KR100486042B1 - Method for preparing chitin hydrolysates having low molecular weight and oligosaccharides by the enzyme treatment - Google Patents

Abstract

The present invention relates to a method for producing chitin low molecular sugar and oligosaccharide by enzyme treatment, and more specifically, adding a diluted organic or inorganic acid to chitosan to obtain a homogenized chitosan solution; Adding an alkaline substance to the chitosan solution to leach and separate the chitosan; Suspending in methanol or acetone to prevent acetylation of the separated chitosan; Adding acetic anhydride to the suspension to obtain acetylated chitin on fibrous; And it relates to a method for producing chitin low-molecular sugar and oligosaccharide by the enzyme treatment comprising the step of hydrolysis by adding chitinase to the chitin. The present invention has an advantage that it is easy to use in food, cosmetics, agriculture, and medical and pharmaceutical fields by synthesizing the chitin on the fibrous and treated by the enzymatic method to prepare a water-soluble low molecular chitin containing chitin oligosaccharides.

Description

Method for preparing chitin hydrolysates having low molecular weight and oligosaccharides by the enzyme treatment

The present invention relates to a method for producing chitin low molecular sugar and oligosaccharide by enzyme treatment, and more specifically, to obtain a water-soluble low molecular sugar containing more effective chitin oligosaccharide, first, a pure fibrous chitin is obtained and treated with an enzyme. A water-soluble chitin low molecular sugar and oligosaccharides.

Chitin is well known as a natural polymer that can be obtained from the natural world, and is better known as a major constituent of the shell of many kinds of crustaceans, such as crabs or shrimp. In particular, it has recently emerged as a biologically specific substance has been used in a variety of fields such as research samples, feed, medicine, pharmacy, agriculture and cosmetic raw materials. However, there is a big disadvantage of insolubility in using chitin, and there are many restrictions on its use.

As a result, many researchers have been developing methods to accept this material, and have already achieved significant results. However, since chitin itself cannot be solvated, classical methods such as chemically formulating and solubilizing it, or treating with strong acid to dissolve, or heating to high temperature to form a low molecular weight material, are still used.

For example, as a prior art for producing water-soluble low molecular weight chitin oligosaccharides, Korean Patent Application No. 98-03031 discloses dispersing chitin in water, adding acid and stirring to swell chitin, and swelling chitin. Disclosed is a method for preparing water-soluble low molecular chitin and chitin oligosaccharides comprising irradiating a solution with ultrasonic waves.

As a method for preparing chitosan oligosaccharides, Japanese Laid-Open Patent Publication No. 1-56755 prepares a low molecular weight water-soluble chitosan using chitosanase, a chitosan degrading enzyme, and Japanese Patent Laid-Open Publication No. 4-99474 to prepare chitosan in an aqueous acetic acid solution. After dissolving, low-molecular-weight water-soluble chitosan oligosaccharides were prepared using chitosanase obtained from Bertsilium AF 9-V-156 in the above solution. Too large, wide molecular weight distribution, and very low yield are pointed out as the biggest problems.

In addition, Korean Patent Application No. 99-32346 discloses chitosan degrading enzyme for the first decomposition step of reacting and neutralizing hydrogen peroxide by adding hydrogen peroxide to a mixture of water, acetic acid and sulfuric acid. And a second decomposition step of decomposing chitosan and a method for producing a low molecular weight water-soluble chitosan oligosaccharide comprising centrifugation, dialysis, and drying the solution.

Recently, microorganism-producing chitinases are used for the production of chitin oligosaccharides as well as chitosan oligosaccharides, but this method also depends on how to make chitin as homogeneous as possible for effective enzyme degradation. I think it can add value.

Therefore, in the present invention, a small but small result was obtained after a long research effort to develop a method for producing a water-soluble low molecular sugar containing more effective chitin oligosaccharides, and the present invention was completed based on this. In other words, the present inventors determined that in order to use the chemical method or the enzymatic method to obtain the chitin oligosaccharide, the chitin of pure fiber is required. In other words, new methods are needed to obtain chitin on pure fibrous fibers. Of course, there are many researchers who find and use chitin solvent as an effort to dissolve chitin easily, but most of the known methods are quite cumbersome, or high concentrations of organic solvents such as sulfuric acid and hydrochloric acid should be used. It is also thought to be not easy.

Accordingly, an object of the present invention is to easily obtain a chitin on a fibrous fiber, and to provide a method for preparing a water-soluble chitin oligosaccharide and a water-soluble chitin low molecular sugar by hydrolyzing it with an enzyme.

The method for preparing chitin low molecular sugar and oligosaccharide by the enzyme treatment of the present invention for achieving the above object comprises the steps of adding a diluted organic acid or inorganic acid to chitosan to obtain a homogenized chitosan solution; Adding an alkaline substance to the chitosan solution to leach and separate the chitosan; Suspending in methanol or acetone to prevent acetylation of the separated chitosan; Adding acetic anhydride to the suspension to obtain acetylated chitin on fibrous; And hydrolyzing the chitin by adding chitinase.

Looking at the present invention in more detail as follows.

As mentioned above, chitin is well known as a major constituent of the shell of numerous types of crustaceans, such as crabs or shrimps. This not only plays an important role in protecting itself from any external attack, but is recently used in various fields as a natural new material. In particular, as the physiological / biological specificity of chitin oligosaccharides is recognized, many researchers are interested in the development of chitin oligosaccharides and water-soluble low molecular sugar production technologies, and are seeking new technologies that are simpler and more reliable. Unfortunately, there are many technical limitations to using chitin.

Chitin is considerably more difficult to dissolve in organic solvents than chitosan (a fully or partially deacetylated substance of chitin), and it is not a small problem in use because it is dissolved in high concentrations of hydrochloric acid, nitric acid, sulfuric acid, or a very specific solvent. It is also true that there are many technical problems with hydrolysis to small molecular weights of specific sizes.

Conventional methods are usually obtained by heating the chitin to a high temperature using a fairly high concentration of hydrochloric acid, but in order to produce oligosaccharides of the most uniform size, it is necessary to first crush the chitin to a minimum size. Even if a small particle of chitin is obtained in the powder state, it may be difficult to say that it is suitable for use in chemical / enzymatic hydrolysis.

Therefore, most researchers make and use colloidal chitin in consideration of the solution of this problem, but this also requires the use of a large amount (40 ~ 60g / 1ℓ) of concentrated hydrochloric acid, safety and environmental friendliness There is no choice but to consider. In addition, recently, a method of hydrolyzing colloidal chitin by using a hydrolase called chitinase has been used, but this method also needs to use a substance called colloidal chitin as a basic substance, thus solving the original problem. .

Therefore, in the present invention, in applying the chemical and enzymatic methods for obtaining water-soluble chitin oligosaccharides and water-soluble low molecular sugars, first, pure fibrous chitin is required.

In other words, most of the commercially available chitin is a very fine powder state, but in fact it is difficult to obtain the oligosaccharide by enzymatic treatment or chemical treatment as it is. Therefore, it is important to obtain a chitin on the fibrous to perform a more efficient reaction. Most conventional methods have been used to produce colloidal chitin by treating chitin with concentrated hydrochloric acid. Also, a method of dissolving chitin by using a chemical functional group or another very specific method and recovering it for a long time has been proposed. However, all of them have a long processing time and use an excessive amount of hydrochloric acid or other chemicals. Therefore, there is a need for a method of producing more efficient fibrous chitin.

According to the method of the present invention, in order to obtain a fibrous chitin, the well-refined chitosan (deacetylation degree of 92.0 to 99%) is first dissolved in a dilute organic or inorganic acid solution, preferably acetic acid solution, stirred for a sufficient time, and completely dissolved. Get As such, by dissolving chitosan in a dilute organic acid, chitosan in a homogeneous state can be obtained, and an efficient reaction can be performed in the subsequent acetylation process. On the other hand, the deacetylation degree of the chitosan may be less than 92.0%, but it takes some time for complete dissolution in the process of dissolving in the organic acid, chitosan having a high degree of deacetylation as possible because there may be an incomplete dissolution chitosan in some cases It is preferable to use.

In the present invention, since chitosan is much more unstable structurally than chitin, chitosan is used instead of chitin as a starting material. In other words, chitin is not soluble in dilute organic or inorganic acids, so it is impossible to maximize the reaction and activity of the enzyme in a subsequent process. Therefore, since chitin as a starting material cannot obtain a chitin of fibrous uniform acetylation, a chitosan solution in which chitosan is dissolved in an organic or inorganic acid solution is used.

Stirring the chitosan solution well and slowly adding a strong, weakly alkaline substance, preferably a Tris-HCl solution, the pH rises over time and the chitosan leaches. A strong, weakly alkaline, preferably tris salt solution is added to precipitate all chitosan until no further leaching is seen.

According to the present invention, strong and weak alkaline substances are used because the basic conditions for the acetylation of chitosan are to maintain the pH at weak alkali. In this case, the pH is preferably about 8.0, and if the pH is increased beyond this, chitosan may be rapidly condensed. The tris salt, which is preferable as the alkaline substance, is a substance that is used very much as a buffer that is usually used for protein purification, and the buffering effect can be expected even in the reaction of adding acetic anhydride.

The precipitated chitosan is recovered using a centrifuge, and then the precipitate is suspended in methanol or acetone solution to prevent acetylation of the chitosan to a hydroxyl group (-OH) at a position other than the free amino group. In this case, high-speed stirring, ultrasonic waves, or vortex may be used to make the suspension as uniform as possible. The pH in this case is also preferably around 8.0. In addition, although the concentration of the methanol or acetone solution is preferably 7 to 15%, even if less than 7% may be sufficient or insufficient in some cases. However, less than 7% may be more likely to be acetylated to the -OH group of the 3 or 6 carbon, and if it exceeds 15%, the acetylation of chitin tends to be lowered.

According to the present invention, the mechanism by which the methanol or acetone solution prevents the acetylation of the functional group possessed by chitosan, that is, the -OH group located at carbon 3 and 6, is not known exactly. However, it is expected that acetic anhydride will be in an unstable state when diluted in dilute solvent, so that it will be in equilibrium. At this time, the organic solvent having lower molecular weight than chitosan can be balanced to favorably balance the -OH group of the organic solvent. have. However, since an appropriate amount of acetic anhydride is added at a pH-adjusted state, acetylation occurs rapidly to the amino group (-NH ₂ ) having a large ion binding capacity, and it is judged that mutual substitution with -OH groups is hard to occur.

Next, acetic anhydride, which is proportional to about 2 times the amount of free amino groups of chitosan, is added very slowly to the suspension while checking the pH. The pH is appropriately maintained with a weak alkali, and it is preferable to use a tris salt as the alkaline substance. After the reaction, acetic anhydride can be added again for complete acetylation of chitosan, and at this time, pH is added and tris salt is added as necessary. That is, under alkaline pH conditions, the free amino group of chitosan (-NH ₂ , located on carbon 2) is not ionized, and the reaction with acetic anhydride is rapid.

After completion of the reaction, the precipitated fibrous acetylated chitin is recovered by centrifugation again, and the precipitate is washed several times with ion-exchanged water to obtain the fibrous acetylated chitin. The fibrous acetylated chitin consists of N-acetyl glucosamine. However, in the conventional colloidal chitin, a certain amount of deacetylation is caused by a forced chemical change, but glucosamine, which is a structural unit of chitosan, exists in part. Accordingly, the acetylated chitin on the fibrous of the present invention is more easily hydrolyzed by enzyme treatment due to its structural specificity than the chitin on colloid.

A hydrolase, preferably chitinase, is added to hydrolyze the chitin obtained from the process. Most commercially available purified enzymes are concentrated to significant concentrations and usually include buffers to maintain enzyme activity.

According to the present invention, the active properties of various commercially available or laboratory-produced chitinases were investigated, and 2 to 4 carbon sugars are produced as the final product of the reaction under the optimal conditions for enzymatic activity. As a result of confirming the reaction product with the reaction time of up to 1 minute unit, the same result was obtained even for such a short reaction time. Thus, much research is needed to obtain higher molecular weight chitin oligosaccharides. Many commercially available chitinases are currently available for purchase, so the present invention does not limit the use of chitinases by type.

On the other hand, the present invention was used by varying the reaction conditions of the enzyme in consideration of the effective use of the enzyme and the diversity of the final reaction product. In order to increase the enzyme activity, it is absolutely necessary to optimize the reaction conditions, but it is necessary to remove unnecessary substances in the future, and as mentioned earlier, it is not necessary to control the decomposition product size, that is, use pure purified water at a suitable temperature instead of using a special enzyme reaction buffer. Dilute acetic acid was used to maintain an appropriate pH (pH 4.0 to 5.0) to carry out the enzyme reaction. Since the enzyme already contains a small amount of buffer components, it is thought to affect the activity of the enzyme somewhat, but the effect is not considered.

The chitin was suspended in an enzyme reaction solution using a vortex, and the suspension was placed in a dialysis membrane passing a molecular weight of 10-kDa to complete preparation of the reaction. Then, the same reaction solution was maintained at a predetermined temperature in a container. The dialysis membrane containing the chitin suspension was transferred thereto, and the reaction was started by the addition of chitinase enzyme. At this time, the vessel containing the external reaction solution was slowly stirred by using a magnetic rod to help the product hydrolyzed by the enzyme to pass through the dialysis membrane, and the enzyme reaction was effectively performed. The reaction was terminated by determining the suspension of chitin in the dialysis membrane. If necessary, the reaction dialysis membrane was transferred to another vessel containing 2 times the volume of purified water, and the reaction was continued. . On the other hand, the method of additionally using the enzyme was avoided depending on whether the chitin remained in the dialysis membrane, and the enzyme was denatured by recovering and distilling from the dialysis membrane to remove the undigested macromolecule and chitinase. The supernatant was included in the final reaction product. At this time, whether or not the chitinase was included in the final reaction product was checked using a commercial protein measurement reagent. On the other hand, the size of the dialysis membrane used in the present invention is not limited.

After completion of the reaction, low molecular sugar was precipitated using acetone in an amount corresponding to 2.5 to 5 times of the reaction solution for simple separation of the oligosaccharide having a low molecular weight and the low molecular sugar water-soluble. At this time, water-soluble low molecular sugar equivalent to an average of 20 to 25% of the total amount produced / recovered can be obtained.

As a result of the above-mentioned volume reduction by thin-wall chromatography and HPLC of the supernatant that was not precipitated by the precipitation method of water-soluble low molecular sugar by acetone as described above, the major hydrolysis products of chitinase were The size of mainly 3-6 tantalum was detected.

In order to use the obtained chitin oligosaccharides for research such as fine chemistry and biochemistry, more precise size-specific purification methods are required, but there is sufficient value for separating and identifying chitin-degrading microorganisms from the natural world in basic biological fields. It is believed that it will not be inferior in use as a food additive. In addition, water-soluble low-molecular sugar is expected to be useful as a raw material for cosmetics, and as many studies have already reported, it is also expected to be used as an alternative fertilizer such as plant cultivation and plant growth promoters.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples.

Example 1

2 g of chitosan (deacetylation degree of about 99%) was added to 10% acetic acid solution, stirred for 1.5 hours, and completely dissolved to prepare a 2% chitosan solution. The solution was stirred well, and 0.5-1 mol of Tris-HCl solution prepared in advance was slowly added thereto. As time passed, the pH was increased and thus chitosan was leached out. Tris salt (Tris-HCl) solution was added to precipitate all chitosan until no further leaching occurred.

In order to recover the precipitated chitosan, the centrifuge was rotated at about 4,000 rpm for 10 minutes to precipitate chitosan, and the supernatant was removed. This precipitate was suspended in 10% methanol solution and stirred at high speed to make the suspension as uniform as possible. At this time, the reaction pH was maintained at about 8.0. Acetic anhydride, which is proportional to 2 times the amount of free anino groups of chitosan, was added very slowly while checking the pH, and a small amount of tris salt was added to maintain the pH as needed.

After 30 minutes, half the amount of acetic anhydride was added again for complete acetylation of chitosan, and at this time, the pH was added while the required amount of Tris salt was added and the reaction was continued for 3 hours.

After completion of the reaction, the precipitated fibrous acetylated chitin was recovered by centrifugation again and the precipitate was washed several times with ion-exchanged water. This gave the fibrous acetylated chitin in an average yield of at least 95%. Acetylation was 99.8%, and the measuring method is as follows.

Measurement method of acetylation degree

A predetermined amount of acetylated chitin was added to the reaction solution (0.5% Picrylsulfonic acid / 5% sodium bicarbonate) and reacted at 37 ° C for 30 minutes, followed by washing with ion-exchanged water. do. Washing was repeated until the absorbance of the wash was near zero in A340. After washing, 1 ml of 70% Perchloric acid was added to dissolve the chitin, and the absorbance was measured using an absorber. In this case, when the absorbance value is 0.25, the number of free amino groups corresponds to 15 nanomoles (nmol). This result is calculated from a calibration curve previously measured using glucosamine.

Example 2

Except for using acetone of 7% instead of 10% methanol solution in Example 1 was carried out in the same manner.

The rate of acetylation was faster than that of Example 1. The determination of the degree of acetylation was determined by the method described above for the amount of unacetylated free amino groups. In addition, when 15% or more of acetone was used, the degree of acetylation was observed. Therefore, good results were obtained when the amount of acetone of 7-10% of the total reaction amount was used. Similar results were obtained with methanol.

Example 3

Method for recovering chitin oligosaccharide and low molecular sugar by hydrolysis of chitin

Commercially available chitinase (Serratia marsecences) was purchased from Sigma Co., Ltd. in order to hydrolyze the chitin obtained from the method of Example 1.

In this example, the reaction conditions of the enzyme were used in consideration of the effective use of the enzyme and the diversity of the final reaction product. Dilute acetic acid (20%) was used in pure purified water to maintain an appropriate pH of 4.0 to 5.0 to carry out the enzyme reaction.

1.0 g of chitin was suspended in an enzyme reaction solution (0.2 L) using high-speed stirring, and the suspension was placed in a dialysis membrane (PIERCE, SnakeSkin dialysis tube) having a molecular weight of 10-kDa. The same reaction solution, which was previously maintained at a temperature of 30 ° C. (5 times the volume of the enzyme reaction solution), was placed in a container, and the dialysis membrane containing the chitin suspension was transferred to the chitinase enzyme (0.1 to 1.0 unit; The reaction was started with the addition of 1.0 mg of N-acetylglucosamine with N-acetylglucosamines / hour, and this unit of enzymatic activity virtually does not apply to this enzyme reaction. At this time, the vessel containing the external reaction solution was slowly stirred by using a magnetic rod to help the product hydrolyzed by the enzyme to pass through the dialysis membrane, and the enzyme reaction was effectively performed. The reaction was terminated by determining the suspension of chitin in the dialysis membrane. If necessary, the reaction dialysis membrane was transferred to another container containing twice the volume of purified water, and the reaction was continued. On the other hand, additional methods of using enzymes were avoided depending on whether chitin remained in the dialysis membrane, and the enzymes were denatured by recovering from the dialysis membrane and steaming for 10 minutes to remove undissolved macromolecules and chitinase. (10,000 rpm) was precipitated for 10 minutes and the supernatant was included in the final reaction product. At this time, whether the chitinase was included in the final reaction product was checked using a commercial protein measurement reagent.

After completion of the reaction, low molecular sugar was precipitated using acetone in an amount corresponding to 2.5 to 5 times of the reaction solution for simple separation of the oligosaccharide having a low molecular weight and the low molecular sugar water-soluble. At this time, water-soluble low molecular sugars corresponding to an average of 20 to 25% of the total amount produced / recovered were obtained. Their average size is estimated to be about 7-12 shots.

As a result of the above-mentioned volume reduction by thin-wall chromatography and HPLC of the supernatant not precipitated by the above-mentioned method of precipitation of water-soluble low molecular sugar by acetone, the major hydrolysis products of chitinase were mainly 3 The size of ˜6 charcoal was detected.

As mentioned above, the methods of the present invention require fibrous chitin to enhance the activity of the enzyme. In the use of chitin low molecular sugars comprising chitin oligosaccharides prepared according to the present invention, it is considered that they are inferior in use to drinking water, food additives, and the like. In addition, water-soluble low-molecular sugar is expected to be greatly used as a raw material for cosmetics, and is also expected to be used as an alternative fertilizer such as plant cultivation and plant growth promoters for obtaining healthy vegetables, and cultivation of special products that create high added value. .

Claims (6)

Adding a dilute organic or inorganic acid to chitosan having a deacetylation degree of 92.0 to 99% to obtain a homogenized chitosan solution; Adding an alkaline substance to the chitosan solution to leach and separate the chitosan; Suspending in methanol or acetone to prevent acetylation of the separated chitosan; Adding acetic anhydride to the suspension to obtain acetylated chitin on fibrous; And Hydrolyzing chitin by adding chitin to the chitin; and a method for producing chitin low molecular sugar and oligosaccharide by enzymatic treatment comprising a. The method of claim 1 wherein the organic acid is acetic acid. The method of claim 1, wherein the weakly alkaline substance is a tris salt, which is used to maintain the overall reaction pH at about 8. 8. The method according to claim 1, wherein the concentration of methanol or acetone is 7 to 15%. The method according to claim 1, wherein the hydrolysis reaction is performed by maintaining a proper pH of 4.0 to 5.0 using dilute acetic acid in pure purified water. The method of claim 1, wherein the method further comprises passing a dialysis membrane having a molecular weight of 10-kDa for selective fractionation of chitin oligosaccharides and low molecular sugars after the hydrolysis step.