JPH07183A - Production of cyclodextrin glucanotransferase produced by corynebacterium and use of the enzyme - Google Patents

Abstract

PURPOSE:To provide a process for the industrial production of CGTase acting at a relatively low temperature to produce CD and having broad receptor specificity. CONSTITUTION:This process for the production of CGTase comprises the culture of a microbial strain belonging to the genus Corynebacterium to produce CGTase and the collection of the produced CGTase from the cultured product. This invention also relates to a process for the production of CD using the enzyme and a process for the production of a sugar transfer product using the enzyme.

Description

Detailed Description of the Invention

[0001]

The present invention relates to cyclodextrin glucanotransferase (EC 2.4.1.19, hereinafter CGT).
ase), a method for producing cyclodextrin (hereinafter referred to as CD) using the enzyme, and a method for producing a glycosyl transfer product using the enzyme.

[0002] More specifically, Corynebacterium (Cory
nebacterium) belonging to the genus CGTase is cultivated, CGTase is produced in the culture, the production method of CGTase which collects this, and the CGTase is allowed to act on a solution such as starch to mainly produce β-CD. The present invention relates to a method for producing a CD to be produced and a method for producing a glycosyl transfer product by allowing the CGTase to act on a sugar donor solution in the presence of an acceptor to produce a glycosyl transfer product.

In CD, 6-8 glucose molecules are α-
It is a non-reducing maltooligosaccharide that is cyclically linked by a 1,4-glucoside bond, and it forms an inclusion compound by incorporating various substances in its molecular cavity, and the physics of the incorporated substances,
The chemical properties can be changed, so that it is possible to stabilize oxidizable compounds and photodegradable compounds, non-volatile volatile compounds, solubilize sparingly soluble compounds, and deodorize odorous substances. Widely used in medicine, cosmetics, agricultural chemicals and food.

[0004] CGTase synthesizes CD by an intramolecular transfer reaction when it acts on starch alone, but it catalyzes an intermolecular glycosyl transfer reaction when it acts on a sugar donor in the presence of a suitable acceptor.
Synthesize the glycosyl transfer product to the receptor.

Recently, various useful substances such as production of coupling sugar, improvement of solubility of hesperidin derivative and rutin, improvement of taste of stevioside and stabilization of L-ascorbic acid by utilizing intermolecular transglycosylation reaction of CGTase. And the properties of useful glycosides have been improved,
CGTases with a wide range of receptor specificity are desired.

[0006]

2. Description of the Related Art Up to now, as a CGTase-producing bacterium, a Bacillus genus has been known.

For example, Bacillus megaterium
us megaterium) CGTase (JP-A-48-40996), Bacillus macerance CGTase (JP-A-56-43212), Bacillus circulans (Bacillus c)
irculans) CGTase (JP-A-48-40996), Bacillus stearothermophilu
s) CGTase (JP-A-60-120984) and the like.

However, from the viewpoint of the intermolecular glycosyl transfer action of CGTase, Bacillus megaterium (Bacill)
us megaterium), Bacillus macerans
cerance) and Bacillus circulance
All of the CGTases of rculans) had narrow receptor specificity and were insufficient for use in the synthesis, modification of physical properties and physical properties of various glycosyl transfer products.

Further, CGTase of Bacillus stearothermophilus is L
-When rhamnose is used as an acceptor, the efficiency of transglycosylation is low,
The amount of transglycosylation products is small.

Moreover, since this enzyme is a thermostable enzyme and has a low reactivity at a relatively low temperature of 40 to 50 ° C. as compared with a reactivity at a high temperature, it is used for the synthesis and modification of substances. Was not always suitable (for example, even if one tries to use this enzyme for glycosyl transfer to L-ascorbic acid, it is difficult to use because L-ascorbic acid is unstable at high temperature). .

[0011]

DISCLOSURE OF THE INVENTION The object of the present invention is to find a microorganism having a CGTase-producing ability that acts at a relatively low temperature to produce CD and, at the same time, has a broad property of receptor specificity. And CGTa in the culture
It is to provide a method for producing CGTase that produces se and collects it.

[0012]

Therefore, the present inventors have
As a result of extensively searching for natural microorganisms having the objective CGTase-producing ability in the natural world, an attempt was made to find out that one strain obtained from soil produced the objective CGTase.

[0013] Then, while knowing that this strain is a new species belonging to Corynebacterium and that there has been no report of producing CGTase in the genus Corynebacterium up to now, the strain was cultured, and CGTase was cultured in the culture. Produce,
The present invention was completed by collecting this.

The mycological properties of the strain of the present invention, which was used in the present invention and newly discovered and isolated from soil, are as follows.

(1) Morphology Cell shape and size: polymorphic bacillus, 1.0 × 1.5 μ Motility: None Spores: None Gram stainability: positive Antiacidity: negative

(2) Growth state in each medium Meat broth agar plate culture: The growth is good, and in the initial stage, it is a pale yellow semi-transparent colony. The surface is slightly scaly.
After a few days, it becomes flat, opaque, and yellow-orange thick colonies. Since the central portion and the peripheral portion have a conical shape or a ridge-like shape, especially that portion shows a strong orange color. Broth agar slant culture: straight, soft, flat and glossy.
The whole circumference is good and the growth is good. Does not produce soluble pigment,
The moss is yellow to pale orange. A slightly alkaline broth agar with an orange color and marked growth. Broth liquid culture: Growth is slightly weak, but uniform turbidity causes membranous precipitation. Litmus milk culture: reduction / digestion

(3) Physiological properties OF test: neither fermentation nor oxidation Attitude toward oxygen: aerobic Gelatin hydrolysis: positive Casein hydrolysis: positive (weak) Starch hydrolysis: positive Tyrosine hydrolysis: negative Esculin Hydrolysis: Negative Tween 80 Hydrolysis: Negative Arginine Hydrolysis: Negative Hippuric Acid Hydrolysis: Negative Catalase: Positive Oxidase: Negative Lecithinase: Negative DNAase: Positive Urease: Negative Hydrogen Sulfide Generation: Negative Indole Formation: Negative 3 Ketolactose production: Negative (yellow) VP test: Negative Nitrate reduction: Negative Utilization of citric acid: Negative Methylene blue reduction: Negative Growth pH range: 7.0-9.4 Growth temperature range: 16-42 ° C (optimum 38 ° C ) Salt growth: 0.5-7.0% positive, 10% negative MacConkey growth: negative Sabouraud agar Growth: Presence or absence of acid production from negative sugars: glucose + L-arabinose-xylose-mannitol-lactose-trehalose + galactose + mannose-maltose + melibiose + sorbit-sucrose + salicin + (weak)

Regarding the above-mentioned mycological properties, Bergey's Man
The characteristics of this bacterium were compared with those of the ual of Determinative Bacteriology 8th edition (1974) and Bergey's Manual of Systematic Bacteriology 2nd volume (1986). Since spores are relatively thermostable bacteria that cannot be detected by staining, Corynebacterium
erium).

Furthermore, from the above-mentioned mycological properties, the present bacterium was found to be a new species of the genus Corynebacterium not described in Bergey's Manual. Therefore, this strain was designated as Corynebacterium sp. ) No.
It was named 9616.

This strain has been deposited as FERM P-13520 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

[0021] In addition, until now, Corynebacterium (Coryne
This is the first strain of bacterium belonging to the bacterium), and there is no report that it has the ability to produce CGTase.

In order to produce CGTase using this strain, the microorganism contains a nutritional source such as a carbon source, a nitrogen source, and an inorganic salt, which are required for good growth of the microorganism and favorable production of the enzyme. It is cultivated in a synthetic or natural medium.

As the carbon source, starch or its composition fraction,
Carbohydrates such as roasted dextrin, modified starch, starch derivatives, physically treated starch and α-starch can be used. Specific examples thereof include soluble starch, corn starch, potato starch, sweet potato starch, dextrin, amylopectin, amylose and the like.

As the nitrogen source, organic nitrogen source substances such as polypeptone, casein, meat extract, yeast extract, corn steep liquor or soybean or soybean meal extract, etc.,
Examples thereof include inorganic salt nitrogen compounds such as ammonium sulfate and ammonium phosphate, and amino acids such as glutamic acid.

As the inorganic salts, phosphates such as 1 potassium phosphate and 2 potassium phosphate, magnesium salts such as magnesium sulfate, calcium salts such as calcium chloride, sodium salts such as sodium carbonate and the like are used.

The culture is carried out under aerobic conditions such as shaking culture or aeration-agitation culture, and the medium is adjusted to a pH range of 7 to 11, preferably a pH range of 8 to 10, and a temperature range of 10 to 40 ° C.
It is preferable to carry out at 25 to 37 ° C., but there is no particular limitation as long as it is a condition under which the microorganism grows and the target enzyme is produced, even under other conditions.

When the culture is carried out in this manner, CGTase is usually produced in the culture medium within 2 to 7 days after the culture is started.

Then, the cells are removed from the culture broth to obtain a culture filtrate, which is desalted and concentrated with an ultrafiltration membrane, and then the enzyme is recovered by salting out with ammonium sulfate or organic solvent precipitation.

The crude CGTase thus obtained can be used as it is for the CD production reaction, but if necessary, it is further adsorbed and eluted by DEAE-Sephadex (trade name, manufactured by Pharmacia) and Sephadex (trade name, It is used after being purified by fractionation (Pharmacia).

The enzymatic chemical properties of the obtained enzyme CGTase are described below.

Action and substrate specificity: 1% soluble starch solution [10 mM acetate buffer (pH 6.0) containing 5 mM CaCl ₂ ] 20 m
Add the enzyme (2 units per 1 g of dry starch) to 1
The reaction was performed at 0 ° C., and the amount of CD produced per starch based on the raw material was measured with time. The result is shown in FIG.

The white squares in the figure show the amount of β-CD produced, the white triangles show the amount of α-CD produced, and the white circles show the amount of γ-CD produced. .

As is clear from FIG. 1, this enzyme acts on starch and mainly produces β-CD, and α-CD and γ-CD.
Also produces.

Receptor specificity: 10 mM phosphate buffer (pH 7.0) containing 10 mM CaCl ₂ containing 10% α-CD and 10% various receptors, respectively, was used as a substrate, and 5 units of this enzyme were used. In addition, the mixture was reacted at 40 ° C. for 20 hours, and the obtained glycosyl transfer product was analyzed by TLC.

At the same time, Bacillus macerans
acerans IFO 3490) -derived CGTase, Bacillus circulans IFO 3329-derived CGTase
And Bacillus stearothermophilus
For each of the CGTases (product of Hayashibara Co.) derived from arothermophilus), the enzyme activity to be added was the same, the receptor specificity was investigated, and the results were shown in Table 1 in comparison with this enzyme (however, The buffer should be 10 mM acetate buffer (p
H5.5) was used. ).

In the symbols in the table, + means that a glycosyl transfer product is produced (the amount of the glycosyl transfer product increases in proportion to the number of +), and (+) means a slight sugar. The case where a transfer product is produced is represented by −, and the case where a glycosyl transfer product is not produced is represented.

[0037]

[Table 1]

As is clear from Table 1, this enzyme produces a glycosyl transfer product with respect to the receptors D-glucose, D-galactose, D-xylose, D-arabinose, L-arabinose and L-rhamnose. Not only do D
-CGs derived from Bacillus macerans and Bacillus circulans because they also produce glycosyl transfer products for mannose, D-ribose, D-fucose and L-fucose
It is clear that there is a broader range of receptor specificity compared to Tase, and also the glycosyl transfer product for L-rhamnose is CG from Bacillus stearothermophilus.
You can also see that it is generating more than Tase.

[0039] When D-glucose, D-xylose and L-rhamnose are used as receptors and the present enzyme, CGTase derived from Bacillus macerans and CGTase derived from Bacillus stearothermophilus are used, the respective glycosyl transfer is performed. The chromatogram of the product analyzed by TLC is shown in FIG.

In the figure, B is a blank (when only the receptor is used)
X is the case of the present enzyme, M is the case of CGTase derived from Bacillus macerans, S is the case of CGTase derived from Bacillus stearothermophilus, and R is glucose and maltooligosaccharides. Indicates.

Optimum pH: The enzyme is allowed to act on a 1.5% soluble starch solution at 40 ° C. under pH conditions of pH 3 to 13 for 30 minutes,
Each activity was measured and the results are shown in FIG.

The white circles in the figure are McIlvain (McIlva
ine) buffer, open triangles are Atkins Pantin (Atkin
s & Pantin) buffer solution pH curve.
As is clear from this, the optimum pH of this enzyme is around 5-6.

Optimum temperature: This enzyme was allowed to act on a 1.5% soluble starch solution at various temperatures for 30 minutes under the condition of pH 6, and the respective activities were measured. The results are shown in FIG.

As is apparent from FIG. 4, the optimum temperature of this enzyme is around 60 ° C.

Stable pH: This enzyme solution was kept at 40 ° C. for 30 minutes under pH conditions of pH 4 to 10, and its residual activity was measured. The results are shown in FIG.

The white circles in the figure represent McIlvain (McIlva
ine) buffer, white triangles are Atkins Pantin (Atki
ns & Pantin) buffer solution stable pH curve. As is clear from FIG. 5, the stable pH range is pH 5.5 to 9.5.
Is.

Temperature stability: The enzyme solution is subjected to various temperatures at various temperatures.
At pH 6.0 [0.1M McIlvain buffer]
After being left for 30 minutes, each residual activity was measured, and the results are shown in FIG.

As can be seen from the solid line in FIG.
The residual activity was 90% at 90 ° C. The enzyme was stabilized by the addition of calcium salt and showed 100% residual activity even after treatment with 50 mM of 10 mM calcium salt (shown by the broken line in FIG. 6).

Activity measurement method: Substrate [1.5% soluble starch,
0.1M Phosphate buffer (pH 7.0)] 0.5ml to which 0.05ml of enzyme solution was added and reacted at 40 ° C for 30 minutes, then 0.1N hydrochloric acid 5m
The reaction was stopped by adding l, and 0.5 ml was taken, and 5 ml of 0.05% iodine-0.05% potassium iodide solution was added to develop color.
The decrease in absorbance at 0 nm was measured.

Under these measurement conditions, the amount of enzyme that reduces the absorbance at 660 nm by 1% per minute was defined as 1 unit.

In order to produce CD by the method of the present invention, for example, the enzyme solution (purified product or crude product) is first added to an aqueous solution containing 1 to 30% of starch (including starch or its composition fraction, modified starch, etc.). 0.5-20 units (product) (per 1 g of dry starch) is added and the enzyme reaction is carried out at pH 4-10 and temperature 20-70 ° C for 1-50 hours.

To produce a glycosyl transfer product by the method of the present invention, for example, 1 to 30% of sugar donor (including starch or its composition fraction, modified starch, CD, etc.) is first contained. 0.5 to 20 units (dry starch 1 g) of this enzyme solution (purified product or crude product) in the presence of 1 to 30% of various sugar receptors in the aqueous solution
In addition, the enzyme reaction is performed at pH 4-10 and temperature 20-70 ° C for 1-50 hours.

The present invention will be specifically described below with reference to examples, but the present invention is not limited to these.

[0054]

【Example】

Example 1 Soluble starch 1.0%, polypeptone 0.5%, yeast extract
0.25%, ammonium sulfate 0.1%, K ₂ HPO ₄ 0.05%, MgS
_{O 4 · 7H 2 O 0.025%} , the medium consisting CaCl ₂ 0.01% (pH7.0)
100 ml is put in a 500 ml Sakaguchi flask and sterilized by a conventional method, and Corynebacterium sp.
p.) No. 9616 FERM-P 13520 was inoculated and cultured at 37 ° C. for 40 hours with shaking.

After culturing, the cultured cells were removed by centrifugation,
2 L of enzyme solution was obtained, and this enzyme solution was added to 5 mM phosphate buffer (pH
After dialysis with 7.0), it was applied to an ultrafiltration membrane (Module SIP, manufactured by Asahi Kasei) to obtain 30 ml of a concentrated solution (CGTase activity was 4.6 units / ml).

Example 2 A solution of soluble starch having a concentration of 5% and 10% [10 mM acetate buffer (pH 6.0) containing 5 mM CaCl ₂ ] in 20 ml of the concentrated solution of CGTase obtained in Example 1 was added to 1.0 ml. Α-C obtained by adding a unit (per 1 g of dry starch) and reacting at 50 ° C. for 47 hours
The yields of D, β-CD and γ-CD (indicated by weight ratio to the substrate) were 16.6% and 3%, respectively, when the starch concentration was 5%.
4.2% and 7.6%, and 10% starch concentration, respectively
It was 13.8%, 27.0% and 6.6%.

Example 3 α-CD was added to 10 mM phosphate buffer (pH 7.0) containing 1 mM CaCl _2.
(10% amount) and D-glucose, D-xylose, L-
Using a substrate containing each of various receptors for rhamnose (each 10% amount), 5 units of the present enzyme was added and reacted at 40 ° C. for 20 hours, and various glycosyl transfer products were separated and obtained by TLC.

[0058]

INDUSTRIAL APPLICABILITY The present invention cultivates a microorganism belonging to Corynebacterium having the ability to produce CGTase, and prepares CGTa in the culture.
It is a method for producing CGTase which produces se and collects this, and a method for producing CD using this enzyme.

The present invention is a method for producing the present enzyme in a high yield, whereby CD can be produced at low cost, and
Since this enzyme has a wide range of receptor specificity and can produce various glycosyl transfer products, it is used in various fields such as food fields, pharmaceutical fields, and industrial fields such as the synthesis and modification of properties of substances. be able to.

[Brief description of drawings]

FIG. 1 shows the relationship between the reaction time when this enzyme is allowed to act on starch and the production amount of various cyclodextrins.

FIG. 2 shows a schematic diagram of chromatograms obtained by TLC analysis of glycosyl transfer products of the present enzyme, CGTase derived from Bacillus macerans and CGTase derived from Bacillus circulans, to various receptors.

FIG. 3 shows an optimum pH curve of this enzyme.

FIG. 4 shows an optimum temperature curve of this enzyme.

FIG. 5 shows a stable pH curve of this enzyme.

FIG. 6 shows a temperature stability curve of this enzyme.

Claims (5)

[Claims] 1. A microorganism belonging to the genus Corynebacterium having a cyclodextrin-glucanotransferase-producing ability is cultivated, and the cyclodextrin
A method for producing cyclodextrin-glucanotransferase, which comprises producing glucanotransferase and collecting the glucanotransferase. 2. A solution of starch, dextrin, amylose, amylopectin, etc. is reacted with cyclodextrin-glucanotransferase produced by the genus Corynebacterium to produce β-cyclodextrin mainly in the reaction solution, which is collected. A method for producing cyclodextrin, which is characterized in that 3. An aqueous solution containing a sugar donor is reacted with a cyclodextrin-glucanotransferase produced by Corynebacterium in the presence of an acceptor to form a sugar transfer product in the reaction solution, which is produced. A method for producing a glycosyl transfer product, which comprises collecting. 4. The sugar donor is any one of starch or a composition fraction thereof, roasted dextrin, processed starch, starch derivative, physically treated starch and carbohydrate such as α-starch, and cyclodextrin. A method for producing the glycosyl transfer product described. 5. The receptor is selected from D-glucose, D-galactose, D-xylose, D-mannose, D-ribose, D-arabinose, L-arabinose, D-fucose, L-fucose and L-rhamnose. The method for producing a glycosyl transfer product according to claim 3, which is any one of the following: