CN111373037B - Chitin-decomposing enzyme composition, chitin decomposition reaction liquid, and method for producing sugar - Google Patents

Abstract

The chitin-degrading enzyme composition comprises: chitinase as a chitinase for hydrolyzing chitin; silica or a silica-containing substance; and a nitrogen-containing heterocyclic compound as a nitrogen-containing heterocyclic compound.

Description

Chitin decomposing enzyme composition, chitin decomposing reaction liquid and method for producing sugar

technical field

The present invention relates to a chitin decomposing enzyme composition, a chitin decomposing reaction liquid and a method for producing sugar.

Background technique

Chitin is contained in exoskeletons of insects and crustaceans, cell walls of fungi, and the like in the biological world, and its annual production amount is estimated to be second only to cellulose, which is a biomass abundantly present on the earth. Therefore, chitin is gaining attention as a biomass resource next to cellulose.

Chitin is an insoluble polysaccharide in which N-acetyl-D-glucosamine is bonded as a monosaccharide, and it is reacted by an enzymatic reaction using chitinase, which is a decomposing enzyme of chitin (also called "chitin decomposing enzyme") , hydrolyzed into low molecular weight chitin polysaccharide (low molecular weight chitin polysaccharide), chitin oligosaccharide derived from chitin polysaccharide, monosaccharide (N-acetyl-D-glucosamine) and the like. In addition, these hydrolyzates are products of enzymatic reactions of chitin.

The products produced in the enzymatic reaction of chitin have excellent antibacterial properties, moisture retention, biocompatibility, safety, and chelating properties. Therefore, utilization in various fields such as medical materials, medicine, cosmetics, fibers, agriculture, water treatment, and food is expected, and research and development are ongoing.

For example, Non-Patent Document 1 describes a technique related to the enzymatic activity of chitinase using silica nanoparticles (SNP). Specifically, it is disclosed that chitinase (Chi9602) derived from Bacillus thuringiensis was immobilized on the surface of SNP by electrostatic adsorption to prepare nano-scale chitinase (SNPC1), and the prepared SNPC1 was used to measure Enzymatic activity of chitinase. In addition, the immobilization rate of silica in the prepared SNPC1 was about 55%.

However, the chitinase activity of SNPC1 was about 43% relative to the chitinase activity of Chi9602 which was 100%, so it was clarified that the activity was reduced by about 57% by the immobilization of silica. That is, it has been shown that in a reaction system in which chitinase is immobilized on silica, sugar can be produced, but the efficiency of the saccharification reaction decreases due to inhibition of enzyme activity, and it is necessary to solve such a problem when using this reaction system. In addition, a complicated reaction system is not desirable from the viewpoint of cost.

prior art literature

non-patent literature

Non-Patent Document 1: X.Qin et al., International Journal of Biological Macromolecules, Vol.82, 2016, p.13-p.21

Contents of the invention

The problem to be solved by the invention

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method capable of improving the efficiency of a saccharification reaction using a chitin-decomposing enzyme through a simple process when silica is used in a reaction system using a chitin-decomposing enzyme. , and a method for producing a chitin decomposing enzyme composition, a chitin decomposing reaction solution, and a sugar that realize high yield of products.

method used to solve the problem

The first aspect of the present invention to achieve the above object is a chitin decomposing enzyme composition characterized in that it contains: chitinase as a chitin decomposing enzyme that hydrolyzes chitin; silicon dioxide or a silicon dioxide-containing substances; and nitrogen-containing heterocyclic compounds that are nitrogen-containing heterocyclic compounds.

A second aspect of the present invention is the chitinase composition according to the first aspect, wherein the chitinase contains at least chitinase A.

A third aspect of the present invention is the chitin decomposing enzyme composition of the first aspect or the second aspect, wherein the nitrogen-containing heterocyclic compound is a nitrogen-containing five-membered heterocyclic compound.

A fourth aspect of the present invention is the chitin decomposing enzyme composition of the third aspect, wherein the nitrogen-containing five-membered heterocyclic compound is any one selected from imidazole and 2-methylimidazole.

A fifth aspect of the present invention for achieving the above object is a chitin decomposition reaction liquid characterized by comprising: chitin; and any one of the chitin decomposing enzyme compositions of the first to fourth aspects.

A sixth aspect of the present invention that achieves the above-mentioned object is a method for producing sugar, characterized in that the saccharide is produced by hydrolyzing chitin using the chitin decomposition reaction solution of the fifth aspect.

A seventh aspect of the present invention is the method for producing sugar according to the sixth aspect, characterized in that the saccharide is produced by hydrolyzing chitin with stirring using the chitin decomposition reaction liquid.

The effect of the invention

According to the present invention, when silica is used in the reaction system using chitin decomposing enzyme, the efficiency of the saccharification reaction using chitin decomposing enzyme can be improved through a simple process, and a high yield of the product can be achieved. A method for producing a liquidized chitin decomposing enzyme composition, a chitin decomposing reaction solution, and sugar.

Detailed ways

(Chitin decomposing enzyme composition)

The chitin decomposing enzyme composition of the present invention improves the efficiency of the saccharification reaction and realizes oligosaccharides, N-acetyl-D-glucosamine, etc. high yield. Hereinafter, the chitin decomposing enzyme composition of this invention is demonstrated in detail.

The chitin decomposing enzyme composition of the present invention is a composition capable of hydrolyzing chitin as a substrate, and contains chitin decomposing enzyme; silica or a substance containing silica; and a nitrogen-containing heterocyclic compound.

Here, chitin as a substrate is a linear high-molecular-weight amino sugar in which a plurality of N-acetyl-D-glucosamine residues are bonded by β-1,4 bonds, and is an insoluble polysaccharide with a firm crystal structure. polysaccharides. Chitin has a chemical structure represented by the following formula (1).

In addition, the deacetylated product of chitin is called chitosan (Chitosan, chitosan), and has a chemical structure represented by the following formula (2).

General chitin has a chitosan structure in which acetyl groups have been partially lost, while chitosan has a chitosan structure in which acetyl groups have been partially acquired. Therefore, chitin in the present invention is a concept including chitin having the above-mentioned chitosan structure and chitosan having a chitin structure. In addition, such chitin may be a chitin-containing substance containing chitin to such an extent that a product in an enzyme reaction can be recovered.

The chitin raw material is not particularly limited, and chitin-based biomass-derived raw materials can be used. Examples of such raw materials include carapaces of shrimps and crabs, carapaces of squid and the like, carapaces or exoskeletons of insects, and cell walls of fungi. Moreover, as a raw material, what originates in a natural product can be used, and a commercial item can also be used. When the raw material is derived from a natural product, one type may be used alone, or two or more types may be used in combination.

As a raw material derived from a natural product, for example, red snow crab can be used. In the case of using red snow crab, it can be dried by crushing the shell of the dried red snow crab, the legs with the trunk removed, and the joints into 3 cm to 5 cm, and then mixed with dilute sodium hydroxide aqueous solution at high temperature and dilute hydrochloric acid at room temperature. In the aqueous solution, soak for 2 hours to 3 hours respectively, remove protein and calcium carbonate, and obtain chitin. In addition, chitosan can be obtained by deacetylating the obtained chitin in a concentrated aqueous solution of sodium hydroxide at high temperature for 8 hours to 20 hours. When the time required for deacetylation is short, the resulting chitosan is a substance having a low degree of deacetylation, that is, chitosan having a chitin structure. The chitin obtained in this way, or the chitosan which has a chitin structure as needed can be used for an enzyme reaction by the process mentioned later using the chitin decomposing enzyme composition of this invention.

In addition, chitin existing in nature is known to have two crystal structures of α-chitin and β-chitin.

The chitin decomposing enzyme composition of the present invention can hydrolyze either structure of α-chitin and β-chitin, or can hydrolyze chitin containing these structures in admixture. In addition, similarly to chitin, it is considered that there are two structures of α and β in chitosan, and chitosan of either structure can be hydrolyzed as long as it has a chitin structure.

In the present invention, a chitinase-based substance was used as the chitinase. Such chitinase refers to acting as an enzyme in an enzyme reaction to hydrolyze chitin to obtain low-molecular-weight chitin polysaccharides (low-molecular chitin polysaccharides), chitin oligosaccharides derived from chitin polysaccharides, Substances such as monosaccharides (N-acetyl-D-glucosamine) and the like.

The source of the above-mentioned chitinase is not particularly limited, and may be, for example, microbial sources, plant sources, insect sources, etc. Among them, chitinases derived from microorganisms are preferred. The microorganisms producing chitinase are not particularly limited, and examples include Serratia (Serratia), Bacillus (Bacillus), Streptomyces (Streptomyces), Alteromonas (Alteromonas), Bacteria such as the genus Coccidioides and the genus Vibrio; fungi such as the genus Aspergillus and the genus Trichoderma.

In addition, the plant that produces chitinase is not particularly limited, and examples include Pararubber tree (Pararubber tree, Hevea brisiliennsis), soybean (Glycine max), tobacco (Nicotiana tabacum) and the like.

Moreover, it does not specifically limit as an insect which produces chitinase, For example, silkworm (Bombyx mori), Spodoptera litura etc. are mentioned.

Among them, chitinases derived from Serratia marcescens, Streptomyces coelicolor, Streptomyces griseus and Bacillus thuringiensis respectively are preferred.

In addition, these chitinases may be artificially altered (for example, cloning in Examples described later). In addition, these chitinases may be used individually by 1 type, and may mix and use 2 or more types. Furthermore, chitinases can be a series of enzyme groups. As such an enzyme group, chitinase (EC 3.2.1.14) etc. are mentioned. In addition, chitinase can be mixed with chitinase from different sources.

In addition, chitinase is known to have a plurality of three-dimensional structures, but the three-dimensional structure is not particularly limited when used as a chitinase decomposing enzyme, and one type may be used alone or two or more types may be used in combination. As a chitinase having such a three-dimensional structure, for example, chitinase A or chitinase B derived from Serratia marcescens, chitinase derived from Bacillus circulans WL-12, etc. Enzyme A1, chitinase derived from pathogenic filamentous fungus Coccidioides immitis, ヘバミン derived from Para rubber tree, chitinase derived from hyperthermia Pyrococcus furiosus, derived from Chitinase C of the actinomycete Streptomyces griseus, etc.

Among them, chitinase A is preferable, and a substance containing at least chitinase A is particularly preferable. In addition, chitinases derived from microorganisms and plants are in a state in which multiple structures are mixed. In this case, chitinase can be purified using a separation method such as size exclusion chromatography. In order to separate the purified chitinase for each structure, it can be realized by, for example, changing the timing of sampling by size exclusion chromatography according to the molecular weight of the chitinase.

In general, most chitinases are enzymes that have optimum enzyme activity in the range of pH 3 to pH 8, but may be enzymes called alkaline chitinases that have optimum enzyme activity in the range of pH 8 to pH 10 or higher. In addition, most chitinases are enzymes that have optimum enzyme activity in the range of reaction temperature from 25°C to 50°C, but those that have optimum enzyme activity in the range of 70°C to 100°C may be referred to as Thermostable chitinase enzyme.

In the present invention, as silica or a substance containing silica, silica, diatomaceous earth, silica sand, quartz, glass, and the like can be used. Among materials containing silica, diatomaceous earth and silica sand are natural products containing silica as the main component. Silica is a general term for compounds containing at least silicon dioxide, and generally a silanol group exists on a part of the surface. The particle shape of the silica may be spherical or non-spherical, the particle structure may be solid or porous, the crystallinity may be amorphous or crystalline, and it may be in the form of powder, suspension, or It can be used in any state such as dispersion liquid. Part of the silica surface may be modified with functional groups other than silanol groups. In addition, a layer of silicon dioxide may be present by reacting with a surface of a compound other than silicon dioxide with a silane coupling agent, a silicon alkoxide, or a silicate ion, or the like. Of these, colloidal silica, diatomaceous earth, and silica sand are particularly preferably used.

Colloidal silica has an average primary particle diameter of 1 nm to 400 nm, preferably 5 nm to 350 nm, and is used as it exists in the chitin decomposition reaction solution described later. The average primary particle diameter was calculated from the specific surface area S (m ² /g) measured by the nitrogen adsorption method (BET method) from the conversion formula (D (nm) = 2720/S). In addition, colloidal silica is used as a dispersion liquid dispersed in a dispersion medium such as water, methanol, ethanol, acetone, methyl ethyl ketone, or ethylene glycol, and the dispersion liquid is called a colloid liquid, a sol, or the like. In the present invention, the dispersion medium can be selected within a range that does not inhibit the activity of the enzyme, but it is preferable to use a dispersion medium such as water or ethanol.

As a method for producing colloidal silica, there are a water glass method using water glass as a raw material, an alkoxide method using a metal alkoxide as a raw material, a gas phase method using a silicon chloride compound as a raw material, and the like. Colloidal silica obtained by any production method may be used, but colloidal silica obtained by a water glass method is preferably used.

In the present invention, the nitrogen-containing heterocyclic compound is a heterocyclic compound containing nitrogen. As such nitrogen-containing heterocyclic compounds, three-membered heterocyclic compounds (nitrogen-containing three-membered heterocyclic compounds) such as methyl aziridine-2-carboxylate, 1-(2-hydroxyethyl) aziridine, etc. compounds); pyrrolidine, 1H-pyrrole, 2H-pyrrole, 3H-pyrrole, imidazole, L-histidine, 2-methylimidazole, 2-ethylimidazole, benzimidazole, pyrazole, 3,5-di Five-membered heterocyclic compounds (nitrogen-containing five-membered heterocyclic compounds) such as methylpyrazole, 1H-1,2,3-triazole, 1,2,4-triazole, 1H-tetrazole; piperidine, 2-methylpiperidine, 4-methylpiperidine, 4-hydroxy-1-methylpiperidine, pyridine, 2-aminopyridine, 2-amino-6-methylpyridine and other six-membered heterocyclic compounds (including nitrogen six-membered heterocyclic compounds); ε-caprolactam, ε-thiocaprolactam, 1,8-diazabicyclo[5.4.0]-7-undecene and other seven-membered heterocyclic compounds (nitrogen-containing seven-membered heterocyclic compounds).

Among them, pyrrolidine, imidazole, L-histidine, 2-methylimidazole, pyrazole, 3,5-dimethylpyrazole, 1,2,4-tris Azole, 2-methylpiperidine, 4-methylpiperidine, 2-aminopyridine, 2-amino-6-methylpyridine, 1,8-diazabicyclo[5.4.0]-7-undeca Carbene is particularly preferably imidazole and 2-methylimidazole.

(Manufacturing method of chitin decomposition reaction solution and sugar)

In the present invention, a chitin decomposition reaction solution is prepared and used when chitin is hydrolyzed. The chitin decomposition reaction liquid of this invention contains chitin as a raw material, and the chitin decomposition enzyme composition of this invention. Details will be described later, but from the viewpoint of improving the efficiency of saccharification reaction, silica or a silica-containing substance and a nitrogen-containing heterocyclic compound are used in combination in the chitin decomposition reaction solution of the present invention.

In general, in an enzyme reaction, a phenomenon in which the reaction rate saturates as the substrate concentration increases, and the reaction rate draws a hyperbola that reaches the saturation maximum rate is observed. This is because the enzyme molecule is often larger than the substrate, and the range of the active center is narrow. Therefore, it can be considered that the substrate and the catalyst (enzyme) react even if they collide (suitable for the active center) compared with metal catalysts. The frequency is also small is the reason. Also, if the substrate concentration is increased, the substrates compete with each other for less active sites of the enzyme, thus saturation occurs.

Therefore, in the chitin decomposing reaction liquid of this invention, what is necessary is just to determine the concentration of chitin decomposing enzyme suitably according to content of chitin. However, if the concentration of the chitin-decomposing enzyme in the chitin-decomposing reaction liquid is too low, the saccharification reaction efficiency of the chitin-decomposing enzyme will decrease, which is not preferable. On the other hand, if the concentration of the chitin decomposing enzyme is too high, not only the saturation phenomenon will occur, but also the chitin decomposing enzyme will be difficult to dissolve in the chitin decomposing reaction solution, which is economically unsuitable.

In addition, in the chitin decomposition reaction solution of the present invention, the concentration of silicon dioxide in silicon dioxide or a material containing silicon dioxide is 0.1 mg/mL to 400 mg/mL, preferably 0.5 mg/mL to 100 mg/mL . If the concentration of silica in the silica or the silica-containing substance is less than 0.1 mg/mL, the efficiency of the saccharification reaction of chitin decomposing enzyme will decrease, which is not preferable. On the other hand, if the concentration of these silicas exceeds 400 mg/mL, not only the dispersibility of the chitin decomposition reaction liquid deteriorates, but also it is economically unsuitable.

In addition, in the chitin decomposition reaction solution, the mass ratio of chitin decomposing enzyme to silicon dioxide or silicon dioxide in a material containing silicon dioxide (chitin decomposing enzyme/silicon dioxide) is 0.0002 to 300, preferably 0.002～30. If the mass ratio of both exceeds the above-mentioned range, the improvement of the saccharification reaction efficiency of chitin decomposing enzyme will not be remarkable.

In addition, in the chitin decomposition reaction liquid, the concentration of the nitrogen-containing heterocyclic compound is 0.005 mg/mL˜100 mg/mL, preferably 0.05 mg/mL˜50 mg/mL. More preferably, it is 0.68 mg/mL to 34 mg/mL. The most preferred range is 6.8 mg/mL to 34 mg/mL. If the concentration of the nitrogen-containing heterocyclic compound is less than 0.005 mg/mL, the efficiency of the saccharification reaction of the chitin decomposing enzyme will be lowered, and on the other hand, if it is higher than 100 mg/mL, not only the dispersibility of the chitin decomposition reaction solution will deteriorate. , and is economically inappropriate.

In addition, in the chitin decomposition reaction liquid, the mass ratio (nitrogen-containing heterocyclic compound/silicon dioxide) of silicon dioxide and nitrogen-containing heterocyclic compound in the silica or the material containing silica is 0.0001～ 100, preferably 0.001-10. If the mass ratio of both exceeds the above-mentioned range, the improvement of the saccharification reaction efficiency of chitin decomposing enzyme will not be remarkable.

In addition, the pH of the chitin decomposition reaction solution is 4-8, preferably 5-7. If the pH is lower than 4, aggregation of silica or a substance containing silica occurs and the efficiency of the saccharification reaction of chitin decomposing enzyme decreases. On the other hand, if the pH is higher than 8, silica or a substance containing silica It is not preferable because it easily dissolves in the chitin decomposition reaction solution. In addition, when alkaline chitinase is used in the chitin decomposition reaction liquid, the solubility of silica or a substance containing silica in the chitin decomposition reaction liquid is adjusted. Accordingly, it can be used as a chitin decomposition reaction solution having a pH higher than 8.

Examples of the pH adjuster for the chitin decomposition reaction solution include inorganic acids such as sulfuric acid, hydrochloric acid, and nitric acid; carboxylic acids such as acetic acid and oxalic acid; hydroxy acids such as citric acid, tartaric acid, and malic acid; phosphoric acid; sodium hydroxide and potassium hydroxide. Other hydroxide salts; ammonia, urea and other nitrogen-containing compounds, etc. The type and concentration are not particularly limited as long as the effect of the present invention is not impaired. In addition, these pH adjusters may be used individually by 1 type, and may mix and use 2 or more types, or may use it in the state of the buffer solution which has buffering action.

In addition, the reaction temperature of the chitin decomposition reaction solution is preferably set according to the optimum temperature of the chitin decomposing enzyme, and is preferably, for example, 10°C to 50°C, particularly preferably 25°C to 40°C. In general, if the reaction temperature is lower than 10°C, the saccharification reaction efficiency of chitin-decomposing enzyme will be significantly lowered, and if it is higher than 50°C, chitin-decomposing enzyme may be inactivated, so it is not preferable. However, when heat-resistant chitinase was used, it was not deactivated even at 70°C to 100°C.

In addition, the pretreatment of the chitin-based biomass-derived raw material may be performed by applying a known treatment method. For example, what is necessary is just to remove protein and calcium carbonate by acid treatment and/or alkali treatment after physical pulverization by a shredder etc., and it may be used as the raw material of chitin.

In addition, the preparation steps of the chitin decomposition reaction liquid are not particularly limited, and silicon dioxide or a substance containing silicon dioxide and a nitrogen-containing heterocyclic compound can be added to the dispersion liquid in which the chitin decomposing enzyme is dispersed to prepare the chitin decomposition reaction solution. The reaction solution. Alternatively, a chitin decomposing enzyme may be added to a dispersion in which silica or a silica-containing substance and a nitrogen-containing heterocyclic compound are dispersed to obtain a chitin decomposing reaction liquid. In these production steps, any order of addition may be used as long as the efficiency of the saccharification reaction of the chitin decomposing enzyme does not decrease. For example, silica or a silica-containing substance and a nitrogen-containing heterocyclic compound may be added simultaneously or separately. At this time, the nitrogen-containing heterocyclic compound may be added in a powder state or in a solution state. Moreover, other additives, such as a pH adjuster, can be added in arbitrary order as long as it is the range which does not impair the effect of this invention.

As explained above, when the chitin decomposition reaction liquid is prepared using the chitin decomposing enzyme composition of the present invention, although the mechanism is not clear, the combination of silica or a substance containing silica and a nitrogen-containing heterocycle The formula compound can further promote the hydrolysis of chitin and improve the efficiency of saccharification reaction using chitin decomposing enzyme. In addition, by improving the efficiency of the saccharification reaction of the chitin decomposing enzyme, the yield of sugar as a product can be increased to achieve a high yield. In addition, the chitin decomposing reaction liquid can reduce the amount of chitin decomposing enzyme used by combining silicon dioxide or a substance containing silicon dioxide and a nitrogen-containing heterocyclic compound. Therefore, it is a simple reaction system with low cost. Also excellent.

Sugar can be produced by hydrolyzing chitin using the chitin decomposition reaction solution prepared using the chitin decomposition enzyme composition of the present invention. When producing sugar, chitin can be hydrolyzed using a chitin decomposition reaction solution under stirring. A specific method for producing sugar will be described in Examples below.

Example

Hereinafter, although it demonstrates in detail based on an Example, this invention is not limited at all by this Example.

(1. Embodiment 1)

(1-1. Preparation of Chitinase A)

Chitinase A used as chitinase decomposing enzyme was prepared according to the following procedure.

Chitinase A (hereinafter referred to as "SmChiA") cloned from Serratia marcescens (refer to T.Watanabe et al., Journal of Bacteriol, Vol.179, No.22, 1997, p.7111-p. 7117), cloned into the plasmid vector pET27b (manufactured by Novagen) for constructing a protein mass expression system using the T7 promoter. When cloning, the carboxy-terminal side of SmChiA was constructed so that a histidine tag was repeated 6 times.

Mass expression of SmChiA using Escherichia coli (E. coli) as a host was carried out by the following procedure.

E. coli BL21(DE3) carrying the above plasmid vector was cultured overnight at 37°C with shaking in LB medium supplemented with 50 μg/mL kanamycin to obtain a culture solution. On the next day, this culture solution was used as an inoculum to inoculate Overnight Express (Overnight Express (registered trademark)) LB medium (manufactured by Novagen) to which 50 μg/mL of kanamycin was added, and cultured with shaking at 30° C. Night. On the next day, bacteria were collected using a centrifuge to produce pellets of bacteria, and the pellets were suspended in Bugbuster (registered trademark) (manufactured by Nobajenn) to which Benzonase (registered trademark) (manufactured by Nobajin) was added. Thus a suspension is obtained. Next, the suspension was left to stand at room temperature for 30 minutes, and then centrifuged again to recover the supernatant fraction.

Next, in order to purify/recover SmChiA from the above-mentioned supernatant fraction, the supernatant fraction was added to a 5×5 mL HisTrap HP column (manufactured by GE ヘルスケア). Then, the above-mentioned HP column was washed with a washing buffer. In addition, a washing buffer was prepared by mixing 20 mM sodium phosphate buffer (pH 7.4), 0.5 M sodium chloride, and 25 mM imidazole.

Next, imidazole was added to the column while forming a concentration gradient such that the concentration became 25 mM to 250 mM (final concentration), to elute SmChiA, and the SmChiA eluted fraction was recovered. The SmChiA eluted fraction was subjected to centrifugal dialysis using Vivaspin (Vivaspin (registered trademark)) 20, and the washing buffer was replaced with 50 mM sodium phosphate buffer (pH 6.0) to obtain a purified enzyme (SmChiA). Quantification of the obtained purified enzyme was carried out by measuring the absorbance at 280 nm. In addition, the molar absorptivity of SmChiA was calculated from the predicted amino acid sequence using "The Proteomics Protocols Handbook" (see J.M. Walker, Humana Press, 2005, p.571-p.607).

(1-2. Measurement of Chitinase Activity of Chitinase Composition)

In addition to SmChiA, which is the purified enzyme obtained by refining in (1-1.) above, a shell was prepared using silica as silica or a substance containing silica, and imidazole as a nitrogen-containing heterocyclic compound. Polysaccharolytic enzyme composition, enzyme activity (chitinase activity) was measured. The determination of the chitinase activity of the reaction system is carried out through the following steps. In addition, the composition of the prepared chitin decomposing enzyme composition is shown in Table 1 below.

First, a rotor was placed in a glass vial, and crystalline chitin (manufactured by Junsei Chemical Co., Ltd.) at a final concentration of 12.5 mg/mL, 20 mM sodium phosphate buffer (pH 6.0), 0.125 mg/mL SmChiA mL, imidazole at a final concentration of 100 mM, and silica sol (manufactured by Nissan Chemical Industries, Ltd., product name: ST-OL, acidic sol) at a final concentration of 50 mg/mL were used to obtain a chitin decomposition reaction solution. The glass vial to which the chitin decomposition reaction liquid was added was placed in a vial stirrer (Vaial Stirrer) HS-10VA (manufactured by Azuwan Co., Ltd.), and placed under the conditions of 25° C., 22 hours, and maximum output. Stir down. Chitinase activity was measured by the following procedure immediately after the reaction.

The determination of chitinase activity is by using PHBAH (p-Hydroxybenzoic acid hydrazide, p-hydroxybenzoic acid hydrazine) method (referring to M.Lever, Analytical Biochemistry, Vol.47, No.1, 1972, p.273-p.279) , The amount of reducing sugars of chitin oligosaccharides and N-acetyl-D-glucosamine as products was obtained. Specifically, the above-mentioned chitin decomposition reaction solution was subjected to centrifugation (4°C, maximum centrifugal acceleration 15780×g, 5 minutes), and the supernatant was collected. Next, a PHBAH solution twice the amount of the supernatant and 2M sodium hydroxide equal to the amount of the supernatant were added to the collected supernatant, and the suspension was sufficiently suspended to obtain a suspension. In addition, the PHBAH solution was obtained by mixing 0.1M PHBAH, 0.2M potassium sodium tartrate, and 0.5M sodium hydroxide.

The resulting suspension was reacted at 98°C for 10 minutes, and then rapidly cooled to 2°C over 10 minutes. Then, the OD value (OD: Optical Density; optical density, optical density) at a wavelength of 405 nm was measured, and the difference in absorbance from the blank was obtained using the OD value. In addition, a calibration curve was prepared using N-acetyl-D-glucosamine, and the amount of reducing sugar was determined from the increase in absorbance in the supernatant, and is shown in Table 1 below. In addition, the amount of reducing sugar shown in the following Table 1 was set as the range of the amount of reducing sugar when n=3.

(2. Embodiment 2～4)

In Examples 2 to 4, as silica or a substance containing silica, silica sol (manufactured by Nissan Chemical Industry Co., Ltd., product name: ST-OZL-35, acid sol), silica Sol (manufactured by Nissan Chemical Industry Co., Ltd., product name: MP-4540M, Na-stable alkaline sol), and fumed silica (manufactured by Ebonick, product name: AEROSIL (registered trademark) OX50, hydrophilic fumed silica silicon), except that, in the same manner as in Example 1, chitinase compositions were prepared, and their chitinase activity was measured. The final concentration of each silica was 50 mg/mL as in Example 1. In addition, the composition of each chitin-degrading enzyme composition and the amount of each obtained reducing sugar were carried out in the same manner as in Example 1, and are shown in Table 1 below. In addition, each reducing sugar amount shown in the following Table 1 was set as the range of the reducing sugar amount when n=3.

(3. Embodiment 5)

In Example 5, except having further added sodium chloride at a final concentration of 100 mM, it was performed in the same manner as in Example 2 to prepare chitinase compositions, and their chitinase activity was measured. In addition, the composition of the chitin decomposing enzyme composition and the amount of reducing sugar obtained were carried out in the same manner as in Example 1, and are shown in Table 1 below. In addition, the amount of reducing sugar shown in the following Table 1 was set as the range of the amount of reducing sugar when n=3.

(4. Embodiment 6)

In Example 6, except having changed the final concentration of imidazole into 500 mM, it carried out similarly to Example 2, prepared the chitin decomposing enzyme composition, and measured the chitinase activity of these. In addition, the composition of the chitin decomposing enzyme composition and the amount of reducing sugar obtained were carried out in the same manner as in Example 1, and are shown in Table 1 below. In addition, the amount of reducing sugar shown in the following Table 1 was set as the range of the amount of reducing sugar when n=3.

(5. Embodiment 7)

In Example 7, the imidazole as a nitrogen-containing heterocyclic compound was changed to 2-methylimidazole, except that, the same operation as in Example 2 was performed to prepare chitin decomposing enzyme compositions, and their chitin content was measured. enzyme activity. In addition, the composition of the chitin decomposing enzyme composition and the amount of reducing sugar obtained were carried out in the same manner as in Example 1, and are shown in Table 1 below. In addition, the amount of reducing sugar shown in the following Table 1 was set as the range of the amount of reducing sugar when n=3.

(6. Comparative examples 1 to 10)

In Comparative Example 1, except that silica, imidazole, and sodium chloride were not added, a chitinase activity was measured in the same manner as in Example 5 to prepare a chitinase enzyme composition.

In Comparative Example 2, except that no silica and sodium chloride were added, a chitinase activity was measured in the same manner as in Example 5 to prepare a chitinase enzyme composition.

In Comparative Example 3, except that no silica was added, a chitinase activity was measured in the same manner as in Example 5 to prepare a chitinase enzyme composition.

In Comparative Example 4, except that no silica and imidazole were added, a chitinase activity was measured in the same manner as in Example 5 to prepare a chitinase enzyme composition.

In Comparative Examples 5-8, except not having added imidazole, respectively, it carried out similarly to Examples 1-4, prepared the chitinase composition, and measured the chitinase activity.

In Comparative Example 9, except not adding imidazole, it carried out similarly to Example 5, prepared the chitin decomposing enzyme composition, and measured the chitinase activity.

In Comparative Example 10, during the reaction, the chitin-degrading enzyme composition was prepared in the same manner as in Comparative Example 3 except that it was left to stand at 25° C. for 22 hours without stirring using the rotor, and measured The chitinase activity.

In addition, in Comparative Examples 1 to 10, the composition of each chitin decomposing enzyme composition and the amount of each obtained reducing sugar were carried out in the same manner as in Example 1, and are shown in Table 1 below. In addition, each reducing sugar amount shown in the following Table 1 was set as the range of the reducing sugar amount when n=3.

(7. Evaluation of chitinase activity)

(7-1. Improvement effect of chitinase activity by addition of imidazole or sodium chloride)

The chitinase activity of Comparative Example 1 was compared with Comparative Examples 2-4, and the improvement effect of chitinase activity by addition of imidazole or sodium chloride was examined.

When the amount of reducing sugar in Comparative Example 1 and Comparative Examples 2-4 is observed, compared with Comparative Example 1, the amount of reducing sugar in Comparative Examples 2-4 is slightly increased. This shows that in Comparative Examples 2 to 4, the hydrolysis reaction of chitin proceeded smoothly and the efficiency of the saccharification reaction was slightly improved, and it became clear that the chitinase activity was slightly improved by the addition of imidazole or sodium chloride.

(7-2. Improvement effect of chitinase activity by addition of silica)

Comparing the chitinase activity of Comparative Example 1 and Comparative Examples 5 to 8, the improvement effect of chitinase activity by the addition of silica was examined.

When looking at the amount of reducing sugar in Comparative Example 1 and Comparative Examples 5 to 8, although some differences were observed in Comparative Example 5 to 8 depending on the form of silica relative to Comparative Example 1, the amount of reducing sugar was all reduced. This indicates that the hydrolysis reaction of chitin did not proceed smoothly in Comparative Examples 5 to 8, and it became clear that chitinase activity was inhibited by the addition of silica. Therefore, it was evaluated here that the chitinase activity of Comparative Examples 5 to 8 was inhibited relative to Comparative Example 1, and the effect of improving chitinase activity was shown in Table 1 below as "inhibition".

The reason why silicon dioxide acts as an inhibitor of chitinase activity is consistent with the results in Figure 5 (SNPC1) of Non-Patent Document 1, so it can be considered that chitinase physically Adsorbed and immobilized on the surface of silica, thereby inhibiting chitinase activity.

(7-3. Improvement effect of chitinase activity by combined use of silica and imidazole)

According to above-mentioned (7-1.) and above-mentioned (7-2.) result, the chitinase activity of embodiment 1～4, embodiment 6 and comparative example 2, comparative example 5～8 is compared, for by two The improvement effect of chitinase activity brought about by the combined use of silica and imidazole was studied.

When observing the amount of reducing sugar in Examples 1 to 4 and Comparative Examples 5 to 8, although some differences were observed in Examples 1 to 4 depending on the form of silica compared to Comparative Examples 5 to 8, the amount of reducing sugar was all the same. increased. Furthermore, when observing the reducing sugar amount of Examples 1-4, 6 and the comparative example 2, compared with the comparative example 2, the reducing sugar amount increased in all of Examples 1-4, 6. The reason is not clear, but it shows that in Examples 1-4, 6, the hydrolysis reaction of chitin proceeded smoothly and the efficiency of the saccharification reaction was improved, and it was clarified that the activity of chitinase was improved by the combined use of silica and imidazole. Therefore, here, it was evaluated that the chitinase activity of Examples 1-4, 6 was improved compared with Comparative Examples 5-8, and the chitinase activity improvement effect was shown in the following Table 1 as "existing".

(7-4. Effect of improving chitinase activity by combined use of silica and sodium chloride)

According to above-mentioned (7-1.) ~ above-mentioned (7-3.) result, the chitinase activity of embodiment 2,5 and comparative example 3,9 is compared, to the combination of silicon dioxide and sodium chloride The improvement effect of chitinase activity brought about by use was studied.

When observing the amount of reducing sugar in Examples 2 and 5, there was almost no change in the amount of reducing sugar in both. Next, when observing the amount of reducing sugar in Example 5 and Comparative Example 3, the amount of reducing sugar in Example 5 increased compared to Comparative Example 3. Next, when looking at the amount of reducing sugar in Example 5 and Comparative Example 9, compared to Comparative Example 9, the amount of reducing sugar in Example 5 increased. From this, it became clear that the cause of the increase in chitinase activity in Example 5 was the combined use of silicon dioxide and imidazole, not the combined use of silicon dioxide and sodium chloride. Therefore, it was evaluated here that the chitinase activity of Example 5 was improved compared to Comparative Example 9, and the effect of improving chitinase activity was shown in Table 1 below as "Yes".

(7-5. Effect of improving chitinase activity by combined use of silica and 2-methylimidazole)

According to above-mentioned (7-1.) ~ above-mentioned (7-4.) result, the chitinase activity of embodiment 7 and comparative example 2, 6 is compared, to the combination of silicon dioxide and 2-methylimidazole The improvement effect of chitinase activity brought about by use was studied.

When observing the amount of reducing sugar in Examples 2 and 7 and Comparative Examples 2 and 6, compared to Comparative Examples 2 and 6, the amount of reducing sugar in both Examples 2 and 7 increased. This shows that, like Example 2 using imidazole, in Example 7 using 2-methylimidazole, the hydrolysis reaction of chitin proceeds smoothly and the efficiency of the saccharification reaction improves. The combined use of 2-methylimidazole increased the activity of chitinase. Therefore, it was evaluated here that the chitinase activity of Example 7 was improved compared to Comparative Example 6, and the effect of improving chitinase activity was shown in Table 1 below as "existing". In addition, from this result, it was suggested that chitinase activity may be improved by the combined use of silica for nitrogen-containing heterocyclic compounds other than imidazole and 2-methylimidazole.

(7-6. Effect of improving chitinase activity by presence or absence of stirring)

According to the result of above-mentioned (7-1.) ~ above-mentioned (7-5.), compare the chitinase activity of comparative example 3 and comparative example 10, the improvement of the chitinase activity brought by the presence or absence of stirring effect was studied.

When the amount of reducing sugar in Comparative Example 3 and Comparative Example 10 was observed, compared to Comparative Example 3, the amount of reducing sugar in Comparative Example 10 decreased, and chitinase activity decreased. Enzyme activity changes depending on the presence or absence of stirring is a phenomenon that may occur in general enzyme reaction systems. Here, it was evaluated that the chitinase activity of Comparative Example 10 was inhibited relative to Comparative Example 3, and the effect of improving chitinase activity was shown in Table 1 below as "inhibition".

Table 1

In addition, the types A and B of nitrogen-containing heterocyclic compounds in Table 1 above are as follows.

A: imidazole (molecular weight 68.08)

B: 2-methylimidazole (molecular weight 82.11)

By above each embodiment and each comparative example, clearly when using chitin decomposing enzyme composition and preparing chitin decomposition reaction liquid, by silicon dioxide, use in conjunction with imidazole or 2-methylimidazole, thereby make by chitin The efficiency of the saccharification reaction by the enzyme A is improved, and the yield of sugar as a product can be increased to achieve a high yield.

industry availability

The present invention can be used to decompose chitin-based biomass containing chitin contained in crustaceans such as crabs and shrimps, insects, and fungi into low-molecular chitin polysaccharides, chitin oligosaccharides derived from chitin polysaccharides, The technical industrial field of sugar such as monosaccharide is used in medical materials, medicine, cosmetics, fiber, agriculture, water treatment, food, etc.

Claims (5)

1. A chitin decomposing enzyme composition, characterized in that it contains: chitinase as a chitin decomposing enzyme that hydrolyzes chitin; silicon dioxide or a material containing silicon dioxide; and a heterocyclic ring containing nitrogen Nitrogen-containing heterocyclic compounds of formula compounds, The nitrogen-containing heterocyclic compound is any one selected from imidazole and 2-methylimidazole as a nitrogen-containing five-membered heterocyclic compound. 2. chitinase composition according to claim 1 is characterized in that, described chitinase contains chitinase A at least. 3. A chitin decomposition reaction liquid, characterized in that it contains: chitin; and the chitin decomposing enzyme composition according to claim 1 or 2. 4. A method for producing sugar, characterized in that the sugar is produced by hydrolyzing chitin using the chitin decomposition reaction solution according to claim 3. 5. The method for producing sugar according to claim 4, wherein the sugar is produced by hydrolyzing chitin with stirring using the chitin decomposition reaction solution.