosides as the sugar acceptor. The yield of sugar-transfer reaction for various glycosides was excellent with 16.9% of sugar-transfer reaction for ascorbic acid only. On the other hand, stevioside, one of the alternative sweeteners, was 42.3%. In naringin and hesperidin, the intermolecular sugar specificity was very low and the intermolecular sugar of L-ascorbic acid was found to be a high specific enzyme. Therefore, the sugar transition with stability to light, heat, and oxygen was identified as a suitable sugar transfer enzyme for the production of L-ascorbic acid-α-glycoside.

Table 2. Substrate Specificity for Glycodonates of Cyclodextrin Glycosyltransferase Produced by Geobacillus sptage-1

Sugar transfer receptor Transfer rate (%) Stevioside 42.3 Hesperidin N.D Rutin N.D L-ascorbic acid 16.9

(3) Substrate specificity for various sugar donors for the preparation of L-ascorbic acid-α-glycoside

Substrate specificity of the sugar donor for intermolecular sugar transfer of L-ascorbic acid using the high glycotransferant cyclodextrin glycosyltransferase derived from Bacillus sp. Biel-12 of the present invention The yield of sugar transition reaction was observed using starch and insoluble suspended swelling starch as sugar donors. As shown in Table 3, cyclodextrins and starch showed relatively high sugar transfer reactions, and especially when insoluble suspended swelled starch was used as a sugar donor, it showed the highest sugar transfer reaction yield.

Table 3. Substrate Specificity of Glycodonates of Cyclodextrin Glycosyltransferases Produced by Geobacilli Spitage-1

Type of donor Transfer rate (%) Maltose (G2) 1.0 Maltotriose (G3) 2.5 Maltotetraose (G4) 2.7 Maltoheptaose (G5) 3.3 Maltohexaose (G6) 5.2 Maltoheptaose (G7) 6.4 Alpha cyclodextrin (α-Cyclodextrin) 7.1 Beta cyclodextrin (β-Cyclodextrin) 9.7 Gamma cyclodextrin (γ-Cyclodextrin) 9.2 Dextrin 10.7 Soluble starch 12.7 Insoluble Suspension Starch 16.9

Example 4 Comparison of Other Bacillus sp. Biel-12 Derived Cyclodextrin Glycosyltransferases with Other Strains

Intermolecular Translocation Specificity Produced by Bacillus sp. Biel-12 Derived from Bacillus macerans , known for its glycemic transfer efficiency to L-ascorbic acid of cyclodextrin glycosyltransferase, and Thermoanaerobacter sp. Comparison was made with industrial cyclodextrin glycosyltransferases derived. The same starch-degrading cyclodextrin glycosyltransferase was added to the reaction mixture of swelling starch as a sugar donor and 5% and 2% (w / v) of L-ascorbic acid as sugar acceptors, respectively. The reaction was carried out to investigate the final sugar transfer yield is shown in Table 4.

      The sugar transfer yield of the cyclodextrin glycosyltransferase produced by Bacillus sp. Biel-12 of the present invention showed a significantly high value of 16.9%. At present, the sugar conversion from Bacillus maserans and Thermoan aerobacter sp., Which is known to have excellent enzyme activity, was significantly higher than the reaction yield of 8.5% and 14.5%. The cyclodextrin glycosyltransferase produced by Bacillus sp. Biel-12 of the present invention is considered to have a high industrial value for preparing L-ascorbic acid-α-glycoside.

Table 4.Comparison of sugar transfer reaction between cyclodextrin glycosyltransferase and commercially available glycotransferase produced by Bacillus sp. Biel-12

Cyclodextrin glucanotransferase Transfer rate (%) Derived from Bacillus SP Biel-12 16.9 From Bacillus maserans 8.5 From Thermoan Aerobics BSP 14.5

<Example 5> Preparation conditions of L-ascorbic acid-α-glycoside using a Bacillus sp. Biel-12-derived cyclodextrin glycosyltransferase

(1) Appropriate amount of cyclodextrin glycosyltransferase

In order to investigate the conversion rate of L-ascorbic acid-α-glycoside according to the amount of enzyme addition, 50 grams / liter of swelled starch was mixed with sugar donor and 20 grams / liter of L-ascorbic acid with sugar acceptor, and cyclodextrin, a sugar transfer enzyme, was used. Glycosyltransferase was added with 100 to 3,000 units of enzyme per gram of L-ascorbic acid followed by a sugar transfer reaction at 50 ° C for 24 hours.

As shown in FIG. 5, the sugar transfer rate continued to increase until the amount of cyclodextrin glycosyltransferase was 100 to 2,000 units per gram of L-ascorbic acid, but did not increase any more. -As much as 2,000 units per gram of ascorbic acid.

(2) Proper mixing ratio of swelling starch as sugar donor and L-ascorbic acid as sugar acceptor

To investigate the mixing ratio of the appropriate sugar donor and sugar receptor, change the insoluble suspended swelled starch from 10 grams / liter to 100 grams / liter, and change the concentration of L-ascorbic acid from 10 grams / liter to 100 grams / liter. The time-transfer reaction after time was performed. As shown in FIG. 6, when the mixing ratio of the sugar donor and the sugar receptor was 5: 2, the maximum amount reached 16.9%. Therefore, the mixing ratio of the appropriate sugar donor and sugar receptor is judged to be 5: 2 (gram starch / gram L-ascorbic acid).

Example 6 Preparation of L-ascorbic acid-α-glycoside using Bacillus sp. Biel-12 derived cyclodextrin glycosyltransferase

In the preparation of L-ascorbic acid-α-glycoside using the cyclodextrin glycosyltransferase of the present invention, the swelling starch as a sugar donor is 50 grams / liter, and the concentration of the sugar acceptor L-ascorbic acid is 20 grams / liter, The amount of enzyme addition was added in 2,000 units per gram of L-ascorbic acid, and then the conversion over time was observed for 24 hours. As a result, as shown in FIG. 6, when the reaction time reached 12 hours, the previous reaction was almost progressed and reached up to 16.9% at 24 hours after the end of the reaction time.

The novel alkalophilic Bacillus sp. Biel-12 strain secured by the present invention is judged to be a new alkalescent microorganism that secretes cyclodextrin glycosyltransferase with a very high sugar transfer activity against L-ascorbic acid. In addition, the cyclodextrin glycosyltransferase secreted by the patented strain Bacillus sp. Biel-12 (KACC 91214P) had the properties of a sugar transfer enzyme suitable for the production of L-ascorbic acid-α-glycoside. In view of these enzymatic properties, the cyclodextrin glycosyltransferase of the present invention is highly suitable for the production of L-ascorbic acid-α-glycoside, which has high stability against light, heat, oxygen, etc. and maintains good physiological activity in vivo. It is expected to be very suitable for the enzyme, so the industrial utilization is expected to be very large.

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Claims (4)

Novel Alkalophilic Bacillus SP Biel-12 ( Bacillus sp. BL-12) isolated from soil registered under KACC 91214P at the Korea Agricultural Microbiological Conservation Center for secreting and producing L-ascorbic acid-specific cyclodextrin glycosyltransferase ). A method for producing stable L-ascorbic acid-α-glycoside using L-ascorbic acid glycotransferase specific cyclodextrin glycosyltransferase enzyme produced by deposited strain KACC 91214P Bacillus sp. Biel-12. delete delete

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