JP2001292791A - Method for producing n-acetyllactosmine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method by which N-acetyllactosamine can be synthesized in a higher yield than that of a conventional method in the enzymic method for synthesizing the N-acetyllactosamine by making the enzyme having galactose transfer actions act on N-acetylglucosamine and lactose used as a substrate. SOLUTION: This method for producing the N-acetyllactosamine is to make the enzyme having the galactose transfer actions act on the lactose and N- acetylglucosamine and the method is characterized by making a glucose oxidase coexist when the enzyme having the galactose transfer actions is made to act on the lactose and N-acetylglucosamine.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to lactose and N
The present invention relates to a method for producing N-acetyllactosamine by using -acetylglucosamine as a substrate and allowing an enzyme having a galactose transfer activity to act thereon.

[0002]

2. Description of the Related Art N-acetyllactosamine is a disaccharide in which D-galactose and N-acetylglucosamine have a β-1,4-glycosidic bond, and is composed of human milk oligosaccharides, lipopolysaccharides, various glycoproteins and glycolipids. It is an oligosaccharide that is present in the sugar chains and is very important biochemically.

[0003] N-acetyllactosamine has a growth activity of bifidobacteria, which is a kind of intestinal bacteria, and exhibits an excellent intestinal regulating action. Therefore, N-acetyllactosamine is a highly nutritious food such as infant formula. It is expected to be used for foods for specified health use. In recent years, attention has also been focused on the physiological activity of glycoconjugate sugar chains containing N-acetyllactosamine.
For example, it is becoming important as a raw material in the synthesis of sialyl Le ^X sugar chains having cell adhesion inhibitory activity.

[0004] As a method for synthesizing N-acetyllactosamine, a chemical synthesis method and an enzyme synthesis method are known. However, since the target substance can be obtained by a one-step reaction, the synthesis method on an industrial scale is as follows. Research on enzyme synthesis methods has been extensively conducted. As a method of synthesizing N-acetyllactosamine using an enzymatic reaction, a method using galactosyltransferase and a method using β-galactosidase have been reported.

As a method using galactosyltransferase, for example, N-acetylglucosamine and uridine diphosphate reported by Brew et al.
Method for synthesizing acetyllactosamine (Proc. Natl. Ac
ad. Sci. USA, 59, 491, 1968), but uridine diphosphate galactose used as a substrate is expensive,
It was not feasible as a synthetic method on an industrial scale.

As a method of solving this problem, Ikeda et al. Co-exist with galactosyltransferase and dried cells of the yeast of the genus Torulopsis in order to synthesize galactose uridine diphosphate in the reaction system.
A method for enzymatically synthesizing N-acetyllactosamine from uridine-5'-monophosphate, galactose and N-acetylglucosamine has been invented (JP-A-62-134096).
No.). Specifically, 0.1 mol per mol of galactose.
We have successfully obtained 2 moles of N-acetyllactosamine. However, in this method, uridine diphosphate galactose must be first synthesized in the reaction system, so that it takes as long as five days until the reaction is completed, and further devising as an industrial scale enzyme synthesis method is required.

On the other hand, as another approach, Baci
llus circulans, Diplococcus pneumoniae (currently St
A method using a galactose transfer reaction by β-galactosidase in cells using β-galactosidase or yeast derived from a microorganism such as reptcoccus pneumoniae) has been reported.

For example, Usui et al. Reported a method for producing N-acetyllactosamine by allowing β-galactosidase derived from Bacillus circulans to act on lactose and N-acetylglucosamine. Lactose 1 which is a galactose donor is reported. This indicates that 0.42 mol of N-acetyllactosamine per mol is synthesized in the reaction solution (JP-A-3-49692).

Ajizaka et al. Also reported that Diplococcus pneumoniae, a β-galactosidase with high β-1,4-linkage selectivity,
A method for producing N-acetyllactosamine using an enzyme (now renamed Streptcoccus pneumoniae) and a phenylgalactopyranoside derivative as a galactose donor has been reported (JP-A-6-335395). This method does not produce the reaction by-product N-acetylallolactosamine produced in the method of Usui et al.
This is a method for synthesizing N-acetyllactosamine. However, the productivity of N-acetyllactosamine in this method is 60 to 80 mM, which is lower than that of Usui et al., And the reaction yield per 1 mol of the substrate is low instead of using an expensive substrate. Not suitable for use on a scale.

[0010] As described above, N-
The acetyllactosamine enzyme synthesis method was not suitable for use on an industrial scale because the yield of N-acetyllactosamine per mole of substrate was low. Accordingly, a method for synthesizing N-acetyllactosamine, which has a high yield of N-acetyllactosamine per mole of substrate and is suitable for production on an industrial scale, has been desired.

[0011]

SUMMARY OF THE INVENTION The present invention relates to a conventional method for synthesizing N-acetyllactosamine by using N-acetylglucosamine and lactose as substrates and reacting an enzyme having a galactose transfer activity. An object is to provide a method capable of synthesizing N-acetyllactosamine with higher yield.

[0012]

Means for Solving the Problems The present inventors have made intensive studies to achieve the above object, and as a result, have found that N-acetylglucosamine and a substrate containing lactose can be used.
In the method for synthesizing acetyllactosamine, glucose, which is a reaction by-product, has an action of inhibiting a reaction for transferring a galactose residue of lactose to N-acetylglucosamine (hereinafter, also referred to as “galactose transfer reaction”). Focusing on, a method for efficiently removing glucose from the reaction system was studied. As a result, the inhibition of the synthesis reaction is released by converting glucose to gluconic acid by glucose oxidase, the gluconic acid generated by the decomposition does not affect the galactose transfer reaction, and glucose oxidase coexists in the reaction system. However, they have found that they do not affect the N-acetyllactosamine synthesis reaction, and have completed the present invention.

That is, the present invention relates to a method for producing N-acetyllactosamine by allowing an enzyme having a galactose transfer activity to act on lactose and N-acetylglucosamine, wherein glucose oxidase is allowed to coexist with the enzyme. A method is provided.

According to the method of the present invention having the above characteristics, glucose produced as a reaction by-product is continuously converted to gluconic acid, the inhibition of the galactose transfer reaction by glucose is reduced, and N-acetyl is obtained in high yield. Lactosamine can be synthesized.

In the method of the present invention, when an enzyme having a galactose transfer activity is allowed to act on lactose and N-acetylglucosamine, the concentration of glucose mixed in the substrate solution is preferably 80 mM or less.

As the glucose oxidase used in the method of the present invention, glucose oxidase derived from a microorganism belonging to the genus Aspergillus or Penicillium can be preferably used, and glucose oxidase derived from Aspergillus niger can be particularly preferably used.

The enzymes having a galactose transfer activity used in the method of the present invention include those belonging to the genus Bacillus and Rhod.
β-galactosidase derived from a microorganism belonging to the genus otorula, Sterigmatomyces or Streptococcus can be preferably used.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail. In the method of the present invention, when lactose and N-acetylglucosamine are used as substrates, an enzyme having a galactose transfer action is acted on, and when enzymatically synthesizing N-acetyllactosamine, glucose oxidase is allowed to coexist and a high yield is obtained. Is a method for synthesizing N-acetyllactosamine.

In the method of the present invention, lactose and N-
The enzymatic synthesis of N-acetyllactosamine using a substrate containing acetylglucosamine is specifically performed by reacting a substrate containing lactose and N-acetylglucosamine with an enzyme having a galactosyltransferring activity. Do it by waking up. Then, glucose oxidase is allowed to coexist in the reaction.

The substrate containing lactose and N-acetylglucosamine used in the above reaction is usually used as an aqueous solution or a buffer solution containing lactose and N-acetylglucosamine, and the content and content ratio of each substance are as described above. It is not particularly limited as long as it is not inconvenient for the enzymatic reaction, but preferably, lactose is about 0.3 to 1 M, more preferably about 0.5 M, and N-acetylglucosamine is 0.5.
It is used at a concentration of about 5 to 1.5 M, more preferably about 1 M. The molar ratio of lactose to N-acetylglucosamine is about 1: 5 to 1: 1, preferably about 1: 2 to 1: 1.

In the present invention, the term "enzyme having a galactose transfer activity" refers to an enzyme having an activity of catalyzing a reaction for transferring a galactose residue from lactose to N-acetylglucosamine, and is an enzyme having such an activity. There is no particular limitation if it exists. As such an enzyme, in addition to the purified enzyme, quiescent cells, dried cells, crushed cells, and the like having the action of the enzyme can also be used.

As specific enzymes, Bacillus circula
genus Bacillus, Rhodotorula minuta and Rhodotorula
genus Rhodotorula such as lactosa, Streptcoccus pneumoniae
Genus such as Streptcoccus sp.
β from the genus Sterigmatomyces such as igmatomyces circulans
-Galactosidase purified enzyme or partially purified enzyme,
Alternatively, a substance containing the enzyme (static cells, dried cells, disrupted cells, etc.) can be preferably used. N-acetyllactosamine is a disaccharide in which D-galactose and N-acetylglucosamine are linked by β-1,4-glycosidic bonds, so that β-acetyllactosamine has high specificity for β-1,4-glycosidic bonds.
Galactosidase is preferred. In particular, Bacillus circula
Highly specific β for β-1,4 glycosidic bond from ns
-A purified or partially purified enzyme of galactosidase or a substance containing the enzyme (static cells, dried cells, disrupted cells, etc.) is preferred.

The concentration of the enzyme can be appropriately set by those skilled in the art so that the above-mentioned reaction proceeds suitably. For example, when β-galactosidase is used, the final concentration is 0.1%.
0 U / ml to 10 U / ml, preferably 0.10 U / ml to 0.20
More preferably, it is U / ml. The enzyme activity of 1 U of β-galactosidase was determined by adding the enzyme to a solution of p-nitrophenyl-β-galactopyranoside (pH 6.0) at 30 ° C.
When the reaction is carried out for 10 minutes, the amount of enzyme that releases 1 μmol of p-nitrophenol per minute is defined.

"Glucose oxidase" used in the present invention is an enzyme having an action of converting glucose to gluconic acid. In addition to a purified enzyme or a partially purified enzyme of glucose oxidase, a quiescent cell, a dried cell, a crushed cell, and the like having the enzyme action can be used. Although the origin of the enzyme is not particularly limited, Aspergillus
niger or other Aspergillus genus or Penicillium no
and those derived from the genus Penisillium such as tatum. In particular
Those derived from Aspergillus niger are preferred.

Regarding the glucose oxidase concentration,
Those skilled in the art can appropriately set such that the reaction of converting glucose to gluconic acid proceeds appropriately. However,
The method of the present invention is characterized by suppressing the inhibition of galactose transfer reaction by glucose generated by an enzymatic reaction between lactose and N-acetylglucosamine by converting generated glucose to gluconic acid with glucose oxidase. It is preferable to set the reaction conditions so as to keep the glucose concentration in the system as low as possible. Specifically, the glucose concentration in the reaction system during the enzymatic reaction is preferably about 80 mM or less, and more preferably 40 mM or less. In order to obtain such a glucose concentration, when β-galactosidase is used at the above concentration, the concentration of glucose oxidase is, for example, 2 to 10 U / ml in final concentration.
It is preferable that In general, gluconic acid is known to exist as an equilibrium mixture of glucono-1,4-lactone, glucono-1,5-lactone and free gluconic acid in an aqueous solution. Gluconic acid produced by oxidase is present in solution as an equilibrium mixture of glucono-1,4-lactone, glucono-1,5-lactone and free gluconic acid.

Both the "enzyme having a galactose transfer activity" and the "glucose oxidase" are enzymes extracted and purified from natural products such as non-recombinant microorganisms capable of producing these enzymes, and have the enzyme activities. Microbial cells, etc., and gene DNAs encoding these enzymes
May be produced by genetic engineering techniques, such as by growing cells of microorganisms or the like into which is introduced. It may be an enzyme having a galactose transfer reaction or a polypeptide containing an active site of glucose oxidase, and such a polypeptide can be produced by a genetic engineering technique.

In the enzymatic reaction in the production method of the present invention, a solution (for example, an aqueous solution,
Buffer solution), an enzyme having a galactose transfer effect is acted on lactose and N-acetylglucosamine to produce N-acetyllactosamine and glucose,
In parallel, glucose oxidase acts on the produced glucose to convert it to gluconic acid. As long as these two enzyme reactions proceed, the mode in which both enzymes act, reaction conditions and the like are not limited, but the optimal pH of the enzyme having a galactose transfer activity and glucose oxidase to be used.
It is preferable to perform the reaction in a buffer at an optimum temperature. For example, when β-galactosidase derived from Bacillus circulans and glucose oxidase derived from Aspergillus niger are used, the pH is 5 to 7 and 30 ° C.
It is preferable to carry out the reaction in a buffer solution that can hold H.

In the present invention, it is important that the enzyme synthesis reaction of N-acetyllactosamine and the reaction of enzymatically converting glucose, which is a galactose transfer inhibitor produced by the reaction, into gluconic acid are performed in the same reaction system. It is. Therefore, the form in which these enzyme reactions are carried out must be a form in which the galactosyltransferase reaction and the enzyme reaction for converting glucose to gluconic acid are performed almost in parallel in the same reaction system. There is no particular limitation as long as they are satisfied, and those skilled in the art can appropriately set them. For example, a method of adding an enzyme preparation in which β-galactosidase and glucose oxidase are combined to a substrate solution containing N-acetylglucosamine and lactose, or using β-galactosidase and glucose oxidase on the same or different carriers (beads, membranes, etc.) A method using a fixed substance, a method in which β-galactosidase and glucose oxidase are immobilized on an ultrafiltration membrane, and the like can be considered.

[0029]

EXAMPLES The present invention will be described more specifically with reference to examples, but the present invention is not limited to the following examples.

[0030]Reference example 1  Measurement of β-galactosidase activity p-nitrophenyl-β-galactopyranoside at 5 mM concentration
In 100 mM sodium acetate buffer (pH 6.0)
To give a substrate solution. Buffer with 100 μl substrate solution
Β-galactosidase enzyme solution diluted to various concentrations
The solution was added in an amount of 20 μl, and reacted at 30 ° C. for 10 minutes. 880μ
1% K of l_TwoBO_Four・ 7H_TwoReaction was stopped by adding 0 aqueous solution
After that, the concentration of released p-nitrophenol is determined by its specific absorption
It was determined by measuring the absorbance at 400 nm. 1U
Enzyme activity is 1 μmol p-nitrophenol per minute
Was defined as the amount of enzyme that liberates

[0031]Reference example 2  Determination of enzyme reaction conditions Commercial lactose (Wako Pure Chemical Industries, Ltd.) and N-
Til glucosamine (Nacalai Tesque) as shown in Table 1.
100 mM sodium acetate buffer (pH
 6.0) to prepare a substrate solution, and add Bacillus circula
ns-derived β-galactosidase (Daiwa Kasei Co., Ltd.)
The solution was added to a concentration of 0.15 U / ml,
The reaction was performed for 17 hours under each temperature condition of 60 ° C. and 60 ° C. reaction
The mixture was heated in a boiling bath for 5 minutes to inactivate the enzyme
After that, dilute it to 1/10 with distilled water and use high-performance liquid chromatography.
The reaction product was analyzed by the above method. Table 1 shows the results.

[0032]

[Table 1] From the results shown in Table 1, the production amount of N-acetyllactosamine increases depending on the substrate concentration, but it is almost flat at a concentration of lactose which is a galactose donor of 0.5 M or more. It was found that the higher the reaction temperature and the higher the reaction temperature, the higher the proportion of the isomer N-acetylallolactosamine produced. From these results, unless otherwise noted, the enzyme concentration was 0.15 U / N in the N-acetyllactosamine synthesis reaction in the subsequent experiments.
ml, the reaction temperature was 30 ° C., the substrate concentrations of lactose and N-acetylglucosamine were 0.5 M and 1 M, respectively, and the reaction time was 17 hours.

[0033]Example 1  Glucose for enzymatic synthesis of N-acetyllactosamine
Of Bacillus circulans depending on the conditions determined in Reference Example 2
of N-acetyllactosamine by β-galactosidase
Perform an enzyme synthesis reaction, and add
(1: 1, Wako Pure Chemicals) column (1.5 x 45 cm)
(500 ml) and 30% methanol (500 ml)
Elution by gradient method and purification
did. The elution pattern is shown in FIG. Of the eluted fractions, 21
UV absorption at 0 nm (absorption of acetyl group) and E690 nm
The first peak at which absorption was detected at
Glucosamine fraction, fraction number in Fig. 1
8 to 34 are collected, lyophilized and dried with N-acetylglucosa.
A monosaccharide fraction consisting of min and glucose was obtained. This collection
Using the obtained monosaccharide fraction as a substrate, the N-A
An enzymatic synthesis reaction of cetyl lactosamine was performed. The result
As a result, the monosaccharide fraction recovered from the enzyme synthesis reaction solution was used.
In the case, commercially available N-acetylglucosamine (Nacalaiite
From N-acetyllactosamine
Production dropped by 30%.

Further, N due to the difference in glucose concentration
The following experiment was performed to investigate the effect on -acetyllactosamine enzyme synthesis. Glucose was added at various concentrations shown in Table 2 to a substrate solution prepared in the same manner as in Reference Example 2 so that the N-acetylglucosamine concentration was 990 mM or 480 mM, and N-acetyllactosamine synthesis was performed in the same manner as in Reference Example 2. The reaction was performed. Table 2 shows the results.

[0035]

[Table 2] From the results shown in Table 2, it was found that the N-acetyllactosamine synthesis reaction was not so strongly inhibited up to a glucose concentration of 80 mM or less. Dixon plot analysis revealed that the mode of inhibition by high concentrations of glucose was antagonistic, with a Ki value of 190 mM.

[0036]Example 2  Monosaccharides for enzymatic synthesis of N-acetyllactosamine
Of N-acetyllactosamine under the conditions determined in Reference Example 2
When the enzyme synthesis reaction is performed, glucose of 250 to 300 mM is obtained.
Is generated. Removes glucose from inside the reaction system during the reaction
Glucose oxidase or glucose
Isomerase converts glucose to gluconic acid or fruc
There are several ways to convert to tooth, but these methods are not
The following experiment was performed to evaluate whether or not it was available.

Glucose, fructose or gluconic acid was added to a substrate solution prepared in the same manner as in Reference Example 2 for 30 minutes each.
Three kinds of substrates added to a concentration of 0 mM were prepared, and N-acetyllactosamine enzyme synthesis was performed under the conditions of Reference Example 2. Table 3 shows the results.

[0038]

[Table 3] The enzyme synthesizing activity of N-acetyllactosamine using a solution to which gluconic acid, a reaction product of glucose oxidase, was added was the same as that in the case where the same substrate solution as in Reference Example 2 was used as a control. The reaction was hardly inhibited by the acid. However, fructose, a reaction product of glucose with glucose isomerase, exhibited galactose transfer reaction inhibitory activity equivalent to that of glucose.

[0039]Example 3  Synthesis of N-acetyllactosamine The substrate solution prepared according to Reference Example 2 had a concentration of 0.15 U / ml.
Add β-galactosidase from sea urchin Bacillus circulans
And glucose glucose from Aspergillus niger.
Sidase (156 units / mg specific activity, manufactured by Kurabo Industries, Ltd.)
Add to a final concentration of 5 U / ml and add 15% ammonia
While controlling the pH to 5 to 7 with water, N-acetyllactosami
An enzyme synthesis reaction was performed. As the substrate concentration, N-acetyl
Luglucosamine concentration was fixed at 1M, but lactose concentration
The degree was set to 0.5M and 1M. 17 hours after the reaction
Table 4 shows the amount of N-acetyllactosamine produced.

[0040]

[Table 4] Add glucose oxidase and reduce lactose concentration to 1
When M was used, the amount of N-acetyllactosamine production increased by 31% as compared with the standard method shown in Reference Example 2.

[0041]

According to the present invention, N yield can be increased with higher yield than the conventional method.
N-acetyllactosamine can be synthesized, N-acetylglucosamine and lactose are used as substrates, and an enzyme having a galactose transfer action is acted on. A method suitable for the production of lactosamine is provided.

[Brief description of the drawings]

FIG. 1 is a diagram showing a sugar elution pattern in activated carbon / celite chromatography of a mixture obtained in an enzymatic synthesis reaction of N-acetyllactosamine by β-galactosidase from Bacillus circulans.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) (C12P 19/26 (C12P 19/26 C12R 1: 645) C12R 1: 645) (C12P 19/26 (C12P 19/26 C12R 1:80) C12R 1:80) (C12P 19/26 (C12P 19/26 C12R 1: 685) C12R 1: 685)

Claims (5)

[Claims] 1. An enzyme having a galactose transfer activity is acted on lactose and N-acetylglucosamine.
-A method for producing acetyllactosamine, wherein glucose oxidase is allowed to coexist when the enzyme is allowed to act. 2. The method according to claim 1, wherein when an enzyme having a galactose transfer activity is allowed to act on lactose and N-acetylglucosamine, the concentration of glucose mixed in the substrate solution is 80 mM or less. 3. The method according to claim 1, wherein the glucose oxidase is Aspergillus.
The method according to claim 1 or 2, which is a glucose oxidase derived from a microorganism belonging to the genus s or Penicillium. 4. The method according to claim 1, wherein the glucose oxidase is Aspergillus.
s niger-derived glucose oxidase.
A method according to any one of claims 1 to 3. 5. An enzyme having a galactose transfer activity,
Genus Bacillus, Rhodotorula, Sterigmatomyces or
The method according to any one of claims 1 to 4, which is β-galactosidase derived from a microorganism belonging to the genus Streptococcus.