WO2023084967A1 - IMMOBILIZED NSPCS AND γ-EC PRODUCTION METHOD USING SAME - Google Patents

Abstract

The present invention provides a production method of γ-glutamylcysteine (γ-EC) that is applicable to industrial production, and immobilized NsPCS enzyme (immobilized NsPCS) to be used in the production method. This production method, which is for producing γ-EC and/or Gly, comprises a step for contacting NsPCS, which is immobilized by amide bonding with carbonyl groups introduced into hydroxyl groups of a porous cellulose carrier, with GSH under the condition of pH 4-10 to thereby synthesize γ-EC and Gly.

Description

Immobilized NsPCS and method for producing γ-EC using the same

The present invention relates to a method for producing γ-glutamylcysteine (also referred to as "γ-EC" in the present invention) applicable to industrial production. More specifically, it relates to a method for producing γ-EC using a PCS-like enzyme (also referred to as “NsPCS” in the present invention) that is similar to phytochelatine synthase (also referred to as “PCS” in the present invention). The present invention also relates to an NsPCS-immobilized enzyme (also referred to as "immobilized NsPCS" in the present invention) used in the production method.

γ-EC (γ-Glu-Cys) exists in a high concentration in the body, and glutathione (in the present invention, It is a dipeptide that functions as an intermediate for the biosynthesis of GSH (also called "GSH") (Fig. 1).
It has been reported that γ-EC, when administered intravenously or orally to laboratory animals, enhances the action of GSH by increasing GSH levels in vivo (Non-Patent Documents 1 and 2). In addition, since it has a similar structure to GSH, it is expected to have the same beneficial action as GSH (Non-Patent Document 3). Furthermore, recently, it has been reported that it has a mitigating effect on dementia and Alzheimer's disease in experimental animals (Non-Patent Documents 4 and 5). Since GSH cannot pass through the brain-blood vessel barrier, γ-EC is drawing attention because the above action indicates that the administered γ-EC itself passes through the brain-blood vessel barrier and exerts its function in the brain.

However, the rate-limiting step in GSH synthesis in vivo is the γ-EC synthesis reaction catalyzed by glutamic acid-cysteine ligase (hereinafter also referred to as “GCL”) (Fig. 1), and the synthesized γ-EC is immediately converted into glutathione. It is converted to GSH by a synthetase (hereinafter also referred to as "GS"). Therefore, the intracellular concentration of GSH is as high as 0.5-10 mM (Non-Patent Document 6), whereas the intracellular concentration of γ-EC is very low as 0.5-10 μM (Non-Patent Document 7). Therefore, unlike GSH, γ-EC is difficult to ferment and produce using living organisms such as yeast, and although chemically synthesized products are available on the market, it is extremely expensive, and its reagent level is 1000 times more expensive than GSH. reach.

In addition to chemical synthesis, several biochemical methods are known for γ-EC production.
For example, a method of producing γ-EC using a yeast mutant strain that has been genetically engineered to weaken GS activity, suppress the conversion of γ-EC to GSH, and accumulate γ-EC (Patent Document 1 , Non-Patent Document 8), an enzymatic method for synthesizing γ-EC by transferring a γ-glutamyl group of a γ-glutamyl donor to a cysteine group using γ-glutamyltransferase (hereinafter also referred to as “GGT”) ( Patent Document 2) and an enzymatic method of synthesizing γ-EC from glutamic acid and cysteine using GCL (Patent Document 3) are known. Some studies on the physiological effects of γ-EC have used γ-EC prepared using GGT (Non-Patent Documents 9 and 10). There is no example where the produced γ-EC is marketed as a product.

Phytochelatine synthase (PCS) is an enzyme that is activated by heavy metals such as cadmium to produce phytokeratin (PC), a heavy metal-conjugated peptide, using GSH as a substrate, mainly in eukaryotes such as higher plants. is. The synthesis reaction of PC by this enzyme consists of (a) a reaction in which GSH is cleaved into γ-EC and glycine (Gly), and (b) a reaction in which the generated γ-EC is bound to the N-terminus of GSH. consists of reactions that synthesize (γ-EC) ₂ -Gly (PC ₂ ), a short PC of In addition, in the reaction of (b), not only GSH but also _PC2 synthesized in (b) was used as a substrate, and γ-EC was bound to the N-terminus to form (γ-EC) ₃ -Gly (PC ₃ ). is synthesized. This reaction is then repeated to synthesize (γ-EC) _n+1 -Gly from (γ-EC) _n -Gly, where n is usually about 2-4.

A PCS-like enzyme (NsPCS) is known as an enzyme similar to the PCS. NsPCS is a PCS-like enzyme encoded by the alr0975 gene from the prokaryotic cyanobacterium Nostoc sp. PCC7120, which has high homology to the PCS gene. NsPCS, unlike PCS, does not require heavy metal activation, and among the reactions catalyzed by PCS, only a) catalyzes the synthesis of γ-EC and Gly using GSH as a substrate. (see Patent Document 4 above). Therefore, by using NsPCS, GSH can be used as a raw material (substrate) to produce γ-EC that has excellent physiological activity and is useful as a raw material for pharmaceuticals, functional foods, or highly functional cosmetics. In Patent Document 4, as a method for producing γ-EC using NsPCS, the alr0975 gene encoding NsPCS is highly expressed in host cells such as E. coli, and NsPCS produced in the host cells uses GSH as a substrate to produce γ-EC. A method for producing -EC and a method for producing γ-EC from GSH by an in vitro enzymatic reaction using NsPCS have been proposed.

However, these methods cannot produce γ-EC continuously, and the γ-EC produced must be separated and purified from other components (the enzyme NsPCS used, the substrate GSH, and the by-product Gly). It is not suitable for industrial production of γ-EC because a separate process is required.

WO2008/126784A1 WO2006/102722A1 WO2016/017631A1 JP-A-2005-137235 WO2016/159334A1 WO2020/022524A1

An object of the present invention is to provide a method for producing γ-EC that enables industrial production. More specifically, the object is to provide a method for efficiently and/or continuously producing γ-EC using an immobilized enzyme in which NsPCS is immobilized on a carrier (immobilized NsPCS).
Another object of the present invention is to provide immobilized NsPCS used in the production method, a method for producing the same, and a method for storing the same.

In order to solve the above problems, the present inventors have made extensive studies.
Specifically, in preparing immobilized NsPCS, binding via glutaraldehyde to the surface of glass beads generally used for enzyme immobilization (crosslinking method), embedding in calcium alginate (inclusive method), and An immobilization method using adsorption onto a porous cellulose carrier (physical adsorption method) was performed under various immobilization and reaction conditions. As a result, the immobilization on glass beads deactivated the enzyme during the immobilization process, and the embedding in calcium alginate gel and adsorption on porous cellulose caused the enzyme to be released during the reaction process. I did not go.

On the other hand, porous cellulose is used as a carrier for immobilizing enzymes, and an active esterifying agent having an NHS ester group is used as a carbonyl group-introducing reagent to introduce carbonyl groups into the hydroxyl groups. Using the method of forming an amide bond with an amino group, neither deactivation of the enzyme (NsPCS) nor detachment of the enzyme during the reaction process was observed, indicating that NsPCS can be stably immobilized on the porous cellulose carrier. Found it. In addition, by using NsPCS immobilized by this method (immobilized NsPCS), as a result of the stable immobilization of NsPCS, the substrate GSH can be converted to γ-EC at a high yield and efficiently. It was confirmed that γ-EC can be obtained, and that it can be used repeatedly and continuously without significantly lowering the conversion efficiency to γ-EC. Furthermore, the immobilized NsPCS prepared by the above method can be stored stably by freezing with liquid nitrogen in a state of being immersed in a buffer solution containing an antifreeze solution, and stable distribution in the market is possible. It was confirmed.

The present invention has been completed through further studies based on these findings, and has the following embodiments.
(I) Method for producing γ-EC and/or Gly (I-1) NsPCS immobilized by amide bonding with the carbonyl group introduced into the hydroxyl group of the porous cellulose carrier is added at pH 4 to 10, preferably pH 7. A method for producing γ-EC and/or Gly, comprising a step (Step 1) of contacting GSH with GSH under conditions of 1 to 9.
(I-2) γ-EC or/and according to (I-1), further comprising a step (step 2) of isolating and recovering γ-EC or/and Gly from the product obtained in step 1 above. Gly manufacturing method.
(I-3) Described in (I-2), wherein the step 2 is a step of contacting the product obtained in the step 1 with an ion exchanger to separate γ-EC and Gly. A method for producing γ-EC and/or Gly.
(I-4) The ion exchanger is a cation exchanger, and the solution containing the product obtained in step 1 is acidified and then brought into contact with the cation exchanger to exchange γ-EC and Gly. The method for producing γ-EC and/or Gly according to (I-3), which is a method for separation.
(I-5) A method in which the ion exchanger is a strong anion exchanger, and the solution containing the product obtained in step 1 is brought into contact with the strong anion exchanger to separate γ-EC and Gly. The method for producing γ-EC and/or Gly according to (I-3), wherein
(I-6) The porous cellulose carrier according to any one of (I-1) to (I-5), wherein the porous cellulose carrier is cellulose filter paper, cellulose sponge, cellulose monolith, or aggregate of powdery porous cellulose. A method for producing γ-EC and/or Gly.

(II) Immobilized NsPCS
(II-1) Immobilized NsPCS in which NsPCS is immobilized on porous cellulose by amide bonding between carbonyl groups introduced into hydroxyl groups of porous cellulose and primary amino groups of NsPCS.
(II-2) The immobilized NsPCS of (II-1), wherein the porous cellulose is a cellulose filter paper, a cellulose sponge, a cellulose monolith, or an aggregate of powdery porous cellulose.

(III) Method for producing immobilized NsPCS (III-1) Method for producing immobilized NsPCS according to (II-1) or (II-2), comprising the following steps:
(1) a step of introducing a carbonyl group to the hydroxyl group of the porous cellulose;
(2) A step of immobilizing NsPCS on the surface of the porous cellulose by amide bonding the primary amino group of NsPCS to the carbonyl group introduced in step (1) above.
(III-2) The step (1) is a step of producing an activated ester of porous cellulose using an active esterifying agent having an NHS ester group as a carbonyl group-introducing reagent (III-1). The immobilized NsPCS production method described in .
(III-3) The method for producing immobilized NsPCS according to (III-2), wherein the active esterifying agent having an NHS ester group is N,N'-disuccinimidyl carbonate.
(III-4) The immobilization according to any one of (III-1) to (III-3), wherein the porous cellulose is cellulose filter paper, cellulose sponge, cellulose monolith, or aggregate of powdery porous cellulose. Chemical NsPCS manufacturing method.

(IV) Method for storing immobilized NsPCS A method for storing immobilized NsPCS according to (IV-1), (II-1) or (II-2), wherein the immobilized NsPCS is immersed in a buffer containing antifreeze. The above method, characterized in that the product is stored at an ultra-low temperature of -150°C or lower in the state of being frozen.
(IV-2) The method for storing immobilized NsPCS according to (IV-1), wherein the cryopreservation is cryopreservation in liquid nitrogen.

The present invention relates to an immobilized enzyme (immobilized NsPCS) obtained by immobilizing a cyanobacteria-derived enzyme NsPCS that synthesizes γ-EC using GSH as a substrate (raw material) on a porous cellulose carrier (immobilized NsPCS), and γ-EC using the same. manufacturing method. By selecting a porous cellulose carrier from among many solid phase carriers, NsPCS can be stably immobilized without the problem of deactivation or detachment of NsPCS. As a result, according to the method for producing γ-EC of the present invention using the immobilized NsPCS, γ-EC can be produced efficiently. In addition, since NsPCS is stably immobilized, immobilized NsPCS can be used repeatedly, and γ-EC can be produced economically and efficiently. Therefore, according to the present invention, it is possible to provide a production method effective for industrial production and stable supply of γ-EC having excellent physiological activity. In addition, immobilized NsPCS can be stably preserved by freezing in a state of being immersed in a buffer containing antifreeze. Therefore, it can be commercially distributed, and the labor and burden of preparation at the time of use are small.

The biosynthetic reaction scheme of γ-glutamylcysteine (γ-EC) and glutathione (GSH) in a living body is shown. In the figure, "GCL" means glutamate-cysteine ligase, "GS" means glutathione synthetase, and "NsPCS" means a PCS-like enzyme encoded by the alr0975 gene. As an example of using DSC (disuccinimidyl carbonate), which is an NHS ester, as a carbonyl group-introducing reagent, (i) porous cellulose (denoted as "(M)-OH" in the figure) (ii) the carbonyl group introduced into the hydroxyl group of the porous cellulose is amide-bonded to the primary amino group of NsPCS to form a porous An outline of the reaction for immobilizing NsPCS on the hydroxyl groups of cellulose is shown. 4 is a scanning electron microscope (SEM) image of the inside of the cellulose monolith produced in Production Example 2. FIG. As a result of the method for producing γ-EC in Experimental Example 1, the reaction time (h) and the amount of substrate (GSH) (-▲-) and reaction product (γ-EC) (-●-) in the reaction solution ( mM). The results of Experimental Example 2 (repeated use of immobilized NsPCS) are shown. Shown is the relative ratio (specific production rate (%)) of the 2nd to 5th production rates when the 1st γ-EC production rate is assumed to be 100%. A schematic diagram of the reactor used in Experimental Example 3 (continuous production of γ-EC using immobilized NsPCS) is shown. The schematic of the reactor used in Experimental example 4 is shown. 3 shows the results of measuring % conversion from GSH to γ-EC by changing the flow rate (rpm) of the substrate solution supplied to the immobilized NsPCS in Experimental Example 3. FIG. In Experimental Example 3 (small scale), substrate solution was continuously supplied to immobilized NsPCS at a rate of 0.25 mL/h for 10 days, and the % conversion rate from GSH to γ-EC was measured over time. . In Experimental Example 4 (scale-up), the substrate solution was continuously supplied to the immobilized NsPCS at a rate of 0.1 mL/h for 10 days, and the % conversion rate from GSH to γ-EC was measured over time. . (1) Time course of γ-EC conversion rate (%) in four reactions (37_1 to 37_4) at 37°C. (2) The change over time in the γ-EC conversion rate (%) in four reactions (25_1 to 25_4) at a temperature of 25°C is shown (Experimental Example 5). A schematic diagram of the reactor used in Experimental Example 6 (production of γ-EC) is shown.

(I) Method for Producing γ-EC and/or Gly The main object of the present invention is to provide a method for producing γ-EC, but the production method of the present invention also produces Gly at the same time. . Therefore, the method for producing γ-EC of the present invention can be said to be a method for producing γ-EC and a method for producing Gly at the same time. Therefore, the production method of the present invention can be said to be a production method of γ-EC and/or Gly.
Hereinafter, when γ-EC and Gly are referred to without distinction, they may be referred to as "this target product" or "this product". Also, the method for producing γ-EC and/or Gly is referred to as "this production method".

In this production method, NsPCS having an amide bond with a carbonyl group introduced into a hydroxyl group of a porous cellulose carrier is brought into contact with GSH, and the enzymatic action of NsPCS cleaves GSH to produce γ-EC and Gly (step 1).

(1) NsPCS
As described above, NsPCS is a PCS-like enzyme encoded by the alr 0975 gene, which is highly homologous to the PCS gene derived from the prokaryotic cyanobacterium Nostoc sp. PCC7120 (see Patent Document 4). The alr 0975 gene (SEQ ID NO: 1) corresponds to the nucleotide sequence alr 0975 in the genome of cyanobacteria Nostoc sp.PCC7120. The cyanobacterium Nostoc sp. PCC 7120, from which this gene is derived, is commercially available from the Pasteur Institute in France under the catalog number PCC 7120. Cloning of the alr 0975 gene can be carried out with reference to the description in Example 4 of Patent Document 4. Also, NsPCS encoded by the alr0975 gene can be produced and obtained with reference to the description of Example 4 of Patent Document 4. These details will be described in an example (manufacturing example 1) described later.

In addition, the NsPCS targeted by the present invention includes not only the alr 0975 gene consisting of the nucleotide sequence shown in SEQ ID NO: 1, but also enzymes encoded by genes having substantially the same function as this. The gene having substantially the same function as the alr 0975 gene is a gene consisting of DNA that hybridizes under stringent conditions with the DNA consisting of the base sequence of SEQ ID NO: 1, and the protein encoded by the gene is Genes with enzymatic activity that catalyze reactions that produce γ-EC and Gly from GSH are included. The above gene also includes a gene encoding an amino acid sequence (SEQ ID NO: 2) encoded by the alr 0975 gene (SEQ ID NO: 1) in which one or several amino acids are deleted, substituted, or added, Also included is a gene having an enzymatic activity that catalyzes a reaction in which a protein consisting of the amino acid sequence produces γ-EC and Gly from GSH.

Genes included in these so-called equivalent ranges can be obtained by sequence searches using databases such as NCBI-BLAST. It can also be obtained by colony or plaque hybridization using the nucleotide sequence shown by SEQ ID NO: 1 or a portion thereof as a probe. The above-mentioned "stringent conditions" refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. More preferably, the conditions can be such that only DNAs having 95% or more of homology specifically hybridize. Such conditions can be set by adjusting the salt concentration, temperature conditions, etc. of the hybridization solution. Also, a gene encoding an amino acid sequence in which one or several amino acids are deleted, substituted or added in SEQ ID NO: 2, wherein the protein consisting of said amino acids reacts to produce γ-EC and Gly from GSH. Genes with catalytic enzymatic activity can be obtained by substituting DNA sequences using commercially available kits such as Site-Directed Mutagenesis Kit (manufactured by Takara Bio) and QuichChange Site-Directed Mutagenesis Kit (manufactured by STRATAGENE). be able to.

(2) Porous Cellulose Carrier In this production method, the NsPCS is used in a state of being immobilized on porous cellulose.
The porous cellulose used to immobilize NsPCS is water-insoluble porous cellulose having hydroxyl groups on its surface. Examples of such porous cellulose include, but are not limited to, cellulose filter paper, cellulose sponge, cellulose monolith, aggregates of powdery porous cellulose, and the like.

Among them, cellulose filter paper and cellulose sponge are well-known substances as porous cellulose and are widely commercially available. Cellulose filter paper, regardless of whether it is for quantitative or qualitative use, should comply with JIS P3801 standards (α cellulose 90% or more, copper value 1.6 or less, pH 5.0 to 8.0). For example, Advantec Toyo Co., Ltd. Any company can be exemplified without limitation. Cellulose sponge is a 100% natural material made from pulp-derived cellulose (cellulose) and natural fibers such as cotton added as reinforcing fibers. A cellulose sponge having the following properties is available from Toray Fine Chemicals Co., Ltd.

The powdery porous cellulose may be porous powder of cellulose having hydroxyl groups on its surface. The porous powder includes, for example, powdery crystalline cellulose. Examples of commercially available powdery crystalline cellulose include, but are not limited to, Avicel (trademark) (powder grade) (manufactured by Asahi Kasei), microcrystalline cellulose (for column chromatography) (manufactured by Merck Millipore), and the like. can.
Avicel is a product obtained by subjecting high-purity natural cellulose to acid hydrolysis under controlled conditions, removing the non-crystalline regions and extracting only the pure crystalline portion, followed by purification and drying. In particular, powder grades are non-fibrous porous particles that are secondary aggregates of fine cellulose crystals (Microcrystalline Cellulose: MCC) due to hydrogen bonding (irregular secondary aggregates). is known (see Non-Patent Documents 15 and 16).
Such pulverulent porous cellulose aggregates include aggregates in powder form (i.e., the final form is a powder), but also powders that have been compacted or compressed, so long as the porosity is not compromised. It also includes those having a granule shape or a tablet shape prepared by processing such as tableting. Preferably the final form is in powder form.

In general, a block of porous structure having a skeleton of a three-dimensional network structure and voids, respectively, is called a "monolith" or a "monolith structure." Cellulose monoliths are those in which the skeleton forming the monolith is mainly made of cellulose material (for example, 90% by mass or more of the skeleton, preferably 100% by mass), and the three-dimensional network formed by the cellulose skeleton and its voids ( It is a three-dimensional porous body in which continuous pores are integrated.
Here, a continuous hole (also referred to as a through-hole) means a continuous void, not a cavity that is independent of each other. Whether the pores are continuous pores can be determined from images of the outside and inside of the cellulose monolith taken using a scanning electron microscope (SEM). The shape of the pores inside or outside the cellulose monolith is preferably circular, elliptical, or similar, but is not limited thereto. By having such continuous pores, the cellulose monolith used in the present invention can introduce an arbitrary amount of functional group (a functional group having a carbonyl group, which will be described later) on its surface. By appropriately adjusting the amount of functional groups introduced and the amount of NsPCS immobilized thereon, the enzymatic reaction efficiency of the NsPCS can be appropriately adjusted.

Cellulose monoliths can be produced using phase separation of polymer solutions. As such phase separation methods, the non-solvent induced phase separation method (NIPS method), which induces phase separation by incorporating a non-solvent (water) (solvent exchange), and the thermally induced phase separation method (TIPS method), which induces phase separation by cooling method), a phase separation method using a mixed solvent, and various other methods are known. All of these phase separation methods for producing cellulose monoliths are known. For example, methods for producing cellulose monoliths using the NIPS method are described in US Pat. A method for producing a cellulose monolith using the TIPS method is described in Patent Document 14. Furthermore, in the phase separation method using a mixed solvent, cellulose acetate, which has excellent solubility, is used as a raw material, and a monolith is synthesized by a phase separation method using a mixed solvent of DMF and 1-hexanol. It is a method of converting to cellulose monolith by alkaline decomposition at , the details of which are described in Non-Patent Documents 12 and 13.

The cellulose monolith used in the present invention can be produced by these known methods. A phase separation method using a mixed solvent is preferred.
Cellulose acetate used as a raw material in the production of cellulose monoliths may be commercially available as long as a monolith can be synthesized by a phase separation method using a mixed solvent with 1-hexanol. Preferably, it can be appropriately selected from typical physical properties (degree of substitution, degree of polymerization) of cellulose acetate.
The degree of substitution is a numerical value indicating how many of the hydroxyl groups of one glucose residue of cellulose are substituted with acetyl groups, and serves as an index indicating the degree of acetylation of cellulose acetate. Although the distinction between cellulose monoacetate (cellulose monoacetate), cellulose diacetate (cellulose diacetate), and cellulose triacetate (cellulose triacetate) according to the degree of acetylation is not clear, the degree of acetyl substitution is 0.5 or more. It can be classified as cellulose monoacetate when it is less than 5, cellulose diacetate when it is 1.5 or more and less than 2.7, and cellulose triacetate when it is 2.7 or more. In the present invention, cellulose diacetate can be preferably used because of its excellent solubility in organic solvents.
Although the degree of polymerization is not limited, it is preferably 50 or more in terms of mass average in order to increase the mechanical strength of the resulting cellulose monolith and to prevent elution into solvents or the like during use. There is no particular upper limit.

As described above, the phase separation method using a mixed solvent consists of two steps: step 1 to prepare a cellulose acetate monolith (CA monolith) from cellulose acetate and step 2 to prepare a cellulose monolith from the CA monolith.

In step 1, N,N-dimethylformamide (DMF) and 1-hexanol are used as solvents for dissolving cellulose acetate. Cellulose acetate is first dissolved in DMF and then 1-hexanol is added. Although the amount of cellulose acetate to be dissolved in MDF is not limited, it can be, for example, 100-500 mg/mL. It is preferably 100-300 mg/mL, more preferably 150-250 mg/mL. The amount of 1-hexanol added is set so that the mixing ratio (volume ratio) of DMF and 1-hexanol in the mixed solvent is in the range of DMF:1-hexanol = 1:0.5 to 1:5. be able to. A preferred mixing ratio is DMF:1-hexanol=1:1 to 1:3, more preferably 1:1 to 1:2.
Next, the mixture is heated at about 70° C. until it becomes transparent, and then allowed to stand still at 20° C. for about 24 hours to phase-separate into a solid phase consisting of a cellulose acetate monolith (CA monolith) and an aqueous phase.

In step 2, the CA monolith produced in step 1 is deacetylated to obtain a cellulose monolith.
Deacetylation can be carried out by hydrolyzing the CA monolith in a hydrous alcohol containing a basic substance such as sodium hydroxide at room temperature. As the alcohol, a lower alcohol having 1 to 5 carbon atoms can be used, preferably methanol. The concentration of the basic substance used for deacetylation can be appropriately set within the range of 0.001 to 1% by mass, preferably within the range of 0.005 to 0.5% by mass.
A cellulose monolith produced by deacetylation can be used as a porous cellulose carrier for immobilizing the NsPCS by successively rinsing with water and a lower alcohol and drying.

The cellulose monolith used in the present invention includes porous bodies having continuous pores with an average pore size of 0.01 to 20.0 μm.
The flat pore size can be obtained by the NLDFT method (Non-Local Density Functional Theory), which is one of the pore size distribution analysis methods. The NLDFT method conforms to ISO standards (ISO 15901-3:2007, Pore size distribution and porosity of solid materials by mercury porosimetry and gas adsorption-Part 3: Analysis of micropores by gas adsorption) and JIS standards (JIS Z 8831-3). Measurement methods for pore size distribution and pore characteristics of powders (solids) - Part 3: Measurement methods for micropores by gas adsorption), software supplied with commercial gas adsorption devices and pore size analyzers available by

The porous cellulose used in the present invention includes porous bodies having a specific surface area of 10 m ² /g or more, preferably 20 to 50 m ² /g. The specific surface area can be determined by the BET formula, preferably the multipoint BET formula. The BET formula can be used by software attached to commercially available gas adsorption devices and specific surface area analyzers.

The porous cellulose used in the present invention also includes porous bodies with a high porosity, for example, 20% by volume or more, preferably 30 to 90% by volume.

The shape and size of the porous cellulose used in the present invention are not particularly limited, and various shapes such as spherical, granular, sheet-like, plate-like, cubic, cuboid, cylindrical, conical, cylindrical and rod-like. can have any size.

(3) Immobilized NsPCS and method for producing the same Porous cellulose has alcoholic hydroxyl groups on its surface, which is a characteristic of cellulose. Therefore, it is possible to bond various atomic groups through chemical reactions. However, proteins including NsPCS do not have a group that directly binds to a hydroxyl group, so an atomic group having binding reactivity with proteins, preferably a carbonyl group, is first introduced into the hydroxyl group of cellulose, and then reacted with NsPCS. Thus, an amide bond can be formed between the carbonyl group and the primary amino group of NsPCS.
In other words, the immobilized NsPCS used in this production method is obtained by immobilizing NsPCS on the porous cellulose by forming an amide bond between the carbonyl group introduced into the hydroxyl group of the porous cellulose and the primary amino group of NsPCS. is an enzyme.

The method for producing immobilized NsPCS can be carried out by the following two steps, as described above:
(i) introducing carbonyl groups to the hydroxyl groups of the porous cellulose;
(ii) A step of immobilizing NsPCS on the porous cellulose by amide bonding the primary amino group of NsPCS to the carbonyl group introduced in step (i) above.

The method for producing immobilized NsPCS may be any method as long as the above steps (i) and (ii) can be performed. It is not limited, and can be appropriately set based on the common technical knowledge in the industry.

(i) step can also be carried out by using a carbonyl group-introducing reagent. Examples of carbonyl group-introducing reagents include active esters of carboxy groups (carbonyl agents) that react with primary amines of proteins to form amide bonds. These carbonyl group-introducing reagents include NHS esters and water-soluble sulfo-NHS. NHS esters include N,N'-disuccinimidyl carbonate (DSC) and disuccinimidyl suberate (DCC). Examples of sulfo-NHS include bis(disuccinimidyl)suberate (BS3). NHS esters are preferred, and DSCs are more preferred.

As an example, the reaction scheme (outline) of the steps (i) and (ii) is shown in FIG. 2, using DSC, which is an NHS ester, as a carbonyl group-introducing reagent.
In the step (i), DSC and a strong base (nucleophilic agent) having a nucleophilic action as a catalyst are dissolved in an organic solvent (eg, acetonitrile) in advance, and porous cellulose is added thereto to react at room temperature. By doing so, an active ester of porous cellulose is produced by an ester reaction.
The nucleophile may be any one that is compatible with the organic solvent, and is preferably N,N-dimethyl-5-aminopyridine or dimethylaminopyridine (DMAP) such as N,N-dimethyl-5-aminopyridine. ) can be used. DSC and DMAP are preferably used in an amount of 1 to 3 equivalents, preferably 1.5 to 2.5 equivalents, relative to the porous cellulose.
Next, in step (ii), the produced active ester of cellulose is reacted with NsPCS in a buffer solution of pH 7-9, preferably pH 7.2-8.5, under conditions of 4° C. to room temperature. By doing so, the active ester of cellulose produced in step (i) is hydrolyzed to liberate N-hydroxysuccinimide, and the primary amino group of NsPCS binds to the carbonyl group introduced into the hydroxyl group of cellulose. NsPCS is immobilized on the porous cellulose.
The buffer solution may have a buffering capacity in the range of pH 7 to 9, preferably pH 7.2 to 8.5. Its salts, HEPES and the like can be exemplified.
The immobilized NsPCS obtained in step (ii) can be used for production of γ-EC or/and Gly of the present invention, for example, after washing with the same buffer as above. In addition, when not used immediately, they can be frozen and stored in a state of being immersed in the above-mentioned buffer solution to which an antifreeze solution has been added without drying. Although the cryopreservation is not limited, it is preferably cryopreservation in liquid nitrogen, and examples thereof include cryopreservation at -150°C or lower, preferably -180°C or lower.

(4) Production method of γ-EC and/or Gly The production method targeted by the present invention is characterized by using the above-described immobilized NsPCS as an enzyme that degrades GSH into γ-EC and Gly. That is, this production method has a step (Step 1) of bringing GSH into contact with immobilized NsPCS and reacting NsPCS with GSH to produce γ-EC and Gly from GSH.

The conditions for contacting GSH with immobilized NsPCS, that is, the conditions for reacting GSH with NsPCS may be conditions under which γ-EC and Gly are produced from GSH. A method in which the reaction is performed in a buffered solution can be mentioned. Examples of the pH of the buffer include pH 7-9, preferably pH 7.2-8.5. Examples of buffers include, without limitation, phosphate buffers, carbonate buffers, bicarbonate buffers, borate buffers, HEPES and the like. A phosphate buffer is preferred, and a potassium phosphate buffer is more preferred. The concentration of the buffer solution can be set as long as it does not interfere with the above reaction.
Also, the above reaction is preferably carried out in the presence of a reducing agent. Examples of the reducing agent include, but are not limited to, tris(2-carboxyethyl)phosphine hydrochloride and the like.
Specifically, it can be carried out by dissolving GSH as a substrate in the buffer solution to which the reducing agent has been added and bringing this into contact with the immobilized NsPCS. The concentration of GSH dissolved in the buffer is desirably set within a range that does not precipitate during the reaction (during the manufacturing process), taking into consideration the solubility of GSH in water (approximately 160 mM at 25°C). Preferred examples include 50 to 110 mM, more preferably 80 to 100 mM. The reaction temperature (contact temperature, buffer solution temperature) is 15 to 37°C, preferably 30 to 37°C.

The above reaction can be carried out in a batch mode in a container (batch reaction), or a buffer solution containing GSH is continuously sent to a column or tube filled with immobilized NsPCS, It can also be carried out in a flow mode in which the reaction is carried out continuously in a column or tube (flow reaction). In the batch-type reaction, although it is necessary to separate the reaction products (γ-EC, Gly) from the immobilized NsPCS, it is possible to obtain a large amount of It is a useful method in that the present product can be obtained in The flow reaction is a useful method in that it does not require separation of the reaction products (γ-EC, Gly) from the immobilized NsPCS, and the product can be obtained continuously.
Since the immobilized NsPCS of the present invention is stably immobilized on the porous cellulose without deactivation of NsPCS, there is no separation of NsPCS, and it is suitable for repeated reactions in both batch and flow modes. can do.

γ-EC and/or Gly can be obtained by isolating and recovering γ-EC and/or Gly from the reaction product obtained in step 1 above.

(Isolation and recovery of γ-EC)
Although the method for isolating and recovering γ-EC from the reaction product (this product) is not limited, liquid chromatography can be used as one method.
The conditions are not particularly limited as long as γ-EC can be separated from coexisting GSH and Gly, preferably γ-EC, GSH and Gly can be separated from each other. The following conditions can be mentioned as.

[HPLC conditions]
Post-column-HPLC method, reversed-phase column (Inertsil ODS-3 5 μm 4.6×250 nm, GL Sciences)
・Eluent Eluent A: 7.5 mM sodium octanesulfonate aqueous solution containing 0.02% trifluoroacetic acid Eluent B: 0.02% trifluoroacetic acid and 30% acetonitrile aqueous solution containing 7.5 mM sodium octanesulfonate ・Gradient: 0 min → 1 Minutes: Eluent A 100% → Eluent A 87% + Eluent B 13%
1 minute → 20 minutes: Eluent A 87% + Eluent B 13% → Eluent A 30% + Eluent B 70%
20 minutes → 21 minutes: Eluent A 30% + Eluent B 70% → Eluent B 100%
21 minutes → 28 minutes: Eluent B 100% maintained 28 minutes → 29 minutes: Eluent B 100% → Eluent A 100%, maintained for 3 minutes.
・Flow rate: 1.5ml/min

Under these conditions, GSH elutes with a retention time of about 12.56 minutes, γ-EC with a retention time of about 13.01 minutes, and Gly with a retention time of about 3.57 minutes, so γ-EC is separated from GSH and Gly. can be recovered. In addition, by collecting the Gly fraction in parallel, Gly can be isolated and obtained at the same time as γ-EC. The recovery of the Gly fraction (isolation and recovery of Gly) can also be carried out again from the residue from which γ-EC has been isolated and recovered. The reaction products (γ-EC, Gly) can be quantified under the HPLC conditions described above using a calibration curve prepared using known amounts of γ-EC and Gly. As described above, Gly can be recovered separately from GSH and γ-EC under the HPLC conditions described above, and is therefore useful as a method for isolating and recovering Gly. In this case also, apart from the isolation and recovery of Gly, the γ-EC fraction may be separated and recovered in parallel with Gly, or the γ-EC fraction may be recovered again from the residue from which Gly was isolated and recovered. (Isolation and recovery of γ-EC) may be performed.

As another method for isolating and recovering γ-EC from the reaction product, for example, a method of connecting a column or tube filled with an ion exchanger to a column or tube filled with immobilized NsPCS can be used. can. According to this method, γ-EC can be directly recovered, so that a column or tube packed with immobilized NsPCS can be effectively used as a flow reactor for γ-EC.

For example, when GSH is 100% converted to γ-EC, Gly is present in the reaction solution in an amount equivalent to γ-EC. When this reaction solution is passed through a column packed with a cation exchanger that strongly repels anions such as sulfone groups, γ-EC and Gly behave as follows:
The pKa and pKb of γ-EC, which is classified as a neutral amino acid having an acidic amino acid Glu and an SH group, are 2.21 and 11.78, respectively. Therefore, it repels the strongly negative sulfone group and does not change the passage speed of the column.
On the other hand, Gly, which is a neutral amino acid and has pKa and pKb of 2.35 and 9.78, respectively, reduces the column passage speed due to its interaction with the sulfone group.
Therefore, by connecting a column or tube filled with immobilized NsPCS to a column filled with a cation exchanger, if a buffer solution containing GSH is sent to this column, γ-EC is first generated as a reaction product. Elution can be obtained. In this case, when using a buffer solution of pH 8.0, the separation of γ-EC and Gly is not clear. Since Gly can be strongly retained on this, the separation from γ-EC can be performed more clearly. To change the pH of the reaction solution, create a branch between the column or tube packed with immobilized NsPCS and the column or tube packed with a cation exchanger, and run an acid or highly concentrated acidic buffer with another pump. It can be carried out by mixing with the reaction liquid and adjusting the pH.

On the other hand, in a column packed with a strong anion exchanger that has a quaternary ammonium group, γ-EC is strongly retained and the passage speed is very slow, but Gly repels this and the passage speed does not change. Therefore, if a column or tube filled with immobilized NsPCS is connected to a column filled with a strong anion exchanger, and a buffer solution containing GSH is sent to this column, Gly is first eluted as a reaction product. γ-EC can be eluted and obtained later. In particular, when a pH 8.0 buffer, which is the same condition as the reaction solution, is used as the mobile phase, there is a large difference in the column passage speeds of γ-EC and Gly, enabling clearer separation. Elution of γ-EC from an anion exchange column is also effective for its concentration, enabling efficient production at various substrate concentrations.

In this way, the immobilized NsPCS can also be used as a flow reactor for stable, continuous, and efficient production of γ-EC or/and Gly from GSH. and is useful for industrial production of γ-EC and/or Gly.

(II) Storage method of immobilized NsPCS Although the above-mentioned immobilized NsPCS of the present invention was inactivated and could not be stored in a freeze-dried state as shown in Experimental Example 7 described later, it was stored in a buffer solution containing an antifreeze solution. It can be stored stably while maintaining its enzymatic activity by immersing it in and storing it at -150°C or below, preferably in liquid nitrogen (-196 to -150°C) at an ultra-low temperature.
Therefore, as a method for preserving the immobilized NsPCS of the present invention, the immobilized NsPCS of the present invention is immersed in a buffer containing an antifreeze solution at -150°C or lower, preferably in liquid nitrogen (-196 to -150°C). To provide a method of cryopreservation. The antifreeze is preferably one that does not affect the effect of the immobilized NsPCS during use, and glycerol, for example, can be used. Preferably, the immobilized NsPCS are subjected to the above-described treatment in a 100-300 mM buffer at pH 7-9, preferably pH 7.2-8.5, containing 10-30% by weight, preferably 20-30% by weight of glycerol. A method of preservation under ultra-low temperature conditions can be mentioned. The buffer solution may have a buffering capacity in the pH range described above, and examples include phosphoric acid or its salts, carbonic acid or its salts, bicarbonate or its salts, boric acid or its salts, and HEPES. can be done.

As used herein, the terms "include" and "contain" include the meanings of "consisting of" and "consisting essentially of".

In the following, the present invention will be described using experimental examples in order to facilitate understanding of the configuration and effects of the present invention. However, the present invention is not limited by these experimental examples. Unless otherwise specified, the following experiments were performed at room temperature (25±5° C.) and atmospheric pressure conditions. Unless otherwise specified, "%" described below means "% by mass", and "part" means "parts by mass".

Materials used in Production Examples and Experimental Examples to be described later and their sources are as follows.
N,N'-Disuccinimidyl carbonate/glucose unit: Tokyo Chemical Industry Co., Ltd.
N,N-dimethyl-5-aminopyridine glucose unit: Wako Pure Chemical Industries, Ltd. Cellulose diacetate (Mn = 3.0 × 10 ⁴ ; 39.3-40.3 wt% acetyl content): Sigma-Aldrichs glutathione (GSH): γ-Glutamylcysteine (γ-EC) manufactured by Nacalai Tesque (for standard): Glycine manufactured by Sigma-ardrich (for standard): Cellulose filter paper manufactured by Nacalai Tesque: Circular quantitative filter paper No. 5B (retention particle diameter 4 μm, thickness 0.21 mm, JIS P3801 Type 5 B): Cut ADVANTEC into a circle with a diameter of 5 mm. Cellulose powder: Cellulose microcrystalline for column chromatography (Cat. No. 1.02331.0500): Merck Millipore

Quantification of γ-EC and Gly produced in Experimental Examples described later was carried out according to the following method.
[Common method]
The reaction solution is subjected to centrifugation (15,000 x g, 4°C, 10 minutes), and after collecting 20 μL of the supernatant, add ultrapure water to make the liquid volume 200 μL, dilute 10-fold, and then dilute 5-fold as follows. Measured by the method shown.
The supernatant was subjected to a post-column-HPLC method using a reversed-phase column (Inertsil ODS-3 5 μm 4.6×250 nm, GL Sciences). A 7.5 mM sodium octanesulfonate aqueous solution containing 0.02% trifluoroacetic acid was used as the eluent A, and a 30% acetonitrile aqueous solution containing 0.02% trifluoroacetic acid and 7.5 mM sodium octanesulfonate was used as the eluent B. Start feeding 100% eluent A and 0% eluent B at a flow rate of 1.5 ml/min. After 1 minute, 87% eluent A and 13% B. After 20 minutes, 30% eluent A and 30% eluent. B 70%, eluent A 0%, eluent B 100% after 21 minutes, maintained for 7 minutes, eluent A 100%, eluent B 0% for 1 minute, maintained for 3 minutes.

[Quantification of γ-EC]
The thiol compounds separated by the above post-column-HPLC method were treated with 50 mM phosphate buffer in 10% acetonitrile solution containing 75.7 μM 5'-dethiobis(2-nitrobenzoic acid) and heated to 40°C by the post-column method. The groups were derivatized and absorbance detected at 412 nm. Quantitation was performed using a calibration curve prepared using known amounts of γ-EC.

[Gly determination method]
Gly was detected by UV in the above post-column-HPLC method and quantified using a calibration curve prepared using known amounts of Gly.

Production Example 1 Production of NsPCS Based on the description of Example 4 of Patent Document 4, NsPCS was produced by the following method. Note that the description of Example 4 of Patent Document 4 can be incorporated into this specification by reference.
(1) Cloning of alr 0975 gene Using the genomic DNA extracted from Nostoc sp.PCC 7120 as a template, the alr 0975 gene (SEQ ID NO: 1) was amplified by PCR using the following primers.
NsF1 (5'-CTTCATATGATAGTTATGAAACTTCTTTATC-3': SEQ ID NO: 3)
NsR1 (5'-ATCGGATCCTAATCTTGTGTTTTACTTACG-3': SEQ ID NO: 4)
The resulting DNA fragment was treated with restriction enzymes NdeI and BamHI, inserted into a pET25b(+) vector (purchased from Novagen) (pET25b-alr 0975), and subjected to sequence analysis. It was confirmed. The resulting plasmid was used to transform E. coli BL21 (DE3) cells.

(2) Purification of recombinant alr 0975 protein (NsPCS) The resulting transformant was liquid-cultured in LB medium containing 50 µg/mL carbenicillin, and isopropyl-β-D- Protein synthesis was induced by adding thiogalactopyranoside (IPTG). After IPTG treatment, cells cultured for 6 hours were collected by centrifugation and suspended in a buffer (100 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA and 1 mM β-mercaptoethanol). The cell suspension was sonicated to extract a soluble fraction, which was dialyzed overnight against a 20 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA and 1 mM β-mercaptoethanol. After that, the sample was applied to a DEAE-Toyopearl column (5 cm x 15 cm; Tosho) and the flow-through fraction was collected. This was dialyzed overnight using 20 mM phosphate buffer (pH 6.0) containing 1 mM EDTA. The resulting protein fraction was applied to a HiTrap SP column (Pharmacia Biotech, Uppsala, Sweden) equipped with an AKTA protein purification system for purification. Purified protein was obtained by a gradient program from 0 to 10 mM NaCl in 20 mM phosphate buffer (pH 6.0).

Purity, molecular weight, and activity of the resulting purified recombinant protein were determined according to the description in Example 4 of Patent Document 4.
As a result, this protein was confirmed to be the desired NsPCS, since it exhibited activity to catalyze the reaction that produces γ-EC and Gly using GSH as a substrate.

Production Example 2 Production of Cellulose Monolith A cellulose monolith was produced according to the method described in Non-Patent Document 12. The manufacturing method described in Non-Patent Document 12 can be incorporated herein by reference. Non-Patent Document 12 describes that a cellulose monolith having continuous pores with an average pore diameter of 11.2 nm and a non-surface area of 42.3 m ² /g can be obtained by this production method.
Specifically, first, cellulose diacetate powder (0.20 g) was completely dissolved in N,N-dimethylformamide (DMF) (1.0 mL) at room temperature. 1-Hexanol (1.5 mL) was added dropwise to this while stirring gently. The mixture was then heated at 70°C until it became clear. Next, this solution was allowed to stand still at 20° C. for 24 hours to undergo phase separation into a liquid phase and a solid phase. The solvent (liquid phase) was exchanged with ethanol three times, followed by drying the solid phase in vacuum to obtain a cellulose acetate monolith (CA monolith).
The resulting CA monolith was then hydrolyzed to prepare a cellulose monolith. Specifically, first, the CA monolith (50 mg) was immersed in 2.0 mL of methanol. After degassing for 5 minutes, 0.15 L of 2M NaOH aqueous solution was added to methanol to initiate hydrolysis reaction at room temperature. Three hours after initiation, the hydrolysis was stopped by neutralizing the solution with 1M aqueous HCl. Cellulose monoliths produced by hydrolysis were rinsed successively with water and methanol and dried in vacuum.
By subjecting the obtained cellulose monolith to a scanning electron microscope (SEM) (Hitachi S-3000 N instrument, 15 kV), it was confirmed that a monolith having a relatively uniform internal morphology was formed. A SEM image is shown in FIG.

Production Example 3 Production of immobilized NsPCS To dehydrated acetonitrile, N,N'-disuccinimidyl carbonate (DSC)/glucose units and N,N-dimethyl- 5-Aminopyridine (DMAP) and glucose units were added and dissolved, and the porous cellulose [cellulose monolith produced in Production Example 2 (Experimental Examples 3 and 4: 0.0317 g, Example 5: 0.3182 g), Cellulose filter paper (Experimental Examples 1 and 2: 0.00165 g/piece) and cellulose sponge (Experimental Example 5: 0.03375-0.03875 g/piece x 4 pieces)] were added, degassed under reduced pressure for 10 minutes, and then at room temperature for 24 hours. Stirred. Then, it was washed with dehydrated acetonitrile three times, dried under reduced pressure, and washed with 20 mM potassium phosphate buffer (pH 8.0).
Next, the resulting porous cellulose (activated ester) was immersed in an NsPCS solution (dissolved in phosphate buffer, diluted with ultrapure water, pH 8.0) adjusted to a concentration of 220 μg/ml for 30 minutes. By doing so, the primary amino group of NsPCS and the carbonyl group introduced to the hydroxyl group of the porous cellulose form an amide bond, forming the surface of the porous cellulose carrier (both the outer surface and the pore surface are collectively referred to as the "surface"). ) was immobilized with NsPCS. Then, it was washed three times with 20 mM potassium phosphate buffer (pH 8.0).
This was used in the following experimental examples as NsPCS-immobilized porous cellulose (immobilized NsPCS).

Experimental Example 1 Method for producing γ-EC The immobilized NsPCS prepared in Production Example 3 using cellulose filter paper as a porous cellulose carrier was added to 1 mM Tris at a rate that the NsPCS concentration in the reaction solution was 26 mg/L. It was reacted with 100 mM GSH at 37° C. in 200 mM potassium phosphate buffer (pH 8.0) in the presence of (2-carboxyethyl)phosphine hydrochloride (TCEP) (reducing agent). Next, the reaction was stopped by adding an appropriate amount of 3.6 N hydrochloric acid, and the reaction solution from which the immobilized NsPCS had been removed was subjected to high-performance liquid chromatography under the conditions described above, and the substrate (GSH) and the reaction product (γ-EC , Gly) were separated and quantified. Since the solubility of GSH in water is about 160 mM, 100 mM is considered to be the maximum concentration at which GSH does not precipitate during the production process.

FIG. 4 shows changes in reaction time (h) and amounts (mM) of substrate (GSH) and reaction product (γ-EC) in the reaction solution. As shown in FIG. 4, 100 mM GSH completely disappeared after about 13 hours, and about 100 mM γ-EC was obtained. This result indicates that the immobilized NsPCS of the present invention can almost completely convert GSH at a high concentration of 100 mM to γ-EC.
Since the concentration of NsPCS in this reaction solution was 26 mg/L, the production rate of γ-EC was about one-third that when free NsPCS was used. In contrast, since it can be used repeatedly, the production amount of γ-EC can be improved as a result. In addition, since it is possible to further increase the amount of immobilized enzyme in the cellulose monolith produced in Production Example 2, by increasing the amount of NsPCS immobilized on the porous cellulose, γ- EC production speed can also be increased.

Experimental Example 2 Confirmation of repeated use of immobilized NsPCS In the same manner as in Experimental Example 1, γ-EC was produced using NsPCS immobilized on cellulose filter paper as porous cellulose (immobilized NsPCS) using GSH as a raw material. rice field. After completion of the reaction, the immobilized NsPCS from which the reaction solution was removed was washed with 20 mM potassium phosphate buffer (pH 8.0). . This was repeated four times, and the ratio of the 2nd to 5th reaction rates to the γ-EC production rate in the first reaction was determined.

Fig. 5 shows the relative ratio (%) of the 2nd to 5th generation rates when the 1st γ-EC generation rate is 100%. As shown in FIG. 5, it was confirmed that the γ-EC production rate gradually decreased with each repetition, but was maintained at about 80% even after the fifth repetition. On the other hand, the conversion rate of GSH to γ-EC did not change, and almost 100% conversion was achieved in the second to fifth reactions, which was the same as in the first reaction. From these results, the immobilized NsPCS of the present invention showed no significant reduction in the production rate even when the reaction was repeated up to at least 5 times using 100 mM GSH as a substrate, and almost completely γ- It was confirmed that it can be converted to EC. From this, it was confirmed that the immobilized NsPCS of the present invention is very stable physically (immobilized state) and enzymatically (activity).

Experimental Example 3 Continuous production of γ-EC using immobilized NsPCS (small scale)
Using the cellulose monolith produced in Production Example 2 as a porous cellulose carrier and the immobilized NsPCS produced in Production Example 3, γ-EC was continuously produced using GSH as a starting material.
Specifically, first, 0.29 mL of immobilized NsPCS (total weight of immobilized NsPCS: 84.5 μg) was placed in a laminate tube, and heat-treated to form a cylinder (inner diameter: 5 mm, length: 15 mm). A reactor having the configuration shown in FIG. 6 was assembled and used to produce γ-EC using GSH as a starting material. As a substrate solution, 10 mM GSH, 1 mM TCEP, and 200 mM potassium phosphate buffer (pH 8.0) were used as in Experimental Example 1.

As a result of adjusting the supply rate of the substrate solution in the range of about 0.25 mL/h to 12.5 mL/h by the peristaltic pump, as shown in Table 1 and FIG. conversion rate (GSH→γ-EC) was obtained. These results indicated that the immobilized NsPCS of the present invention enabled continuous production of γ-EC using GSH as a substrate.

In this experiment, a small volume of immobilized NsPCS of about 0.3 mL was used, but theoretically, if this experimental system is scaled up, mass production of γ-EC is possible. Specifically, for example, when using 5 mL of immobilized NsPCS, the flow rate is 2.5 mL/h, and when using 5 L of immobilized NsPCS, the flow rate is 2.5 L/h. It is believed that γ-EC can be obtained from GSH with a yield of 10% or more. Theoretically, 100% conversion can be achieved by further reducing the substrate supply rate. In addition, by increasing the amount of NsPCS immobilized on the porous cellulose, it is possible to achieve a conversion of 90% or more, or even 100%, in a shorter time. Efficient production suitable for

Next, we tested how long γ-EC can be produced continuously.
As a result, as shown in Fig. 9, when the substrate was supplied at a rate of 0.25 mL/h in the above experimental system, the conversion rate (GSH → γ-EC) was 90% or more for 240 hours (10 days). maintained. From this result, it was found that the production of γ-EC from GSH using the immobilized NsPCS of the present invention can be carried out continuously for at least 10 days without deterioration of NsPCS.

Experimental Example 4 Continuous production of γ-EC using immobilized NsPCS (scale-up)
Instead of the cylindrical column (inner diameter 5 mm, length 15 mm) used in Experimental Example 3, 2.2 mL of immobilized NsPCS prepared in Production Example 3 using cellulose monolith (total weight of NsPCS: 261 μg) was placed in a laminate tube. γ-EC was produced using GSH as a raw material using a reaction apparatus having the configuration shown in FIG.
As a result, when using a 100 mM GSH solution, it was possible to almost completely convert GSH to γ-EC by reacting it with immobilized NsPCS at a supply rate of about 0.2 mL/h. As a result, 100 mM GSH could be almost completely converted to γ-EC by increasing the volume of immobilized NsPCS about 10-fold and increasing the amount of NsPCS by 3-fold compared to Experimental Example 3. .
As in Experimental Example 3, this reactor was used to test how long γ-EC can be continuously produced. As a result, as shown in FIG. 10, when the substrate was supplied at a rate of about 0.2 mL/h in the above experimental system, the conversion rate (GSH → γ-EC) was 90% or more for 240 hours (10 days). was maintained.

From this result, the following trial calculation can be made.
・100 mM GSH was completely converted to γ-EC and Gly at a flow rate of 0.2 mL/h.
From this, it is estimated that 0.2 mL x 24 = 4.8 mL of 100 mM γ-EC can be obtained in 24 hours (1 day). Its production volume is 250(g/mol) x 0.1 mol/L x 0.0048 L = 0.12 g/day.

Experimental Example 5 Production of γ-EC using immobilized NsPCS and evaluation of durability of immobilized NsPCS (1) Production of γ-EC using immobilized NsPCS Production example using cellulose sponge as a porous cellulose carrier The immobilized NsPCS prepared in 3 was added to 200 mM It was reacted with 100 mM GSH at 37°C in a potassium phosphate buffer (pH 8.0). Next, the reaction was stopped by adding an appropriate amount of 3.6 N hydrochloric acid, and the reaction solution from which the immobilized NsPCS had been removed was subjected to high-performance liquid chromatography under the conditions described above, and the substrate (GSH) and the reaction product (γ-EC , Gly) were separated and quantified.
As a result, it was confirmed that γ-EC and Gly were produced at a rate of 100% from GSH after 72 hours of reaction (conversion rate 100%). No deterioration was observed in the cellulose sponge after 72 hours of reaction.

(2) Evaluation of durability of immobilized NsPCS The durability of the immobilized NsPCS prepared in Production Example 3 using cellulose sponge as a porous cellulose carrier was evaluated.
Specifically, according to the method described in (1) above, the reaction at a temperature of 37° C. for 48 hours was repeated four times, or the reaction at a temperature of 25° C. for 48 hours was repeated four times. In each reaction system, the conversion rate (%) to γ-EC in each of 1 to 4 reactions was measured over time.
Figure 11 (1) shows the γ-EC conversion rate (%) in four reactions (37_1 to 37_4) at a temperature of 37 °C over time, and in four reactions (25_1 to 25_4) at a temperature of 25 °C FIG. 11(2) shows the change in the γ-EC conversion rate (%) over time.
As shown in FIGS. 11 (1) and (2), the first reaction (γ-EC conversion rate: 97%) at 37 ° C. for 48 hours and the fourth reaction (γ-EC conversion rate: 81.5%) were 15.5 % decrease, and a 21.5% decrease was observed in the 4th reaction (γ-EC conversion rate: 54.9%) compared to the first reaction (γ-EC conversion rate: 76.4%) at 25 ° C for 48 hours, but it is certain It was confirmed that γ-EC was obtained in In addition, no deterioration of the cellulose sponge was observed in any of the reaction systems after 4 reactions. From these results, it was confirmed that the immobilized NsPCS prepared using the cellulose sponge is durable against repeated use (continuous reaction) as a carrier and as an enzyme.

Experimental Example 6 Preparation of Immobilized NsPCS and Production of γ-EC Using the Same (1) Preparation of Immobilized NsPCS Using crystalline cellulose powder as a porous cellulose carrier, immobilized NsPCS was prepared by the following method.
Specifically, according to the procedure of Production Example 3, dehydrated acetonitrile was added with twice the amount of N,N'-disuccinimidyl carbonate (DSC)/glucose unit and N,N with respect to the number of moles of the crystalline cellulose powder. -Dimethyl-5-aminopyridine (DMAP)·glucose unit was added and dissolved, to which about 100 mg of crystalline cellulose powder was added and stirred at room temperature for 24 hours. Then, it was washed with dehydrated acetonitrile three times, dried under reduced pressure, and washed with 20 mM potassium phosphate buffer (pH 8.0).
Next, the resulting crystalline cellulose powder (activated ester) was immersed in 4 mL of NsPCS solution (diluted with ultrapure water) adjusted to a concentration of 220 μg/ml for 30 minutes. By doing so, the primary amino group of NsPCS and the carbonyl group introduced to the hydroxyl group of the porous cellulose form an amide bond, forming the surface of the porous cellulose carrier (both the outer surface and the pore surface are collectively referred to as the "surface"). ) was immobilized with NsPCS. Then, it was washed three times with 20 mM potassium phosphate buffer (pH 8.0).
Table 2 shows the amount of immobilized enzyme per volume of immobilized NsPCS obtained.

The amount of enzyme immobilized per volume is about 2.5 times that of cellulose monolith, and it was confirmed that a large amount of enzyme NsPCS can be efficiently immobilized on crystalline cellulose powder.

(2) Production of γ-EC using immobilized NsPCS Using the immobilized NsPCS prepared above, γ-EC was continuously produced using GSH as a starting material.
Specifically, first, 2.5 mL of immobilized NsPCS (total weight of immobilized NsPCS: about 1460 μg) was placed in a glass tube whose lower end was sealed with a glass wool plug, and a reactor having the configuration shown in FIG. 12 was assembled. γ-EC was produced using GSH as a raw material. As a substrate solution, 100 mM GSH, 1 mM TCEP, and 200 mM potassium phosphate buffer (pH 8.0) were used as in Experimental Example 4.

As a result of adjusting the feed rate of the substrate solution in the range of about 0.10 mL/h to 3.0 mL/h with a peristaltic pump, a conversion rate of about 100% (GSH → γ-EC) was obtained at a feed rate of about 0.15 mL/h. was gotten. These results indicate that the immobilized NsPCS enables stable and efficient continuous production of γ-EC using GSH as a substrate.

Experimental Example 7 Storage Stability of Immobilized NsPCS The storage stability of immobilized NsPCS was evaluated.
Specifically, the immobilized NsPCS prepared in Production Example 1 were either stored at ultra-low temperature in liquid nitrogen (-196°C) or stored in a dry state after freeze-drying, and changes in activity before and after storage were examined. . More specifically, ultra-low-temperature storage in liquid nitrogen is carried out by immersing the immobilized NsPCS produced in Production Example 2 in a 200 mM potassium phosphate buffer (pH 8.0) containing 25% glycerol as an antifreeze solution. Stored in nitrogen for 2 days. In addition, dry storage by freeze-drying is performed by immersing the immobilized NsPCS produced in Production Example 2 in 200 mM potassium phosphate buffer (pH 8.0) containing 25% glycerol, treating it with liquid nitrogen, and freezing it. Freeze-dried in a desiccator and stored for 2 days.
Thereafter, according to the method described in Experimental Example 2, γ-EC was produced using GSH as a starting material, and the production rate of γ-EC before storage was compared.

Table 3 shows the relative ratio (%) of the γ-EC production rate after storage when the γ-EC production rate before storage is taken as 100%.

As shown in Table 3, when immersed in a buffer containing antifreeze solution and stored in liquid nitrogen, the activity was maintained at 90% or more, and although the reaction time was somewhat prolonged, GSH was almost 100% γ- Converted to EC. On the other hand, the enzymatic activity was lost after freeze-drying, and no γ-EC was produced. Based on these results, NsPCS was immobilized on porous cellulose by covalent bonding, and when it was immersed in a buffer solution containing antifreeze solution and stored at ultra-low temperature in liquid nitrogen, the enzymatic activity was maintained for a long period of time. It was confirmed that it can be stored stably at

The immobilized NsPCS of the present invention and the method for producing γ-EC using the same have high industrial applicability in at least one of the following points.
1) By using the immobilized NsPCS of the present invention, the raw material GSH can be completely converted into γ-EC and Gly. 2) This production method does not require magnesium or expensive ATP as cofactors.
3) According to the present invention, γ-EC can be easily separated from substrates, enzymes and the like.
4) Unlike Patent Document 2, this production method does not use cells, so the reaction solution does not contain cell-derived contaminants (including amino acids, peptides, etc.), making it easy to separate and purify γ-EC. is.
5) It is possible to stably and/or continuously produce γ-EC while maintaining a high conversion rate from GSH to γ-EC for a period of at least 10 days.
6) The immobilized NsPCS can be stably stored without deactivation under cryogenic storage conditions in liquid nitrogen in the presence of an antifreeze solution.
7) Gly is an amino acid that is difficult to produce by a fermentation method that is usually used for industrial production of amino acids, but according to this production method, an equal amount of Gly can be produced simultaneously with γ-EC from GSH.
8) According to the continuous production method using immobilized NsPCS of the present invention, similarly to γ-EC, Gly can be stably and continuously produced without the need for separation from substrates and enzymes. It can also be easily separated from γ-EC by passing the reaction product through an ion exchange resin.

SEQ ID NO: 1 is the nucleotide sequence of the alr 0975 gene, which has high homology with the PCS gene derived from Cyanobacteria Nostoc sp. PCC7120; SEQ ID NO: 2 is the amino acid sequence of the PCS-like enzyme (NsPCS) encoded by the alr 0975 gene. SEQ ID NOs: 3 and 4 show the nucleotide sequences of the primer set (NsF1, NsrR1) used when the alr 0975 gene was amplified by PCR using the genomic DNA extracted from Nostoc sp.PCC 7120 as a template.

Claims (14)

1. A step of contacting NsPCS immobilized by amide bonding with a carbonyl group introduced into a hydroxyl group of a porous cellulose carrier with GSH under conditions of pH 4 to 10 to generate γ-EC and Gly (Step 1). A method for producing γ-EC or/and Gly.
2. The method for producing γ-EC and/or Gly according to claim 1, further comprising a step (step 2) of isolating and recovering γ-EC and/or Gly from the product obtained in step 1 above.
3. γ-EC or/and Gly according to claim 2, wherein said step 2 is a step of contacting the product obtained in said step 1 with an ion exchanger to separate γ-EC and Gly. manufacturing method.
4. The ion exchanger is a cation exchanger, and the solution containing the product obtained in step 1 is acidified and then brought into contact with the cation exchanger to separate γ-EC and Gly. , a method for producing γ-EC or/and Gly according to claim 3.
5. The method for separating γ-EC and Gly by contacting the solution containing the product obtained in step 1 with the strong anion exchanger, wherein the ion exchanger is a strong anion exchanger. 3. A method for producing γ-EC or/and Gly according to 3.
6. The method for producing γ-EC or/and Gly according to claim 1 or 2, wherein the porous cellulose carrier is cellulose filter paper, cellulose sponge, cellulose monolith, or aggregate of powdered porous cellulose.
7. Immobilized NsPCS, in which NsPCS is immobilized on porous cellulose by amide bonding between the carbonyl groups introduced into the hydroxyl groups of porous cellulose and the primary amino groups of NsPCS.
8. The immobilized NsPCS according to claim 7, wherein the porous cellulose is a cellulose filter paper, a cellulose sponge, a cellulose monolith, or an aggregate of powdered porous cellulose.
9. A method for producing immobilized NsPCS according to claim 7 or 8, comprising the steps of:
   (1) a step of introducing a carbonyl group to the hydroxyl group of the porous cellulose;
   (2) A step of immobilizing NsPCS on the porous cellulose by amide bonding the primary amino group of NsPCS to the carbonyl group introduced in step (1) above.
10. 10. The immobilized NsPCS production according to claim 9, wherein the step (1) is a step of producing an activated ester of porous cellulose using an active esterifying agent having an NHS ester group as a carbonyl group-introducing reagent. Method.
11. The method for producing immobilized NsPCS according to claim 10, wherein the active esterifying agent having an NHS ester group is N,N'-disuccinimidyl carbonate.
12. The method for producing immobilized NsPCS according to claim 9 or 10, wherein the porous cellulose is a cellulose filter paper, a cellulose sponge, a cellulose monolith, or an aggregate of powdered porous cellulose.
13. 9. The method for preserving the immobilized NsPCS according to claim 7 or 8, wherein the immobilized NsPCS is immersed in a buffer solution containing an antifreeze solution and stored at a low temperature of -150°C or less, the aforementioned method.
14. The method for preserving immobilized NsPCS according to claim 13, wherein the cryopreservation is cryopreservation in liquid nitrogen. 

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