KR900003746B1 - Method for purification of cyclo dextringlycosyl transferase - Google Patents

Abstract

A method for purifying cyclodextrin glycosyltransferase comprises (a) conjugating alpha-cyclodextrin to Eupergit-C with acetate buffer soln. (10 mM, pH 5.5) at 4 deg. C and filling into a column, (b) passing cyclodextrin glycosyltransferase soln. through the column to conjugate, and (c) desorbing with cyclodextrin mixt. aq. soln.. The cyclodextrin glycosyltransferase is useful for preserving vitamines, deodoring food smells.

Description

Method for Purifying Cyclodextrin Glycosyltransferase

The present invention relates to a method for purifying cyclodextrin glycosyltransferase (hereinafter referred to as CD enzyme).

CD enzyme is an enzyme that can be obtained by culturing Bacillus macerans, B. megaterium, B. circulans, and the like. S. Kitabata et al., J. Jap Soc. starch Sci., vol 29, No. 1, 13-18 (1982), and cyclodextrins (α, β, r) are synthesized from starch. Cyclodextrin (hereinafter referred to as CD) is a non-reducing maltooligosaccharide in which 6-12 glucose molecules are cyclically bound by a-1,4-glucoside bonds, and industrially useful are 6,7,8 alpha, β, r-CD. CD uses include stabilization of medicines and vitamins that are easily affected by heat and air, prevention of volatilization such as fragrances, deodorization of food, stabilization of emulsifiers and active ingredients in cosmetics, improvement of pesticide properties, and chemical reaction catalysts. have. General Method of CD Enzyme Purification S. Kobayash et al, Carbohydrate research, 61, 229-238 (1978), adsorbed the CD enzyme of supernatant obtained by centrifugation of the culture onto starch (24 ° C. 24 hours) and washed with 30% ethanol. The CD enzyme was desorbed from the starch obtained by centrifugation with a buffer solution. The resultant was filtered to remove the starch, and then pure CD enzyme was obtained through ion exchange chromatography and fractionation chromatography. Therefore, this method takes more than 72 hours and recovery rate is less than 60% is difficult to effectively purify.

The present inventors have studied to purify CD enzymes in a short time, and as a result, the binding of alphacyclodextrin (α-CD), which is a substrate of CD enzymes, to oxirane of Eugergit-C (Rohm Pharma, Darmstadt) In a single process, more than 90% of pure CD enzymes could be recovered in a single step. Testing of a matrix suitable for binding to α-CD showed that Eugene-C. Sepharose-4B (pharmacia) and Sepharose-6B The reaction conditions were simpler than that of the pharmacia, resulting in a faster flow rate and a shorter regeneration time when used repeatedly.

The whole process of purification is carried out at 4 ℃, equilibrate the column with affinity gel, fill with 10 mM acetic acid buffer solution (PH 5.5), and pass through the enzyme solution. CD enzyme binds to α-CD of the substitution gel. And other proteins are released. After confirming that all other proteins were lost by UV detector (278nm), equilibrate with 10mM acetate buffer solution (PH 5.5), and pass CD mixture aqueous solution through the column. Purification takes place.

The CD enzymes obtained by culturing (B. macerans), (B. megaterium), and (B. circulans) according to the present invention were purified in a short time with the Eufer-C + α-CD affinity gel. After removing the heterologous protein by selectively attaching only CD enzyme to the affinity gel, an α-CD solution or a CD mixture solution may be used to desorb the CD enzyme from the affinity gel. The α-CD is required to be above a certain concentration and the amount depends on the amount of CD enzyme attached to the affinity gel. Determination of enzyme activity of CD enzymes includes iodine method, glucoamylase method and cyclodextrin-trichloroethylene precipitation method. N. Nakamura et al., Agr. Biol, chem., 40 (5), 935-941, (1976) One unit of enzyme of the iodine method is defined as the amount of enzyme that causes a 10% decrease in absorbance at 700 nm for 1 minute at 40 ° C. One unit of glucoamylase is defined as the amount of enzyme that produces lumole glucose for one minute at 40 ° C. CD enzyme solution with total activity of 11,000 units and inactive 11 units / mg was purified using conventional methods such as starch adsorption, desorption, ion exchange chromatography, and fractionation chromatography. 3,850 units were recovered, recovering 35% and inactive 770 units / mg. The fold increased 70-fold, but 9920 units were recovered using the Purified-C + α-CD matrix, yielding a 90-fold recovery and a 100-fold increase in inactive 1100 units / mg. The following examples further illustrate the present invention, but the present invention is not limited thereto.

Example 1

Enzyme solution was prepared using B. macerans (KFCC 32408) and B. megaterium (KFCC 32320) in a medium containing 1.5% starch, 1.5% bact-peptone, 0.025% calcium chloride, 0.5% potassium chloride, and 0.5% potassium phosphate. The inoculum was incubated at 1% and maintained at 30 ° C. for 24 hours. The supernatant was centrifuged to prepare an enzyme solution. In the case of B. macerans, the protein amount of the supernatant was 10.2 mg / ml, the enzyme activity was 120 units / ml. In the case of B. megaterium, the protein amount was 4 mg / ml and the enzyme activity was 70 units / ml. Enzyme activity was measured by glucoamylase and protein determination by Lowry method.

Example 2

Preparation of the Matrix

(1) Euferzil-seed + α-CD 0.1N sodium hydroxide solution 100ml of euferzil-seed, 0.5gr, α-CD 0.5gr and reacted at 45 ℃ for 16 hours, filtered and washed with distilled water to buffer 10mM acetic acid Add 100 ml of solution (PH 5.5).

(2) Sepharose-4B + 1,4-butanediol diglycidyl ether + α-CD (PDG Dean et al., Affinity Chromtography, IRL Press, 1985) sodium borohydride concentration adjusted to 2 mg / ml To 100 ml of 0.6 N sodium hydroxide solution, add 5.0 ml of dried Sepharose-4B and 5.0 ml of 1,4-butanediol diglycinyl ether, and react for 8 hours at 25 ° C. After filtration and washing, 100 ml of 0.1 N sodium hydroxide solution was added. After adding to 0.5gr of α-CD, and reacted at 45 ℃ for 16 hours, washed with distilled water and put into 100m1 of 10mM acetic acid buffer solution (PH 5.5).

(3) Sepharose-6B + 1,4-butanediol diglycinyl ether + alpha -CD It is manufactured by the same method as Sepharose-4B.

Enzyme solution prepared by culturing B. macerans (KFCC 32408) by filling columns (1), (2) and (3) with a diameter of 1 cm and a height of 12 cm (protein amount of 10.2 mg / ml, enzyme activity 120 units) / ml) and B. megaterium (KFCC 32320) incubated with 100ml of enzyme solution (protein amount 4.0mg / ml, enzyme activity 70units / ml) through the column to attach CD enzyme and α-CD The enzyme was purified by passing 50 ml of 3% aqueous CD solution, and the recovery rate was 90% and fold 100 times (1), (2) and (3). The flow rate of (1) is 2.0 ml / min, and the flow rates of (2) and (3) are 0.5 ml / min or less, and (1) is the most economical in the required time.

Euferzyl-C + α-CD (hereinafter referred to as 1), Sepharose-4B + 1,4-butanediol diglycidyl ether + α-CD (hereinafter referred to as 2), Sepharose-6B + 1,4- As a result of continuous use of butanediol diglycidyl ether + α-CD (hereinafter referred to as 3), (1) can be used continuously up to three times, but (2) and (3) should be regenerated after one use and the play time ( 1) was enough for 1 hour, but (2) and (3) took more than 2 hours and were unstable.

Example 2

Example 2,3 but using a CD mixture aqueous solution having a viscosity of 200-1500cps at 4 ℃ in place of α-CD aqueous solution, the results were similar to those of Examples 2,3. (Α-CD is a very expensive material, so Example used a low-cost aqueous solution of CD mixture prepared by the general enzyme reaction)

Claims (2)

Bind α-cyclodextrin to Euphorgit-C using 10 mM acetic acid buffer solution (PH 5.5) at 4 ° C, fill the column, and pass cyclodextrin glycosyltransferase solution to cyclodextrin glycosyl A method for purifying cyclodextrin glycosyltransferase, characterized in that the transferase is combined and then desorbed with an aqueous solution of a cyclodextrin mixture. The method for purifying cyclodextrin glycosyltransferase according to claim 1, wherein the aqueous solution of cyclodextrin glycosyl mixture has a viscosity of 200-1500 cps at 4 ° C.