JP7120559B2 - A carrier for protein immobilization, a complex, and a method for producing the same. - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act http://www. c-linkage. co. jp/icc2017/index. html, https://www. c-linkage. co. jp/icc2017/program. html, https:/www. c-linkage. co. jp/icc2017/common/files/program_for_poster_session. pdf Presented in the program of The 4th International Cellulose Conference published on September 13, 2017 on the website at the above address

TECHNICAL FIELD The present invention relates to protein-immobilizing carriers, complexes, and methods for producing them.

In recent years, the development of bioreactors using enzymes, biosensors, and the like has become popular.

In these developments, complexes in which proteins are immobilized on carriers are used.
For example, when the immobilized protein is an enzyme, the use of this complex makes it possible to decompose the object to be treated by an enzymatic reaction.

More specifically, for example, when the enzyme is a carbohydrate-degrading enzyme such as glucoamylase, the use of the complex makes it possible to decompose starch contained in rice, for example, in the brewing process. Further, for example, when the enzyme is a proteolytic enzyme such as trypsin, the use of the above-described complex enables proteome analysis technology to decompose the protein, which is the object to be processed.

Techniques using cellulose fibers as a carrier for forming such a composite have been proposed (see Patent Documents 1 to 3).

Japanese Patent No. 5734106 JP-A-8-33708 Japanese Patent No. 5781321

However, it is difficult to say that the techniques disclosed in Patent Documents 1 to 3 sufficiently exhibit the activity of the protein immobilized on the carrier.
There is also a demand for simple production of such a simple substance.

In view of the above circumstances, the present invention provides a protein-immobilizing carrier that can sufficiently exhibit the activity of the protein to be immobilized and that is easy to manufacture, a complex using the same, and a method for producing the same. The challenge is to

（ｂ）前記セルロースが、セルロースＩ型結晶構造を有する、
（ｃ）前記セルロースナノファイバーの数平均繊維径が３～１０００ｎｍ、平均アスペクト比が１０～１０００である。 The carrier for protein immobilization according to the present invention is
comprising cellulose nanofibers formed by cellulose,
The cellulose nanofiber satisfies the following (a) to (c) and is for immobilizing protein amino groups.
(a) the cellulose has an ester structure represented by the following general formula (1) at the C6 position of the glucose unit;

(b) the cellulose has a cellulose type I crystal structure;
(c) The cellulose nanofibers have a number average fiber diameter of 3 to 1,000 nm and an average aspect ratio of 10 to 1,000.

According to such a configuration, the protein-immobilizing carrier is provided with the specific cellulose nanofibers having the specific substituent, and is brought into contact with the protein to remove the succinimide group from the substituent and remove the alkyl carboxyl remaining at the end. The group can be covalently attached to amino groups of proteins.
By immobilizing the protein by this binding, a complex between the carrier for protein immobilization and the protein can be formed.
The complex thus formed has a higher activity of the immobilized protein than the complex formed using other carriers comprising cellulose nanofibers.
The cellulose nanofiber has a number average fiber diameter of 3 to 1000 nm and an average aspect ratio of 10 to 1000, and the C6 position of the glucose unit of cellulose has an ester structure represented by the general formula (1). Thus, it becomes possible to introduce many of the above specific ester structures as substituents into the cellulose unit. This makes it possible to immobilize a larger amount of protein and to have higher dispersion stability than other cellulose nanofibers. Therefore, the affinity between the immobilized protein and the object (substrate, etc.) treated by the protein does not decrease, and high activity can be expressed.
In this way, the activity of the immobilized protein can be fully exhibited.
As described above, the carrier for immobilization can sufficiently exhibit the activity of the protein to be immobilized, and is easy to manufacture.

In the carrier for protein immobilization having the above configuration,
The number of moles of the ester structure may be 25% or more of the total number of moles of carbon atoms at the C6 position of the glucose unit of the cellulose.

According to such a configuration, the number of moles of the ester structure is 25% or more with respect to the total number of moles of carbons at the C6 position of the glucose unit of cellulose, so that in the subsequent contact with the protein, the amino group of the protein It is possible to increase the immobilized amount by forming an amide bond and to retain more enzyme activity. In addition, the protein-fixing carrier can be produced more easily.

In the carrier for protein immobilization having the above configuration,
It may be possible to be solid-liquid separated.

According to such a configuration, the protein-immobilizing carrier can be subjected to solid-liquid separation, so that it can be easily recovered from the reaction solvent during production. Moreover, when it is dispersed in a solvent and used after production, it can be separated from the solvent and recovered after use. Therefore, it can be collected efficiently. In addition, since it is possible to wash with a solvent after being collected, reuse becomes possible.
The complex formed using such a carrier can be separated from the reaction solvent and washed after use to reduce impurities and unreacted proteins, and to reduce aggregation and self-digestion of the complex. Therefore, the activity of the immobilized protein is higher than that of complexes formed using other carriers having cellulose nanofibers.

In the carrier for protein immobilization having the above configuration,
The protein may be an enzyme.

According to such a configuration, since the protein to be immobilized is an enzyme, it can be used in the step of degrading starch in the sake brewing process and in the step of degrading protein into peptides and amino acids in proteome analysis.
In addition, since the enzymes used for such applications can be immobilized, the carrier on which the enzymes are immobilized can be separated, recovered, and reused by filtration after being used for the above applications. , can be used efficiently.
Furthermore, although enzymes are prone to deactivation and denaturation, immobilization of such enzymes makes it difficult for enzymes to deactivate and denature.
In addition, an immobilized carrier on which an enzyme is immobilized can be used as a packing material for column chromatography, thereby making it possible to use it as a stationary phase for column chromatography.
Thus, the protein-immobilized carrier becomes more useful.

In the protein immobilization carrier having the above configuration,
The enzyme may be a proteolytic enzyme or a carbohydrate degrading enzyme.

Here, in proteome analysis, proteolytic enzymes are used in the decomposition step of degrading proteins into peptides and amino acids. As a result, there is a risk of deactivation or degeneration over time. For example, trypsin is prone to deactivation and denaturation over time due to its self-digesting property.
However, by immobilizing such a proteolytic enzyme on the carrier for immobilizing proteins, it becomes possible to suppress the decomposition of the proteolytic enzyme itself. becomes possible.
On the other hand, carbohydrate degrading enzymes are used for saccharification of starch in food processing processes. For example, carbohydrate-degrading enzymes such as glucoamylase, although not self-degrading, may contaminate foods after processing. When mixed with food, it may be treated as an undesirable foreign substance.
However, by immobilizing such a carbohydrate-degrading enzyme on the protein-immobilizing carrier, it becomes possible to separate and recover the carbohydrate-degrading enzyme by filtration after being used in the food processing process. It is possible to suppress contamination of the carbohydrate-degrading enzyme into the inside.

The composite according to the present invention is
A complex in which a protein is immobilized on the protein-immobilizing carrier,
The amino group of the protein is covalently bonded to the terminal alkylcarboxy group obtained by removing the succinimide group from the substituent of the protein-immobilizing carrier, and solid-liquid separation can be performed.

According to such a configuration, the amino group of the protein is covalently bonded to the alkyl carboxy group of the protein-immobilizing carrier and immobilized, so that the protein is immobilized more efficiently than other carriers having cellulose nanofibers. The activity of the protein becomes high.
In addition, since the bond between the protein-immobilizing carrier and the protein is a covalent bond, the bond is less susceptible to the pH, salt concentration, and ionic strength of the buffer solution. and elution are difficult to occur. Therefore, the immobilized protein can withstand elution and washing, so that the protein-immobilizing carrier can be repeatedly used with the protein immobilized thereon, resulting in improved durability.
Here, when the binding between the protein-immobilizing carrier and the protein is a binding other than a covalent bond, that is, for example, when the binding is due to ionic bond or biological affinity, Since it is easily affected by pH and salt concentration, the conditions under which protein can be sufficiently bound are limited, and depending on the conditions, protein desorption and elution may easily occur. Further, for example, in the case of binding by hydrophobic interaction, although it is easy to bind highly hydrophobic proteins, the binding force is weaker than that of covalent binding, and the types of proteins that can be sufficiently bound are limited. Therefore, it is difficult to say that it can bind a wide range of proteins (sufficient generality).
However, when the binding between the protein-immobilizing carrier and the protein is a covalent bond, a wide variety of proteins can be more sufficiently bound to the protein-immobilizing carrier.
In addition, since the complex can undergo solid-liquid separation, it can be easily recovered from the reaction solvent during production. Moreover, when it is dispersed in a solvent and used after production, it can be separated from the solvent and recovered after use. Therefore, it can be collected efficiently. In addition, after being collected, it can be washed with a solvent, so that it can be reused.
Such complexes can be separated from the reaction solvent and washed after use to reduce impurities and unreacted proteins, and also to reduce aggregation and self-digestion of the complexes. Immobilized proteins are more active than conjugates with supports containing fibers.
Therefore, the activity of the immobilized protein can be fully exhibited, and the production is simple.

In the composite with the above configuration,
The amount of the protein immobilized on the protein-immobilizing carrier may be 10 mg/g or more per mass of the cellulose.

According to this configuration, the amount of protein immobilized on the protein-immobilizing carrier is 10 mg/g or more per mass of cellulose, so that the protein-immobilized complex has a higher activity per mass. Therefore, enzymatic activity can be expressed by applying a small amount of the complex to the object to be treated. Therefore, the activity of the protein immobilized on the complex becomes higher. For example, the interaction and reaction points between the immobilized protein and the object treated by the protein are increased, and the reaction is promoted, resulting in high activity of the protein.

In the composite with the above configuration,
The covalent bond may be an amide bond.

According to such a configuration, there is an advantage that it is difficult to be decomposed or hydrolyzed by alkalis, acids, and water, and durability can be exhibited.

（ｂ）前記セルロースが、セルロースＩ型結晶構造を有する、
（ｃ）前記セルロースナノファイバーの数平均繊維径が、３～１０００ｎｍ、平均アスペクト比が１０～１０００である。 The method for producing a protein-immobilizing carrier according to the present invention comprises:
A method for producing the protein-immobilizing carrier,
a step of oxidizing cellulose with a co-oxidizing agent in the presence of an N-oxyl compound to obtain cellulose nanofibers having alkylcarboxy groups;
a step of reacting the cellulose nanofibers having the alkylcarboxy group, a condensing agent, and a succinimide compound;
By performing solid-liquid separation of the reaction suspension obtained in the reacting step,
A protein-immobilizing carrier that satisfies the following (a) to (c) and is provided with cellulose nanofibers for immobilizing amino groups of proteins is produced.
(a) The cellulose has an ester structure represented by the following general formula (1) at the C6 position of its glucose unit.

(b) the cellulose has a cellulose type I crystal structure;
(c) The cellulose nanofibers have a number average fiber diameter of 3 to 1,000 nm and an average aspect ratio of 10 to 1,000.

According to such a configuration, a protein-immobilizing carrier comprising the specific cellulose nanofibers having the specific substituents can be easily produced.
The protein-immobilizing carrier produced in this manner is provided with the specific cellulose nanofibers having the specific substituent, is brought into contact with the protein, the succinimide group is removed from the substituent, and the alkyl remaining at the end is removed. Carboxy groups can be covalently attached to protein amino groups.
By immobilizing the protein by this binding, a complex between the carrier for protein immobilization and the protein can be formed.
Further, by performing the step of solid-liquid separation of the reaction suspension obtained in the step of reacting, the carrier for protein immobilization can be obtained as a solid content, which is convenient. It also becomes possible to wash the separated carrier for protein immobilization.
The complex thus formed can be separated from the reaction solvent and washed to reduce impurities and unreacted proteins, and to reduce aggregation and self-digestion of the complex. becomes expensive.
The produced cellulose nanofibers have a number average fiber diameter of 3 to 1000 nm and an average aspect ratio of 10 to 1000, and the C6 position of the glucose unit has an ester structure represented by the above general formula (1). By having it, it becomes possible to introduce many of the above specific ester structures as substituents into the cellulose unit. This makes it possible to immobilize a larger amount of protein and to have higher dispersion stability than other cellulose nanofibers. Therefore, the affinity between the immobilized protein and the object (substrate, etc.) treated by the protein does not decrease, and high activity can be expressed.
Thus, a protein-immobilizing carrier capable of sufficiently exhibiting the activity of the protein to be immobilized can be easily produced.

The method for producing a composite according to the present invention comprises:
A method for producing the composite,
In the reaction suspension obtained in the step of reacting in the method for producing a protein-immobilizing carrier, the succinimide group is removed from the substituent of the protein-immobilizing carrier contained in the reaction suspension to form a terminal. covalently attaching an amino group of a protein to the alkyl carboxy group;
and solid-liquid separation of the reaction suspension.

According to such a configuration, a complex in which the protein is immobilized on the specific protein-immobilizing carrier can be easily produced.
The complex produced in this way is immobilized by covalently bonding the amino group of the protein to the alkyl carboxy group of the protein-immobilizing carrier, and can be subjected to solid-liquid separation. and washing can reduce impurities and unreacted proteins, and reduce aggregation and autolysis of complexes. Therefore, the activity of the immobilized protein becomes high.
In addition, as described above, the cellulose nanofibers possessed by the carrier contained in the composite have a number average fiber diameter of 3 to 1000 nm and an average aspect ratio of 10 to 1000, and the C6 position of the glucose unit is the above general formula ( By having the ester structure represented by 1), it is possible to immobilize more proteins and to have higher dispersion stability than other cellulose nanofibers. Therefore, the activity of the immobilized protein is higher than when it is immobilized on a carrier having other cellulose nanofibers.
In this way, a complex that can fully exhibit the activity of the immobilized protein can be easily produced.

INDUSTRIAL APPLICABILITY As described above, according to the present invention, a protein-immobilizing carrier capable of sufficiently exhibiting the activity of a protein to be immobilized and which is easy to manufacture, a complex using the same, and a method for producing the same are provided. be done.

FIG. 1 shows a reaction for obtaining a protein-immobilizing carrier according to one embodiment of the present invention. A diagram showing a reaction to obtain a complex according to one embodiment of the present invention. A diagram showing the reaction of Scheme 1 in Comparative Example 1 A diagram showing the reaction of Scheme 2 in Comparative Example 1 A diagram showing the reaction of Scheme 3 in Comparative Example 1 Diagram showing the reaction of Scheme 4 in Comparative Example 1 Graph showing the relationship between substrate concentration and enzyme activity Graph showing the relationship between pH and activity rate Graph showing relationship between temperature and activity rate

Embodiments of the protein-immobilizing carrier of the present invention, complexes using the same, and methods for producing them will be described below.

First, a carrier for protein immobilization and a method for producing the same will be described.

（ｂ）前記セルロースが、セルロースＩ型結晶構造を有する、
（ｃ）前記セルロースナノファイバーの数平均繊維径が３～１０００ｎｍ、平均アスペクト比が１０～１０００である。 The protein-immobilizing carrier of the present embodiment is
comprising cellulose nanofibers formed by cellulose,
A carrier for protein immobilization, wherein the cellulose nanofibers satisfy the following (a) to (c) and are for immobilizing amino groups of proteins:
(a) The cellulose has an ester structure represented by the following general formula (1) at the C6 position of its glucose unit.

(b) the cellulose has a cellulose type I crystal structure;
(c) The cellulose nanofibers have a number average fiber diameter of 3 to 1,000 nm and an average aspect ratio of 10 to 1,000.

Hereinafter, the specific cellulose nanofibers provided in the protein-immobilizing carrier of the present embodiment may be referred to as cellulose nanofibers for immobilization.

The cellulose nanofiber for immobilization is a cellulose nanofiber formed using cellulose having a carboxy group at the C6 position of the glucose unit in the cellulose molecule (hereinafter sometimes referred to as anion-modified cellulose).
More specifically, the cellulose nanofiber for immobilization uses the anion-modified cellulose nanofiber as a raw material, and a succinimide group is bonded to the carboxy group so as to have the substituent represented by the general formula (1). It is a cellulose nanofiber.

The anion-modified cellulose nanofibers have carboxyl groups obtained by an oxidation method using an N-oxyl compound as a catalyst.
The anion-modified cellulose nanofiber may have at least one of an aldehyde group and a ketone group in addition to the carboxy group.

In the oxidation method using an N-oxyl compound as a catalyst, oxidation is performed using a co-oxidizing agent in the presence of the N-oxyl compound. Oxidation using a co-oxidizing agent in the presence of an N-oxyl compound oxidizes natural cellulose such as kraft pulp derived from softwood in the presence of an N-oxyl compound such as 2,2,6,6-tetramethylpiperidine (TEMPO). , a method of oxidation using a co-oxidant.

Examples of the cellulose (cellulose raw material) for forming the anion-modified cellulose nanofibers include cellulose nitrate, cellulose sulfate, cellulose phosphorylation, and carboxymethyl cellulose.

The anion-modified cellulose containing carboxyl groups produced by oxidation using the N-oxyl compound as a catalyst has a cellulose type I crystal structure.
That is, the anion-modified cellulose nanofiber is a fiber formed by surface-oxidizing a naturally-derived solid cellulose raw material having a type I crystal structure and pulverizing it.

In the process of biosynthesis of natural cellulose, nanofibers called microfibrils are first formed almost without exception, and these bundles are bundled to form a higher-order solid structure. In order to weaken the hydrogen bonding between the surfaces involved, a portion of the hydroxyl groups (hydroxyl groups at the C6 position of each glucose unit in the cellulose molecule) are oxidized and converted to at least carboxy groups, optionally aldehyde groups and converted to at least one of the ketone groups.

Here, the fact that the anion-modified cellulose has a type I crystal structure means that, for example, in the diffraction profile obtained by wide-angle X-ray diffraction image measurement, 2 theta = 14 to 17 ° and 2 theta = 22 to 23. It can be identified by having typical peaks at two positions around °.

The anion-modified cellulose nanofiber preferably has a carboxy group content of 1.2 to 2.5 mmol/g.
In addition, when the anion-modified cellulose nanofiber has at least one of an aldehyde group and a ketone group, and the content of the aldehyde group or the ketone group is high, active esterification using a condensing agent (condensing agent) and a succinimide compound is not promoted, making it difficult to obtain the desired immobilized carrier. Aldehyde groups and ketone groups may combine with amino groups of proteins to produce Schiff bases by-products or inhibit enzymatic activity. Considering this point, it is preferred that the total content of aldehyde groups and ketone groups is 0.3 mmol/g or less as measured by the semicarbazide method, and that no aldehyde groups are detected by Fehling's reagent. When the total content of aldehyde groups and ketone groups is 0.3 mmol/g or less, the desired immobilized carrier can be easily obtained, and by-products of Schiff bases and inhibition of enzyme activity can be suppressed.

If the amount of carboxyl groups is too small, the cellulose nanofibers for immobilization to which the succinimide groups are bound may precipitate or aggregate. It may be too much.
On the other hand, when the content of carboxy groups (the amount of carboxy groups) is in the range of 1.2 to 2.5 mmol/g, the sedimentation and aggregation of the cellulose nanofibers for fixing can be suppressed. It can also prevent the sexuality from becoming too strong.
Considering this point of view, it is more preferable that the content of carboxy groups (the amount of carboxy groups) is 1.5 to 2.0 mmol/g.

For measuring the amount of carboxy groups, for example, 60 ml of a 0.5 to 1% by mass slurry is prepared using the above anion-modified cellulose nanofiber sample whose dry mass was precisely weighed and water, and the pH is adjusted with a 0.1 M hydrochloric acid aqueous solution. After about 2.5, 0.05M sodium hydroxide aqueous solution is added dropwise and the electrical conductivity is measured. Measurements are continued until the pH is approximately 11. From the amount (V) of sodium hydroxide consumed in the neutralization step of the weak acid whose electric conductivity changes slowly, the amount of carboxyl groups is obtained according to the following formula (Equation 1).
Carboxy group amount (mmol/g) = V (ml) x [0.05/cellulose mass (g)] (Formula 1)

The amount of carboxyl groups is adjusted by controlling the amount of co-oxidizing agent added and the reaction time used in the oxidation step.

Measurement of the total content of aldehyde groups and ketone groups by the semicarbazide method is performed, for example, as follows. That is, exactly 50 ml of a 3 g/l semicarbazide hydrochloride aqueous solution adjusted to pH=5 with a phosphate buffer is added to the dried anion-modified cellulose nanofiber sample, and the mixture is sealed and shaken for two days. Next, 10 ml of this solution is accurately collected in a 100 ml beaker, 25 ml of 5N sulfuric acid and 5 ml of 0.05N potassium iodate aqueous solution are added, and stirred for 10 minutes. Then, 10 ml of a 5% aqueous potassium iodide solution is added, and immediately titrated with a 0.1N sodium thiosulfate solution using an automatic titrator. A carbonyl group content (total content of aldehyde groups and ketone groups) can be obtained. Semicarbazide reacts with an aldehyde group or a ketone group to form a Schiff base (imine), but does not react with a carboxy group. Therefore, it is considered that only the aldehyde group and the ketone group can be quantified by the above measurement.

Carbonyl group amount (mmol/g) = (D-B) x f x [0.125/w] (Formula 2)
D: Titration volume of sample (ml)
B: Titration volume of blank test (ml)
f: factor of 0.1N sodium thiosulfate solution (-)
w: sample amount (g)

In the anion-modified cellulose nanofiber, only the hydroxyl group at the C6 position of each glucose unit in the cellulose molecule on the fiber surface is selectively oxidatively modified to form a carboxy group and optionally at least an aldehyde group and a ketone group. Either. Whether or not only the hydroxyl group at the C6 position in the glucose unit on the surface of this anion-modified cellulose nanofiber is selectively oxidized can be confirmed, for example, by a ¹³ C-NMR chart. That is, the 62 ppm peak corresponding to the C6 position of the primary hydroxyl group of the glucose unit that can be confirmed in the ¹³ C-NMR chart of cellulose before oxidation disappears after the oxidation reaction, and instead the peak derived from the carboxy group etc. (178 ppm peak derived from a carboxyl group) appears. In this way, it can be confirmed that only the hydroxyl group at the C6 position of the glucose unit is oxidized to a carboxyl group or the like.

Further, the detection of aldehyde groups in the anion-modified cellulose nanofibers can also be performed using Fehling's reagent, for example. That is, for example, a Fehling reagent (a mixed solution of sodium potassium tartrate and sodium hydroxide and an aqueous solution of copper sulfate pentahydrate) was added to a dried sample and then heated at 80°C for 1 hour. It can be judged that aldehyde groups were not detected when the cellulose nanofibers were blue and the cellulose nanofibers were dark blue, and aldehyde groups were detected when the supernatant was yellow and the cellulose nanofibers were red. .

When the anion-modified cellulose nanofiber has at least one of aldehyde groups and ketone groups generated by the oxidation reaction, these aldehyde groups and ketone groups are preferably reduced with a reducing agent. This is because the fixing cellulose nanofibers containing the anion-modified cellulose can be easily obtained. From this point of view, it is more preferable that the reduction by the reducing agent is by sodium borohydride (NaBH ₄ ).

In the general formula (1), n is an integer of 0 or more. n is not particularly limited as long as it is within a range where the shape of the nanofibers can be maintained. For example, n may be 0, 1, 2, or an integer of 3 or greater.

The anion-modified cellulose nanofibers may be fibrous cellulose having a number average fiber diameter of 3 nm to 1000 nm. The number average fiber diameter of the anion-modified cellulose nanofibers is not particularly limited as long as it is within the above range. For example, the number average particle diameter may be 3 nm or more, 10 nm or more, 20 nm or more, 50 nm or more, or 1000 nm or less, 500 nm or less, 400 nm or less, 200 nm or less, 100 nm or less, or 80 nm or less. Moreover, those combinations may be sufficient. The average aspect ratio of the anion-modified cellulose nanofibers may be, for example, 10-1000, 50-1000, or 100-1000.
When the anion-modified cellulose nanofibers have a number average fiber diameter of 3 to 1000 nm and an average aspect ratio of 10 to 1000 nm, the fixing cellulose nanofibers have a number average fiber diameter of 3 to 1000 nm and an average aspect ratio of 3 to 1000 nm. becomes 10 to 1000.

When the number average fiber diameter of the anion-modified cellulose nanofibers is smaller than 3 nm, the number average fiber diameter of the fixing cellulose nanofibers is also smaller than 3 nm accordingly. Due to this, when producing the cellulose nanofibers for fixation, the cellulose nanofibers for fixation may be highly dispersed in the solvent, and in this case, centrifugation may become difficult. In addition, the cellulose nanofibers for fixation may essentially dissolve in the dispersion medium, and in this case, separation by filtration and washing may become difficult. If fractionation such as centrifugation and filtration becomes difficult, the yield will decrease, and it will be difficult to sufficiently express the function of the cellulose nanofibers for immobilization. On the other hand, when the number average fiber diameter of the anion-modified cellulose nanofibers is larger than 1000 nm, the number average fiber diameter of the fixing cellulose nanofibers is also larger than 1000 nm accordingly. In this case, although the recovery by centrifugation or filtration is facilitated, the dispersion stability in the solvent may be lowered and precipitation may occur. Moreover, when the average aspect ratio of the anion-modified cellulose nanofibers is less than 10, the dispersion stability may deteriorate.
Therefore, the number-average fiber diameter of the anion-modified cellulose nanofibers should be such that the dispersion stability of the cellulose nanofibers for fixation is not lowered and the operability of solid-liquid separation by centrifugation or filtration, recovery, washing, etc. is not impaired. From this point of view, 3 nm to 1000 nm is preferable as described above. From the same point of view, the number average fiber diameter of the anion-modified cellulose nanofibers is preferably 10-1000.

The average aspect ratio of the anion-modified cellulose nanofibers can be measured, for example, by the following method. That is, from the TEM image (magnification: 10,000 times) obtained by casting the cellulose nanofibers on a hydrophilized carbon film-coated grid and negatively staining with a 2% by mass uranyl acetate aqueous solution, the short width of the cellulose nanofibers Observe the number average width and the number average width of the long width. At that time, assume an arbitrary image width axis in the obtained image (TEM image), and adjust the sample and observation conditions (magnification, etc.) so that 20 or more fibers intersect with that axis. do. After obtaining an observation image that satisfies this condition, two random axes are drawn vertically and horizontally from each image, and the fiber diameters of fibers crossing the axes are visually read. In this way, at least three images of non-overlapping surface portions are taken with an electron microscope, each showing the short width (short width) fiber diameter and the long width ( (Long width) is read (thus, at least 20 x 2 x 3 = 120 fiber diameter information can be obtained), and the average value (number average width) of the obtained values is calculated. In this way, the number average width of the short width and the number average width of the long width are calculated. Using these values, the average aspect ratio can be calculated according to the following formula.
Average aspect ratio = number average width of long width (nm) / number average width of short width (nm)
The average aspect ratio of the fixing cellulose nanofibers is also measured in the same manner.

The number average fiber diameter of the anion-modified cellulose nanofiber is a value measured as follows.
That is, an aqueous dispersion of the anion-modified cellulose nanofibers having a solid content of 0.05 to 0.1% by mass is prepared, and the dispersion is cast on a hydrophilized carbon film-coated grid, It is used as a sample for observation with a transmission electron microscope (TEM). When anion-modified cellulose nanofibers with a large fiber diameter are included, a scanning electron microscope (SEM) image of the surface cast on glass may be observed. Observation with an electron microscope image is performed at a magnification of 5,000, 10,000, or 50,000 times depending on the size of the constituent fibers. At that time, an arbitrary image width axis is assumed in the obtained image, and the sample and observation conditions (magnification, etc.) are adjusted so that 20 or more fibers intersect the axis. After obtaining an observation image that satisfies this condition, two random axes are drawn vertically and horizontally from each image, and the fiber diameters of fibers crossing the axes are visually read. In this way, a minimum of three non-overlapping surface portion images are taken with an electron microscope and the short width (minor width) fiber diameter values of the fibers crossing each of the two axes are read (thus, the minimum 20 × 2 × 3 = 120 fiber diameter information is obtained), and the average value (number average width) of the obtained values is calculated. Data on the number average width of the short width thus obtained is calculated as the number average fiber diameter.
The number average fiber diameter of the cellulose nanofibers for fixation is also measured in the same manner.

The anion-modified cellulose nanofiber can be produced by, for example, performing (1) an oxidation reaction step, (2) a reduction step, (3) a purification step, (4) a dispersion step (miniaturization treatment step), and the like. .

(1) Oxidation Reaction Step After dispersing natural cellulose and an N-oxyl compound in water (dispersion medium), a co-oxidizing agent is added to initiate the reaction. During the reaction, a 0.5 M sodium hydroxide aqueous solution is added dropwise to maintain the pH at 10 to 11, and the reaction is considered completed when no change in pH is observed. Here, the co-oxidizing agent is not a substance that directly oxidizes the hydroxyl groups of cellulose, but a substance that oxidizes the N-oxyl compound used as an oxidation catalyst.

The natural cellulose means purified cellulose isolated from biosynthetic systems of cellulose such as plants, animals, and bacteria-produced gels. More specifically, softwood pulp, hardwood pulp, cotton pulp such as cotton linter and cotton lint, non-wood pulp such as straw pulp and bagasse pulp, bacterial cellulose (BC), cellulose isolated from sea squirt, seaweed and cellulose isolated from. These are used alone or in combination of two or more. Among these, cellulose isolated from softwood pulp, hardwood pulp, cotton pulp such as cotton linter and cotton lint, straw pulp, bagasse pulp and other non-wood pulp is preferred, and softwood pulp and hardwood pulp are preferred. It is more preferable that the cellulose isolated from is subjected to a treatment such as beating to increase the surface area, because the reaction efficiency can be improved and the productivity can be improved. In addition, if the microfibril bundles are stored without drying (never dry) after isolation and purification, the microfibril bundles are in a state that easily swells. It is more preferable because the average fiber diameter can be reduced.

The dispersion medium for natural cellulose in the above reaction is water, and the concentration of natural cellulose in the aqueous reaction solution is arbitrary as long as the reagent (natural cellulose) can sufficiently diffuse. Usually, it is about 5% by mass or less based on the mass of the aqueous reaction solution, but the reaction concentration can be increased by using an apparatus with a strong mechanical stirring force.

Examples of the N-oxyl compounds include compounds having nitroxy radicals that are generally used as oxidation catalysts. The N-oxyl compound is preferably a water-soluble compound, more preferably a piperidine nitroxyoxy radical, particularly 2,2,6,6-tetramethylpiperidinooxy radical (TEMPO) or 4-acetamido-TEMPO. preferable. A catalytic amount of the N-oxyl compound is sufficient, and it is preferably added to the aqueous reaction solution in a range of 0.1 to 4 mmol/l, more preferably 0.2 to 2 mmol/l.

Examples of the co-oxidizing agent include hypohalous acid or salts thereof, halogenous acid or salts thereof, perhalogenous acids or salts thereof, hydrogen peroxide, and perorganic acids. These are used alone or in combination of two or more. Among them, alkali metal hypohalites such as sodium hypochlorite and sodium hypobromite are preferable.
When sodium hypochlorite is used, the reaction is preferably carried out in the presence of an alkali metal bromide such as sodium bromide from the viewpoint of reaction rate.
The amount of the alkali metal bromide to be added is about 1 to 40 times, preferably about 10 to 20 times, the molar amount of the N-oxyl compound.

The pH of the aqueous reaction solution is preferably maintained in the range of about 8-11. The temperature of the aqueous solution can be anywhere from about 4 to 40°C, but the reaction can be carried out at room temperature (25°C) and no particular temperature control is required. In order to obtain the desired amount of carboxy groups, etc., the degree of oxidation is controlled by the amount of co-oxidizing agent added and the reaction time. Generally, the reaction time is about 5-120 minutes, and is completed within 240 minutes at most.

(2) Reduction Step In the production of the anion-modified cellulose nanofibers, it is preferable to carry out a reduction reaction after the oxidation reaction. Specifically, the finely oxidized cellulose after the oxidation reaction is dispersed in purified water, the pH of the aqueous dispersion is adjusted to about 10, and reduction reaction is performed with various reducing agents. As the reducing agent used in the present embodiment, it is possible to use common reducing agents, but LiBH ₄ , NaBH ₃ CN, NaBH ₄ and the like are preferred. Of these, NaBH ₄ is preferred from the viewpoint of cost and availability.

The amount of the reducing agent is preferably in the range of 0.1 to 4% by mass, particularly preferably in the range of 1 to 3% by mass, based on the finely oxidized cellulose. The reaction is carried out at room temperature or a temperature slightly higher than room temperature, usually for 10 minutes to 10 hours, preferably 30 minutes to 2 hours.

After completion of the above reaction, the pH of the reaction mixture is adjusted to about 2 with various acids, and solid-liquid separation is performed with a centrifugal separator while purified water is sprinkled to obtain cake-like finely oxidized cellulose. Solid-liquid separation is performed until the electric conductivity of the filtrate becomes 5 mS/m or less.

(3) Purification Step Next, purification is performed for the purpose of removing unreacted co-oxidizing agents (hypochlorous acid, etc.) and various by-products. Since the reactant fibers (reactant fibers) are not normally dispersed to nanofiber units at this stage, they can be purified to a high degree of purity ( 99% by mass or more) of the reactant fiber and water.

As the purification method in the above purification step, any device such as a method using centrifugal dehydration (for example, a continuous decanter) may be used as long as the device can achieve the above-described purpose. The aqueous dispersion of reactant fibers thus obtained has a solid content (cellulose) concentration in the range of approximately 10% to 50% by weight when squeezed. Considering the subsequent dispersion step, a solid content concentration higher than 50% by mass is not preferable because extremely high energy is required for dispersion.

(4) Dispersion process (miniaturization process)
For example, an appropriate amount of a base is added to the reactant fibers impregnated with water (aqueous dispersion) obtained in the above purification step, and dispersed in a dispersion medium for dispersion treatment. The type of base to be added is not particularly limited as long as it does not affect the dispersing step or subsequent immobilization of the enzyme. The viscosity increases with the treatment, and a finely divided cellulose fiber dispersion can be obtained. The amount of the base to be added is not particularly limited, but it is desirable that the amount is such that the water dispersion of the cellulose fibers that have been subjected to the pulverization treatment is uniformly dispersed in the dispersion medium. In addition, if the amount of base added is small, the immobilized enzyme may precipitate or aggregate in subsequent enzyme immobilization or activity evaluation of the immobilized enzyme, resulting in a decrease in enzyme activity. In order to suppress the decrease in activity, it is desirable to add an amount such that the immobilized enzyme is evenly dispersed. Thereafter, the dispersion of cellulose fibers is dried to obtain the anion-modified cellulose nanofibers, which are used for bonding with succinimide groups. The anion-modified cellulose nanofibers may be used for bonding with the succinimide groups in the form of a dispersion without drying the dispersion of the cellulose fibers.

Commercially available products can be used as such anion-modified cellulose nanofibers, and examples thereof include Rheocrysta (manufactured by Daiichi Pharmaceutical Co., Ltd.).
A carboxy group may form a salt with a cation.

The above-mentioned cellulose nanofibers for fixing are prepared by subjecting the anion-modified cellulose nanofibers obtained in (1) to (4) above to (5) a succinimide group-introducing step of binding succinimide groups to the anion groups, and (6) a reaction suspension. It can be produced by performing a separation step of solid-liquid separation of the turbid liquid. The steps (5) and (6) will be described below.

(5) Step of introducing succinimide group In the anion-modified cellulose nanofiber obtained in (4), the carboxyl group at the C6 position of the glucose unit of each cellulose is reacted with a condensing agent (condensing agent) and a succinimide compound. The substituent represented by general formula (1) is present at the C6 position of the glue unit. That is, the cellulose nanofiber for fixation is obtained in which the C6 position of the glucose unit has an ester structure represented by the general formula (1).
Specifically, for example, as shown in FIG. 1, the anion-modified cellulose nanofibers, a condensing agent as a catalyst, and a succinimide compound are reacted at room temperature for 1 hour to form the fixing cellulose nanofibers. obtain.
Condensing agents include 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDAC) and its hydrochloride, N,N'-dimethylcarbodiimide (DIC), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide and toluenesulfonates thereof, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and its methiozide, and carbodiimide compounds such as N,N'-dicyclohexylcarbodiimide. The condensing agent can be used singly or as a mixture of two or more.
This reaction is preferably carried out under alkaline conditions. Alkaline conditions can be achieved, for example, by adding sodium hydroxide.
Succinimide compounds include N-hydroxysuccinimide, N-hydroxysulfosuccinimide and the like. The reaction product may be appropriately washed with a buffer solution or the like. Buffers include 2-(N-morpholino)ethanesulfonic acid (MES) buffer, trishydroxymethylaminomethane (Tris)-hydrochloric acid buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ( HEPES) buffer, piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) buffer, and the like.

By the step of introducing a succinimide group, the carboxyl group at the C6 position reacts with the succinimide compound to produce an active ester. Since this active ester can be covalently bonded to amino groups of proteins, this covalent bond enables the protein-immobilizing carrier of the present embodiment to be bonded to proteins.
In this respect, the reaction performed in the succinimide group-introducing step corresponds to an active esterification reaction.

(6) Separation step The reaction suspension obtained in the step of introducing succinimide (that is, the step of reacting the carboxy group at the C6 position of the glucose unit of each cellulose with a condensing agent (condensing agent) and a succinimide compound) is solid-liquid To separate. Methods for solid-liquid separation include centrifugation, filtration, and the like, and these methods may be combined.

The cellulose nanofibers for fixation obtained by introducing the succinimide groups may be mixed with a dispersing medium such as water or a mixed solution of water and an organic solvent. That is, in the carrier for protein immobilization of the present embodiment, the cellulose nanofibers for immobilization may be provided with a dispersion medium such as water or a mixed solution of water and an organic solvent.

（ｂ）前記セルロースが、セルロースＩ型結晶構造を有する、
（ｃ）前記セルロースナノファイバーの数平均繊維径が、３～１０００ｎｍ、平均アスペクト比が１０～１０００である。 As described above, the method for producing a protein-immobilizing carrier of the present embodiment includes:
A method for producing the protein-immobilizing carrier,
a step of oxidizing cellulose with a co-oxidizing agent in the presence of an N-oxyl compound to obtain cellulose nanofibers having alkylcarboxy groups (anion-modified cellulose nanofibers);
a step of reacting the cellulose nanofibers having the alkylcarboxy group, a carbodiimide compound, and a succinimide compound;
By performing solid-liquid separation of the reaction suspension obtained in the reacting step,
A method for producing a protein-immobilizing carrier that satisfies the following (a) to (c) and comprises cellulose nanofibers (cellulose nanofibers for immobilization) for immobilizing amino groups of proteins.
(a) the cellulose has an ester structure represented by the following general formula (1) at the C6 position of the glucose unit;

(b) the cellulose has a cellulose type I crystal structure;
(c) The cellulose nanofibers have a number average fiber diameter of 3 to 1,000 nm and an average aspect ratio of 10 to 1,000.

The protein-fixing carrier of the present embodiment contains, in addition to the fixing cellulose nanofibers and the acid separating medium, an acid separating agent, a plasticizer, an organic solvent, a preservative, and an antifoaming agent within a range that does not impair the effects of the present invention. , thickeners, lubricants, inorganic salts, and other conventionally known additives may be appropriately blended as necessary.

In the protein-immobilizing carrier of the present embodiment, proteins include enzymes, peptides, polypeptides, antigens, polyclonal antibodies, monoclonal antibodies, single-chain antibodies, antibody fragments (e.g., F(ab), F(ab')2 , and Fv fragments), protein A, protein G, etc., glycoproteins, nucleic acids (DNA, RNA, etc.), glycans, small organic molecules, cells, viruses, bacteria, probes, receptor molecules, etc. can be immobilized. However, in this embodiment, it is preferred that the protein is an enzyme.
Enzymes include proteolytic enzymes, lipolytic enzymes, oxidases, carbohydrate-degrading enzymes, and the like.
In addition to immobilizing proteins, substances other than proteins may also be immobilized on the protein-immobilizing carrier of the present embodiment.

Preferably, the enzyme is a proteolytic enzyme or a carbohydrate-degrading enzyme.
Proteolytic enzymes include proteases such as trypsin, chymotrypsin, pepsin, rennet, elastase, papain, carboxypeptidase, aminopeptidase, alkaline protease, serine protease, and glutaminase.
Carbohydrate degrading enzymes include glucoamylase, saccharase, maltase, lactase, dextranase, amylopullulanase, glucanase and the like.

In addition, the protein-immobilizing carrier of the present embodiment may be capable of solid-liquid separation. That is, it may be possible for the solid-liquid separation as a water-insoluble component.

Next, the composite of this embodiment and the manufacturing method thereof will be described.

The complex of this embodiment is
A complex in which a protein is immobilized on the protein-immobilizing carrier of the present embodiment,
The amino group of the protein is covalently bonded to the terminal alkylcarboxy group obtained by removing the succinimide group from the substituent of the protein-immobilizing carrier, and solid-liquid separation can be performed.

In the complex of this embodiment, the covalent bond is preferably an amide bond.
According to such a configuration, there is an advantage that it is difficult to be decomposed or hydrolyzed by alkalis, acids, and water, and durability can be exhibited.

The composite of this embodiment is formed, for example, according to the scheme shown in FIG.
That is, glucoamylase and MES buffer were added to the protein immobilization carrier of the present embodiment, and the C6 position of the glucose unit in the cellulose nanofiber for immobilization was shaken using an incubator at 4°C for 24 hours. A carboxy group and an amino group of glucoamylase are covalently bonded (amide bond in FIG. 2). Thereafter, the complex of the present embodiment is obtained by solid-liquid separation of the reaction suspension of such covalent bonds.

That is, in the method for producing a complex of the present embodiment, in the reaction suspension obtained in the step of reacting in the method for producing a protein-immobilizing carrier of the present embodiment, the protein contained in the reaction suspension is immobilized. a step of removing the succinimide group from the substituent of the carrier for protein and covalently binding the amino group of the protein to the terminal alkyl carboxy group;
and solid-liquid separation of the reaction suspension.
Methods for solid-liquid separation include centrifugation, filtration, and the like, and these methods may be combined.

In the method for producing a complex of the present embodiment, as described above, the covalent bond is preferably an amide bond.

（ｂ）前記セルロースが、セルロースＩ型結晶構造を有する、
（ｃ）前記セルロースナノファイバーの数平均繊維径が３～１０００ｎｍ、平均アスペクト比が１０～１０００である。 As described above, the protein-immobilizing carrier of the present embodiment is
comprising cellulose nanofibers formed by cellulose,
The cellulose nanofiber satisfies the following (a) to (c) and is for immobilizing protein amino groups.
(a) the cellulose has an ester structure represented by the following general formula (1) at the C6 position of the glucose unit;

(b) the cellulose has a cellulose type I crystal structure;
(c) The cellulose nanofibers have a number average fiber diameter of 3 to 1,000 nm and an average aspect ratio of 10 to 1,000.

According to such a configuration, the protein-immobilizing carrier is provided with the specific cellulose nanofibers having the specific substituent, and is brought into contact with the protein to remove the succinimide group from the substituent and remove the alkyl carboxyl remaining at the end. The group can be covalently attached to amino groups of proteins.
By immobilizing the protein by this binding, a complex between the carrier for protein immobilization and the protein can be formed.
The complex thus formed has a higher activity of the immobilized protein than the complex formed using other carriers comprising cellulose nanofibers.
The cellulose nanofiber has a number average fiber diameter of 3 to 1000 nm and an average aspect ratio of 10 to 1000, and the C6 position of the glucose unit of cellulose has an ester structure represented by the general formula (1). Thus, it becomes possible to introduce many of the above specific ester structures as substituents into the cellulose unit. This makes it possible to immobilize a larger amount of protein and to have higher dispersion stability than other cellulose nanofibers. Therefore, the affinity between the immobilized protein and the object (substrate, etc.) treated by the protein does not decrease, and high activity can be expressed.
In this way, the activity of the immobilized protein can be fully exhibited.
As described above, the carrier for immobilization can sufficiently exhibit the activity of the protein to be immobilized, and is easy to manufacture.

Here, when an attempt is made to produce a protein-fixing carrier using non-anion-modified (unmodified) cellulose nanofibers, schemes 1 to 3 in FIGS. Three reactions need to be performed.
In contrast, as in the present embodiment, when the anion-modified cellulose nanofibers are used to produce a protein-immobilizing carrier, two reactions shown in FIGS. 1 and 2 may be performed.
Therefore, the protein-immobilizing carrier of the present embodiment is easier to produce.
In addition, the protein-immobilizing carrier produced using the anion-modified cellulose nanofibers of the present embodiment is more efficient than the protein-immobilizing carrier produced using unmodified cellulose nanofibers when the protein is immobilized. , the activity of the immobilized protein can be more fully exerted.

In the carrier for protein immobilization of this embodiment,
The number of moles of the ester structure may be 25% or more of the total number of moles of carbon atoms at the C6 position of the glucose unit of the cellulose.

According to such a configuration, the number of moles of the ester structure is 25% or more with respect to the total number of moles of carbons at the C6 position of the glucose unit of cellulose, so that in the subsequent contact with the protein, the amino group of the protein It is possible to increase the immobilized amount by forming an amide bond and to retain more enzyme activity. In addition, the protein-fixing carrier can be produced more easily.

In the carrier for protein immobilization of this embodiment,
It may be possible to be solid-liquid separated.

According to such a configuration, the protein-immobilizing carrier can be subjected to solid-liquid separation, so that it can be easily recovered from the reaction solvent during production. Moreover, when it is dispersed in a solvent and used after production, it can be separated from the solvent and recovered after use. Therefore, it can be collected efficiently. In addition, since it is possible to wash with a solvent after being collected, reuse becomes possible.
The complex formed using such a carrier can be separated from the reaction solvent and washed after use to reduce impurities and unreacted proteins, and to reduce aggregation and self-digestion of the complex. As a result, the activity of the immobilized protein is higher than that of complexes formed using other carriers with cellulose nanofibers.

In the carrier for protein immobilization of this embodiment,
The protein may be an enzyme.

According to such a configuration, since the protein to be immobilized is an enzyme, it can be used in the step of degrading starch in the sake brewing process and in the step of degrading protein into peptides and amino acids in proteome analysis.
In addition, since the enzymes used for such applications can be immobilized, the carrier on which the enzymes are immobilized can be separated, recovered, and reused by filtration after being used for the above applications. , can be used efficiently.
Furthermore, although enzymes are prone to deactivation and denaturation, immobilization of such enzymes makes it difficult for enzymes to deactivate and denature.
In addition, an immobilized carrier on which an enzyme is immobilized can be used as a packing material for column chromatography, thereby making it possible to use it as a stationary phase for column chromatography.
Thus, the protein-immobilized carrier becomes more useful.

In the carrier for protein immobilization of this embodiment,
The enzyme may be a proteolytic enzyme or a carbohydrate degrading enzyme.

Here, in proteome analysis, proteolytic enzymes are used in the decomposition step of degrading proteins into peptides and amino acids. As a result, there is a risk of deactivation and degeneration over time. For example, trypsin is prone to deactivation and denaturation over time due to its self-digesting property.
However, by immobilizing such a proteolytic enzyme on the carrier for immobilizing proteins, it becomes possible to suppress the decomposition of the proteolytic enzyme itself. becomes possible.
On the other hand, carbohydrate-degrading enzymes are used for saccharification of starch in food processing processes. For example, carbohydrate-degrading enzymes such as glucoamylase, although not self-degrading, may contaminate foods after processing. When mixed with food, it may be treated as an undesirable foreign substance.
However, by immobilizing such a carbohydrate-degrading enzyme on the protein-immobilizing carrier, it becomes possible to separate and recover the carbohydrate-degrading enzyme by filtration after being used in the food processing process. It is possible to suppress contamination of the carbohydrate-degrading enzyme into the inside.

The complex of this embodiment is
A complex in which a protein is immobilized on the protein-immobilizing carrier,
The amino group of the protein is covalently bonded to the terminal alkylcarboxy group obtained by removing the succinimide group from the substituent of the protein-immobilizing carrier, and solid-liquid separation can be performed.

According to such a configuration, the amino group of the protein is covalently bonded to the alkyl carboxy group of the protein-immobilizing carrier and immobilized, so that the protein is immobilized more efficiently than other carriers having cellulose nanofibers. The activity of the protein becomes high.
In addition, since the bond between the protein-immobilizing carrier and the protein is a covalent bond, the bond is less susceptible to the pH, salt concentration, and ionic strength of the buffer solution. and elution are difficult to occur. Therefore, the immobilized protein can withstand elution and washing, so that the protein-immobilizing carrier can be repeatedly used with the protein immobilized thereon, resulting in improved durability.
Here, when the binding between the protein-immobilizing carrier and the protein is a binding other than a covalent bond, that is, for example, when the binding is due to ionic bond or biological affinity, Since it is easily affected by pH and salt concentration, the conditions under which proteins can be sufficiently bound are limited, and depending on the conditions, desorption and elution of proteins may easily occur. Further, for example, in the case of binding by hydrophobic interaction, although it is easy to bind highly hydrophobic proteins, the binding force is weaker than that of covalent binding, and the types of proteins that can be sufficiently bound are limited. Therefore, it is difficult to say that it can bind a wide range of proteins (sufficient generality).
However, when the binding between the protein-immobilizing carrier and the protein is a covalent bond, a wide variety of proteins can be more sufficiently bound to the protein-immobilizing carrier.
In addition, since the complex can undergo solid-liquid separation, it can be easily recovered from the reaction solvent during production. Moreover, when it is dispersed in a solvent and used after production, it can be separated from the solvent and recovered after use. Therefore, it can be collected efficiently. In addition, after being collected, it can be washed with a solvent, so that it can be reused.
The complex prepared using such a carrier can be separated from the reaction solvent and washed after use to reduce impurities and unreacted proteins, and to reduce aggregation and self-digestion of the complex. As a result, the activity of the immobilized protein is higher than that of complexes formed using other carriers with cellulose nanofibers.
Therefore, the activity of the immobilized protein can be fully exhibited, and the production is simple.

In the complex of this embodiment,
The amount of the protein immobilized on the protein-immobilizing carrier may be 10 mg/g or more per mass of the cellulose.

According to this configuration, the amount of protein immobilized on the protein-immobilizing carrier is 10 mg/g or more per mass of cellulose, so that the protein-immobilized complex has a higher activity per mass. Therefore, enzymatic activity can be expressed by applying a small amount of the complex to the object to be treated. Therefore, the activity of the protein immobilized on the complex becomes higher. For example, the interaction and reaction points between the immobilized protein and the object treated by the protein are increased, and the reaction is promoted, resulting in high activity of the protein.

In the complex of this embodiment,
The covalent bond may be an amide bond.

According to such a configuration, there is an advantage that it is difficult to be decomposed or hydrolyzed by alkalis, acids, and water, and durability can be exhibited.

（ｂ）前記セルロースが、セルロースＩ型結晶構造を有する、
（ｃ）前記セルロースナノファイバーの数平均繊維径が、３～１０００ｎｍ、平均アスペクト比が１０～１０００である。 The method for producing the protein-immobilizing carrier of the present embodiment comprises
A method for producing the protein-immobilizing carrier,
a step of oxidizing cellulose with a co-oxidizing agent in the presence of an N-oxyl compound to obtain cellulose nanofibers having alkylcarboxy groups;
a step of reacting the cellulose nanofibers having the alkylcarboxy group, a condensing agent, and a succinimide compound;
By performing solid-liquid separation of the reaction suspension obtained in the reacting step,
A protein-immobilizing carrier that satisfies the following (a) to (c) and is provided with cellulose nanofibers for immobilizing amino groups of proteins is produced.
(a) The cellulose has an ester structure represented by the following general formula (1) at the C6 position of its glucose unit.

(b) the cellulose has a cellulose type I crystal structure;
(c) The cellulose nanofibers have a number average fiber diameter of 3 to 1,000 nm and an average aspect ratio of 10 to 1,000.

According to such a configuration, a protein-immobilizing carrier comprising the specific cellulose nanofibers having the specific substituents can be easily produced.
The protein-immobilizing carrier produced in this manner is provided with the specific cellulose nanofibers having the specific substituent, is brought into contact with the protein, the succinimide group is removed from the substituent, and the alkyl remaining at the end is removed. Carboxy groups can be covalently attached to protein amino groups.
By immobilizing the protein by this binding, a complex between the carrier for protein immobilization and the protein can be formed.
Further, by performing the step of solid-liquid separation of the reaction suspension obtained in the step of reacting, the carrier for protein immobilization can be obtained as a solid content, which is convenient. It also becomes possible to wash the separated carrier for protein immobilization.
The complex thus formed can be separated from the reaction solvent and washed to reduce impurities and unreacted proteins, and to reduce aggregation and self-digestion of the complex. becomes expensive.
The produced cellulose nanofibers have a number average fiber diameter of 3 to 1000 nm and an average aspect ratio of 10 to 1000, and the C6 position of the glucose unit has an ester structure represented by the above general formula (1). By having it, it becomes possible to introduce many of the above specific ester structures as substituents into the cellulose unit. This makes it possible to immobilize a larger amount of protein and to have higher dispersion stability than other cellulose nanofibers. Therefore, the affinity between the immobilized protein and the object (substrate, etc.) treated by the protein does not decrease, and high activity can be expressed.
Thus, a protein-immobilizing carrier capable of sufficiently exhibiting the activity of the protein to be immobilized can be easily produced.

The method for producing the composite of this embodiment includes:
A method for producing the composite,
In the reaction suspension obtained in the step of reacting in the method for producing a protein-immobilizing carrier, the succinimide group is removed from the substituent of the protein-immobilizing carrier contained in the reaction suspension to form a terminal. covalently attaching an amino group of a protein to the alkyl carboxy group;
and solid-liquid separation of the reaction suspension.

According to such a configuration, a complex in which the protein is immobilized on the specific protein-immobilizing carrier can be easily produced.
The complex produced in this way is immobilized by covalently bonding the amino group of the protein to the alkyl carboxy group of the protein-immobilizing carrier, and can be subjected to solid-liquid separation. and washing can reduce impurities and unreacted proteins, and reduce aggregation and autolysis of complexes. Therefore, the activity of the immobilized protein becomes high.
In addition, as described above, the cellulose nanofibers contained in the composite have a number average fiber diameter of 3 to 1000 nm and an average aspect ratio of 10 to 1000, and the C6 position of the glucose unit is represented by the above general formula (1). By having the represented ester structure, it is possible to immobilize a larger amount of protein and to have higher dispersion stability than other cellulose nanofibers. Therefore, the activity of the immobilized protein is higher than when it is immobilized on a carrier having other cellulose nanofibers.
In this way, a complex that can fully exhibit the activity of the immobilized protein can be easily produced.

As described above, according to the present embodiment, a protein-immobilizing carrier that can sufficiently exhibit the activity of a protein to be immobilized and that is easy to manufacture, a complex using the same, and a method for producing the same are provided. provided.

The carrier for immobilizing proteins, the complex, and the method for producing them of the present embodiment are, for example, bioreactors in food processing processes, immobilized enzymes for saccharification in brewing and sake brewing, biosensors, fillers for column chromatography, and proteome analysis. It can be suitably used for applications such as fillers for medical applications, biological tests, diagnostic devices and chips.
For example, the complex of the present embodiment can be packed in a column or the like and used for the above applications.
Moreover, when the immobilized protein is an enzyme, and the enzyme is, for example, a proteolytic enzyme, it can be applied to, for example, proteome analysis technology for the development of biopharmaceuticals.
For example, when the enzyme is a carbohydrate-degrading enzyme, it can be applied to food manufacturing techniques such as food processing and sake brewing.

The protein-immobilizing carrier, the composite, and the method for producing the same of the present embodiment are as described above. It is not particularly limited.

EXAMPLES The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples as long as the gist thereof is not exceeded. In the examples, "%" means mass basis unless otherwise specified.

[Production of aqueous cellulose dispersion B1]
(1) Oxidation Step 0.5 g (0.08 mmol/g) of TEMPO and 5.0 g (1.215 mmol/g) of sodium bromide were dissolved in 1600 g of purified water and cooled to 10°C. After dispersing bleached softwood kraft pulp (NBKP) equivalent to 200 g in dry mass in this solution, add 15.0 g (5 mmol / g) of 12% sodium hypochlorite aqueous solution in terms of solid content to react. started. Since the pH decreased with the progress of the reaction, the pH was adjusted to 10 to 10.5 by adding a 24% NaOH aqueous solution as needed, and the reaction was allowed to proceed for 2.0 hours.

(2) Reduction step After solid-liquid separation of the obtained reaction product was performed using a centrifuge, purified water was added to adjust the solid content concentration to 4%. After that, the pH of the slurry was adjusted to 10 with a 24% NaOH aqueous solution. The temperature of the slurry was set to 30° C., 0.3 g (0.2 mmol/g) of NaBH ₄ was added, and reacted for 2 hours.

(3) Purification step 1M HCl was added to the obtained reactant to adjust the pH to 2, and then filtered through a glass filter. Thereafter, washing with a sufficient amount of ion-exchanged water and filtration were performed, and the electric conductivity of the obtained filtrate was measured. The purification process was terminated when the electric conductivity of the filtrate did not change even after repeated washing with water. Thus, an anion-modified cellulose fiber containing water and having a solid content of 20% was obtained.

(4) Dispersing step An appropriate amount of water and sodium hydroxide are added to the obtained anion-modified cellulose fibers to prepare a slurry having a solid content of 2%, and a high-pressure homogenizer is used to perform two passes of refining treatment at 150 MPa to obtain an anion. An aqueous dispersion of modified cellulose nanofibers (aqueous cellulose dispersion) B1 was obtained.
When measured by the method described later, the carboxyl group content of the obtained cellulose aqueous dispersion B1 was 1.97 mmol/g, and the carbonyl group content was 0.10 mmol/g. I was not able to admit. The number average fiber diameter of the anion-modified cellulose nanofibers contained in the aqueous cellulose dispersion B1 was 4 nm, and the average aspect ratio was 280. The crystalline structure of the cellulose contained in the modified cellulose nanofibers was found to have a type I crystal structure. In addition, the peak at 62 ppm corresponding to the C6 position of the primary hydroxyl group of the glucose unit, which can be confirmed in the ¹³ C-NMR chart of cellulose before oxidation, disappears after the oxidation reaction, and is instead derived from the carboxy group at 178 ppm. A peak had appeared. Therefore, it was confirmed that only the hydroxyl group at the C6 position of the glucose unit was oxidized to a carboxyl group or the like.

[Production of aqueous cellulose dispersion B2]
In the dispersing step, a homomixer (Primex Co., Ltd.) is used instead of the high-pressure homogenizer, and the aqueous cellulose dispersion is prepared in the same manner as in the production of the aqueous cellulose dispersion B1, except that the micronization treatment is performed at 12000 rpm for 10 minutes. B2 was obtained.
When measured by the method described later, the anion-modified cellulose nanofibers contained in the obtained aqueous cellulose dispersion B2 had a carboxy group content of 1.97 mmol/g and a carbonyl group content of 0.10 mmol/g. , on the other hand, no detection of aldehyde groups was observed. The number average fiber diameter of the anion-modified cellulose nanofiber contained in the aqueous cellulose dispersion B2 was 400 nm, and the average aspect ratio was 20. The crystalline structure of the cellulose contained in the anion-modified cellulose nanofibers was found to have a type I crystal structure. In addition, the peak at 62 ppm corresponding to the C6 position of the primary hydroxyl group of the glucose unit that can be confirmed in the ¹³ C-NMR chart of the cellulose before oxidation disappears after the oxidation reaction, and instead a peak derived from the carboxy group at 178 ppm. was appearing. Therefore, it was confirmed that only the hydroxyl group at the C6 position of the glucose unit was oxidized to a carboxyl group or the like.

Using the produced anion-modified cellulose nanofiber (cellulose aqueous dispersion B1 or B2) as a sample, each characteristic was measured as follows.

[Measurement of carboxy group content]
A water dispersion of 60 ml was prepared by dispersing 0.25 g of the sample in water, and after adjusting the pH to about 2.5 with a 0.1 M hydrochloric acid aqueous solution, a 0.05 M sodium hydroxide aqueous solution was added dropwise to increase the electrical conductivity. was measured. Measurements were continued until the pH was approximately 11. The amount of carboxyl groups was obtained according to the following formula (Formula 1) from the amount (V) of sodium hydroxide consumed in the neutralization step of the weak acid whose electrical conductivity changes slowly.
Carboxy group amount (mmol/g) = V (ml) x [0.05/cellulose mass (g)] (Formula 1)

[Measurement of carbonyl group content (semicarbazide method)]
About 0.2 g (dry mass) of a sample was accurately weighed, and exactly 50 ml of an aqueous solution of 3 g/l of semicarbazide hydrochloride adjusted to pH 5 with a phosphate buffer was added, tightly stoppered, and shaken for two days. Next, 10 ml of this solution was accurately collected in a 100 ml beaker, 25 ml of 5N sulfuric acid and 5 ml of 0.05N potassium iodate aqueous solution were added and stirred for 10 minutes. Then, 10 ml of 5% potassium iodide aqueous solution is added and immediately titrated with 0.1N sodium thiosulfate solution using an automatic titrator. A carbonyl group content (total content of aldehyde groups and ketone groups) was determined.
Carbonyl group amount (mmol/g) = (DB) x f x [0.125/w] (Formula 2)
D: Titration volume of sample (ml)
B: Titration volume of blank test (ml)
f: factor of 0.1N sodium thiosulfate solution (-)
w: sample amount (g)

[Detection of aldehyde group]
0.4 g of the sample was precisely weighed, Fehling's reagent (5 ml of a mixed solution of sodium potassium tartrate and sodium hydroxide, and 5 ml of copper sulfate pentahydrate aqueous solution) prepared according to the Japanese Pharmacopoeia was added, and then heated at 80°C for 1 hour. Heated for hours. When the supernatant was blue and the sample portion (solid content) was dark blue, it was judged that no aldehyde group was detected, and was evaluated as "none". In addition, when the supernatant was yellow and the sample portion was red, it was judged that an aldehyde group was detected, and was evaluated as "yes".

[Number average fiber diameter]
The number average fiber diameter of the aqueous cellulose dispersion was observed using a transmission electron microscope (TEM) (manufactured by JEOL Ltd., JEM-1400). That is, after casting the sample on a hydrophilized carbon film-coated grid, the number average fiber diameter was determined from the TEM image (magnification: 10,000 times) negatively stained with a 2% uranyl acetate aqueous solution according to the method described above. Calculated.

[Average aspect ratio]
The average aspect ratio of the aqueous cellulose dispersion was measured as follows. That is, after casting the sample on a hydrophilized carbon film-coated grid, the TEM image (magnification: 10000 times) negatively stained with a 2% by mass uranyl acetate aqueous solution was used to determine the anion-modified cellulose according to the method described above. Observe the number average width of the shorter width (short width) and the number average width of the longer width (long width) of the nanofibers, and from these values, according to the method described above, the average aspect ratio is calculated. Calculated.

〔Crystal structure〕
Using an X-ray diffractometer (RINT-Ultima3, manufactured by Rigaku Co., Ltd.), the diffraction profile of the sample was measured, and was typically observed at two positions near 2 theta = 14 to 17° and 2 theta = 22 to 23°. A crystal structure (type I crystal structure) was evaluated as "presence" when such a peak was observed, and was evaluated as "absence" when no peak was observed.

[Selective oxidation at C6 position]
A ¹³ C-NMR chart was used to confirm whether only the hydroxyl group at the C6 position of the glucose unit on the sample surface was selectively oxidized to a carboxyl group or the like.

(1) Example 1
A succinimide group was bound to the carboxyl group at the C6 position of the glucose unit of cellulose in the anion-modified cellulose nanofiber to produce a protein-immobilizing carrier, and a protein was immobilized on the obtained protein-immobilizing carrier to produce a complex. . Details are as follows.

(1-1) Anion-Modified Cellulose Nanofibers Used Cellulose aqueous dispersion B1 was used as anion-modified cellulose nanofibers.

(1-2) Production of carrier for protein immobilization The following raw materials were used.
Cellulose aqueous dispersion B1
EDAC (1-ethyl-3 (3-dimethylaminopropyl) carbodiimide, manufactured by Wako Pure Chemical Industries, Ltd.)
NHS (N-hydroxysuccinimide, manufactured by Wako Pure Chemical Industries, Ltd.)

As shown in FIG. 1, activated esterification of cellulose aqueous dispersion B1 was carried out.
20.17 g (wet weight) of cellulose aqueous dispersion B1, 20 mL of 16 mmol of NHS aqueous solution, and 20 mL of 16 mmol of EDAC aqueous solution were added to a 50 mL Erlenmeyer flask and stirred at room temperature for 1 hour using a magnetic stirrer (200 rpm). As a result, the carboxy group at the C6 position of the glucose unit in the cellulose of the aqueous cellulose dispersion B1 and NHS are esterified to form cellulose nanofibers having an activated ester at the C6 position (activated esterified cellulose nanofibers). A protein-immobilizing carrier was produced.
The resulting reaction suspension was centrifuged at 10000 rpm for 15 minutes in a centrifuge, the supernatant was collected by decantation, and the residue was washed twice with MES buffer and then collected. A protein-immobilizing carrier (cellulose C6-position active ester carrier) was obtained. The cellulose C6-position active ester carrier could be easily separated and washed under the above centrifugal conditions.

(1-3) Production of Complex of Protein-Immobilizing Carrier and Protein The following raw materials were used.
Cellulose C6-position active ester carrier produced in the production of the protein immobilization carrier Sodium hydroxide (NaOH) (Nacalai Tesque Co., Ltd.)
MES (2-(N-morpholino) ethanesulfonic acid) (manufactured by Dojindo Laboratories)
Glucoamylase (manufactured by Tokyo Chemical Industry Co., Ltd.)

As shown in FIG. 2, glucoamylase was immobilized on a cellulose C6-position active ester carrier.
The cellulose C6-position active ester carrier obtained above is placed in a separate Erlenmeyer flask, 20 mL of 1000 μg/mL glucoamylase-MES buffer is added, and an incubator is used at 4° C. and 100 rpm for 24 hours. By shaking, the C6-position active ester of the glucose unit of the cellulose C6-position active ester carrier and glucoamylase (protein) undergo an amidation reaction to form a complex of the cellulose C6-position active ester carrier and glucoamylase. rice field. In addition, the MES buffer was prepared by the following method.
The resulting reaction suspension was centrifuged at 10,000 rpm for 15 minutes in a centrifuge, and the supernatant was collected by decantation and washed, after which the complex was obtained as a residue. A complex of cellulose C6-position active ester carrier and glucoamylase could be easily separated by the above centrifugation conditions, and the desired complex was obtained by washing.
The resulting complex was stored cold (4° C.) in pure water and used for evaluation of enzymatic activity, which will be described later.
On the other hand, the supernatant was used to determine the amount of enzyme immobilized, which will be described later.

[Method for preparing MES buffer]
After preparing a 0.05 M MES aqueous solution and a 0.05 M sodium hydroxide aqueous solution in the amounts shown in Table 1 below, the sodium hydroxide solution obtained is gradually added to 300 mL of the obtained MES aqueous solution (reference 120 mL) and adjusted to pH 6.0 with a pH meter.

(2) Example 2
Using the cellulose C6-position active ester carrier obtained in Example 1, trypsin (derived from porcine pancreas, manufactured by Wako Pure Chemical Industries, Ltd.) was immobilized on this carrier instead of glucoamylase to produce a complex, which will be described later. It was evaluated so as to do.
Fixation of trypsin to the cellulose C6-position active ester carrier was carried out as follows.

The cellulose C6-position active ester carrier obtained in Example 1 was placed in a different Erlenmeyer flask from the above, 50 mL of 1000 μg/mL trypsin-MES buffer was added, and the mixture was incubated at 4° C. and 100 rpm in an incubator for 24 hours. After shaking for a period of time, the active ester at the C6 position of the glucose unit of the cellulose C6-position active ester carrier and trypsin were amidated to form a complex of the cellulose C6-position active ester carrier and trypsin. The resulting reaction suspension was centrifuged at 10,000 rpm for 15 minutes in a centrifuge, and the supernatant was collected by decantation to obtain the complex as a residue. After washing the resulting complex, it was stored cold (4° C.) in pure water and used for evaluation of enzymatic activity described later.
On the other hand, the supernatant was used to determine the amount of enzyme immobilized, which will be described later.
Thus, a complex of cellulose C6-position active ester carrier and trypsin was produced.

(3) Example 3
Trypsin was immobilized on the protein-immobilizing carrier by the same procedure as in Example 1, except that the cellulose dispersion B2 was used, to produce a complex of the protein-immobilizing carrier and trypsin. Since the cellulose C6-position active ester carrier of cellulose dispersion B2 has a larger number average fiber diameter than the cellulose C6-position active ester carrier of cellulose aqueous dispersion B1 of Examples 1 and 2, the same centrifugation as in Examples 1 and 2 was performed. Depending on the separation conditions, compared to Examples 1 and 2, the cellulose-derived residue could be separated, recovered, and washed. In addition, the complex of the cellulose C6-position active ester carrier and trypsin was separated from the reaction suspension more easily than the complexes of Examples 1 and 2 under the above centrifugal conditions, as described later. It can be recovered, washed, and washed to give the desired conjugate.

(4) Comparative Example 1
Using cellulose nanofibers in which the C6 position of the glucose unit has not been anion-modified (unmodified), the hydroxyl group at the C6 position of the glucose unit of the cellulose is reacted with epichlorohydrin, followed by the reaction with sodium glutamate, Next, a succinimide group was bound to produce a protein-immobilizing carrier. In addition, a protein was immobilized on the obtained carrier for protein immobilization to produce a complex. Details are as follows.

(4-1) Undenatured cellulose nanofiber Celish KY-100S (undenatured cellulose nanofiber, manufactured by Daicel Finechem Co., Ltd.) was used as the undenatured cellulose nanofiber.
The characteristics of the Celish KY-100S used were as follows.
Number average fiber diameter: 100 nm
Average aspect ratio: 5000
Crystal structure: "Yes" type I crystal structure

(4-2) Production of carrier for protein immobilization The following raw materials were used.
Celish KY-100S
Sodium hydroxide (manufactured by Nacalai Tesque Co., Ltd.)
Epichlorohydrin (manufactured by Wako Pure Chemical Industries, Ltd.)
Boric acid (H ₃ BO ₃ ) (manufactured by Nacalai Tesque Co., Ltd.)
Potassium chloride (KCl) (manufactured by Kishida Chemical Co., Ltd.)

(Scheme 1) Activation epoxidation of unmodified cellulose nanofibers As shown in Fig. 3, activation epoxidation of unmodified cellulose fibers was performed.
In a 500 mL separable flask, 20.1 g (wet mass) of unmodified cellulose nanofibers in a wet state and 100 mL of 5% sodium hydroxide aqueous solution are added, and stirred in a water bath at 30 ° C. using a magnetic stirrer (200 rpm). Stirred for an hour. Furthermore, 100 mL of epichlorohydrin was added, and the mixture was further stirred in a water bath at 30° C. for 2 hours to generate cellulose nanofibers having an activated epoxy at the C6 position of the glucose unit.
The resulting cellulose nanofibers were filtered through silk and washed with pure water until the smell of epichlorohydrin disappeared.
Then, it was washed with 100 mM borate buffer. In addition, the borate buffer was prepared by the following method.
The resulting activated epoxidized cellulose nanofibers (cellulose nanofibers of scheme 1) were used immediately in the next scheme (modification with sodium glutamate).

[Method for preparing borate buffer]
After preparing an aqueous boric acid solution to which potassium chloride was added and an aqueous sodium hydroxide solution according to the formulation shown in Table 2 below, the sodium hydroxide solution obtained was gradually added to 629 mL of the obtained boric acid aqueous solution (reference 120 mL) and adjusted to pH 9.0 with a pH meter.

(Scheme 2) Modification of Cellulose Nanofibers of Scheme 1 with Sodium Glutamate As shown in FIG. 4, the cellulose nanofibers of Scheme 1 were modified with glutamic acid.
Using the epoxidized cellulose nanofibers obtained in Scheme 1 and sodium glutamate, a composite of epoxidized cellulose nanofibers and sodium glutamate was produced.
The following raw materials were used.
Cellulose nanofibers in scheme 1 (produced in scheme 1)
L(+)-sodium glutamate (manufactured by Wako Pure Chemical Industries, Ltd.)
Sodium hydroxide (NaOH) (manufactured by Nacalai Tesque Co., Ltd.)

9.36 g of sodium glutamate and 100 mL of 0.01 M sodium hydroxide aqueous solution were added to a 200 mL Erlenmeyer flask to dissolve sodium glutamate.
Further, the cellulose nanofibers of Scheme 1 were added and shaken at 30° C. and 200 rpm for 24 hours using an incubator to produce sodium glutamate-modified cellulose nanofibers (cellulose nanofibers of Scheme 2).
The obtained cellulose nanofibers were filtered through silk, washed with pure water, and then used in the next scheme (activation esterification).

(Scheme 3) Activated Esterification of Cellulose Nanofibers of Scheme 2 As shown in FIG. 5, the cellulose nanofibers of Scheme 2 were subjected to activated esterification.
The cellulose nanofibers of Scheme 2, EDAC, and NHS were used to produce activated esterified cellulose nanofibers in which NHS was bound to the carboxy groups (COONa) of the cellulose nanofibers of Scheme 2.

The following raw materials were used.
Cellulose nanofibers in scheme 2 (produced in scheme 2)
EDAC (1-ethyl-3 (3-dimethylaminopropyl) carbodiimide manufactured by Wako Pure Chemical Industries, Ltd.)
NHS (N-hydroxysuccinimide, manufactured by Wako Pure Chemical Industries, Ltd.)
Sodium chloride (NaCl) (manufactured by Nacalai Tesque Co., Ltd.)
Sodium hydroxide (NaOH) (manufactured by Nacalai Tesque Co., Ltd.)
MES (2-(N-morpholino) ethanesulfonic acid) (manufactured by Dojindo Laboratories)

The cellulose nanofibers of Scheme 2 were washed with 1.5 M NaCl aqueous solution, 0.1 M NaOH aqueous solution, and 50 mM MES buffer. The MES buffer was prepared by the method described above.
Cellulose nanofibers of Scheme 2, 20 mL of 12 mmol of EDAC aqueous solution, and 20 mL of 12 mmol of NHS aqueous solution are added to a 50 mL Erlenmeyer flask, and the carboxyl groups of Scheme 2 ( COONa) produced active-esterified cellulose nanofibers.
Cellulose nanofibers (cellulose nanofibers in Scheme 3) are suction-filtered from the obtained reaction suspension, washed with pure water, then washed with MES buffer, and collected to obtain a protein-immobilizing carrier. As a sodium glutamate modified cellulose-active ester carrier was obtained and used immediately in the following schemes.

(Scheme 4) Immobilization of glucoamylase to sodium glutamate-modified cellulose-active ester carrier As shown in Figure 6, glucoamylase was immobilized to a sodium glutamate-modified cellulose-active ester carrier.
Using a sodium glutamate-modified cellulose-active ester carrier and glucoamylase, the activated ester of the sodium glutamate-modified cellulose-active ester carrier and the amino group of glucoamylase are amidated to form a sodium glutamate-modified cellulose-active ester. Glucoamylase was immobilized on the carrier.

The following raw materials were used.
Sodium glutamate modified cellulose-active ester carrier (prepared in scheme 3)
Glucoamylase (manufactured by Tokyo Chemical Industry Co., Ltd.)
MES (2-(N-morpholino) ethanesulfonic acid) (manufactured by Dojindo Laboratories)
Sodium hydroxide (NaOH) (manufactured by Nacalai Tesque Co., Ltd.)
Sodium chloride (NaCl) (manufactured by Nacalai Tesque Co., Ltd.)

Add 20 mL of sodium glutamate-modified cellulose-active ester carrier and 1000 μg/mL glucoamylase-MES buffer to a 50-mL Erlenmeyer flask, shake at 4° C. and 200 rpm for 24 hours using an incubator to obtain sodium glutamate-modified cellulose- A complex of sodium glutamate-modified cellulose-active ester carrier and glucoamylase was produced by amidating the activated ester of the active ester carrier and the amino group of glucoamylase.
The complex was filtered from the resulting reaction suspension, washed with 50 mM MES buffer, washed with pure water, stored cold (4° C.) in pure water, and used for enzymatic activity evaluation described later. Using.
On the other hand, the filtrate was used for determination of enzyme immobilization amount described later.

(5) Comparative Example 2
Using cellulose nanofibers in which the C6 position of the glucose unit has not been anion-modified (unmodified), the hydroxyl group at the C6 position of the glucose unit of the cellulose is reacted with epichlorohydrin to introduce an epoxy group for protein fixation. A carrier was produced, and a protein was immobilized on the obtained protein-immobilizing carrier to produce a complex. Details are as follows.
Immediately after obtaining the activated epoxidized cellulose nanofiber (epoxy group-modified cellulose carrier) of Comparative Example 1 as a carrier for protein immobilization, it was placed in an Erlenmeyer flask separate from the above, and 1000 µg/mL glucoamylase-MES buffer was added. 20 mL of the liquid was added and shaken at 4° C. and 200 rpm for 24 hours using an incubator to produce a complex of the epoxy group-modified cellulose carrier and glucoamylase.
The complex was filtered from the resulting reaction suspension, washed with 50 mM MES buffer, washed with pure water, stored cold (4° C.) in pure water, and used for enzymatic activity evaluation described later. Using.
On the other hand, the filtrate was used for determination of enzyme immobilization amount described later.

(6) Comparative Example 3
Trypsin was immobilized on the protein-immobilizing carrier in the same manner as in Comparative Example 1, except that trypsin was used as the protein, to produce a complex of the protein-immobilizing carrier and trypsin. Details are as follows.

The glutamic acid-modified cellulose-active ester carrier of Comparative Example 1 was used as a protein-immobilizing carrier, placed in a separate Erlenmeyer flask, 50 mL of 1000 μg/mL trypsin-MES buffer was added, and incubated at 4° C. using an incubator. Shaking at 100 rpm for 24 hours causes an amidation reaction between the active ester at the C6 position of the glucose unit of the glutamic acid-modified cellulose-active ester carrier and trypsin, resulting in a complex of the glutamic acid-modified cellulose-active ester carrier and trypsin. was generated. The resulting reaction suspension was centrifuged at 10,000 rpm for 15 minutes in a centrifuge, and the supernatant was collected by decantation to obtain the complex as a residue. After washing the resulting complex, it was stored cold (4° C.) in pure water and used for the evaluation of enzymatic activity described later.
On the other hand, the supernatant was used to determine the amount of enzyme immobilized, which will be described later.

(7) Comparative Example 4
Trypsin was immobilized on the protein-immobilizing carrier by the same procedure as in Comparative Example 2, except that trypsin was used as the protein, to produce a complex of the protein-immobilizing carrier and trypsin. Details are as follows.

Using the epoxy group-modified cellulose carrier of Comparative Example 2, put it in a different Erlenmeyer flask from the above, add 50 mL of 1000 μg/mL trypsin-MES buffer, and shake at 4° C. and 200 rpm for 24 hours using an incubator. , prepared a complex of an epoxy group-modified cellulose carrier and trypsin.
The complex was filtered from the resulting reaction suspension, washed with 50 mM MES buffer, washed with pure water, stored cold (4° C.) in pure water, and used for enzymatic activity evaluation described later. Using.
On the other hand, the filtrate was used for determination of enzyme immobilization amount described later.

(8) Comparative Example 5
Anion-modified cellulose beads formed using cellulose in which the C6 position of the glucose unit is anion-modified is used as a protein-immobilizing carrier, trypsin is immobilized on the protein-immobilizing carrier, and a complex of the protein-immobilizing carrier and trypsin is formed. manufactured. Cellufine C-500 (manufactured by JNC Co., Ltd.) having CH ₂ OCH ₂ COOH at the C6 position was used as the cellulose beads (average particle size: 40 to 130 μm).
Details are as follows.

(8-1) Production of Complex of Anion-Modified Cellulose Beads and Protein The following raw materials were used.
Cellufine C-500 (manufactured by JNC Co., Ltd.)
EDAC (1-ethyl-3 (3-dimethylaminopropyl) carbodiimide manufactured by Wako Pure Chemical Industries, Ltd.)
NHS (N-hydroxysuccinimide, manufactured by Wako Pure Chemical Industries, Ltd.)
Sodium chloride (NaCl) (manufactured by Nacalai Tesque Co., Ltd.)
Sodium hydroxide (NaOH) (manufactured by Nacalai Tesque Co., Ltd.)
MES (2-(N-morpholino) ethanesulfonic acid) (manufactured by Dojindo Laboratories)

Activated esterification of Cellufine C-500 was carried out.
12.00 g of Cellufine C-500, 20 mL of 0.6 M NHS aqueous solution, and 20 mL of 0.6 M EDAC aqueous solution were added to a 50 mL Erlenmeyer flask and stirred at room temperature for 1 hour using a magnetic stirrer (200 rpm). As a result, the carboxyl group of Cellufine C-500 and NHS were esterified to produce a protein-immobilizing carrier, which is a cellulose bead having an activated ester (activated esterified cellulose bead).
The resulting reaction suspension was centrifuged at 10000 rpm for 15 minutes in a centrifuge, the supernatant was collected by decantation, and the residue was washed twice with MES buffer and then collected. A carboxymethylated cellulose-active ester carrier was obtained as a carrier for protein immobilization.

The carboxymethylated cellulose-active ester carrier obtained above was placed in a separate Erlenmeyer flask, 20 mL of 500 μg/mL glucoamylase-MES buffer was added, and the mixture was placed in an incubator at 4° C. and 100 rpm. After shaking for 24 hours, the active ester of the carboxymethylated cellulose-active ester carrier and glucoamylase were amidated to form a complex of the carboxymethylated cellulose-active ester carrier and glucoamylase.
The resulting reaction suspension was centrifuged at 10,000 rpm for 15 minutes in a centrifuge, and the supernatant was collected by decantation to obtain the complex as a residue.
After washing the resulting complex, it was stored cold (4° C.) in pure water and used for evaluation of enzymatic activity described later. On the other hand, the supernatant was used to determine the amount of enzyme immobilized, which will be described later.

(9) Comparative Example 6
Trypsin was immobilized on the protein-immobilizing carrier by the same procedure as in Comparative Example 5, except that trypsin was used as the protein, to produce a complex of the protein-immobilizing carrier and trypsin.
Details are as follows.

The carboxymethylated cellulose-active ester carrier obtained in Comparative Example 5 was placed in a different Erlenmeyer flask from the above, 20 mL of 1000 μg/mL trypsin-MES buffer was added, and the conditions were 4° C. and 100 rpm using an incubator. for 24 hours, the active ester of the carboxymethylated cellulose-active ester carrier and trypsin were amidated to form a complex of the carboxymethylated cellulose-active ester carrier and trypsin.
The resulting reaction suspension was centrifuged at 10,000 rpm for 15 minutes using a centrifuge, and the supernatant was collected by decantation to obtain the above complex as a residue.
After washing the resulting complex, it was stored cold (4° C.) in pure water and used for the evaluation of enzymatic activity described later. On the other hand, the supernatant was used to determine the amount of enzyme immobilized, which will be described later.

(10) Comparative Example 7
Undenatured cellulose beads formed using undenatured cellulose having a hydroxyl group at the C6 position of the glucose unit are used as a protein-immobilizing carrier, glucoamylase is immobilized on the protein-immobilizing carrier, and the protein-immobilizing carrier is complexed with trypsin. manufactured the body. Cellufine GC-15 (manufactured by JNC Co., Ltd.) was used as the cellulose beads (average particle size: 50 to 125 μm).

(10-1) Production of Complex of Undenatured Cellulose Beads and Protein The following raw materials were used.
Cellufine GC-15 (manufactured by JNC Co., Ltd.)
Epichlorohydrin (manufactured by Wako Pure Chemical Industries, Ltd.)
Sodium hydroxide (NaOH) (manufactured by Nacalai Tesque Co., Ltd.)

An epoxy group was introduced into Cellufine GC-15.
Put 16.00 g of wet Cellufine GC-15 and 100 mL of 5% sodium hydroxide borate buffer in a 50 mL Erlenmeyer flask, stir at 30° C. for 1 hour (200 rpm), and add 160 mL of epichlorohydrin. and further stirred at 30° C. for 2 hours (200 rpm). The resulting epoxidized cellulose particles were filtered on silk, washed with ultrapure water until the smell of epichlorohydrin disappeared, and then washed with methanol to obtain an epoxy group-modified carrier as a protein immobilization carrier. Cellulose beads were obtained. 20 mL of a borate buffer solution containing epoxy group-modified cellulose beads and 500 μg/mL glucoamylase was placed in a 50 mL Erlenmeyer flask, and stirred at 4° C. and 200 rpm for 1 hour in an incubator to allow the epoxy group-modified cellulose beads and protein to react. A complex was obtained. The resulting composite was filtered, washed three times with 100 mM borate buffer, and then washed three times with ultrapure water, and the composite particles were stored cold in ultrapure water.

(11) Comparative Example 8
The enzymatic activity of glucoamylase was evaluated using only glucoamylase without using a protein-immobilizing carrier, as described later.

(12) Comparative Example 9
The enzymatic activity of trypsin was evaluated using only trypsin without using a protein-immobilizing carrier, as described later.

(13) Evaluation Enzyme immobilization (enzyme modification) amount for Examples 1 to 3 and Comparative Examples 1 to 7, enzyme activity for Examples 1 to 3 and Comparative Examples 1 to 9, Example 1 and Comparative Examples 1 and 2 , 5 and 8, and the activity rate against temperature for Example 1 and Comparative Examples 1, 5 and 8 were measured as follows.

[Enzyme immobilization (enzyme modification) amount]
The amount of glucoamylase and trypsin immobilized (modified) in the complex was measured using the Bradford method.
The Bradford method is a quantification method that utilizes the shift of the absorbance of a CBB solution from 465 nm to 595 nm due to the adsorption of Coomassie brilliant blue (CBB, the structural formula of which is shown below) to proteins.
Using this Bradford method, when the reaction for immobilizing glucoamylase and trypsin on the protein-immobilizing carrier was carried out in Examples 1 to 3 and Comparative Examples 1 to 7, the concentrations of glucoamylase and trypsin remaining in the reaction solution were examined. The amount of glucoamylase and trypsin immobilized on the protein immobilization carrier (immobilized amount) was determined by the method.

Measurement of residual glucoamylase and trypsin concentrations in the reaction solution (test solution) using the Bradford method was performed according to the microplate assay method of protein assay CBB solution (5-fold concentration). The following reagents were used.
Test solution: reaction solution (filtrate obtained by filtering the reaction suspension when glucoamylase and trypsin were immobilized on the protein-immobilizing carrier in Examples 1 to 3 and Comparative Examples 1 to 7)
Protein assay CBB solution (5 times concentrated) (manufactured by Nacalai Tesque Co., Ltd.)

160 mL of test solution and 40 mL of protein assay CBB solution were added to the microplate.
The resulting mixture was stirred at 30°C for 10 minutes using a microplate reader (manufactured by BioTek SYNERGY), and the absorbance (595 nm) of the stirred mixture was measured.
The absorbance of a blank (pure water) was measured, and the difference between this measurement result and the absorbance of the test solution was taken as the absorbance of the test solution.
In addition, glucoamylase and trypsin are dissolved in MES buffer or borate buffer, and the concentrations of glucoamylase and trypsin are adjusted to 3.9, 7.8, 31.25, 62.5, 125, 250, and 500 mg/mL. Adjusted standard solutions were prepared, the absorbance (595 nm) of each standard solution was measured in the same manner as above, and a calibration curve was created with the horizontal axis representing the glucoamylase and trypsin concentrations and the vertical axis representing the absorbance.
Using this calibration curve, the glucoamylase and trypsin concentrations of the test solution were calculated.
Tables 5 and 6 show the results.

[Enzyme activity]
(1) Evaluation of glucoamylase activity by the Somogyi-Nelson method The Somogyi-Nelson method is a method for quantifying reducing sugars using a copper reagent, and Nelson's coloring reagent is used to finally measure the absorbance. The method.
Using this Somogyi-Nelson method, the complexes of Example 1 and Comparative Examples 1, 2, and 5 were used as samples to decompose soluble starch (substrate) to produce glucose, and the produced glucose was reduced by copper. After performing, the absorbance (660 nm) was measured. Using the glucoamylase (without complex) of Comparative Example 8 as a sample, soluble starch was similarly decomposed and the absorbance (660 nm) was measured (Comparative Example 8).

The following reagents were used.
Soluble starch (manufactured by Wako Pure Chemical Industries, Ltd.)
Somogi liquid (manufactured by Wako Pure Chemical Industries, Ltd.)
Nelson solution (manufactured by Wako Pure Chemical Industries, Ltd.)
Disodium hydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.)
Sodium dihydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.)
Glucose (manufactured by Wako Pure Chemical Industries, Ltd.)

1 mL of the starch solution and 1 mL of pH 6.87 phosphate buffer were added to the test tube and soaked in a constant temperature bath at 40° C. for 10 minutes.
1 mL of a mixture of a sample and water adjusted to 50 mg/mL was added and allowed to react for exactly 10 minutes.
After 10 minutes, it was immediately heat-quenched in boiling water.
1 mL of the resulting decomposition reaction solution and 1 mL of Somogi solution were added to another test tube, and a reduction reaction with copper was performed in boiling water for 20 minutes under the following conditions.
(Reaction conditions)
Enzyme concentration: 50 mg/mL, reaction time: 10 min
Reaction temperature: 40°C, pH 6.87

After the reaction, the mixture was quenched with ice water, 1 mL of Nelson's solution was added, and the mixture was allowed to stand for 30 minutes.
22 mL of pure water was added to the obtained reduction reaction solution to fill up to 25 mL, and the mixture was allowed to stand still for 15 minutes, and the absorbance (660 nm) was measured. The absorbance of pure water was measured as a blank.
For glucose aqueous solutions of 400, 300, 200, 100, 50, and 25 mg/mL instead of the decomposition reaction solution, the operations from reduction reaction to absorbance measurement were performed in the same manner as above, and the horizontal axis was the glucose concentration and the vertical axis was the absorbance. A glucose calibration curve was created.
From this calibration curve, the glucose concentration of the diluted reduction reaction solution was calculated.
Using the following formula (Formula 3) from the calculated glucose concentration, the enzymatic activity of glucoamylase (1 Unit (U), that is, the titer for liberating 1 mg of glucose per minute) was calculated.

(2) Evaluation of enzymatic activity of glucoamylase by Michaelis Menten kinetics
Each complex obtained in Example 1 and Comparative Examples 1, 2 and 5 was added to water so that the concentration of the complex was 50 mg/mL. For Comparative Example 3, glucoamylase was added to water such that the concentration of glucoamylase was 50 mg/mL.
On the other hand, the enzymatic activity against the substrate (soluble starch) at each concentration (0.0625, 0.125, 0.25, 0.5, 1, 2, 3, 4%) was measured by the above-mentioned "Glycogenase by the Somogyi-Nelson method. Activity evaluation of amylase", and the glucoamylase immobilized on the complex (Example 1 and Comparative Examples 1, 2, 5) and the non-immobilized glucoamylase (Comparative Example 8) were measured according to the following formula ( The Michaelis Menten kinetics shown in formula 4) was examined using the Hanes Woolf plot shown in the following formula (formula 5). The results are shown in FIG. 7, Tables 5 and 6, and the calculated Michaelis Menten kinetics coefficients are shown in Table 3. In FIG. 7, Example 1 is shown as CNF(RH)-GA, Comparative Example 1 as CNF(Celish)-GA, and Comparative Example 8 as Free GA.

(3) Evaluation of enzymatic activity of trypsin by quantifying the amount of free tyrosine Using casein as an object (substrate) to be treated with protein, a complex of protein-immobilizing carrier and trypsin and enzymatic activity evaluation of trypsin were evaluated. C. for 10 minutes, and after stopping the reaction using trichloroacetic acid as a reaction terminator, the amount of liberated tyrosine was quantified.
Details are as follows.

A 40 mg/mL casein solution was prepared using the following reagents.
Casein (derived from milk, manufactured by Wako Pure Chemical Industries, Ltd.)
Disodium hydrogen phosphate/12 water (manufactured by Wako Pure Chemical Industries, Ltd.)
Citric acid monohydrate (manufactured by Nacalai Tesque Co., Ltd.)

2 g of casein and 30 mL of 0.2 M disodium hydrogen phosphate aqueous solution were added to a 100 mL Erlenmeyer flask, and the casein was dissolved in a water bath at 65 to 70°C. The resulting solution was made up to 50 mL with a pH 7.0 Mcllvaine buffer (pH was adjusted using a 0.2 mol/L sodium dihydrogen phosphate aqueous solution and a 0.1 mol/L citric acid aqueous solution). , to obtain a 40 mg/mL casein solution.

Using the resulting 40 mg/mL casein solution and the following reagents, the complex of the protein-immobilizing carrier and trypsin and the enzymatic activity of trypsin were evaluated.
Trichloroacetic acid (manufactured by Wako Pure Chemical Industries, Ltd.)
Sodium carbonate (manufactured by Katayama Chemical Industry Co., Ltd.)
Folin reagent (manufactured by Nacalai Tesque Co., Ltd.)

1 mL of ultrapure water and 1 mL of casein solution were placed in a 20 mL Erlenmeyer flask and stirred at 200 rpm for 10 minutes in an incubator set at 37°C. After adding 1 mL of a complex of a protein-immobilizing carrier and trypsin and reacting at 200 rpm for 10 minutes, 3 mL of trichloroacetic acid was added to stop the reaction (generation of white precipitate), and the mixture was stirred at 200 rpm for 30 minutes. After collecting the solution containing the precipitate generated by adding trichloroacetic acid with a cylinder, the precipitate was removed with an 80 μm filter (ADVANTEC DISMIC-25cs, Cellulose Acetate 0.80 μm), and the reaction solution was collected in a test tube. . 30 μL of the collected reaction solution, 150 μL of sodium carbonate aqueous solution, and 30 μL of 1.8-fold diluted Folin reagent were put into a microplate. After allowing this microplate to stand still at 37° C. for 30 minutes, the absorbance (actual value) of the reaction solution at 750 nm was measured. On the other hand, using ultrapure water as a blank instead of the reaction solution, the same operation as above was performed, and the absorbance of the blank at 750 nm was measured. The difference between the blank absorbance and the absorbance of the reaction solution (measured value) was defined as the absorbance of the reaction solution (measurement result). From the absorbance of this reaction solution, the tyrosine concentration was calculated using a tyrosine calibration curve prepared by the method described below. Then, the complex of the protein-immobilizing carrier and trypsin, and the enzymatic activity of trypsin (1 Unit (U) = titer for liberating 1 μg of tyrosine per minute) were calculated by the following formula (Formula 6).

A standard curve for tyrosine was constructed as follows.
In the complex of the protein-immobilizing carrier and trypsin, and the enzymatic activity manipulation of trypsin, tyrosine solutions of 100, 50, 25, 12.5, and 6.25 μg/mL are used instead of the reaction solution as described above. A standard curve for tyrosine was prepared by plotting tyrosine concentration on the horizontal axis and absorbance on the vertical axis (obtained by subtracting the absorbance of the blank).

(4) Evaluation of enzymatic activity of trypsin by Michaelis-Menten kinetics Enzymatic activity was evaluated by the following procedure.
For the complexes of Examples 2, 3, Comparative Examples 3, 4, 6, 7, and trypsin not immobilized on a protein immobilizing carrier (Comparative Example 9), each concentration (1.875 mg / mL to 40 mg / mL ) against casein (the amount of free tyrosine) was measured according to the above-mentioned “Evaluation of enzymatic activity of trypsin by quantification of the amount of free tyrosine”, and the above formula (Formula 4) Michaelis-Menten kinetics, the above formula (Formula 5) A Hanes-Woolf plot was used to investigate. Tables 5 and 6 show the results.

[Activity rate for pH]
The complexes of Example 1 and Comparative Examples 1, 2 and 5 were examined for enzyme activity (activity rate) with respect to pH.
On the other hand, the enzymatic activity with respect to pH was measured in the same manner for non-immobilized glucoamylase (Comparative Example 8).

Reagent used 0.1 M acetic acid (manufactured by Nacalai Tesque Co., Ltd.)
Sodium acetate (manufactured by Nacalai Tesque Co., Ltd.)
Sodium chloride (manufactured by Nacalai Tesque Co., Ltd.)
Disodium hydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.)
Sodium dihydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.)
Tris (manufactured by SERVA)
0.1 M hydrochloric acid (manufactured by Wako Pure Chemical Industries, Ltd.)
Soluble starch (manufactured by Wako Pure Chemical Industries, Ltd.)
Somogi liquid (manufactured by Wako Pure Chemical Industries, Ltd.)
Nelson solution (manufactured by Wako Pure Chemical Industries, Ltd.)
Phosphoric acid (manufactured by Wako Pure Chemical Industries, Ltd.)
Glucoamylase (manufactured by Tokyo Chemical Industry Co., Ltd.)

Buffers of pH 2-9 were prepared in 100 mL portions by adding pure water to the formulations shown in Table 4 below.
For the complexes of Example 1 and Comparative Examples 1, 2, and 5, only the pH was changed in the range of pH 2.0 to 9.0 without changing other conditions, and the above-mentioned " Enzyme activity was measured in accordance with Somogyi-Nelson Method for Evaluation of Glucoamylase Activity.
On the other hand, the enzymatic activity of non-immobilized glucoamylase (Comparative Example 8) was similarly measured at each pH.
Enzyme concentration: 50 mg/mL, substrate concentration: 3%
Reaction time: 10 min, reaction temperature: 40°C
8, Tables 5 and 6 show graphs in which the enzyme activity was obtained from the measurement result of the absorbance (660 nm), the horizontal axis was pH, and the vertical axis was the activity rate of the enzyme. The results of Example 1 and Comparative Examples 1 and 8 are shown in FIG. In FIG. 8, the highest enzyme activity value is 100%, and the percentage of this value is shown as the activity rate of the enzyme. In FIG. 8, Example 1 is shown as CNF(RH)-GA, Comparative Example 1 as CNF(Celish)-GA, and Comparative Example 8 as Free GA.

[Activity rate against temperature]
For the complexes of Example 1 and Comparative Examples 1 and 5, the enzyme activity rate was measured at each temperature.
For the non-immobilized glucoamylase (Comparative Example 8), the activity rate of the enzyme at each temperature was similarly measured.

Reagents used pH 4 acetate buffer (prepared with the above [activity ratio for pH])
pH 5 acetate buffer (prepared with the above-mentioned [activity ratio for pH])
Soluble starch (manufactured by Wako Pure Chemical Industries, Ltd.)
Somogi liquid (manufactured by Wako Pure Chemical Industries, Ltd.)
Nelson solution (manufactured by Wako Pure Chemical Industries, Ltd.)
Glucoamylase (manufactured by Tokyo Chemical Industry Co., Ltd.)

For the composites of Example 1 and Comparative Examples 1 and 5, the following reaction was performed by changing only the reaction temperature to 20, 25, 30, 40, 50, 60, 70, and 80°C without changing other conditions. Enzyme activity was measured under each of the conditions according to the "Evaluation of glucoamylase activity by the Somogyi-Nelson method" described above.
On the other hand, the enzymatic activity at each temperature was similarly measured for non-immobilized glucoamylase (Comparative Example 8).
Enzyme concentration: 50 mg/mL, substrate concentration: 3%
Reaction time: 10min
pH: pH 5.0 for Example 1 and Comparative Examples 1 and 5, pH 4.0 for Comparative Example 8
9, Tables 5, and 6 show graphs in which the enzyme activity was determined from the absorbance (660 nm) measurement results, with the horizontal axis representing pH and the vertical axis representing the activity rate of the enzyme. The results of Example 1 and Comparative Example 1 are shown in FIG. In FIG. 9, the highest enzyme activity value is 100%, and the percentage of this value is shown as the activity rate of the enzyme. In FIG. 9, Example 1 is indicated as CNF(RH)-GA, and Comparative Example 8 is indicated as Free GA.

[Solid-liquid separation evaluation by centrifugation]
From the reaction suspension obtained when producing the cellulose C6-position active ester carrier in Examples 1 to 3, the cellulose C6-position active ester carrier was separated from the water-insoluble solid component by centrifugation at 10,000 rpm for 15 minutes. Solid-liquid separation was performed, and the degree of separation was evaluated as follows. As the reaction suspension of Example 2, the reaction suspension of Example 1 is used.
Similarly, from the reaction suspension obtained when producing a complex of cellulose C6-position active ester carrier and glucoamylase or trypsin in Examples 1 to 3, the complex was centrifuged at 10,000 rpm for 15 minutes. was subjected to solid-liquid separation as a water-insoluble solid component, and the degree of separation was evaluated as follows.
The degree of separation was evaluated according to the following criteria by visually confirming the separation of water-insoluble solid components after centrifugation. Table 7 shows the results.
・Judgment Criteria If water-insoluble components are deposited in the lower layer, a clear boundary is observed between the sediment in the lower layer and the supernatant liquid in the upper layer, and the supernatant liquid can be decanted, it is extremely good. as "◎".
A water-insoluble component is deposited in the lower layer, and although there is turbidity between the sediment in the lower layer and the supernatant liquid in the upper layer, and no clear boundary is observed, the supernatant liquid can be decanted. is represented as "○".

As described above, it was found that, in the Examples, the activity of the protein can be exhibited more sufficiently over a wide range of pH and temperature than when the protein is not immobilized. Moreover, it was found that the cellulose nanofibers of Examples can immobilize more proteins than the cellulose beads of Comparative Examples. In addition, it was found that the cellulose C6-position active ester carrier and the complex of Examples could be sufficiently well separated into solid and liquid.

Claims (10)

comprising cellulose nanofibers formed by cellulose,
The cellulose nanofiber satisfies the following (a) to (c) and is for immobilizing amino groups of proteins,
A carrier for protein immobilization that is dispersed in a dispersion medium:
(a) the cellulose has an ester structure represented by the following general formula (1) at the C6 position of the glucose unit;

(b) the cellulose has a cellulose type I crystal structure;
(c) The cellulose nanofibers have a number average fiber diameter of 3 to 1,000 nm and an average aspect ratio of 10 to 1,000. 2. The protein-immobilizing carrier according to claim 1, wherein the number of moles of the ester structure is 25% or more of the total number of moles of carbon atoms at the C6 position of the glucose unit of the cellulose. 3. The protein-immobilizing carrier according to claim 1, which is capable of solid-liquid separation. The protein-immobilizing carrier according to any one of claims 1 to 3, wherein said protein is an enzyme. 5. The protein-immobilizing carrier according to claim 4, wherein the enzyme is a proteolytic enzyme or a carbohydrate-degrading enzyme. A complex in which a protein is immobilized on the protein-immobilizing carrier according to any one of claims 1 to 5,
A complex, which is obtained by covalently bonding an amino group of a protein to an alkyl carboxy group, which is a terminal obtained by removing a succinimide group from the ester structure of the protein-immobilizing carrier, and which can be solid-liquid separated. 7. The composite according to claim 6, wherein the amount of said protein immobilized on said protein-immobilizing carrier is 10 mg/g or more per mass of said cellulose. 8. The conjugate according to claim 6 or 7, wherein said covalent bond is an amide bond. A method for producing a protein-immobilizing carrier according to any one of claims 1 to 5,
a step of oxidizing cellulose with a co-oxidizing agent in the presence of an N-oxyl compound to obtain cellulose nanofibers having alkylcarboxy groups;
a step of reacting the cellulose nanofibers having the alkylcarboxy group, a condensing agent, and a succinimide compound;
By performing solid-liquid separation of the reaction suspension obtained in the reacting step,
A method for producing a protein-immobilizing carrier that satisfies the following (a) to (c) and produces a protein-immobilizing carrier comprising cellulose nanofibers for immobilizing amino groups of proteins:
(a) the cellulose has an ester structure represented by the following general formula (1) at the C6 position of the glucose unit;

(b) the cellulose has a cellulose type I crystal structure;
(c) The cellulose nanofibers have a number average fiber diameter of 3 to 1,000 nm and an average aspect ratio of 10 to 1,000. A method for producing a composite according to any one of claims 6 to 8,
In the reaction suspension obtained in the step of reacting in the method for producing a protein-immobilizing carrier according to claim 9, the succinimide group is removed from the ester structure of the protein-immobilizing carrier contained in the reaction suspension. and covalently bonding an amino group of a protein to the terminal alkyl carboxy group;
and a step of solid-liquid separation of the reaction suspension.

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