JPH0279991A - Production of n,n'-diacetyl chitobiose - Google Patents

Abstract

PURPOSE:To obtain N,N'-diacetyl chitobiose in high yield in a short time by reaction between chitin (partial degradation product) and an enzyme produced from N,N'-diacetyl chitobiose-productive Bacillus microorganisms. CONSTITUTION:The objective N,N'-diacetyl chitobiose can be obtained in high yield by reaction between (A) an enzyme produced from N,N'-diacetyl chitobiose- productive Bacillus microorganisms and (B) chitin or a partial degradation product therefrom. The Bacillus microorganisms is pref. Bacillus sp.PI-7S (FERM P-9843).

Description

Detailed Description of the Invention (Industrial Application Field) The present invention provides a method for producing N,N''-diacetylchitobiose, more specifically, a method for producing N,N''-diacetylchitobiose using a microorganism belonging to the genus Bacillus.
The present invention relates to a method for producing N'-diacetylchitobiose.

(Prior art) Generally, in order to produce N,N'-diacetylchitobiose, chitin is used as a raw material and N,N'-diacetylchitobiose is fractionated using acid hydrolysis. Hydrolysis method is adopted.

However, according to this method, it is difficult to purify N,N'-diacetylchitobiose, which is the desired dimer, because the monomer of N-acetylchito-oligosaccharide up to the hexa-hexane is produced. Moreover, the yield also becomes poor.

Therefore, in order to eliminate such drawbacks, Japanese Patent Application Laid-open No. 63
-7792, using microorganisms of the genus Bacillus to
A method for producing , N''-diacetylchitobiose has been developed.

That is, in this method, Bacillus X-7u, which is a microorganism belonging to the genus Bacillus, is cultured in a medium containing chitin, and N,N'-diacetylchitobiose is produced from the culture.

(Problem to be solved by the invention) However, although this method improves the yield compared to the acid hydrolysis method described above, the chitobiose produced is assimilated by microorganisms, so this assimilation is reversed. This causes a decrease in yield, and as a result, the yield from chitin is at most 30 to 4
It was about 0%.

Furthermore, as thermophilic microorganisms are used for culturing at a temperature of approximately 50 degrees Celsius, there is no risk of contamination with other bacteria, but on the other hand, culturing at a high temperature of 50 degrees Celsius is a waste of energy, and moreover, the cultivation time is approximately 50 degrees Celsius. Five days were required.

Furthermore, since the culture solution contains many impurities other than chitobiose, it is necessary to repeatedly operate the activated carbon column, and since the amount of solution is large, the time spent on the column is also increased. Therefore, there is a problem in that the overall working time increases.

The present invention has been made as an improvement to the method for producing N,N'-diacetylchitobiose using microorganisms as described above, in order to solve all of the above-mentioned problems.
The objective is to enable work at a culture temperature close to room temperature, reduce the number of days of culture, and further facilitate purification and significantly improve yield.

(Means for Solving the Problems) The present invention has been made to solve the above-mentioned problems, and the means for solving the problems are as follows: Microorganisms of the genus Bacillus [mainly Bacillus sp, PI-7S (Feikoken Kogyosei No. 9)
No. 843)] is reacted with chitin or a partial decomposition product of chitin to produce N.

The purpose of the present invention is to produce No-diacetylchitobiose.

Another method is to culture Bacillus sp, PI-7S (Feikoken Bacteria No. 9843) in a medium containing chitin or a partial decomposition product of chitin, and to extract N, N'-diacetyl chitobi from the medium. The purpose is to collect and manufacture oath.

Bacillus sp, PI-7S, mainly used in the present invention
(Feikoken Bacteria No. 9843) is a strain collected by the present inventors, and its mycological properties are as follows.

(A) Morphological properties (a) Bacterial morphology Rod (b) Spores oval, swollen (c) Motile (d) Durham staining indeterminate (B) Growth status in each of the following media (a) Meat juice When cultured on an agar medium at 37° C. for 24 to 96 hours, colonies with convex circular edges were formed and swelled over time.

The color is almost cloudy when cultured for 24 hours, but 4
After 8 hours of culture, the color became light yellow or yellowish.

The growth status was recognized as positive.

(b) Meat juice liquid stirring medium was aerobic and became cloudy when cultured at 37°C for 24 hours.

The growth status was recognized as positive.

(C) No growth under anaerobic conditions (D) Physiological properties (a) Production of catalase + (b) VP reaction +W (c) pH 4,8 at VP prosthesis (d) Production of gas from glucose ( e) Acid productivity ■ Glucose + ■ Arabinose ■ Xylose ■ Mannitol + (f) Liquefaction of gelatin + (g) Decomposition of starch + (h) Decomposition of tyrosine (i) Availability of citric acid (j) Egg yolk reaction -(k) Nitrate reduction (1) Indole production (m) Growth at pH 5,7 +(n) 5%
Growth in the presence of NaCl - (o) Growth in the presence of 7% NaCl (p) Growth at 50°C - In the above, + means positive, - means negative, and 10 W means weakly positive.

This strain has already been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on January 28, 1986.

(Example) Examples of the present invention will be described below.

Dear Doctor, This example is an example of a method for producing N,N'-diacetylchitobiose using an enzyme solution.

First, Bacillus sp, PI~7S was mixed with flake chitin 0.
The cells were inoculated into a medium containing 5%, and liquid culture was performed for about 72 hours. This culture filtrate has a chitinase activity of 90 to 120ml.
/ml and chitosanase activity 0.35-0.40/2m
l was present, but almost no chitobiase activity was included.

Next, this culture filtrate was concentrated by ammonium sulfate fractionation, and ammonium sulfate was removed by dialysis to prepare a desired enzyme solution.

300 ml of the enzyme solution prepared as described above (180 U as chitinase activity) was added to 8 g of N-acetyl chitosan and reacted at 37°C for 20 hours.

Next, the enzyme reaction was stopped by heat treatment to obtain a sugar solution.

High performance liquid chromatography analysis of chitin decomposition products in the sugar solution revealed that the main components were N, N'
- diacetylchitobiose.

Furthermore, in order to increase the purity of this N,N'-diacetylchitobiose, it was purified using an activated carbon-Celite column.

The N,N'-diacetylchitobiose obtained by purification was a white powder with a yield of 86%.

The melting point and Rf value of thin layer chromatography of this purified N,N"-diacetylchitobiose are different from that of standard N,N"-diacetylchitobiose.
'-Diacetylchitobiose showed the same value.

Furthermore, by high-performance liquid chromatography, the above N, N
' - When the purity of diacetylchitobiose was examined,
It was over 96%.

In this way, in the above example, N,N'-diacetylchitobiose was produced by the reaction of the enzyme solution obtained by further preparing the culture solution of microorganisms with N-acetylchitosan, so that assimilation by microorganisms was not possible. (, N in high yield, N”
- Diacetylchitobiose could be obtained.

Moreover, since the culture filtrate described above contained almost no chitobiase activity, purified N
, N'-diacetylchitobiose was not decomposed into N-acetylglucosamine (this was a factor in increasing the -layer formation rate).

The method for measuring chitinase activity and chitosanase activity in the above examples is as follows.

(a) Chitinase activity measurement method 0.1 M McIlivaine buffer
(pH 6,8) 30 N-acetyl chitosan in 2 ml
Suspend (1), add 1 ml of enzyme solution, and react at 37°C for 60 minutes. After stopping the reaction, the reducing sugar produced is determined by the 5chales method. 1 per minute under these conditions. ! /1101
The amount of enzyme that releases the reducing sugar corresponding to N-acetylglucosamine is defined as IU (unit).

(b) Chitosanase activity measurement method 1% soluble chitosan solution (pH 6.0 Hml) and 1 ml of enzyme solution were reacted at 37°C for 10 minutes, the reaction was stopped by boiling, and reducing sugar was determined by the 5 chales method.

The amount of enzyme that releases reducing sugars corresponding to 1 ua+ol of glucosamine per minute under these conditions was defined as 1 tJ (unit).

(c) Chitobiase activity assay method N,N'-diacetyl chitobiose containing 0.5
．． I Mcllivaine buffer (p
H6, 8) Add 1 ml of enzyme solution to 1 ml and react at 37°C for 30 minutes.

The generated N-acetylglucosamine was transferred to Morgan-E.
It was quantified by the lson method. Enzyme activity is 1 μmol per minute
The amount of enzyme that decomposes N,N'-diacetylchitobiose was defined as IU (unit).

This example is an example of a method for producing N,N' diacetylchitobiose using a microorganism culture solution.

First, the above Bacillus sp, PI-75 was inoculated into a medium containing 5% colloidal chitin, and incubated at about 37°C for about 4 hours.
Culture was performed for 8 hours.

Next, the supernatant obtained by centrifugation of the culture solution was subjected to high performance liquid chromatography and analyzed, and it was found that N,N'-diacetylchitobiose was contained.

Then, this supernatant liquid was purified using an activated carbon-Celite column to obtain the desired N,N''-diacetylchitobiose.

In Example 1, the enzyme was prepared by culturing microorganisms, and N, by the reaction of the enzyme and N-acetyl chitosan.
Although the production of N'-diacetylchitobiose was performed in a series of operations, it is not necessarily necessary to perform in a series of operations.

For example, using an enzyme solution prepared and stored in advance as described above, N-acetyl chitosan is added to it and N,N
It is also possible to produce '-diacetylchitobiose.

Furthermore, the type of chitin to be added to the enzyme solution is not limited to the N-acetyl chitosan used in the above embodiments, and for example, colloidal chitin or flake chitin may be used.

From the same point of view, the type of chitin contained in the culture medium of Example 1 or Example 2 is not limited to the flake chitin or colloidal chitin of the examples. That is, chitin as used in the present invention is not limited to N-acetyl chitosan, which means chitin in the narrow sense, but has a broader meaning that includes flake chitin and colloidal chitin. Furthermore, it is also possible to use a partially decomposed product, such as chitin oligosaccharide, which has been partially decomposed by some means.

Furthermore, in the above Example 1, the enzyme may be immobilized in advance before adding the enzyme solution to N-acetyl chitosan. In this case, the enzyme can be easily recovered, and the N,
This method has the advantage that the enzyme solution after purification of N'-diacetylchitobiose can be easily reused and used continuously.

Furthermore, in the above example, the enzyme solution was reacted with N-acetyl chitosan, that is, the enzyme was made into a liquid and the enzyme was reacted with N-acetyl chitosan. For example, it is possible to dissolve N-acetyl chitosan in a solvent and add the powder containing the enzyme to the solution.

In addition, in the above-mentioned Example 2, it is also possible to immobilize the microorganisms, and in this case, the collection of the microorganisms becomes easier.
Further, there is an advantage that separation from the target product N,N''-diacetylchitobiose is easy.

Furthermore, although the production method using enzymes of the present invention focuses on using Bacillus sp, PI-7S as mentioned above, the types of microorganisms are not limited to these and other Microorganisms of the genus Bacillus may also be used.

In short, any Bacillus microorganism capable of producing N,N'-diacetylchitobiose may be used.

Further, in Example 1, the enzyme was prepared from the culture filtrate by ammonium sulfate fractionation, but instead of this method, the enzyme may be prepared by ultrafiltration.

In addition, various operating conditions such as the amount of chitin contained in the medium, culture temperature, culture time, purification means, etc. are not limited to the above embodiments, and can be changed as desired.

(Effects of the Invention) (A) As mentioned above, the present invention allows an enzyme produced from a Bacillus microorganism capable of producing N,N''-diacetylchitobiose to react with chitin or a partial decomposition product of chitin. Since this is a method for producing N,N' diacetylchitobiose, the reaction time is shorter than in the conventional production method such as that disclosed in JP-A No. 63-7792, which shortens the overall working time. Moreover, the yield of N,N''-diacetylchitobiose was significantly increased, which was a particularly remarkable effect.

Particularly, when Bacillus sp, PI-7S (Feikoken Bacterial Serial No. 9843) is used as the microorganism, it has the effect of further shortening the working time because it multiplies rapidly.

(b) Also, since the method uses the prepared enzyme, the resulting sugar solution has few impurities and the amount of liquid is small, so the process of purifying N,N''-diacetylchitobiose is greatly simplified. It has the advantage of being standardized.

(c) Furthermore, since microorganisms do not assimilate N,N' diacetylchitobiose as in the past, this further contributes to improving the yield.

(d) Furthermore, in the production method by culturing of the present invention, as the microorganism, the fast-growing Bacillus sp.
Since PI-7S is used, the work time is reduced compared to conventional methods.

Moreover, since the Bacillus sp, PI-75, can be cultured at a temperature close to room temperature, it has the advantage of eliminating wasteful energy consumption.

Claims (1)

[Scope of Claims] 1. N,N'-diacetylchitobiose is produced by reacting an enzyme produced from a Bacillus microorganism capable of producing N,N'-diacetylchitobiose with chitin or a partial decomposition product of chitin. A method for producing N,N'-diacetylchitobiose, which comprises producing tobiose. 2. The Bacillus microorganism is Bacillus sp. PI-7
The method for producing N,N'-diacetylchitobiose according to claim 1, wherein the N,N'-diacetylchitobiose is S (Keikoken Kyoiku No. 9843). 3. Bacillus sp. PI-7S (Meiko Kenzonoyori No. 9843
N,N'-diacetylchitobiose, characterized in that it is produced by culturing N,N'-diacetylchitobiose (No.) in a medium containing chitin or a partial decomposition product of chitin, and collecting N,N'-diacetylchitobiose from the medium. manufacturing method.