JP2019092445A - Compositions of chitin-degrading enzyme, reaction solution for chitin degradation, and methods for preparing saccharides - Google Patents

Abstract

To provide compositions of a chitin-degrading enzyme, a reaction solution for chitin degradation and methods for producing saccharides, which can improve the saccharification efficiency of the chitin-degrading enzyme in a simple process and can achieve a high product yield when silica is applied in the reaction system with a chitin-degrading enzyme.SOLUTION: The chitin-degrading enzyme composition of the invention comprises a chitinase being a chitin-hydrolyzing enzyme, silica or a silica-containing material and a nitrogen-containing heterocyclic compound.SELECTED DRAWING: None

Description

  The present invention relates to a chitinolytic enzyme composition, a chitinolytic reaction solution and a method for producing a sugar.

  In the living world, chitin is contained in the exoskeleton of insects and crustaceans, cell walls of fungi, etc., and its annual production is estimated to be second to cellulose which is abundant biomass on the earth. For this reason, chitin is growing in interest as a biomass resource next to cellulose.

  Chitin is an insoluble polysaccharide to which N-acetyl-D-glucosamine which is a monosaccharide is bound, and chitin which is reduced in molecular weight by an enzyme reaction using chitinase which is a chitin decomposing enzyme (also referred to as a "chitin decomposing enzyme") It is hydrolysed into polysaccharide (lower molecularized chitin polysaccharide), chitin oligosaccharide derived from chitin polysaccharide, monosaccharide (N-acetyl-D-glucosamine) and the like. These hydrolysates are products in the enzymatic reaction of chitin.

  The products of chitin enzymatic reaction have excellent antibacterial, moisturizing, biocompatibility, safety, chelating properties and the like. Therefore, their use in fields such as medical materials, medicines, cosmetics, textiles, agriculture, water treatment, and food are expected, and research and development are being promoted.

  For example, Non-Patent Document 1 describes a technology related to the enzymatic activity of chitinase using silica nanoparticles (SNP). Specifically, chitinase (Chi9602) derived from Bacillus thuringiensis is immobilized on the surface of SNP by electrostatic adsorption, nanoscale chitinase (SNPC1) is prepared, and chitinase is prepared using SNPC1 prepared. It is disclosed that the enzyme activity was measured. The immobilization rate of silica in the prepared SNPC1 is about 55%.

  However, since the chitinase activity of SNPC1 is about 43% with respect to the chitinase activity of Chi9602 about 100%, it became clear that the activity is reduced about 57% by the immobilization of silica. That is, in a reaction system in which chitinase is immobilized on silica, although sugar production is possible, it has been found that the enzymatic activity is inhibited and the glycation reaction efficiency is lowered, and such a reaction system is used It is necessary to solve the problem. Also, from the viewpoint of cost, complicated reaction systems are not desirable.

X. Qin et al. , International Journal of Biological Macromolecules, Vol. 82, 2016, p. 13-p. 21

  The present invention has been made in view of the above circumstances, and when silica is applied in a reaction system using a chitinolytic enzyme, the efficiency of the saccharification reaction by the chitinolytic enzyme is improved in a simple step, and It is an object of the present invention to provide a chitinolytic enzyme composition, a chitinolytic reaction solution and a method for producing a sugar that can realize high yield.

  The first aspect of the present invention for achieving the above object is a chitinase which is a chitinolytic enzyme that hydrolyzes chitin, a silica or a silica-containing substance, and a nitrogen-containing heterocyclic compound which is a nitrogen-containing heterocyclic compound. In a chitinolytic enzyme composition characterized by containing

  A second aspect of the present invention lies in the chitinolytic enzyme composition according to the first aspect, wherein the chitinase comprises at least chitinase A.

  A third aspect of the present invention is the chitinolytic enzyme composition according to the first or 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 chitinolytic enzyme composition according to the third aspect, wherein the nitrogen-containing five-membered heterocyclic compound is any one selected from imidazole and 2-methylimidazole. It is in the thing.

  According to a fifth aspect of the present invention for achieving the above object, there is provided a chitin degrading reaction liquid comprising chitin and the chitinolytic enzyme composition according to any one of the first to fourth aspects. is there.

  A sixth aspect of the present invention for achieving the above object is a method for producing sugar characterized in that sugar is produced by hydrolyzing chitin using the chitin degradation reaction liquid of the fifth aspect.

  A seventh aspect of the present invention is the method for producing a sugar according to the sixth aspect, which comprises producing a sugar by hydrolyzing chitin using the chitin degradation reaction liquid under stirring.

  According to the present invention, when silica is applied in a reaction system using a chitinolytic enzyme, it is possible to improve the efficiency of the saccharification reaction by the chitinolytic enzyme in a simple step and to achieve high yield of the product. The present invention can provide a chitinolytic enzyme composition, a chitinolytic reaction solution, and a method for producing sugar.

(Chitin degrading enzyme composition)
The chitinolytic enzyme composition of the present invention improves the saccharification reaction efficiency in an enzyme reaction using a chitinase which is a chitinolytic enzyme to increase the yield of oligosaccharides and N-acetyl-D-glucosamine as a product To achieve Hereinafter, the details of the chitinolytic enzyme composition of the present invention will be described.

  The chitinolytic enzyme composition of the present invention is a composition capable of hydrolyzing the substrate chitin and comprises a chitinolytic enzyme, a silica or a silica-containing substance, and a nitrogen-containing heterocyclic compound. It is.

  Here, chitin which is a substrate is a linear high molecular weight amino sugar to which many N-acetyl-D-glucosamine residues are β-1,4 bonded, and is an insoluble polysaccharide having a strong crystal structure. Chitin has a chemical structure represented by the following formula (1).

  Moreover, the deacetylated thing of chitin is called chitosan and has a chemical structure represented by following formula (2).

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

  Although it does not restrict | limit especially as a raw material of the above-mentioned chitin, The raw material derived from chitin type | system | group biomass can be used. As such raw materials, for example, crustaceans such as shrimps and crabs, soft shells such as squid, crustaceans and exoskeletons such as insects, cell walls of fungi and the like can be mentioned. Moreover, a thing derived from a natural product may be used as a raw material, and a commercial item may be used. When the raw materials are derived from natural products, one type may be used alone, or two or more types may be mixed and used.

  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, in addition to dried red snow crab shell, ground leg and ground part are crushed to 3 cm to 5 cm, and heated hot dilute sodium hydroxide aqueous solution and dilute hydrochloric acid aqueous solution for 2 hours each Chitin can be obtained by soaking for 3 hours to remove proteins and calcium carbonate. In addition, chitosan can be obtained by deacetylating the obtained chitin in high temperature concentrated sodium hydroxide aqueous solution over 8 hours-20 hours. If the time required for deacetylation is short, the resulting chitosan will be one with a low degree of deacetylation, ie chitosan having a chitin structure. The chitin thus obtained, or the chitosan having a chitin structure as required, can be subjected to an enzyme reaction according to the procedure described later using the chitinolytic enzyme composition of the present invention.

  In addition, it is known that chitin existing in nature has two kinds of crystal structures of α-chitin and β-chitin.

  The chitinolytic enzyme composition of the present invention can be hydrolyzed with any structure of α-chitin and β-chitin, or even with chitin containing a mixture of these structures. It can be disassembled. As with chitin, it is considered that both α and β structures exist in chitosan, but even chitosan having any structure can be hydrolyzed if it has a chitin structure.

  In the present invention, chitinase is mainly used as a chitinolytic enzyme. Such a chitinase functions as an enzyme in an enzyme reaction and hydrolyzes chitin to reduce the molecular weight of chitin polysaccharide (reduced molecular weight chitin polysaccharide), chitin oligosaccharide derived from chitin polysaccharide, monosaccharide (N-acetyl-D) Glucosamine) etc. means products from which products can be obtained.

  Although the origin of the above-mentioned chitinase is not particularly limited, it may be, for example, a microorganism, a plant, an insect or the like, and among these, a microorganism-derived chitinase is preferable. The microorganism producing chitinase is not particularly limited, and examples thereof include, but are not limited to, Serratia, Bacillus, Streptomyces, Alteromonas, Coccidioides, Vibrio, for example. Bacteria such as genus; fungi such as Aspergillus and Trichoderma.

  Further, the plant producing chitinase is not particularly limited, and examples thereof include Para rubber tree (Hevea brisilien nsis), soybean (Glycine max), tobacco (Nicotiana tabacum) and the like.

  Moreover, the insect which produces chitinase is not particularly limited, and examples thereof include Bombyx mori and Spodoptera litura.

  Among these, chitinases derived respectively from Serratia marcescens (Serratia marcescens), Streptomyces coelicolor, Streptomyces griseus, and Bacillus thuringiensis are preferable.

  In addition, these chitinases may be artificially modified (for example, cloning of Examples described later). Also, these chitinases may be used alone or in combination of two or more. The chitinase may also be a series of enzymes. Such enzymes include chitinases (EC 3.2.1.14) and the like. In addition, chitinases may be used as a mixture of chitinases of different origin.

  In addition, although it is known that chitinase has a plurality of three-dimensional structures, when it is used as a chitinolytic enzyme, the three-dimensional structure is not particularly limited, and two or more kinds are mixed even if one type is used alone. You may use it. Examples of chitinases having such a three-dimensional structure include chitinase A and chitinase B derived from Serratia marcescens, chitinase A1 derived from Bacillus circulans WL-12, and Coccidioides imimitis, a pathogenic filamentous fungus. And the like. Chitinase derived from Hevea brasiliensis, Hebamine derived from Hevea brasiliensis, chitinase derived from Pyrococcus furiosus of hyperthermia, and chitinase C derived from Streptomyces griseus actinomycete.

  Among these, chitinase A is preferable, and one containing at least chitinase A is particularly preferable. The microorganism-derived or plant-derived chitinase is in a state in which a plurality of structures are mixed. In this case, the chitinase can be purified using a separation method such as, for example, size exclusion chromatography. In order to separate the purified chitinase into structures, for example, the timing of sampling of size exclusion chromatography can be realized by changing the molecular weight of the chitinase.

  Most of the chitinases generally have an optimum enzyme activity in the range of pH 3 or more and pH 8 or less, but are so-called alkaline chitinases having an optimum enzyme activity in the range of pH 8 to pH 10 or more It is also good. In addition, many of the chitinases have optimum enzyme activity in the reaction temperature range of 25 ° C. or more and 50 ° C. or less, but heat resistance having optimum enzyme activity in the range of 70 ° C. or more and 100 ° C. or less It may be called sexual chitinase.

  In the present invention, silica, diatomaceous earth, silica sand, quartz, glass and the like can be used as the silica or silica-containing material. Among silica-containing materials, diatomaceous earth and silica sand are natural products mainly composed of silica. Silica is a general term for compounds containing at least silicon dioxide, and generally, silanol groups are present on part of the surface. The silica may have a spherical or non-spherical particle shape, may have a solid or porous particle structure, or may be amorphous or crystalline, and may be in the form of powder, suspension, dispersion, etc. You may use in any state. A part of the silica surface may be modified with other functional groups other than silanol groups. In addition, it may be in a form in which a layer of silica is made to react on the surface of a compound other than silica with a silane coupling agent, silicon alkoxide, or silicate ion. Among them, application of colloidal silica, diatomaceous earth and silica sand is particularly preferable.

Colloidal silica has an average primary particle diameter of 1 nm or more and 400 nm or less, preferably 5 nm or more and 350 nm or less, and is used by being present in a chitin decomposition reaction solution described later. The average primary particle size is calculated from a specific surface area S (m ² / g) measured by a nitrogen adsorption method (BET method) by a conversion formula (D (nm) = 2720 / S). Colloidal silica is used as a dispersion liquid dispersed in a dispersion medium such as water, methanol, ethanol, acetone, methyl ethyl ketone and ethylene glycol, and the dispersion liquid is called a colloid liquid, sol or the like. In the present invention, the dispersion medium may be selected as long as the activity of the enzyme is not inhibited, but the application of a dispersion medium such as water or ethanol is preferred.

  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, and a vapor phase method using a silicon chloride compound as a raw material. Although the colloidal silica obtained by any manufacturing method may be used, application of the colloidal silica obtained by the water glass method is preferable.

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

  Among these, pyrrolidine, imidazole, L-histidine, 2-methylimidazole, pyrazole, 3,5-dimethylpyrazole, 1,2,4-triazole, 2-methylpiperidine, 4- 4-pyrrolidine, imidazole, L-histidine, 2-methylimidazole, and the like. Preferred are methyl piperidine, 2-aminopyridine, 2-amino-6-methylpyridine and 1,8-diazabicyclo [5.4.0] -7-undecene, and in particular imidazole and 2-methylimidazole.

(Method for producing chitin degradation reaction liquid and sugar)
In the present invention, when hydrolyzing chitin, a chitin degradation reaction solution is prepared and used. The chitin degradation reaction liquid of the present invention contains chitin as a raw material and the chitin degradation enzyme composition of the present invention. Although the details will be described later, from the viewpoint of achieving the effect of improving the saccharification reaction efficiency, in the chitin decomposition reaction liquid of the present invention, a silica or a silica-containing substance and a nitrogen-containing heterocyclic compound are used in combination.

  In general, in the enzyme reaction, a phenomenon is observed in which the reaction rate saturates as the substrate concentration increases, and the reaction rate draws a hyperbola leading to the saturation maximum rate. This is because the enzyme molecule is larger than the substrate in many cases, and the range of the active center is narrow, so even if the substrate and the catalyst (enzyme) collide (compare with the active center), the reaction occurs. It is considered that the cause of the problem is small. And, when the concentration of the substrate is increased, the saturation phenomenon occurs because the substrate becomes compatible with less active centers of enzymes.

  Therefore, in the chitinolytic reaction solution of the present invention, the concentration of the chitinolytic enzyme may be appropriately determined according to the content of chitin. However, if the concentration of the chitinolytic enzyme in the chitinolytic reaction solution is too low, the saccharification reaction efficiency of the chitinolytic enzyme is unfavorably lowered. On the other hand, when the concentration of chitinolytic enzyme is too high, not only the saturation phenomenon occurs, but the chitinolytic enzyme becomes difficult to dissolve in the chitinolytic reaction solution, which is economically unsuitable.

  In the chitin degradation reaction liquid of the present invention, the concentration of silica in the silica or silica-containing substance is 0.1 mg / mL or more and 400 mg / mL or less, preferably 0.5 mg / mL or more and 100 mg / mL or less is there. If the concentration of silica in the silica or silica-containing material is less than 0.1 mg / mL, the efficiency of the saccharification reaction of the chitinolytic enzyme is unfavorably reduced. On the other hand, when the concentration of these silicas is higher than 400 mg / mL, not only the dispersibility of the chitin decomposition reaction solution is deteriorated but also it is economically unsuitable.

  In the chitin degradation reaction solution, the mass ratio of chitin degrading enzyme to silica or silica in the silica-containing substance (chitin degrading enzyme / silica) is 0.0002 or more and 300 or less, preferably 0.002 or more and 30 It is below. When the mass ratio of the both is out of the above range, the improvement of the saccharification reaction efficiency of the chitinolytic enzyme is not remarkable.

  In the chitin degradation reaction solution, the concentration of the nitrogen-containing heterocyclic compound is 0.005 mg / mL or more and 100 mg / mL or less, preferably 0.05 mg / mL or more and 50 mg / mL or less. More preferably, they are 0.68 mg / mL or more and 34 mg / mL or less. Most preferably, it is 6.8 mg / mL or more and 34 mg / mL or less. If the concentration of the nitrogen-containing heterocyclic compound is lower than 0.005 mg / mL, the saccharification reaction efficiency of the chitinolytic enzyme is reduced, which is not preferable. On the other hand, if it is higher than 100 mg / mL, the dispersibility of the chitin decomposition reaction solution is only deteriorated. Not economically unfit.

  Moreover, in the chitin decomposition reaction liquid, the mass ratio (nitrogen-containing heterocyclic compound / silica) of silica and nitrogen-containing heterocyclic compound in the silica or silica-containing substance is preferably 0.0001 or more and 100 or less, preferably It is 0.001 or more and 10 or less. When the mass ratio of the both is out of the above range, the improvement of the saccharification reaction efficiency of the chitinolytic enzyme is not remarkable.

  Moreover, the pH of the chitin decomposition reaction liquid is 4 or more and 8 or less, preferably 5 or more and 7 or less. When the pH is lower than 4, aggregation of the silica or the silica-containing substance occurs to reduce the saccharification reaction efficiency of the chitinolytic enzyme, while when the pH is higher than 8, the silica or the silica-containing substance is easily dissolved in the chitin decomposition reaction solution Unfavorable. In addition, when using an alkaline chitinase for a chitin decomposition reaction liquid, the solubility with respect to the chitin decomposition reaction liquid of a silica or a silica containing material is adjusted. Thereby, it can use as a chitin decomposition reaction liquid whose pH is higher than eight.

  Mineral acids such as sulfuric acid, hydrochloric acid and nitric acid as pH adjusters of chitin decomposition reaction liquid; carboxylic acids such as acetic acid and oxalic acid; hydroxy acids such as citric acid, tartaric acid and malic acid; phosphoric acid; sodium hydroxide, hydroxide Examples include hydroxide salts such as potassium; and nitrogen-containing compounds such as ammonia and urea. As long as the effects of the present invention are not impaired, the type and concentration thereof are not particularly limited. These pH adjusters may be used alone or in combination of two or more, or may be used in the form of a buffer having a buffering action.

  The reaction temperature of the chitin degradation reaction solution is preferably set according to the optimum temperature of the chitin degradation enzyme, for example, 10 ° C. or more and 50 ° C. or less, particularly 25 ° C. or more and 40 ° C. or less Is preferred. In general, if the reaction temperature is lower than 10 ° C., the saccharification reaction efficiency of the chitinolytic enzyme is significantly reduced, and if it is higher than 50 ° C., the chitinolytic enzyme may be inactivated, which is not preferable. However, when heat-resistant chitinase is used, it does not inactivate even if it is 70 degreeC or more and 100 degrees C or less.

  The pretreatment of the chitin-based biomass-derived material may be performed by applying a known treatment method. For example, after physical pulverization with a cutter mill or the like, protein and calcium carbonate may be removed by acid treatment and / or alkali treatment, and this may be used as a chitin raw material.

  Although the preparation procedure of the chitin degradation reaction liquid is not particularly limited, the silica or the silica-containing substance and the nitrogen-containing heterocyclic compound may be added to the dispersion in which the chitin degradation enzyme is dispersed to form a chitin degradation reaction liquid. Alternatively, a chitinolytic enzyme may be added to the dispersion in which the silica or silica-containing substance and the nitrogen-containing heterocyclic compound are dispersed, to form a chitinolytic reaction solution. In these adjustment procedures, the addition order does not matter as long as the saccharification reaction efficiency of the chitinolytic enzyme is not reduced. For example, the silica or silica-containing substance and the nitrogen-containing heterocyclic compound may be added simultaneously or separately. At that time, the nitrogen-containing heterocyclic compound may be added in the form of powder or may be added in the form of solution. The other additives such as pH adjusters may be added in any order as long as the effects of the present invention are not impaired.

  As described above, when preparing a chitinolytic reaction solution using the chitinolytic enzyme composition of the present invention, the mechanism is not clear, but by using silica or a silica-containing substance and a nitrogen-containing heterocyclic compound in combination The hydrolysis of chitin can be further promoted to improve the efficiency of the saccharification reaction by the chitinolytic enzyme. Further, by improving the saccharification reaction efficiency of the chitinolytic enzyme, it is possible to improve the yield of the product sugar and to realize high yield. In addition, since this chitin decomposition reaction liquid can reduce the amount of chitinolytic enzyme used by the combined use of silica or a silica-containing substance and a nitrogen-containing heterocyclic compound, it is a simple reaction system and excellent in cost. ing.

  A sugar can be produced by hydrolyzing chitin using a chitinolytic reaction solution prepared using the chitinolytic enzyme composition of the present invention. When producing sugars, chitin degradation reaction solution may be used to hydrolyze chitin under agitation. Specific sugar production methods will be described in the following examples.

  The present invention will be described in more detail based on examples given below, but the invention is in no way limited by these examples.

(1. Example 1)
(1-1. Preparation of chitinase A)
The chitinase A used as a chitinolytic enzyme was prepared according to the following procedure.
Chitinase A (hereinafter referred to as "SmChiA") cloned from Serratia marcescens (see T. Watanabe et al., Journal of Bacteriol, Vol. 179, No. 22, 1997, p. 7111-p. 7117) Then, it was cloned into plasmid vector pET27b (manufactured by Novagen) for constructing a large amount of protein expression system using T7 promoter. During cloning, it was constructed so that a Histidine tag repeated six times was attached to the carboxyl terminal side of SmChiA.

  Large-scale expression using SmChiA as a host in Escherichia coli (E. coli) was performed according to the following procedure.

  E. coli carrying the above plasmid vector E. coli BL21 (DE3) was cultured with shaking overnight at 37 ° C. in LB medium supplemented with 50 μg / mL kanamycin to obtain a culture solution. The following day, using this culture solution as an inoculum, the cells were inoculated into Overnight Express (Overnight Express (registered trademark)) LB medium (manufactured by Novagen) to which 50 μg / mL kanamycin was added, and shake culture was performed overnight at 30 ° C. The next day, bacteria were collected using a centrifuge to prepare a pellet of bacteria, and this pellet was added to Benzonase (Benzonase (registered trademark)) (manufactured by Novagen) Bugbuster (Bugbuster (registered trademark)) (Novagen Corporation) Suspension) to obtain a suspension. The suspension was then allowed to stand at room temperature for 30 minutes and centrifuged again, and the supernatant fraction was collected.

  Next, in order to purify and recover SmChiA from the supernatant fraction, the supernatant fraction was added to 5 × 5 mL of HisTrap HP column (manufactured by GE Healthcare). Thereafter, the HP column was washed with washing buffer. The 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 applying a concentration gradient so that the concentration was 25 mM to 250 mM (final concentration), SmChiA was eluted, and the SmChiA-eluted fraction was collected. The SmChiA elution fraction was subjected to centrifugal dialysis using Vivaspin (registered trademark) 20, and the above washing buffer was replaced with 50 mM sodium phosphate buffer (pH 6.0), and the purified enzyme (SmChiA) was used. I got The quantification of the obtained purified enzyme was performed by measuring the absorbance at 280 nm. The molar absorption coefficient 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 chitinolytic enzyme composition)
In addition to SmChiA which is a purified enzyme obtained by purification in the above (1-1.), A chitinolytic enzyme composition is prepared using silica as the silica or silica-containing substance and imidazole as the nitrogen-containing heterocyclic compound. The enzyme activity (chitinase activity) was measured. The measurement of the chitinase activity of this reaction system was carried out according to the following procedure. The composition of the prepared chitinolytic enzyme composition is shown in Table 1 below.

  First, one rotator is placed in a glass vial, and crystalline chitin (made by Junsei Chemical Co., Ltd.) with a final concentration of 12.5 mg / mL, 20 mM sodium phosphate buffer (pH 6.0), 0.125 mg / mL. A Chitin degradation reaction solution was obtained by adding mL of SmChiA, a final concentration of 100 mM of imidazole, and a final concentration of 50 mg / mL of silica sol (Nissan Chemical Industry Co., Ltd., product name: ST-OL, acidic sol). The glass vial to which the chitin degradation reaction solution was added was arranged in a vial stirrer (Vaial Stirrer) HS-10VA (manufactured by As One Corporation), and stirred at 25 ° C. for 22 hours under the conditions of maximum output. Immediately after completion of the reaction, the chitinase activity was measured by the following procedure.

  The measurement of chitinase activity is a product using PHBAH (p-Hydroxybenzoic acid hydrazide) method (M. Lever, Analytical Biochemistry, Vol. 47, No. 1, 1972, p. 273-p. 279). It carried out by calculating | requiring the reducing sugar amount of chitin oligosaccharide and N-acetyl-D-glucosamine. Specifically, the chitin degradation reaction solution was subjected to centrifugation (4 ° C., maximum centrifugal acceleration 15780 × g, 5 minutes), and the supernatant was recovered. Next, to the collected supernatant, a PHBAH solution twice as much as the supernatant and 2M sodium hydroxide equivalent to the above supernatant were added, and well suspended to obtain a suspension. The PHBAH solution is obtained by mixing 0.1 M PHBAH, 0.2 M potassium sodium tartrate, and 0.5 M sodium hydroxide.

  The resulting suspension was reacted at 98 ° C for 10 minutes and then quenched to 2 ° C in 10 minutes. Thereafter, an 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 determined using this OD value. Further, 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 above supernatant, and the results are shown in Table 1 below. In addition, the amount of reducing sugar shown in the following Table 1 was made into the range of the amount of reducing sugar in n = 3.

(2. Examples 2 to 4)
In Examples 2 to 4, silica sol (manufactured by Nissan Chemical Industries, Ltd., product name: ST-OZL-35, acidic sol), silica sol (manufactured by Nissan Chemical Industries, Ltd., product name: MP-4540M, as the silica or the silica-containing substance) Chitinolytic enzyme composition in the same manner as in Example 1 except that Na-stable alkaline sol) and fumed silica (manufactured by Evonik, product name: AEROSIL (registered trademark) OX 50, hydrophilic fumed silica) were respectively applied Were prepared, and their chitinase activity was measured. The final concentration of each silica was 50 mg / mL in the same manner as in Example 1. The composition of each chitinolytic enzyme composition and the amount of each reducing sugar determined are shown in the following Table 1 in the same manner as in Example 1. Moreover, each reducing sugar amount shown in the following Table 1 was made into the range of the reducing sugar amount in n = 3.

(3. Example 5)
In Example 5, chitinolytic enzyme compositions were prepared in the same manner as in Example 2 except that sodium chloride at a final concentration of 100 mM was further added, and their chitinase activities were measured. The composition of the chitinolytic enzyme composition and the determined amount of reducing sugar are shown in the following Table 1 in the same manner as in Example 1. In addition, the amount of reducing sugar shown in Table 1 below was in the range of the amount of reducing sugar at n = 3.

(4. Example 6)
In Example 6, chitinolytic enzyme compositions were prepared in the same manner as in Example 2 except that the final concentration of imidazole was changed to 500 mM, and their chitinase activities were measured. The composition of the chitinolytic enzyme composition and the determined amount of reducing sugar are shown in the following Table 1 in the same manner as in Example 1. In addition, the amount of reducing sugar shown in Table 1 below was in the range of the amount of reducing sugar at n = 3.

(5. Example 7)
In Example 7, chitinolytic enzyme compositions were prepared in the same manner as in Example 2 except that imidazole which is a nitrogen-containing heterocyclic compound was changed to 2-methylimidazole, and their chitinase activities were measured. The composition of the chitinolytic enzyme composition and the determined amount of reducing sugar are shown in the following Table 1 in the same manner as in Example 1. In addition, the amount of reducing sugar shown in Table 1 below was in the range of the amount of reducing sugar at n = 3.

(6. Comparative Examples 1 to 10)
In Comparative Example 1, a chitinolytic enzyme composition was prepared in the same manner as in Example 5 except that silica, imidazole and sodium chloride were not added, and the chitinase activity was measured.

  In Comparative Example 2, a chitinolytic enzyme composition was prepared in the same manner as in Example 5 except that silica and sodium chloride were not added, and the chitinase activity was measured.

  In Comparative Example 3, a chitinolytic enzyme composition was prepared in the same manner as Example 5 except that silica was not added, and the chitinase activity was measured.

  In Comparative Example 4, a chitinolytic enzyme composition was prepared in the same manner as in Example 5 except that silica and imidazole were not added, and the chitinase activity was measured.

  In Comparative Examples 5 to 8, a chitinolytic enzyme composition was prepared in the same manner as in Examples 1 to 4 except that the imidazole was not added, and the chitinase activity was measured.

  In Comparative Example 9, a chitinolytic enzyme composition was prepared in the same manner as Example 5 except that no imidazole was added, and the chitinase activity was measured.

  In Comparative Example 10, a chitinolytic enzyme composition was prepared in the same manner as Comparative Example 3 except that the reaction was allowed to stand at 25 ° C. for 22 hours without stirring using a rotor during the reaction, and this chitinase activity was Was measured.

  In Comparative Examples 1 to 10, the composition of each chitinolytic enzyme composition and the calculated amount of each reducing sugar are shown in the following Table 1 in the same manner as in Example 1. Moreover, each reducing sugar amount shown in the following Table 1 was made into the range of the reducing sugar amount in n = 3.

(7. Evaluation of chitinase activity)
(7-1. Improvement effect of chitinase activity by addition of imidazole or sodium chloride)
The chitinase activities of Comparative Example 1 and Comparative Examples 2 to 4 were compared, and the improvement effect of the chitinase activity by addition of imidazole or sodium chloride was examined.

  As for the reducing sugar amounts in Comparative Example 1 and Comparative Examples 2 to 4, the reducing sugar amounts in Comparative Examples 2 to 4 in Comparative Example 1 were slightly increased. This indicates that the hydrolysis reaction of chitin was smoothly performed in Comparative Examples 2 to 4 and the saccharification reaction efficiency was slightly improved, and it was apparent that the chitinase activity was slightly improved by the addition of imidazole or sodium chloride. became.

(7-2. Improvement effect of chitinase activity by addition of silica)
The chitinase activities of Comparative Example 1 and Comparative Examples 5 to 8 were compared, and the improvement effect of the chitinase activity by the addition of silica was examined.

  The amount of reducing sugars of Comparative Example 1 and Comparative Examples 5 to 8 is different from that of Comparative Example 1 in Comparative Examples 5 to 8, although the difference in the degree of reduction is seen depending on the form of silica. It was decreasing. This indicates that Comparative Examples 5 to 8 did not smoothly carry out the hydrolysis reaction of chitin, and it became clear that the chitinase activity was inhibited by the addition of silica. Therefore, it evaluated that the chitinase activity of Comparative Examples 5-8 with respect to the comparative example 1 was inhibited here, and showed the chitinase activity improvement effect in following Table 1 as "inhibition."

  As the reason why silica is a factor that inhibits chitinase activity, it is consistent with the result of FIG. 5 (SNPC1) of Non-Patent Document 1, so in a state where chitinase and silica coexist, chitinase physically adsorbs on the silica surface It is thought that it is immobilized and chitinase activity is inhibited.

(7-3. Improvement effect of chitinase activity by combined use of silica and imidazole)
Based on the results of the above (7-1.) And the above (7-2.), The chitinase activities of Examples 1 to 4, 6 and Comparative Example 2 and Comparative Examples 5 to 8 are compared, and silica and imidazole are compared. We examined the improvement effect of chitinase activity by the combined use of

  As for the reducing sugar amounts of Examples 1 to 4 and Comparative Examples 5 to 8, Examples 1 to 4 are different from Comparative Examples 5 to 8 in any degree depending on the form of silica. The amount of reducing sugar was increasing. Furthermore, when the reduced sugar amount of Examples 1 to 4 and 6 and Comparative Example 2 is examined, the reduced sugar amount is increased in each of Examples 1 to 4 and 6 relative to Comparative Example 2. Although the reason is not clear, this indicates that the hydrolysis reaction of chitin was smoothly performed in Examples 1 to 4 and the saccharification reaction efficiency was improved, and the combination of silica and imidazole improved the chitinase activity It became clear that it did. Therefore, it evaluated that the chitinase activity of Examples 1-4 and 6 with respect to Comparative Examples 5-8 improved here, and showed the chitinase activity improvement effect in following Table 1 as "presence".

(7-4. Improvement effect of chitinase activity by combined use of silica and sodium chloride)
Based on the above results (7-1.) To (7-3.), The chitinase activities of Examples 2 and 5 and Comparative Examples 3 and 9 are compared, and improvement of chitinase activity by the combined use of silica and sodium chloride We examined the effect.

  As for the amounts of reducing sugars in Examples 2 and 5, the amounts of reducing sugars in both were hardly changed. Next, looking at the amounts of reducing sugars of Example 5 and Comparative Example 3, the amount of reducing sugar of Example 5 was higher than that of Comparative Example 3. Next, when the reducing sugar amount of Example 5 and Comparative Example 9 is seen, the reducing sugar amount of Example 5 compared with Comparative Example 9 was increasing. From these facts, it was clarified that the factor in which the chitinase activity was improved in Example 5 is the combined use of silica and imidazole, not the combined use of silica and sodium chloride. Therefore, it was evaluated here that the chitinase activity of Example 5 with respect to Comparative Example 9 was improved, and the chitinase activity improvement effect was shown in the following Table 1 as "presence".

(7-5. Improvement effect of chitinase activity by combined use of silica and 2-methylimidazole)
Based on the above results (7-1.) To (7-4.), The chitinase activities of Example 7 and Comparative Examples 2 and 6 are compared, and improvement of chitinase activity by the combined use of silica and 2-methylimidazole We examined the effect.

  As for the reducing sugar amounts of Examples 2 and 7 and Comparative Examples 2 and 6, the reducing sugar amounts of Examples 2 and 7 were both higher than those of Comparative Examples 2 and 6. This indicates that the hydrolysis reaction of chitin was smoothly carried out and the saccharification reaction efficiency was improved also in Example 7 using 2-methylimidazole, as in Example 2 using imidazole. It was revealed that chitinase activity was improved by the combined use of silica and 2-methylimidazole. Therefore, it was evaluated here that the chitinase activity of Example 7 with respect to Comparative Example 6 was improved, and the chitinase activity improvement effect is shown in Table 1 below as “Present”. From these results, it was suggested that the nitrogen-containing heterocyclic compounds other than imidazole and 2-methylimidazole may be improved in chitinase activity by combined use with silica.

(7-6. The improvement effect of the chitinase activity by the presence or absence of stirring)
Based on the above results (7-1.) To (7-5), the chitinase activities of Comparative Example 3 and Comparative Example 10 were compared, and the improvement effect of the chitinase activity depending on the presence or absence of stirring was examined.

  As for the reducing sugar amounts of Comparative Example 3 and Comparative Example 10, the reducing sugar amount of Comparative Example 10 was lower than that of Comparative Example 3 and the chitinase activity was reduced. The change in enzyme activity with or without agitation is a phenomenon that can occur in a general enzyme reaction system. Here, it evaluated that the chitinase activity of the comparative example 10 with respect to the comparative example 3 was inhibited, and showed the chitinase activity improvement effect in following Table 1 as "inhibition."

In addition, types A and B of the nitrogen-containing heterocyclic compound in the above Table 1 are as shown below.
A: imidazole (molecular weight 68.08)
B: 2-methylimidazole (molecular weight 82.11)

  According to each of the above Examples and Comparative Examples, when preparing a chitinolytic reaction solution using a chitinolytic enzyme composition, the glycation reaction by chitinase A can be achieved by using silica and imidazole or 2-methylimidazole in combination. It has become clear that the efficiency can be improved and the yield of the product sugar can be improved to realize a high yield.

  The present invention decomposes chitin-based biomass containing chitin contained in crustaceans such as crabs and shrimps, insects, fungi and the like into saccharides such as low molecular weight chitin polysaccharides, chitin oligosaccharides derived from chitin polysaccharides and monosaccharides It can be used in the industrial field to which the technology is applied, for example, medical materials, medicines, cosmetics, fibers, agriculture, water treatment, food and the like.

Claims (7)

  A chitinolytic enzyme composition comprising chitinase which is a chitinolytic enzyme that hydrolyzes chitin, a silica or a silica-containing substance, and a nitrogen-containing heterocyclic compound which is a nitrogen-containing heterocyclic compound .   The chitinase composition according to claim 1, wherein the chitinase contains at least chitinase A.   The chitinolytic enzyme composition according to claim 1 or 2, wherein the nitrogen-containing heterocyclic compound is a nitrogen-containing five-membered heterocyclic compound.   The chitinolytic enzyme composition according to claim 3, wherein the nitrogen-containing five-membered heterocyclic compound is any one selected from imidazole and 2-methylimidazole.   A chitin degrading reaction liquid comprising chitin and the chitin degrading enzyme composition according to any one of claims 1 to 4.   A method for producing a sugar comprising producing a sugar by hydrolyzing chitin using the chitin degradation reaction liquid according to claim 5. The method for producing a sugar according to claim 6, wherein the sugar is produced by hydrolyzing chitin using the chitin degradation reaction liquid under stirring.