JPS6345800B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing a cyclodextrin-glucosyl-transferase starch adsorbent by adsorbing cyclodextrin-glucosyl-transferase on treated starch treated in an aqueous medium at an initial starch gelatinization initiation temperature. . As for cyclodextrin glucosyl transferase, for example, cyclodextrin glucosyl transferase produced by Bacillus magerans is well known.
Published in 2015, WS Hale, L C Rawlins, Cereal
Chem., 28, 49 (1951), S. Schwimmer, J.A.
Garibaldi, Cereal Chem., 29, 108 (1952), Japanese Patent Publication No. 56-43212, etc.) act on starches such as horse starch, corn starch, and small horse starch, or their processed starches, to produce 6 glucose molecules. It is a useful enzyme that produces cyclodextrins such as α-cyclodextrin consisting of 7 glucose units, β-cyclodextrin consisting of 7 glucose units, and γ-cyclodextrin consisting of 8 glucose units. Conventionally, in order to obtain cyclodextrin by applying the enzyme to starch, etc., which is a raw material, the enzyme has been used as it is, or it has been used as an adsorbent of the enzyme which has been adsorbed onto raw starch or cooked starch. However, when using the enzyme as it is, there is a problem of contamination with impurities, and it is necessary to purify the enzyme. It has come to be. However, when using raw starch to obtain this adsorbate, the adsorption efficiency of the enzyme is poor;
Furthermore, in the case of steamed starch, it is extremely difficult to separate and collect it due to the gelatinization phenomenon during steaming. The present inventors conducted various studies on useful cyclodextrin glucosyl transferase adsorbates for cyclodextrin production, and found that, quite unexpectedly, treated starch treated at the initial gelatinization starting temperature of starch By adsorbing cyclodextrin glucosyl transferase, it has a high titer enzyme activity, and the adsorbed material adsorbs the enzyme extremely well, so there is no loss of the enzyme, which is extremely good. I learned that. Furthermore, in particular, the treated starch is preliminarily treated with cyclodextrin, glucosyl,
It has been found that by using treated starch treated with transferase, the hypersensitivity of the raw material treated starch has been improved and a better adsorbent can be obtained. The present invention has been completed based on the above findings, and is characterized by adsorbing cyclodextrin glucosyl transferase to treated starch treated in an aqueous medium at the initial gelatinization initiation temperature of starch. dextrin glucosyl
This is a method for producing a transferase adsorbate. First, the treated starch used in the present invention may be a treated starch obtained by heating starch such as horse starch, corn starch, wheat starch, etc. at each initial gelatinization start temperature. In addition, the initial gelatinization start temperature for each starch varies somewhat depending on the type of starch, but for example, the gelatinization temperature for horse starch is approximately
56~66℃, corn starch gelatinization temperature about 62~72
℃, the gelatinization temperature of wheat starch is about 52 to 63 degrees Celsius, etc., and the initial start temperature of each gelatinization temperature is preferably about 55 to 58 degrees Celsius. The initial gelatinization starting temperature of corn starch is about 60 to 63°C, and the initial gelatinization starting temperature of wheat starch is about 50 to 53°C. In addition, in obtaining this treated starch, for example,
A 20W/V% starch dispersion aqueous solution is prepared and treated at the initial gelatinization starting temperature of each starch, or starch is dispersed in an aqueous solution kept at the initial gelatinization starting temperature in advance and maintained at a constant temperature. Furthermore, the treatment time is usually 2 hours or more, preferably 8 hours or more. In order to obtain a more suitable treated starch,
This treated starch may be treated with cyclodextrin glucosyl transferase, and in this enzyme treatment, the enzyme may be added in advance to either the starch or its aqueous dispersion; The amount of enzyme used is usually 100u per 100g of starch (Tilden Hudson
unit) or more, preferably 400~500u
used to some extent. In addition, the enzyme is used in the following ways:
The enzyme may be used as it is, in a solution form, or as an enzyme adsorbent adsorbed on an adsorbent such as starch. Further, the treatment conditions for the enzyme may be the same as the treatment conditions at the initial gelatinization start temperature. Furthermore, the amount of these treated starches to be used is 0.5 to 5.0% based on the water content of the aqueous medium used and the cyclodextrin glucosyl transferase-containing solution described below.
It may be used as a concentration of a 1% solution, especially in order to obtain a high titer adsorbent, it is better to use a 1% solution or less, and in order to adsorb the enzyme without loss, it is recommended to use a solution at a temperature of about 3 to 5 °C. Use is preferred. Next, the desired cyclodextrin glucosyl transferase is added to the thus obtained treated starch or the starch treated with the cyclodextrin glucosyl transferase action, and is adsorbed. Dextrin glucosyl transferase may be any enzyme that has the ability to produce cyclodextrin from starch or the like, such as Bacillus magerans, alkalophilic Bacillus, Bacillus megaterium, Bacillus circulans, Bacillus spp. Examples include cyclodextrin glucosyl transferase produced by S. stearothermophilis and the like. Generally, it is used as a cyclodextrin glucosyl transferase-containing solution, which is the culture solution. Furthermore, for adsorption, in an aqueous medium, preferably an aqueous solution under cooling at 10°C or less,
It is sufficient to stir it for about one night. Furthermore, the cyclodextrin glucosyl transferase starch adsorbate thus obtained can be easily separated from the aqueous medium by solid-liquid separation means such as filtration or centrifugation, and the target adsorbate obtained can be If necessary, it may be dehydrated and washed with ethanol etc. and stored in a cool place. The cyclodextrin glucosyl transferase starch adsorbate of the present invention obtained in this manner is a starch adsorbate that improves the loss of the enzyme and has a high titer, and is particularly preferable as a method for producing the same. . Furthermore, the adsorbate of the present invention thus obtained is usefully used as an enzyme for producing cyclodextrin. EXAMPLES Next, the present invention will be specifically described with reference to Examples, but the present invention is not limited by these in any way. Example 1 (Experiment 1) 100 ml of water was added to each 20 g of horse starch, and cyclodextrin glucosyl transferase (enzyme titer 450/ g of cyclodextrin glucosyl transferase starch adsorbate
0.2g) was added, and each treated starch was heated at each temperature and time shown in Table 1 to obtain each treated starch. Seitz filtration was performed to collect the starch, and the elapsed time and amount recovered were measured. . Then this treated starch 0.5
g (40% water) to 50ml (20U/
ml; enzyme from Bacillus magellans), stirred overnight at 5°C, and filtered to obtain cyclodextrin.
Glucosyl transferase starch adsorbate and its liquid were obtained. As a result, the results of the heating treatment at each temperature and time were as shown in the first table. Also, enzyme titer (U/g) of cyclodextrin glucosyl transferase starch adsorbent
was calculated according to the following formula. Enzyme titer (U/g) = [20U/ml - (liquid enzyme titer/ml)] x 50ml/0.5g
×100−moisture content rate/100〕

[Table] (Experiment 2) Add 100 ml of water to each 20 g of Mareishiyo starch, heat it at each temperature and time shown in Table 2 to obtain each treated starch (40% moisture), and then apply this treatment. Using 0.5 g of starch, the same procedure as in Experiment 1 above was carried out to separate the starch adsorbed material and the liquid. The above measurement results were as shown in Table 2.

[Table] **: Cannot be used due to gelatinized state that cannot be filtered.
(Experiment 3) The treated starch (moisture
40%) was used in each amount shown in Table 3 below, and the adsorption ability of cyclodextrin glucosyl transferase was measured for each amount used. For cyclodextrin glucosyl transferase, use 50 ml of 20 U/ml enzyme solution.
The enzyme titer of the starch adsorbate was calculated according to the calculation method described above. The results were as shown in Table 3.

[Table] From the above experimental results, the gelatinization temperature of Mareishiyo starch is approximately 56-66℃, the initial gelatinization start temperature is 55℃,
The enzyme-starch adsorbent made using treated starch obtained by processing at 57°C has better adsorption of enzymes than that of treated starch at a temperature lower than that temperature, and especially for cyclodextrin, glucosyl, Starch treated with transferase action is better and does not show gelatinization,
Furthermore, as is clear from Experiment 3, in order to obtain a starch adsorbent with a high titer, it is preferable to use less than 1.0 g of treated starch, that is, less than 1% solution, and in order to make almost all of the enzyme adsorbed to the treated starch. It has been found that it is preferable for the treated starch to have a solubility of 2.0 g or more, that is, a solubility of 4% or more. Example 2 (Experiment 1) Using corn starch instead of horse starch in Example 1, the following experiment was conducted in the same manner as in Experiment 1 in Example 1 to determine the enzyme starch adsorption under each treatment condition (temperature, time). The enzyme titer of the sample was measured. The results were as shown in Table 5.

[Table] From the above results, it can be seen that the enzyme-starch adsorbent obtained by using the starch treated with the treatment condition of the initial gelatinization start temperature of 62°C according to the present invention at the gelatinization temperature of corn starch of about 62 to 72°C has It showed good adsorption properties with good titer. Example 3 (Experiment 1) The same procedure as in Experiment 1 of Example 1 was conducted using wheat starch instead of the horse starch of Example 1, and the adsorption of the enzyme starch was determined under each treatment condition (temperature, time). The enzyme titer of the sample was measured. The results were as shown in Table 6.

[Table] ***: Unusable due to gelatinized state From the above results, the gelatinization temperature of wheat starch is approximately 52 to 63
The enzyme-starch adsorbent using the treated starch according to the present invention, which had an initial gelatinization starting temperature of 52° C., exhibited a significantly high titer and good adsorption properties. Example 4 (Preparation of cyclodextrin glucosyl transferase) Seed culture medium 20 consisting of 3% bran, 0.5% ammonium sulfate, 0.5% calcium carbonate, and 0.1% dehydrator was added to 30 volumes of jar fermenters (2 bottles). )
plus Bacillus macerans Schardinger IFO3490
[Deposited as NRRLB-388 at the Agricultural Research Culture Collection, USA] was inoculated and cultured with aeration at 40°C for 13 hours. Next, this culture was added to medium 1200 (2 ton tanks, 2 bottles) consisting of the same composition as above, and 40
Cultured at ~°C for 52 hours. After culturing, each tank culture was combined, cold water (5°C) and supernatant were added, and filtered through a drum filter to give a concentration of 20 U/ml.
A solution containing cyclodextrin glucosyl transferase was obtained. (Adjustment of treated starch) In a 500 volume tank, 56℃ water 400℃ (55~56℃
(approximately 56℃ constant), 100kg of horse starch (moisture content)
17.5%) and 450,000 U of cyclodextrin glucosyl transferase (1 kg of 450 U/g oxygen starch adsorbent) were added and stirred for 16 hours at a constant temperature of approximately 56°C, then taken out in a basket and washed with water. The treated starch obtained was then stored under refrigeration. (Preparation of cyclodextrin-glucosyl-transferase starch adsorbent) The cyclodextrin-glucosyl-transferase-containing solution obtained above was put into a 3-ton tank, and the above-mentioned treated starch was added thereto. The mixture was kept at 5° C. and cooled and stirred overnight to allow adsorption. Thereafter, this was passed through a basket to remove the produced cyclodextrin glucosyl transferase starch adsorbate, and then heated at 5°C.
Rinse with cold water of 2,000 ml, and then add alcohol
16 (twice) to obtain the target cyclodextrin glucosyl transferase starch adsorbate 121.1Kg (380U/g, dry weight 70.8Kg,
An enzyme titer of 1.1 U/ml) was obtained.

Claims (1)

[Scope of Claims] 1. A cyclodextrin glucosyl transferase characterized in that the cyclodextrin glucosyl transferase is adsorbed onto treated starch treated in an aqueous medium at an initial temperature for starch gelatinization. Method for producing starch adsorbent. 2. The treated starch treated at the initial starch gelatinization initiation temperature is the treated starch treated at the initial starch gelatinization initiation temperature of cyclodextrin glucosyl transferase action. Manufacturing method.