JPH0655151B2 - Method for producing low molecular weight chitosan - Google Patents

Description

Detailed Description of the Invention [Industrial applications]

The present invention relates to a method for producing low molecular weight chitosan by an enzymatic method. More specifically, it relates to a method for efficiently producing low molecular weight chitosan having an arbitrary molecular weight, which is expected to be applied to foods, cosmetics, pharmaceuticals, etc. by degrading chitosan with an enzyme.

[Prior art]

Chitosan is a basic homopolysaccharide in which 2-amino-2-deoxy-D-glucose (glucosamine) is linearly linked by β- (1 → 4), and is industrially usually found in the shells of crabs and shrimps. It is obtained by deacetylating the contained natural chitin. This chitosan has protein aggregating power and is used as an aggregating agent for industry. Recently, its application in the fields of food, cosmetics, pharmaceuticals, etc. has been drawing attention. However, chitosan obtained by deacetylating natural chitin generally has a high molecular weight and has a drawback that it is difficult to handle because of its high viscosity when made into a solution and its use is limited. Therefore, various studies have been conducted to improve the utility of chitosan by chemically or enzymatically decomposing chitosan to make it low-molecular weight and easily water-soluble. Examples of the method for producing low molecular weight chitosan include, for example, hydrolysis with hydrochloric acid (ST Horowitz et al., Journal of Ameri.
can Chemical Society, Vol.79, p5046-5049 (1957)), decomposition by nitrous acid (F. Yaku et al., Cellulose Chemistry and
Technology, Vol.11, p421-430 (1977)), decomposition with hydrogen peroxide (Japanese Patent Publication No. 56-33401, Japanese Patent Laid-Open No. 1-185301), decomposition with chlorine gas (Japanese Patent Laid-Open No. 60-186504), sodium perborate There are chemical decomposition methods such as decomposition (JP-A-61-40303),
There is a problem of handling complexity such as the danger of chemicals used, waste liquid treatment, the molecular weight distribution of the low molecular weight chitosan obtained is wide, and it is difficult to obtain chitosan controlled to a specific narrow molecular weight range, There are drawbacks such as deamination of chitosan and browning tendency. On the other hand, various attempts have been made to reduce the molecular weight of chitosan using an enzyme. The chitosanase as a degrading enzyme includes a chitosanase (Y. Tomi) produced by Bacillus sp. R-4.
naga et al., Bio-chimica et Biophysica Acta, Vol.410, p14.
5-155 (1975)), chitosanase produced by Bacillus genus 99-5 (Horiuchi, Japan Society for Agricultural Chemistry, 1984 Conference, Abstracts, p550), Chitosanase produced by Bacillus No.7-M (special 1-56755), Bacillus. B. cereus
Of chitosanase (Japanese Patent Laid-Open No. 60-180585), Chitosanase of Bacillus circulans (Japanese Patent Laid-Open No. 63-94971), Bacillus pamilus (B. pu)
milus) produced chitosanase (JP-A-63-63382),
Chitosanase produced by Alcaligenes sp. MHK-1 (JP-A-62-201571), chitosanase produced by Strepto-myces sp. No. 6 (JSPrice et al., Jounal of Bacteriology, Vol. 124, p157
4-1585 (1975)), Nocardia Orientalis (Noca
chitosanase produced by rdia orientaris (Katsumi et al.,
Japan Society for Agricultural Chemistry, 1989 Annual Conference, Lecture Summary p609),
Penicillium islan
Chitosanase produced by d-icum (DMFenton et al., Jour
nal of General Microbiology, Vol.126, p151-165 (198
1)) is known. In addition, the molecular weight of chitosan has been reduced by using an enzyme other than chitosanase. For example, a method of degrading chitosan by at least one of papain, cellulase and acid protease
1-57958), a method of decomposing with an enzyme agent containing pectinase as a main component (JP-A 1-291799), and a method of decomposing with cellulase (JP-A 2-20292).
However, although these decomposition reactions using enzymes other than chitosanase may be caused by the chitosanase mixed in the enzyme preparation, they have not been studied in detail.

[Problems to be Solved by the Invention]

Such an enzymatic decomposition method has advantageous characteristics such as milder reaction conditions and easier control of the molecular weight of the obtained chitosan as compared with the above-mentioned chemical decomposition method. However, with conventional enzymes, enzyme production, purification,
The problem of cost related to the reaction process and the browning of chitosan during the reaction and heat deactivation of the enzyme have become a bottleneck, and it has not been industrialized. Therefore, the present invention has been made for the purpose of providing a method capable of efficiently producing a low-molecular-weight chitosan that is industrially applicable while maintaining the advantages of the method for producing a low-molecular-weight chitosan using an enzyme. is there.

[Means for Solving the Problems]

That is, the method for producing a low-molecular-weight chitosan of the present invention comprises adding a chitosan solution to the genus Verticillium (Verticillium sp.).
It is characterized by acting on chitosanase produced by a microorganism belonging to the group to lower the molecular weight. The raw material chitosan used in the present invention is a high molecular weight chitosan obtained by deacetylating natural chitin by an ordinary method such as alkali treatment, or a chitosan obtained by further chemically or enzymatically degrading it to a certain extent, especially as to its molecular weight. There is no limitation, and chitosan having any molecular weight can be used as a raw material. Also, colloidal chitosan prepared by dispersing and suspending chitosan in water under basic or neutral conditions can be used as a raw material. Further, both chitosan having a high degree of deacetylation (95 to 100%) and commonly used chitosan having a low degree of deacetylation (65 to 95%) can be used as raw materials. The enzyme used in the present invention is not limited as long as it is an enzyme produced by a microorganism belonging to the genus Verticillium,
Among them, the chitosanase produced by Verticillium genus AF9-V-156 strain (Ministry of Industrial Science and Technology No. 11377) and its mutant strain is most preferable. Regarding the method for producing this enzyme, a patent application has already been filed by the same applicant as this application (Japanese Patent Application No. 2-
81531). The pH at which the enzymatic reaction is carried out can be appropriately selected depending on the optimum pH of the chitosanase used, the solubility of the starting chitosan, etc., but the reaction under acidic conditions is preferred due to the nature of the basic polysaccharide chitosan. To dissolve chitosan, usually suspend chitosan in water, add an acid such as acetic acid to dissolve it, and then add an alkali such as sodium hydroxide.
The process of adjusting the pH is taken. However, chitosan often precipitates in the process of adding the alkali, and it takes time to redissolve, so that it takes a long time to adjust the pH, which is industrially inefficient. Therefore, it is preferable to dissolve chitosan with an acid and carry out the enzymatic reaction at the pH as it is. Especially when using chitosanase produced by Verticillium genus AF9-V-156 strain or its mutant strain, pH 3.
If it is 0 or more, the reaction can be performed efficiently, so by adjusting the amount of acid added when dissolving chitosan and maintaining the pH after dissolution at 3.0 or more, it is possible to carry out the enzyme reaction as it is without adding alkali. You can The reaction temperature can be selected in the range of 30 to 80 ° C., which is usually the action temperature of chitosanase, but the reaction at a high temperature promotes browning of chitosan, so a reaction at a low temperature is desirable to avoid browning as much as possible. In the case of chitosanase produced by Verticillium genus AF9-V-156, the enzymatic reaction can be efficiently performed at 30 to 37 ° C.

【Example】

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples. Example 1. Low viscosity chitosan LLWP (trade name, manufactured by Kimitsu Chemical Industry Co., Ltd .; Deacetylation degree: 75-85%) 0.5 g in acetate buffer (0.1 M,
(pH 5.0) 100 ml was added and stirred to dissolve the chitosan and bring it to 0.
It was a 5% chitosan solution. Berticillium AF9
The chitosanase produced by the -V-156 strain was added at 10 mU / ml and reacted at 37 ° C for 2 hours. After deactivating the enzyme by heat treatment at 80 ° C. for 15 minutes, the degree of depolymerization of chitosan was examined by gel filtration method. That is, 0.4 ml of chitosan solution after enzymatic reaction was diluted to 1 ml, and gel filtration was performed using a column (15 mm × 44 mm) packed with a carrier for gel filtration Sephacryl S-200 (trade name of Pharmacia) to analyze the molecular weight distribution. did. 510,00 as a molecular weight marker
Dextran (manufactured by Sigma) of 0, 71,000, 39,000 and 9,000, chitohexaose (molecular weight of about 1,200, manufactured by Seikagaku Corporation), and glucosamine hydrochloride were used. As a result, it was revealed that the chitosan of the enzyme reaction solution obtained in this Example was composed of a component having a molecular weight of about 40,000 and two peaks of oligosaccharide from glucosamine monosaccharide. Example 2. 5 g of chitosan "Flownac C" (trade name of Kyowa Yushi Kogyo Co., Ltd.) was suspended in 90 ml of pure water and stirred for 10
3.5 ml of M lactic acid was added to dissolve chitosan. After completely dissolving, the liquid volume was adjusted to 100 ml to prepare a 5% chitosan solution.
The pH of this solution was 3.8. Chitosanase produced by Verticillium genus AF9-V-156 strain was added to this at 40 mU / ml and reacted at 30 ° C. for 18 hours. 80
After deactivating the enzyme by heat treatment at 15 ° C for 15 minutes, the degree of depolymerization of chitosan was examined. The degree of reduction of the molecular weight of chitosan after the enzymatic reaction was examined by the gel filtration method as in Example 1. As a result, it was found that the chitosan in the enzyme reaction solution obtained in this example was distributed mainly in glucosamine hexamer.

【The invention's effect】

As described above, according to the present invention, it is possible to efficiently produce a low molecular weight chitosan having an arbitrary molecular weight using general-purpose chitosan as a raw material, and therefore it can be industrially sufficiently applied. Furthermore, the low molecular weight chitosan produced by the method of the present invention can be widely applied to foods, cosmetics, pharmaceuticals and the like.

Claims (2)

[Claims] 1. A solution of chitosan in the genus Verticillium (Ve
rticillium sp.) belonging to the present invention, a chitosanase produced by a microorganism belonging to rticillium sp. 2. The method for producing a low molecular weight chitosan according to claim 1, wherein the microorganism belonging to the genus Verticillium is Verticillium AF9-V-156 (Microtechnical Laboratory No. 11377).