DE2361878C2 - Process for the production of cyclodextrin glycosyltransferase - Google Patents

Description

The present invention relates to a method for producing the enzyme cyclodextrin glycosyltransferase using Bacillus macerans (cf. the two claims).

It has long been known that Bacillus macerans produces an enzyme commonly referred to as cyclodextrin glycosyltransferase (CGT), but which is also known as Bacillus macerans amylase, Chardinger dextrinogenase and cyclodextrin transglycosidase. This enzyme is capable of this , Strength to a number of degrade non-reducing, cyclic D-glycosyl polymers known as cyclodextrins or Schardinger dextrins. Cyclodextrins are capable of forming inclusion complexes with a variety of other molecules form, and this property enables application in a number of chemically oriented industries, z. B. in the food industry as well as in the petrochemical and pharmaceutical industries. One of the drawbacks to the widespread use of cyclodextrins in the past has been their difficulty to produce the important conversion enzyme CGT. Known methods of making CGT include the cultivation of Bacillus macerans in media containing potato pieces, potato extract, oatmeal or wheat bran. However, these methods have the disadvantage that long cultivation times are required (e.g. up to two weeks) to generate the CGT. In addition, the media used are not chemically accurate defines attempts to produce CGT in well-defined media that are free of natural starchy Materials, so far have proven to be less successful.

These problems can be overcome by the method characterized in the claims. The term "soluble starch," as it is used here, means strength produced by the action has been modified by oxidizing substances, acids, glycerine or enzymes, so that the product is in hot Water is made soluble or dispersible.

The initial concentration of the soluble starch in the liquid medium is preferably 10-20 Wt% / v.

One should preferably let the Bacillus grow as much as possible before the starch concentration drops to one The extent to which growth is restricted or no longer occurs at all, i.e. i.e. that

maximum yields of dry biomass available from the process range between 1.5 to 5.0% by weight / o / v, and accordingly the Bacillus is allowed to grow until it has a concentration of 1.5 to 5.0

Wt.- ° / o / V reached (based on the dry biomass) before the starch concentration drops to a level at

which growth is largely stopped. At this stage in particular, the CGT is generated quickly, and you should not allow the starch concentration to increase until the desired amount of CGT is present. the

CGT is generated within a period of 10 to 50 hours from the point at

which the growth of Bacillus is greatly slowed down by the drop in starch concentration.

CGT is produced in considerable quantities by the Bacillus when the growth rate increases reducing the concentration of the starch solution to a value in the range from 0.2 to 10.0% by weight / v is restricted.

Although one can apply other conditions, it has been found that a temperature of 32 to 40 ° C, for example 39 ⁰ C and a pH value of 6.5 to 7.5, for example 7.1, is according to the invention for the Appropriate procedure. The growth of the Bacillus is an aerobic process and therefore air and oxygen should be added to the process during the growth stages of the Bacillus. It is preferable to stir the medium as well.

The liquid medium preferably contains diammonium hydrogen phosphate and ammonium dihydrogen phosphate as sources for nitrogen and phosphorus, the amount of (NH ^) ₂ HPO ₄ 1 to 20 g / l and the amount of NH ₄ H ₂ PO «0.5 to 10 g / l amounts to.

The metals magnesium and calcium and the trace elements iron and manganese are preferably added in the form of their chlorides. The preferred amount of FeCl ₂ · 4 H ₂ O is 2 to 15 mg / 1 and the preferred amount of MnCl ₂ · 4 H ₂ O 2 to 20 mg / 1. The medium can also contain 0.5 to 5 mg / l ZnO.

The medium contains the vitamin biothin preferably in an amount of 0.5 to 15 μg / l and the vitamin Thiamine as Hydrochloric !, preferably in an amount of 0.1 to 2 mg / l.

A particularly suitable liquid medium for use with the erfindungsgemäöen method is one containing soluble starch, thiamine, biotin, zinc, ammonium, phosphate, sulfate, chloride, citrate and b ^r> molybdate ions as well as sources of magnesium Includes calcium, potassium, manganese and iron. This medium is new and, as such, is within the scope of the present invention.

The process according to the invention can be carried out batchwise or continuously. In the case of discontinuous operation, the liquid medium also contains a small amount, e.g. B. ö, 5 g / l, an additional

Chen, easily assimilable carbon source, ζ. Β. Maltose.

In the case of continuous operation, according to the invention, the dilution ratio should be under constant conditions preferably below 0.5 h -1, and preferably from 0.02 to 0.1 h -1 in order to achieve high yields To achieve CGT.

A particularly preferred continuous procedure consists of a two-stage modification, the Dilution rate in the second stage is lower than in the first stage, e.g. B. in the first stage 0.1 h- 'and in the second stage 0.C33 h-'

The CGT can be obtained from the liquid medium by any means. That Enzyme can e.g. B. be precipitated by using a cold organic precipitant such as acetone, or by evaporation in a vacuum with subsequent dialysis and freeze-drying.

One can use a number of methods to analyze CGT based on various properties of the enzyme. These are summarized below

1. Amylase (dextrinization) activity

This method was first described by Manning and Campbell (J. biol. Former, 1961,236,2952) and is based on the changing color intensity of the starch-iodine complex in standard starch solutions, the treated with the enzyme and compared to untreated controls. However, this method is not specific to the CGT.

2. Cyclodextrin glycosyl transferase (CT) activity

The method of Thoma et al. (Analyt Biochem, 1965, 13, 91) for measuring the activity of glycolsyl transferase is based on the ability of the enzyme, «-cyclodextrin to a non-reducing co-substrate,« -methylglucoside, to couple, whereby one receives linear «-Methyl-maltooligosaccharide, which are not recycled can. These saccharides are then hydrolyzed with pancreatic dr-amylase and the resulting sugars become measured by common methods. The method requires the absence of the enzyme cyclo-dextrinase, which the -cyclodextrin to be reduced sugars would degrade in the absence of the co-substrate.

3. Cyclodextrin synthetase (CS) activity

The method described by Tilden and Hudson (J. Bact, 1942, 43, 527) is a widely used one specific method for determining CGT. The ability of the enzyme to convert starch into cyclodextrins, is used by mixing samples with iodine solution at intervals on a microscope glass and the edges of the evaporating droplet under a low magnification microscope indicate the presence of the characteristic complex of -cyclodextrin-iodine. More practical details of the methods 1 to 3 are also described by Lane and Pirt (J. Appl. Chem. BiotechnoL, 1971, 21, 330).

4. Another and new method for determining the CS activity of CGT preparations has been developed, to replace the aforementioned method 3, which is tedious and time consuming. This method for Measurement of the ability of CGT, starch in cyclodextrins, to catalyze the hydrolysis of esters. That The procedure is as follows:

A 3.3% solution of standardized, soluble, high quality starch is made from 9 parts of 0.055 M phosphate buffer pH 6.2, and 1 part 5 χ 10 ^-3 M CaC ^ is added when the solution is cold. Per 4.5 cm ³ and 0.5 cm ³ starch solutions Stärkeiösungen and 0.5 cm ³ CGT preparation are equilibrated at 40 ° C and mixed. After 10 minutes, 1.5 cm ^{3 is} removed and mixed with 3.0 cm ³ were 0.3 m CO ₃ - ² ZHCOrPuffer, pH mixed 10.6 3 cm ³ of this mixture was placed in a cuvette (1 cm light path), and to 25 brought. 10 μΐ of a solution of 3,5-dimethylphenyl acetate or 1-naphthyl acetate (3 × 10 ~ ² m) in acetonitrile are placed in the inner part of the cuvette. The cuvette is then inverted three times as quickly as possible and placed in a registering spectrophotometer held at 25 °. The hydrolysis of the ester is then followed at 240 or 330 nm. The observed rate constant for the first order reaction (k _o ) is calculated from the expression:

where S ₀ , S, and S _{1x are} absorption immediately after addition of the ester after t minutes or at the end of the reaction.

The calculation is repeated for five values of 5, and t , whereupon the average value for k _o is obtained. The first order constant for the base rate of hydrolysis of the ester in the absence of cyclodextrin (k _u ) is measured by replacing CGT with distilled water in the mixture. The value

for -—— was then calculated. The concentration of cyclodextrins [CD] in the cuvette is then obtained from a calibration curve _k _ _k versus T ^ ™. which is formed by using solutions of dried, chromatographically pure -cyclodextrin from 10 ~ ² m to 5 χ \ 0 ~ < in in 3% Van Linge starch. The unit of CS activity is defined as that which produces a total of 1 m of α-cyclodextrin under the test conditions.

The process according to the invention is further illustrated in the following examples.

Example 1 (submitted later) Production of the vaccination culture (inocula)

Bacillus macerans NCIB 9368 was placed on nutrient glucose agar and incubated at 37 ° C. for 2 days. About five colonies were placed on a medium consisting of 1.5 g of crushed wheat and 0.6 g of calcium carbonate in 45 cm ^{3 of} water, incubated at 37 ° C for 5 days, and then stored at room temperature for use as a stock culture.

One breeds «! Inocula from the stock culture either in a conical 250 or 2000 cm ³ flask containing ίο 10 cm ³ or 100 cm ^{3 of} the medium. The stock culture (5% v / v) was added to the flask, which was incubated at 37 ° C on a rotating shaker at 200 rpm. The culture was then used as a vaccine (5% v / v) for the cultures of Bacillus.

Example 2 Breeding of B. macerans

The growth medium used in the following cultivations of B. macerans is called DM and has the composition given in Table I.

a) Discontinuous breeding

A CeCaw fermenter apparatus with an operating volume of 2 liters was filled with the medium which had been inoculated with a culture of B. macerans. The temperature during the cultivation was kept at 39 ° C and the pH at 7.1 by adding 1 M H ₂ SO ₄ or 3 or 5 M NaOH.

The culture was started by stirring at 1200 rpm and blowing air into the upper space of the fermentor of 600 cmVmin ventilated. Foaming was suppressed by adding polypropylene glycol

b) Continuous (chemostat) breeding

These were carried out under similar conditions as the discontinuous cultivations with the Except that the operating volume of the fermenter was 1.25 liters and the air was at one speed of 1 l / min was introduced. The dilution ratio used was 0.05 or 0.03 h ~ '.

Table I.

c) Two-stage (chemostat) breeding

Two fermenters of the »porton type« with working volumes of 1 liter and 3 liters were used for the first and second stage applied The fermentor of the first stage was stirred at 1200 rpm and rotated at a speed

aerated rate of 2 l / min. The air was also used to transport the culture from the first to the second stage. The DM medium was added to the first stage in two separate streams, one solution containing the phosphates and K ₂ SO * and the other containing the starch, vitamins and inorganic salts. The second fermentor was stirred at 900 rpm and the dilution ratio in the second stage was determined by monitoring the flow; the medium in the first stage and the flow of culture from the first stage to one

Waste container regulated The difference between these two resulted in the amount that went through in the second stage the airflow is brought. The conditions of pH, temperature, and foam control have already been set described in (a) above.

For each of the above cultivars, the culture was tested for CGT activity by one or more of the

Composition of DM Amount per liter (NH ₄ I ₂ HPO ₄ 5.0 g NH ₄ H ₂ PO ₄ 2.0 g K ₂ SO ₄ O ^ g MgCl ₂ · _{6H 2} O 03 g CaCl ₂ · 2 H ₂ O 0.5 g Citric acid (adjusted to pH 7 with 1.3 g NaOH) Soluble starch 6-3Og Maltose (only for discontinuous 0.5 g Cultures) Thiamine HCl 2 mg Biotin 15 µg ZnO 4 mg MnCi ₂ · _{4H 2} O 15 mg FeCl ₂ · 4 H ₂ O 10 mg (NH ₄ J ₂ MoO ₄ · 2 H ₂ O 2 mg

methods described above. In addition, the concentration of the dry biomase and the Determined residual starch concentration.

a) The results obtained in typical discontinuous cultivations are shown in FIGS. 1 to 3 specified. These show the changes over time, the yield of dry biomass, residual starch concentration and CGT (amylase) activity in a number of cultivars using starting con - ■> strength concentrations of 6, 10, 20 or 30 g / l.

The following points emerge from these results.

1) Biomass yields using this medium do not exceed around 5 g / l,

2) Substantial CGT activity does not occur until the starch concentration of the medium is low Level has been reduced,

3) the greatest CGT activity is observed using an initial starch concentration of 20 g / l.

b) The results of the chemostat cultivations at different dilution ratios using an initial concentration of 10 g / l are shown in FIG. 4 compiled. One can clearly see that the CGT activity increases rapidly when the dilution ratio is reduced below 0.5 h ~ '. The figures in Table II shows the effect of various initial strength concentrations on CGT activity in Chemostat cultures and evidences the greater CGT activity that comes with the greater initial starch concentration connected is.

c) The results of the typical two step chemostat cultivations are given in Table III. D \ and

Eh mean the dilution ratios in the first and second fermentors, and F shows an additional |

Feeding starch at a rate of 0.08 g / g dry biomass χ h to the second stage. The following conclusions can be drawn with reference to the results in Tables II and III.

1) When working in the first stage at a dilution ratio that gives the maximum biomass productivity results (g biomase / I χ h), CGT activities are negligible in the second stage generated,

2) the CGT activity is increased by 57% for Ο, = 0.1 h- 'and £ »2 = 0.033 h-' when additional starch is added takes place in the second stage.

Table Il

; s- Initial starch concentration g / l

Example 3 Extraction of CGT from the supernatant solution from the culture

50 cm ^{3 of} a solution with 10 g of Na ₂ HPO * and 4 g of KH ₂ PO *, which is adjusted to a pH of 7.0, were added to one liter of the supernatant solution from the culture at 2 °. The solution was cooled to -3 ° C and stirred vigorously while 1.5 volumes of cold acetone (2 ° C) containing acetic acid were slowly added. The amount of acetic acid, which was determined in a test precipitation, was sufficient to push the pH down to 6.5 to 6.6 at the end. The precipitate was allowed to settle for 10 minutes. The clear liquid was pressure tested and the precipitated fraction by brief centrifugation at 2 ° gesammelL The precipitate was washed three times with 10 cm ³ of 0.01 M phosphate buffer of pH 6.2 extracts the 5 χ 10 ~ ⁴ jn CaCl ₂ and The extract was dialyzed at 2 ° against the same buffer. Yields of up to 91% were obtained.

Dilution
relationship
(n- ¹ ) Initial starch concentration g / l
10
Biomass (g / l) amylase CS
Activity activity 13th
Biomass (g / l) Amylase
activity CS
activity 0.03
0.05
Tabel 3.6 270 26.3
III 3.6
4.3 660
416 66.6 step Dilution ratio
(h- ¹ ) Elapsed time
(0 CGT activity
(Amylase)

1 D ₁ = O ₁ I. 118 192 2 D ₂ = 0.033 118 204 2 D ₂ = 0.033 F 269 320 1 D ₁ = 0.5 394 0 2 D ₂ = 0.033 394 50 2 D, = 0.033 F 486 17th

For this purpose 4 sheets of drawings

Claims (2)

Patent claims: 1. A method for producing cyclodextrin glycosyltransferase (CGI} by growing Bacillus macerans, characterized in that Bacillus macerans is grown in a liquid medium tet, the initially 5 to 30 wt .-% / V of a soluble starch as a carbon source, one or more It contains up to sources of nitrogen and phosphorus, essential minerals and the vitamins biotin and thiamine he has reached a concentration of 1.5 to 5.0 wt .-% / V (based on the dry biomass), then the The starch concentration in the medium falls to a level at which the growth of the Bacillus macerans is largely stopped, then the Bacillus macerans the formation of CGT within a period of 10 is allowed up to 50 hours and the CGT is then obtained from the liquid medium 2. The method according to claim 1, characterized in that the starch concentration is reduced to a level in Can drop in the range from 0.2 to 10.0% by weight / V.