JP5356704B2 - Method for producing disaccharides containing rare sugars - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a rare-sugar-bound disaccharide with at least one rare sugar as a constituent monosaccharide. <P>SOLUTION: The method is characterized by comprising producing the objective rare-sugar-bound disaccharide by using a disaccharide as the raw material (substrate) and converting a monosaccharide as a constituent saccharide into a rare sugar with the disaccharide as it is. The above conversion to monosaccharide as a constituent saccharide into a rare sugar with the disaccharide as it is such a reaction as to proceed via a 3-ketodisaccharide. This reaction includes such a reaction as to form the 3-ketodisaccharide by oxidizing the 3-site of the disaccharide as the raw material (substrate) by microbial reaction, and this reaction is such a reaction as to form the 3-ketodisaccharide followed by reducing it. The above microbial reaction is such as oxidation reaction as to subject an oxidase derived from a microorganism belonging to the genus Agrobacterium having the property to make the 3-ketodisaccharide to the 3-site of the disaccharide, wherein the above microorganism is Agrobacterium tumefaciens. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to a method for producing a rare sugar-linked disaccharide having at least one rare sugar as a constituent monosaccharide.

  Conventional methods for producing disaccharides can be said to have the following two main methods used industrially. The first is a method of producing a disaccharide by degrading a polysaccharide using a hydrolase, which is represented by a method of degrading starch using β-amylase to produce maltose. Cellobiose can be produced from cellulose in a similar manner. The second is production using the transfer activity of sugar hydrolase. This is widely used for the production of various oligosaccharides. For example, decomposition of reducing sugar in oligosaccharide containing α-galactosyl group obtained by allowing α-galactosidase to act on galactose or a substance containing galactose, separation of non-reducing disaccharide containing α-galactosyl group Production method, and α-galactosyl group comprising hydrolysis of Gal1α-1βGal mixed in oligosaccharide containing α-galactosyl group obtained in the same manner as described above in a substance containing galactose and glucose, decomposition of reducing sugar, and separation operation A method for producing a non-reducing disaccharide containing glycan is shown (Patent Document 1).

On the other hand, whether or not the above two methods can be used as a method for producing disaccharides containing rare sugars is considered as follows. The first method, that is, the method for producing disaccharides by decomposing polysaccharides cannot be used. This is because there is no polysaccharide comprising rare sugars as constituent sugars. The second method can produce a disaccharide containing a rare sugar by using the rare sugar as a receptor. For example, a reaction for transferring xylan D-xylose to a rare sugar using xylanase has already been carried out (Patent Document 2).
As a method other than the first and second methods, a disaccharide in which sucrose D-fructose is converted to D-psicose by a method using a reverse reaction of sucrose phosphorylase Has been successfully manufactured (Patent Document 3).

JP 2003-160594 A JP 2006-169124 A JP 2007-91667 A

  An object of the present invention is to provide a method for producing a rare sugar-linked disaccharide having at least one rare sugar as a constituent monosaccharide.

The gist of the present invention is a method for producing a rare sugar-linked disaccharide having at least one rare sugar as described in (1) to ( 5 ) below as a constituent monosaccharide.
(1) Using a disaccharide as a raw material (substrate), the 3-position of the disaccharide is oxidized by a microbial reaction to produce a 3-keto disaccharide, which is further reduced to produce a disaccharide containing a rare sugar. Letting
The above microbial reaction is an oxidative reaction in which an oxidase derived from an Agrobacterium having the property of producing a 3-keto disaccharide is allowed to act ;
A method for producing a rare sugar-linked disaccharide comprising at least one rare sugar as a constituent monosaccharide.
( 2 ) The method for producing a rare sugar-linked disaccharide comprising at least one rare sugar as a constituent monosaccharide according to (1 ), wherein the microorganism is Agrobacterium tumefaciens .
( 3 ) The method for producing a rare sugar-linked disaccharide comprising the at least one rare sugar as described in (1) or (2), wherein the reaction for reducing the 3-keto disaccharide is an organic chemical reduction reaction.
( 4 ) The 3-keto disaccharide is obtained by microbial oxidation of lactitol or lactose, and the rare sugar-binding disaccharide comprises the rare sugar D-gulose as a constituent monosaccharide ( 1 ) to ( 3 ) A method for producing a rare sugar-linked disaccharide comprising at least one rare sugar as defined in any one of 3 ) as a constituent monosaccharide.
( 5 ) The method for producing a rare sugar-linked disaccharide comprising at least one rare sugar as a constituent monosaccharide according to any one of (1) to ( 4 ), wherein disaccharide containing the rare sugar is further separated from the reaction mixture.

  The present invention can provide a method for producing a rare sugar-linked disaccharide having at least one rare sugar as a constituent monosaccharide.

[Features of the present invention]
As a method for producing disaccharides containing rare sugars, a new method other than the method described in the background section has been developed. That is, the conventional method is a method of using a rare saccharide, which is a monosaccharide, as an acceptor. Therefore, first, a rare saccharide as a free monosaccharide was produced, and another monosaccharide was bound thereto by various methods. The novel method of the present invention is based on a completely new idea of the following characteristics.
In the novel method of the present invention, a disaccharide is used as a raw material, and a monosaccharide that is a constituent sugar is converted into a rare sugar without hydrolyzing the disaccharide. This method is effective for the production of disaccharides containing rare sugars, since rare sugars are not bound after production.

[Principle of new method]
The principle of the novel method of the present invention using a disaccharide as a raw material (substrate) and converting a monosaccharide, which is a constituent saccharide without changing the disaccharide, into a rare sugar is shown in FIGS.
It is a reaction via a 3-keto disaccharide, and includes a reaction in which the 3-position of a disaccharide as a raw material (substrate) is oxidized by a microbial reaction to produce a 3-keto disaccharide. More specifically, it is a reaction in which the 3-position of a disaccharide as a raw material (substrate) is oxidized by a microbial reaction to produce a 3-keto disaccharide, which is further reduced. The oxidation and reduction reactions of lactitol, lactose, and trehalose as raw materials (substrates) are shown in FIGS.
Chemical formula 1 shows the reaction in the case of lactitol. It is possible to oxidize the 3-position of non-reducing D-galactose to produce (ii) 3-keto lactitol. That is, the 3-keto disaccharide is obtained by microbial oxidation of lactitol or lactose, and the rare sugar-binding disaccharide uses the rare sugar D-gulose as a constituent monosaccharide. The disaccharide lactitol is a reaction through which the monosaccharide, which is a constituent sugar, is converted into the rare sugar D-gulose as it is but via the 3-keto disaccharide 3-keto-lactitol (see FIG. 1).

Agrobacterium microorganisms have the property of oxidizing the 3-position of the monosaccharide on the non-reducing side of the disaccharide to produce a 3-keto disaccharide. In the case of lactitol, the Agrobacterium microorganism oxidizes the 3rd position of non-reducing D-galactose to produce (ii) 3-keto lactitol.
The novel method of the present invention is a method for producing D-grosyl-D-sorbitol in the case of lactitol by reducing the 3-keto disaccharide. Since this reduction is asymmetric, there is currently no method for the reaction to proceed, so that lactitol is also present in the solution after the reaction.

[Disaccharides as raw material (substrate)]
This basic principle can be applied to any disaccharide as long as it is a disaccharide that oxidizes the 3-position of a microorganism. That is, as the disaccharide containing a rare sugar according to the present invention, various disaccharides can be used as long as the 3-position of the non-reducing monosaccharide is oxidized.
Examples include lactitol, lactose, sucrose, maltose, maltitol, trehalose, and the like. From each disaccharide, it is possible to produce a disaccharide bound to a rare sugar that is epimerized at position 3 of the monosaccharide to be oxidized.
Chemical formula 2 shows the case of trehalose. In this case, there are two places to oxidize, and 3-ketotrehalose which has been oxidized once and 3-diketotrehalose in which both have been oxidized will be obtained. By reducing this oxidized 3-ketotrehalose, three types of disaccharides are obtained. That is, trehalose which is a raw material in which two D-glucoses are combined, one D-glucose is reduced to D-allose, a disaccharide in which D-allose and D-glucose are combined, and D-allose and D-glucose. A disaccharide to which allose is bound will be produced (see FIG. 3).

The method of the invention includes the step of separating disaccharides, including rare sugars, from the reaction mixture.
After completion of the reaction, a disaccharide containing a rare sugar can be separated by a known method as necessary. Thereafter, if desired, disaccharides containing rare sugars can be purified by applying purification means such as gel filtration chromatography or activated carbon column chromatography.

[Microorganisms]
The microorganism having the property of producing the 3-keto disaccharide used in the present invention is a microorganism belonging to the genus Agrobacterium.
The separation of microorganisms that oxidize disaccharides to oxidize the 3-position of non-reducing sugars is described below.
Isolation place: Soil in Faculty of Agriculture, Kagawa University Strain name: M31
Species identification: Agrobacterium tumefaciens (Deposit number NITE P- 489)
That is, the strain Agrobacterium tumefaciens M31 was deposited domestically on February 15, 2008 at the Patent Organism Depositary Center of the National Institute of Advanced Industrial Science and Technology (1-1-1 Higashi 1-1-1 Tsukuba City, Japan). (Deposit number NITE P- 489)

[Culture conditions]
For isolation of the M31 strain, microorganisms were separated at 30 ° C. using an inorganic salt liquid medium (Table 1) containing 2% (w / w) lactitol (D-galactosyl-D-sorbitol) as a single carbon source. went. A strain having the ability to grow under these conditions, oxidize in a disaccharide state, and accumulate the oxide in the fines on culture was isolated. As the agar medium used for the isolation and storage of microorganisms, the 2% TSB (tryptic soy broth) agar medium shown in Table 2 was used.

  The isolated bacterial strain was examined for its enzyme production capacity and enzyme production conditions. As a result, it was found that the target enzyme can be stably induced and produced by adding sucrose, which is a carbon source cheaper than lactitol. That is, it has been found that a large amount of enzyme is produced in a liquid medium in which a small amount of D-lyxose (0.1% to 3%, desirable concentration is 1%) is added to 2% TSB. It has also been confirmed that the enzyme can be stably produced even with cheaper 0.5% yeast extract, 0.5% polypeptone, 0.5% sodium chloride, and 1% sucrose. Activity was also produced with other disaccharides, but the addition of sucrose was most effective. The influence of the cultured carbon source is shown in FIG.

  The production amount of 3-keto-lacitol in the 2% lactitol mineral salt medium of the M31 strain was the most suitable condition after 24 hours from the culture. FIG. 5 shows the results of washing cell reaction using 1% lactitol (A). Although it is converted to almost 100% 3-ketolactitol in 40 hours, a peak is generated in addition to the target product, but it is unknown at this time (B). The washing cell reaction conditions were 50 mM potassium phosphate (pH 7.0), 30 degrees.

Next, Table 3 shows the results of identification of M31, and Table 4 shows the results of ketose production by the cultured carbon source.
The amount of ketose was measured by the cysteine carbazole method. Enzyme activity was performed as before.

Properties of activity (glucoside 3-dehydrogenase) to produce 3-keto disaccharides by washing cell reaction.
1) pH
Activity is shown at pH 6-9, but 7-8 is desirable.
2) Metal ions There are no metal ions that increase the activity in particular, and they are greatly inhibited by divalent copper ions.
3) Influence of oxygen Although it is hardly oxidized under nitrogen conditions, it is sufficiently oxidized only by stirring under air conditions.

[Disaccharide conversion reaction (cultured MSM-sucrose): Transition of ketose production (Fig. 6)]
As shown in FIG.
Maltose: All consumed.
Lactose: The reaction is completed after 5 hours (100%), and after 32 hours a peak other than the product is generated.
Maltitol: The reaction is completed after 5 hours (21 min, 100%), and after 32 hours, 21 min disappears, and the peak at 28 min is dominant.
Lactitol: The reaction is completed after 5 hours (18 min, 100%), almost stable after 32 hours, and a peak occurs at 29 min.

[Example of immobilized microorganisms]
The suspension was suspended in 3.0 g (w / w) of sucrose cultured cells and 5 ml of 50 mM Tris-HCl (pH 7.0), and mixed with the same amount of 4% aqueous sodium alginate solution. Drop into 0.2MCaCl2 solution and leave for 2 hours. Washed with 50 mM Tris-HCl (pH 7.0).
Reaction (FIG. 7): 1% lactitol, 10 mM Tris-HCl (pH 7.0)
The initial speed drops to 1/3 to 1/2 of the free, but is eventually converted to 100%.

  Details of the present invention will be described in the examples. The present invention is not limited to these examples.

Examples of production of rare sugar-containing disaccharides using lactitol are described below.
[culture]
The cells were pre-cultured in 3 ml of medium supplemented with 2% TSB and 1% sucrose at 30 degrees for 10 hours. The whole amount was cultured in a 3 L medium having the same composition. The culture apparatus was a jar fermenter with 5 L and effective volume of 3 L, with an aeration rate of 3 L / min and stirring at 400 rpm. In addition, 3 ml of anti-foaming agent made of Shin-Etsu silicone was added. The cells were cultured at 30 degrees for 15 hours. The cell density after 15 hours was OD ₆₆₀ = 6.74.

[1] Washing cell reaction (FIG. 8)
The cells were centrifuged at 9000 rpm and washed with 10 mM phosphate buffer (pH 7.5). This was prepared so as to OD ₆₆₀ = 30, final concentration 2% lactitol. Under this condition, this time was about 630 ml, and a washing cell reaction was carried out in a shaking culture machine containing about 100 ml of a 500 ml baffled Erlenmeyer flask for sufficient aeration. After about 27 hours, 100% lactitol had been converted to 3 ketolactitol.

  Thereafter, the cells and the reaction solution were separated using a centrifuge, and the reaction was started again under the same conditions. Approximately 20 hours later, it was converted to 100% 3-ketolactitol. Similarly, it was possible to continue these operations a total of 8 times. A total of 96 g of lactitol could be converted into a solution containing 100% 3-ketolactitol. Furthermore, it was possible to continue the washing cell reaction, but this time it was completed 8 times (period is 1 week). It is speculated that the reason why the reaction was possible for such a long time was that the M31 strain slowly metabolized lactitol to obtain a nutrient source. In fact, the washing cell reaction is a frequently used means for producing rare sugars, but there has never been an example of maintaining enzyme activity for such a long time. The recovery rate of the sugar after the actual reaction was 65 g (63 g). FIG. 5 shows the result of the washing cell reaction as a result of HPLC analysis.

[2] Purification after washing cell reaction (FIG. 9)
Since a large amount of proteins and bacterial cell residues are mixed in the sugar solution after the reaction, purification using a hollow fiber filter was performed. Protein was removed using Asahi Kasei Micromodule SLP-1053 (fraction <molecular weight 3000). At this point, the corresponding amount of sugar is 60 g.

  A liquid containing 30 g of purified 3-ketolactitol was prepared to 500 ml (6% w / v), and 3-ketolactitol was reduced by Raney nickel hydrogenation method. The reaction was carried out for 6 hours while maintaining the stirring speed at 700 rpm at a hydrogen pressure of 1.2 MPa (gauge pressure) while heating at 50 degrees using TEM-1000M manufactured by Pressure Glass Industrial Co., Ltd. Raney nickel was activated as follows. 100 g of 20% NaOH aqueous solution was added to 10 g of 50% Raney nickel (manufactured by Nacalai Tesque). After the addition, heating was performed at 80 ° C. for 8 hours. After confirming that the generation of bubbles stopped, the catalyst was washed with distilled water by decantation. The washing was performed until the washing solution reached pH 9.2. FIG. 9 shows the results of purification after washing cell reaction as HPLC analysis results.

(Theoretically, the original lactitol and D-glucyl-D-sorbitol occur at 1: 1, but the two are separated on this column because the structure is very similar only at 17.46 min (RT of lactitol). 26.39 minutes is D-sorbitol. It is estimated that 3-ketolactitol or D-glucyl-D-sorbitol is unstable or decomposed.)
Lactitol and D-glucyl-D-sorbitol were isomerized by the desalting resin, or the bond between disaccharides was cleaved. Although the reason for this has not been clarified, it is generally known that some sugars undergo changes such as isomerization when desalting is performed for a long time. The 3-ketolactitol and D-glucyl-D-sorbitol of the present invention are novel substances, and many of the properties are unknown. However, the elucidation of this cause is not carried out because it is out of the scope of the present invention. D-sorbitol removal was performed to hinder the next step. Using a one-pass chromatographic separation apparatus (NK-26, manufactured by Nisshin Kikai Co., Ltd.), 27 g of the above sugar mixture was separated. Table 5 shows the separation conditions, and FIG. 10 shows the HPLC results after the separation.

  Of the 20 fractions, No. 6 to No. 10 were collected as 100% fractions of lactitol and D-glucyl-D-sorbitol.

  The recovered amount was 9.6 g. Since about 1/3 of 27 g was sorbitol, the remaining 18 g was lactitol and D-glucyl-D-sorbitol, and the recovery rate was about 53%.

[4] Confirmation of composition of lactitol and D-glucyl-D-sorbitol It was investigated what kind of sugar the sugar obtained in [3] above was composed. Acid hydrolysis was performed using dilute hydrochloric acid. The sugar solution was adjusted to 480 ml with a 2% (w / v) concentration, mixed with an equal amount of 1N hydrochloric acid, and treated at 80 ° C. for 6 hours. After the treatment, the solution was neutralized and desalted and analyzed by HPLC. The analysis result is shown in FIG.
As shown in FIG.
The peak per 10 minutes could not be removed
17.53 min peak: D-galactose
21.27 minute peak: D-growth
Peak at 26.45 minutes: D-sorbitol The composition ratio thereof was D-galactose: D-growth: D-sorbitol = 3.7: 1.3: 5. From this, it was found that the sugar produced when hydrogenating 3-ketolactitol obtained was 75% lactitol and 25% D-glucyl-D-sorbitol.

  Theoretically, the original lactitol and D-glucyl-D-sorbitol should occur at 1: 1, but the actual composition was approximately 3: 1. This cause needs further investigation.

[5] Confirmation by D-growth NMR The acid hydrolyzate obtained in [4] above was collected and concentrated to obtain a sugar solution corresponding to about 8.5 g. D-growth was purified using the above-mentioned one-pass chromatographic separation apparatus. Table 6 shows the conditions and results.

  Fractions (No. 10 and No. 11) with a purity of 98% or more were collected. The yield was 0.42g. FIG. 12 shows the analysis result of 13C NMR (Authentic, Product).

  The confirmation of D-growth from this hydrolyzate indicates that the basic concept of the present invention is correct. That is, the 3-position of D-galactose at the non-reducing end of lactitol, which is a disaccharide, is oxidized to 3 ketolactitol. By reducing it, it is reliably shown that disaccharides containing rare sugar D-gulose are produced.

With regard to the production method of rare sugars, research using microbial reactions and enzyme reactions has been advanced, and various researches have been carried out using Izumoring, which is a production strategy for all rare sugars, as a blueprint. Since various rare sugars, which are these monosaccharides, can be produced in large quantities, research on industrial use is progressing.
On the other hand, research on disaccharides containing rare sugars has been developed, such as a method using a glycosyltransferase of a polysaccharide hydrolase and a method using the reverse reaction of sucrose phosphorylase. And using it as a receptor to bind other monosaccharides. For this reason, there is a disadvantage that the production amount is small and the cost is increased because rare sugar is used as a raw material. In the present invention, a disaccharide is used as a raw material, and a monosaccharide which is a constituent sugar is converted to a rare sugar while being bound to the disaccharide, so that mass production is possible.
Since a sufficient amount of disaccharides containing rare sugars has not been obtained so far, little research has been conducted on physiological activity. With the establishment of a method that enables mass production in large quantities according to the present invention, development of applications for foods and the like as completely new carbohydrates can be expected in the future.

It is drawing which shows the chemical formula explaining the oxidation and reduction | restoration reaction which convert the monosaccharide which is a constituent saccharide | sugar into a rare saccharide using lactitol. It is drawing which shows the chemical formula explaining the oxidation and reduction | restoration reaction which convert the monosaccharide which is a component saccharide | sugar into a rare saccharide using lactose as it is. It is drawing which shows the chemical formula explaining the oxidation and reduction | restoration reaction which convert the monosaccharide which is a component saccharide | sugar into a rare saccharide | sugar using trehalose. It is drawing which shows the influence of a culture carbon source about the isolate | separated strain. It is drawing which shows the result of the washing | cleaning microbial cell reaction using 1% lactitol of M31 stock | strain. It is drawing which shows transition of disaccharide conversion reaction (cultured MSM-sucrose): ketose production amount. It is drawing which shows transition of relative activity in the immobilized microorganism. It is drawing which shows the result of the washing | cleaning microbial cell reaction of Example 1 by a HPLC analysis result. It is drawing which shows the result of the purification after washing | cleaning microbial cell reaction by a HPLC analysis result. It is drawing which shows the HPLC analysis result after isolation | separation. It is a drawing showing the composition confirmation HPLC analysis results of lactitol and D-glucyl-D-sorbitol. It is drawing which shows a NMR (Authentic, Product) analysis result.

Claims (5)

Using a disaccharide as a raw material (substrate), oxidizing the 3-position of the disaccharide by a microbial reaction to produce a 3-keto disaccharide, and further reducing this to produce a disaccharide containing a rare sugar,
The above microbial reaction is an oxidative reaction in which an oxidase derived from an Agrobacterium having the property of producing a 3-keto disaccharide is allowed to act;
A method for producing a rare sugar-linked disaccharide comprising at least one rare sugar as a constituent monosaccharide. The above microorganism, Agrobacterium tumefaciens (Agrobacterium tumefaciens, Accession No. NITE P-489) The method of producing a rare sugar binding disaccharide to constituent monosaccharide at least one rare saccharide according to claim 1. The method for producing a rare sugar-linked disaccharide comprising at least one rare sugar as a constituent monosaccharide according to claim 1 or 2 , wherein the reaction for reducing the 3-keto disaccharide is an organic chemical reduction reaction. 3 keto disaccharides, are those obtained by microbial oxidation of lactitol or lactose, a rare sugar binding disaccharide, any one of claims 1 to 3 in which the constituting monosaccharide rare sugar D- gulose A method for producing a rare sugar-linked disaccharide comprising at least one rare sugar according to one item as a constituent monosaccharide. The method for producing a rare sugar-linked disaccharide comprising at least one rare sugar as a constituent monosaccharide according to any one of claims 1 to 4 , wherein a disaccharide containing the rare sugar is further separated from the reaction mixture.