JP2882589B2 - Method for measuring the degree of deacetylation of chitin and chitosan - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for measuring the degree of deacetylation of chitin and chitosan.

(Conventional Techniques and Problems) Chitin is composed of N-acetylglucosamine contained in crustaceans such as crabs and shrimps, insects such as beetles and crickets, and cell walls of fungi, etc., linked by β- (1-4) bonds. Is a kind of polysaccharide having a different structure. On the other hand, chitosan is a chitin derivative deacetylated by treating chitin with a concentrated alkali, and is a polysaccharide in which glucosamine is β- (1-4) -linked. However, in general, chitin is 10-
Few are 20% deacetylated and chitosan is completely deacetylated, and chitosan obtained by deacetylation from chitin is usually 70-95% deacetylated. As described above, the degree of deacetylation of chitin and chitosan varies widely. This degree of deacetylation not only affects the physicochemical properties of chitin, chitosan and its derivatives, but also greatly affects the quality of various products made from these substances. It is also known that the degree of deacetylation greatly affects the physiologically active functions of chitin and chitosan. Therefore, accurate determination of the degree of deacetylation of chitin and chitosan is an important factor in knowing the properties of these substances.

Conventionally, various methods have been devised for measuring the degree of deacetylation of chitin and chitosan. For example, typical methods include colloid titration with polyvinyl potassium sulfate, a method using an infrared absorption spectrum (IR method), and a method of quantifying acetic acid generated when hydrolyzing chitin or chitosan (hydrolysis method). It can be mentioned as a method for measuring the degree of acetylation. However, these conventional methods have several problems. Although the colloid titration method is a simple quantitative method, it can be applied only to chitosan that dissolves in a dilute acid, and there remains a problem in its accuracy. The IR method is a highly accurate deacetylation method that can be applied to both chitin and chitosan, but requires skill in sample preparation and requires a long time when measuring a large number of samples. It is not always a simple method. The hydrolysis method is a highly accurate method because acetic acid can be directly quantified, but has disadvantages such as complicated experimental operations and a long time.

(Means for Solving the Problem) The present inventors have conducted intensive studies on a method for completely hydrolyzing chitin and chitosan to N-acetylglucosamine and glucosamine, which are constituents thereof, using an enzyme. An exo-type β-D-glucosaminidase that acts on chitosan oligosaccharides and chitosan to release the monosaccharide glucosamine and N-monosaccharide used for chitin oligosaccharides and chitin
It has been found that by combining enzymes that release acetylglucosamine, and by coexisting chitinase and chitosanase with these enzymes, chitin and chitosan can be more effectively completely degraded to N-acetylglucosamine and glucosamine. It was completed.

(Constitution of the Invention) The enzyme used in the present invention includes an exo-type β which decomposes chitosan oligosaccharides and chitosan to release glucosamine.
-D-glucosaminidase, β-N-acetylhexosaminidase (or β-N-acetylglucosaminidase) that acts on chitin oligosaccharides and chitin to generate N-acetylglucosamine, chitosanase that degrades chitosan to chitosan oligosaccharide, Examples include chitinase and lysozyme that degrade chitin into chitin oligosaccharide. Among these enzymes, commercially available enzymes can be used for β-N-acetylhexosaminidase, chitosanase, chitinase and lysozyme. For example,
β-N-acetylhexosaminidase (or β-N
-Acetylglucosaminidase) is
Examples of chitinases include those derived from Bacillus R-4 and Bacillus pamilus, and those derived from Streptomyces griseus, Serratia marcescens, and Aeromonas hydrophila. Lysozyme is generally derived from egg white. In addition, it can also be prepared by culturing microorganisms that produce these enzymes. The exo-type β-D-glucosaminidase that degrades chitosan oligosaccharide or chitosan and releases glucosamine used in the present invention can be prepared according to the preparation examples described later.

Since the degradation of chitin and chitosan by enzymes is essential to completely degrade these substances, at least exo-type β-D-glucosaminidase and β-N-acetylhexosaminidase (or β-N- It is essential to use both enzymes (acetylglucosaminidase). Furthermore, in order to make the decomposition faster, it is preferable to use chitosanase for chitosan and chitinase for chitin, and if necessary, both chitosanase and chitinase can be used together. These enzymes are
They are added simultaneously when decomposing chitin and chitosan, but they can also be added separately. The enzyme reaction is not particularly limited as long as it can completely decompose chitin and chitosan into N-acetylglucosamine and glucosamine, which are monosaccharides, but pH, temperature, and enzyme concentration at which a quicker operation can be performed. It is preferable to set conditions and the like.

After complete hydrolysis of chitin or chitosan, the quantification of N-acetylglucosamine and glucosamine produced is as follows:
It can be measured by a method using high performance liquid chromatography or a colorimetric method.

Preparation Example-Preparation of exo-type β-D-glucosaminidase-A culture solution containing 1% glucose, 1% peptone, 0.03% potassium dihydrogen phosphate, 0.07% potassium monohydrogen phosphate and 0.05% magnesium sulfate (pH 7.0 ) 500 ml was put into a 500 ml Erlenmeyer flask for 100 ml each, and Nocardia orientalis IFO-12806 strain was inoculated into the flask and cultured with shaking at 30 ° C for 2 days. Next, the above culture solution was transferred to a jar fermenter containing 1% chitosan, 0.01% yeast extract, and a culture solution 4.5 containing the same inorganic salt as above, and cultured with aeration and stirring at 30 ° C. for 4 days. The culture was centrifuged to remove the cells, and solid supernatant was added to the supernatant to obtain 80% saturation, thereby precipitating the protein. This precipitate was dissolved in a small amount of a 25 mM acetate buffer (pH 4.5), developed on a Sephadex G-25 column equilibrated with the same buffer, and desalted to obtain a crude enzyme solution.

This crude enzyme solution was applied to a CM-Sephadex C-50 column equilibrated with the above buffer, and subjected to ion exchange chromatography. After washing the column with the same buffer, 0 to 0.6M
The target enzyme was eluted using a linear concentration gradient with an acetate buffer containing sodium chloride. Fractions having β-D-glucosaminidase activity were collected and salted out with ammonium sulfate. The resulting precipitate was dissolved in a 50 mM acetate buffer (pH 5.5) and applied to a Toyopearl HW-55S column equilibrated with the same buffer. The protein was eluted with the above buffer and the active fraction was collected. Next, this fraction was applied to a chitotriitol-Sepharose CL-4B column equilibrated with a 50 mM acetate buffer, and subjected to affinity chromatography. After washing the column with the above buffer, the target enzyme was eluted with the same buffer containing 2 mM chitotriitol. The active fraction was collected and the protein was precipitated by ammonium sulfate salting out. The precipitate was dissolved in a small amount of a 50 mM acetate buffer (pH 5.5) and dialyzed with the same buffer to obtain a purified enzyme preparation.

In the present invention, either a crude enzyme solution or a purified enzyme may be used.

Hereinafter, the present invention will be described in more detail with reference to Examples, but it is a matter of course that the present invention is not limited by the description.

(Example) Five kinds of chitosans (AE), whose degree of deacetylation was previously measured by a colloid titration method using polyvinyl potassium sulfate, were dissolved in 300 ml of 0.2% acetic acid. After removing a small amount of insoluble matter, for 2 ml of this chitosan solution,
Exo-β-glucosaminidase, β-N-acetylhexosaminidase and chitosanase were added in 5 units each. Samples D and E had an additional 5 units of chitinase due to the relatively low degree of deacetylation. Reaction solution at 40 ° C
After holding for 12 hours, 1 ml of the reaction solution was freeze-dried. A part of the remaining reaction solution was sampled, and glucosamine and N-acetylglucosamine were separated and colorimetrically determined. The quantification method used the indole-hydrochloric acid method for glucosamine and the method of Leishig et al. For N-acetylglucosamine. The freeze-dried sample was dissolved in 0.1 ml of water, and glucosamine and N-acetylglucosamine were separated and quantified by high performance liquid chromatography (HPLC). The analysis conditions in HPLC are shown below. As a representative example, the analysis pattern of the sample A (see Table 1) by HPLC is shown in the attached figure.

Table 1 shows the values of the degree of deacetylation of each sample measured by the colorimetric method and the HPLC method based on the principle of the present invention and the results measured by the conventional colloid titration method.

 The enzymatic decomposition of each sample was performed three times.

Indole-hydrochloric acid method (quantification of glucosamine) To 0.5 ml of sample (glucosamine, 10-200 μg), add 0.5 ml of 5% sodium nitrite solution and 0.5 ml of 33% acetic acid, stir well, leave for 10 minutes and deamination Perform the reaction. next,
Add 0.5 ml of 12.5% ammonium sulfamate solution and leave for 30 minutes with occasional stirring to eliminate excess nitrous acid. 2 ml of 5% hydrochloric acid and 0.2 ml of 1% indole (ethanol solution) are added thereto, and the mixture is heated in a boiling water bath for 5 minutes.
After cooling, add 2 ml of ethanol and measure the absorbance at 492 nm.

Method of Reissig et al. (Quantification of N-acetylglucosamine) 0.5 ml sample (N-acetylglucosamine, 5-30 μm)
g), add 0.1 ml of a borate solution, and add exactly 5 in a boiling water bath.
Heat for a minute. After cooling with running water, add 3 ml of p-dimethylaminobenzaldehyde (DMAB) reagent and mix well. After accurately maintaining at 37 ° C for 20 minutes, cool to room temperature and
Measure absorbance at m.

Borate solution-0.8 M boric acid-potassium hydroxide solution (pH 9.
1) DMAB reagent Dissolve 10 g of DMAB in 100 ml of acetic acid containing 1N hydrochloric acid at a concentration of 12.5%, and dilute 10 times with acetic acid immediately before use.

HPLC analysis conditions Column: YMC-Pack PA-03 Mobile phase: acetonitrile: water = 3: 1 Flow rate: 0.1 ml / min Detection: differential refractometer Temperature: room temperature (Effect of the Invention) According to the method of the present invention, the degree of deacetylation of chitin and chitosan can be measured simply and with high accuracy.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows patterns when glucosamine and N-acetylglucosamine produced after completely hydrolyzing chitosan (sample A) with an enzyme are analyzed by high performance liquid chromatography.

Claims (1)

(57) [Claims] 1. Deacetylation of chitin or chitosan, comprising decomposing chitin or chitosan into N-acetylglucosamine and glucosamine, which are constituent monosaccharides, by an enzyme, and quantifying the produced monosaccharide. Degree measurement method.