KR19990084278A - Novel cyclodextrin glycosyltransferase with high conversion of gamma-cyclodextrin produced by Bacillus brevis CD162 162 - Google Patents

Abstract

The present invention is a new bacterium that produces a lot of gamma-cyclodextrin from starch. Bacillus brevis CD162 isolated and identified from domestic soil and a novel cyclodextrin glycosyltransferase produced by the bacterium ( cyclodextrin glycosyltransferase). Cyclodextrin has a unique donut-like structure, so that the inner surface of the cyclodextrin molecular cavity in the aqueous solution is hydrophobic and the outer surface is hydrophilic so as to encapsulate various organic and inorganic compounds within the cavity (Szejtli, J. Carbohydrate). Polymers. 12: 375-392.1990). Cyclodextrins, which have been noted as molecular capsules for improving various physical properties by using these clathrates, are characterized by odour masking, taste masking and removal of gourds, stabilization of volatiles, sweetening and stabilizing low-calorie sweeteners, and photolysis. It is widely used in foods, medicines, cosmetics, resin products and pesticides because it has the effect of protecting the substances and oxides, emulsification, stabilization of unstable substances, solubilization of poorly soluble substances, and powdering of deliquescent and adhesive substances. Parrish, MA cyclodextrin-a review. 42. 1393. 1989). Gamma-cyclodextrin in cyclodextrin has a large solubility due to the larger size of the cavity than alpha- and beta-cyclodextrin, so it is very advantageous for industrial use.

However, since gamma-cyclodextrin is difficult to mass-produce and the unit cost is much higher than that of alpha- and beta-cyclodextrin, there are many limitations in practical use. Accordingly, cyclodextrin glycosyltransferase, which produces a large amount of gamma-cyclodextrin developed in the present invention, may be widely used in food, medicine, cosmetics, resin products, and pesticides by solving such problems.

Description

Novel cyclodextrin glycosyltransferase with high conversion of gamma-cyclodextrin produced by Bacillus brevis CD162 162

The present invention provides a novel cyclodextrin glycosyltransferase (CGTase) having high conversion rate of gamma-cyclodextrin produced by Bacillus brevis bacteria and gamma-cyclo using the CGTase. It relates to a method of converting dextrin. Cyclodextrins are cyclic oligosaccharides composed of six or more glucopyranose units linked by α-1,4 glucoside bonds. Cyclodextrins with up to 12 glucose residues are known, but only three cyclodextrins consisting of 6 to 8 glucoses are currently produced industrially, and much research is being conducted on their use. Cyclodextrins are termed alphacyclodextrins by ⁶ glucose, betacyclodextrins by ⁷ , and gamma-cyclodextrins by 8, with cavities of 174, 262 and 427Å3, depending on the number of glucose residues. . In addition, cyclodextrin has a unique donut-like structure, so that the inner surface of the cyclodextrin intramolecular cavity in the aqueous solution is hydrophobic and the outer surface is hydrophilic so as to enclose various organic and inorganic compounds in the cavity (Szejtli, J. Carbohydrate Polymers. 12: 375-392 .. (1990)). Cyclodextrins, which have been noted as molecular capsules for improving various physical properties by using such clathrate action, are characterized by odour masking, taste masking and deodorization, stabilization of volatile substances, enhancement of sweetness and stabilization of low calorie sweeteners, It is widely used in foods, pharmaceuticals, cosmetics, resin products and pesticides because it has the effect of protecting photodegradable substances and oxides, emulsifying, stabilizing unstable substances, solubilizing poorly soluble substances, and powdering deliquescent and adhesive substances. (Parrish, MA Cyclodextrin-a review. 42. 1393. (1989)).

Cyclodextrin can be produced with several types of CGTases, but the enzymes known to date have various drawbacks such as extremely narrow pH ranges, low temperature stability, and low cyclodextrin productivity. To overcome these shortcomings, researchers are conducting a lot of research. Horikoshi (Horikoshi, K. United States US Pat. 3923598. 1975) is described in Bacillus sp. No 135, Bacillus sp. No 169, Bacillus sp. No 13, Bacillus sp. An enzyme capable of producing cyclodextrin from a wide range of pHs (pH 6.0 to 10.5) was obtained from alkalophilic bacteria such as No 17-1. Yu et al. (Yu, EKC, H. Aoki, and M. Misawa.Appl. Microbiol. Biotechnol. 28. 377. 1988) isolate Bacillus amyloliquefaciens AL 35, allowing gelatinized starch to be up to 95% of the total cyclodextrin product. An alpha-cyclodextrin glycosyltransferase was isolated that can predominantly convert to alpha-cyclodextrin. Gamma-cyclodextrin-producing enzymes are described by Kato et al. (Kato, T., and K. Horikoshi. Denpun Kagaku. 33.137.1986). Separated from 313. This enzyme produced only gamma-cyclodextrin and no alpha-cyclodextrin and beta-cyclodextrin, but showed a gamma-cyclodextrin conversion of 5% from amylose and 2-3% from starch. Despite the well-established potential of cyclodextrin in many respects, the cyclodextrin market is still struggling with two important factors. First, the production cost of cyclodextrins is high, and there is a lot of limitations in practical use. Second, the large amount of inclusions is large and the solubility is large compared to alpha- and beta-cyclodextrins. Difficult to do In fact, there are not many reports on gamma-cyclodextrin glycosyltransferase secreting microorganisms that produce only gamma-cyclodextrin. Therefore, in order to produce and supply gamma-cyclodextrin at a low price, studies on separation of excellent strains that mass-produce gamma-cyclodextrin-producing cyclodextrin glycosyltransferase and improvement of cyclodextrin glycosyltransferase productivity are important. . To solve this problem, many researchers have isolated the isolates of cyclodextrin glycosyltransferase producing strains producing high yield of gamma-cyclodextrin, cloning of genes and high expression of cyclodextrin glycosyltransferase, and economical for the cyclodextrin production. In order to satisfy the industrial applications of cyclodextrins, such as the development of phosphorus processes, we continue to research.

In the present invention, in order to actively solve the problem of the production of cyclodextrin, a strain having high gamma-cyclodextrin conversion rate based on the previously reported gamma-cyclodextrin conversion rate of cyclodextrin glycosyltransferase was sampled in soils from all over the country. As a special screening medium was searched for, the strain with the highest gamma-cyclodextrin conversion was selected and identified. In addition, by examining the enzyme production conditions of the selected strains, the produced enzymes are separated and purified to reveal the enzyme characteristics, and utilizing these investigated enzyme characteristics, there is a novelty compared to the cyclodextrin glycosyltransferase reported to date. Judging, the method for producing cyclodextrin using this enzyme was established to complete the present invention.

There is no identifier and content above.

The present invention relates to a novel cyclodextrin glycosyltransferase characterized by Bacillus brevis CD162 (KCTC8885P) and producing a lot of gamma-cyclodextrin from starch produced by the bacterium. The present invention also includes those relating to cyclodextrins produced by novel enzymes.

The present invention will be described in more detail as follows.

The present invention relates to a novel enzyme cyclodextrin glycosyltransferase and the new strain Bacillus brevis CD162, wherein the conversion of gamma-cyclodextrin from starch reaches 9.7%. In addition, the present invention can be applied to the low-cost production of gamma-cyclodextrin from starch using the enzyme produced by the above-described bacterium Bacillus brevis CD162.

First, the isolation and identification of Bacillus brevis CD162 (Bacillus brevis CD162) according to the present invention is as follows.

[Isolation of New Microorganisms]

In order to isolate microorganisms producing large amounts of novel cyclodextrin glycosyltransferases, new microorganisms producing large amounts of cyclodextrin glycosyltransferase were isolated from thousands of strains obtained from soils throughout the country.

[Identification and Naming of New Microorganisms]

1) Morphology and Culture Characteristics

As a result of Gram staining, the strains cultured in the optimal growth medium were found to be Gram-positive bacteria. As shown in FIG. 1, the cell size was 0.4-0.5 X 1.5-2 μm. ), Which is consistent with the morphological characteristics of Bacillus sp.

2) Physiological Characteristics

The physiological characteristics of the strains were positive in catalase experiments and hydrolyzed casein, gelatin, and starch.

3) Chemical Characteristics of Cells

Based on the general characteristics of Bacillus sp. (Bacillus sp.), Which is the result of the above characteristics analysis, the cell wall fatty acid composition of the cell was analyzed to determine the Bacillus sp.

4) Identification and Naming of Mycobacteria CD162

As a result of analyzing the morphological, cultural and physiological characteristics, the isolated patent strain can be confirmed in the buggy's manual [Bergey's Manua1 Systematic Bacteriology Vol 2 (William & Wilkins Co., 1989)]. However, there was no strain that accurately matched the results of analyzing the fatty acid composition of the cell wall with the data of MIDI and the metabolic diversity. However, the bacterium of Bacillus brevis was found to have the most similar result to Bacillus brevis. It is considered a variant. Therefore, the isolated microorganisms were named Bacillus brevis CD162, and as of April 22, 1998, they were deposited in the Genetic Resource Center of the Korea Institute of Science and Technology Biotechnology Research Institute, which is a depository institution for Korean patent strains, and received accession number KCTC8885P.

The present invention relates to a strain for producing a cyclodextrin glycosyltransferase having a novel characteristic and an enzyme produced by the microorganism.

Example 1 Cyclodextrin Glycosyltransferase Production by Bacillus Brevis CD162

The microorganism of this patent is enzymatic production medium. 2.O% soluble starch, O.5% yeast extract, O.5% peptone, O.1% K ₂ HPO ₄ , 0.O2% MgSO ₄ 7H ₂ O, 1 Enzyme was produced by adjusting the medium containing .0% Na ₂ CO ₃ to pH 10.2 and sterilizing for 15 minutes at 121 ° C. and then inoculating 1% of the seed culture in a flask at 30 ° C. for 3 days with the same medium composition. The titer of the enzyme produced was 0.1 ml of 0.1 M KH ₂ PO ₄ -0.2 M Na ₂ B ₄ O ₇ buffer with pH 8.0 and 0.5 ml of 10% soluble starch in 0.1 ml of enzyme solution. 0.11 mL of 0.025M BCG solution and 0.2M citrate buffer of pH 4.2 were added to measure absorbance at 630 nm, and then the amount of gamma-cyclodextrin produced was calculated based on the standard curve. The titer was 1 unit of the amount of enzyme which produces 1 mol of gamma-cyclodextrin per minute. The highest enzyme yield was obtained when the enzyme was incubated at 30 ° C for 96 hours, and the highest enzyme yield was 0.9 unit / ml.

Example 2 Purification and Enzyme Properties of Enzymes

The supernatant obtained by centrifuging the culture solution of Bacillus brevis CD162 produced according to Example 1 for 15 minutes at 12,00 g for 15 minutes was saturated with ammonium sulfate (Ammonium sulfate) at 4 ° C. to precipitate the protein, followed by dialysis. Cyclodextrin glycosyltransferase was purely separated using the Phenyl Superose HR 515 (manufactured by Phamacia, Sweden) column. The molecular weight was found to be about 74,000 Daltons as shown in FIG. This purely isolated enzyme protein was used to examine the enzymatic properties through the following experimental example.

Experimental Example 1 Determination of enzyme protein molecular weight and N-terminal amino acid sequence

The molecular weight of the purified enzyme protein was determined to be 74,000 Daltons by SDS-PAGE electrophoresis, and the N-terminal amino acid sequence was determined using the Protein / peptide Sequencer (Applied Biosystems, USA). As shown in Table 2. As shown in Table 2, the N-terminal amino acid sequence of the existing Bacillus-derived cyclodextrin glycosyltransferase enzymes showed high homology, but the N-terminus of the original cyclodextrin glycosyltransferase was identical to that of the enzyme. Since it is one of the consensus regions, the cyclodextrin glycosyltransferase produced by the Bacillus brevis CD162 of the present invention was determined to be a novel enzyme.

Experimental Example 2) Activity and Stability of Novel Cyclodextrin Glycosyltransferases Against Heat and pH

In order to investigate the effect of temperature and pH on the activity and stability of cyclodextrin glycosyltransferase, the optimum temperature was 55 ° C. In order to measure the stability to heat, the residual activity was measured after 30 minutes at each temperature. As a result, the activity was very stable up to 50 ° C, but the activity suddenly decreased above 55 ° C, but almost lost at 60 ° C or higher. Stability was shown.

As a result of measuring the activity of cyclodextrin glycosyltransferase at different pHs, the optimum pH was 8.0, and the remaining activity of the enzyme was measured after standing at various pHs for 30 minutes to measure stability against pH. It showed very stable activity in the range of .5∼9.0 and showed 83% of its activity at pH 10, but it rapidly decreased at pH 11, showing pH stability of 33%.

[Production of Cyclodextrins Using Enzymes]

Example 3 Production of Cyclodextrins Using Starch

In order to analyze cyclodextrin produced by the novel cyclodextrin glycosyltransferase, 0.1 ml of purified enzyme solution was added to 2.5 ml of 10% soluble starch, reacted at 40 ° C for 24 hours, and then treated with glucoamylase. Sugars other than the cyclodextrin produced by the complete hydrolysis with glucose (glucose) and analyzed by HPLC.

The production ratio of alpha-cyclodextrin: beta-cyclodextrin: gamma-cyclodextrin produced by the present enzyme was found to be 0.2: 4.1: 1.0, and the conversion rate from soluble starch to cyclodextrin was 1.3. %, Beta-cyclodextrin was 33.9%, gamma-cyclodextrin was 9.7%. Among the conversion rates of cyclodextrin, especially the conversion rate of gamma-cyclodextrin, which is expensive, is high when the substrate is soluble starch. The conversion of gamma-cyclodextrin by cyclodextrin glycosyltransferase produced by Bacillus subtilis No.313 (Kato, T., K. Horikoshi. Denpun Kagaku. 33 (2), 137 (1986)) was 2 ˜3%, produced by Bacillus ohbensis (Sin, KA, A. Nakamura, K. Kobayashi, H. Masaki, T. Uozumi. Appl. Microbiol. Biotechnol. 35, 600 (1991)) The conversion of gamma-cyclodextrin by cyclodextrin glycosyltransferase was 5%, and the gamma-cyclodextrin conversion by the novel cyclodextrin glycosyltransferase of the present invention was high when the conversion to gamma-cyclodextrin was compared.

As described above, the present invention utilizes a cyclodextrin glycosyltransferase produced by the new strain Bacillus brevis CD162 to easily convert alpha-cyclodextrin, beta-cyclodextrin and expensive gamma-cyclodextrin from starch. As it can be produced, it can be usefully used industrially.

Claims (2)

Bacillus brevis CD162 (KCTC 8885P) Novel cyclodextrin glycosyltransferase with high conversion of gamma-cyclodextrin produced by claim 1