Gamma-Glutamylcysteine Production Using Phytochelatin Synthase-Like Enzyme Derived from Nostoc sp. Covalently Immobilized on a Cellulose Carrier

Abstract

Gamma-glutamylcysteine (γ-EC) is an intermediate generated in the de novo synthesis of glutathione (GSH). Recent studies have revealed that the administration of γ-EC shows neuroprotective effects against oxidative stress in age-related disorders and chronic diseases like Alzhiemer’s disease in model animals, which is not expected function in GSH. A phytochelatin synthase-like enzyme derived from Nostoc sp. (NsPCS) mediates γ-EC synthesis from GSH. To achieve low-cost and stable commercial level supply, the availability of immobilized NsPCS for γ-EC production was investigated in this study. Among the tested immobilization techniques, covalent binding to the cellulose carrier was most effective, and could convert GSH completely to γ-EC without decreasing the yield. The stable conversion of γ-EC from 100 mM GSH was achieved by both batch repeated and continuous reactions using the immobilized NsPCS on cellulose sheet and column shape monolith, respectively. The immobilization of NsPCS on those carriers is promising alternative technique for high-yielding and cost-effective production of γ-EC on its commercial applications.

INTRODUCTION

It is well known that glutathione (GSH) has multiple important functions, including antioxidant defense and xenobiotic removal.^1,2) In mammals, GSH is synthesized from glutamic acid (Glu), cysteine (Cys), and glycine (Gly) in two ATP-dependent steps, wherein glutamate-cysteine ligase (GCL) catalyzes the formation of γ-glutamylcysteine (γ-EC) from Glu and Cys, and glutathione synthetase (GS) catalyzes the formation of GSH from γ-EC and Gly. Cellular homeostasis of GSH is regulated by non-allosteric feedback inhibition exerted by GSH on GCL activity.³⁾ However, GS generally shows higher activity than GCL and no inhibitory regulation.³⁾ Therefore, intracellular γ-EC levels are markedly lower than those of GSH in mammals, as synthesized γ-EC is immediately converted to GSH by GS.⁴⁾ Gould and Pazdro have reported that alterations in GSH homeostasis are associated with aging process and various age-related disorders and disease states including chronic neuroinflammations, and Alzheimer’s disease.⁵⁾ For many disorders, diseases, and aging, it is suggested that GSH depletion is associated with the onset of dysfunctional GCL regulation with insufficient γ-EC synthesis to supply GSH and maintain appropriate homeostasis.^6–8) However, exogenous GSH administration could not help alleviate those disorders and disease states, as the ability to ensure homeostatic regulation of GSH levels is decreased in those cases.⁹⁾ On the other hand, recent studies have revealed that the administration of γ-EC increases intracellular GSH levels in a human trial pilot study.¹⁰⁾ More recently, administration of γ-EC ameliorates oxidative damage and neuroinflamention induced by amyloid β in human astrocytes¹¹⁾ and exerts neuroprotective effect in Alzheimer’s disease in animal models.¹²⁾ Impact of γ-EC on GSH status is promising for various potential health outcomes in preventing and improving age-related disorders and chronic diseases.^13,14)

At the commercial level, however, γ-EC is difficult to obtain and very expensive because commercially available γ-EC is currently produced by chemical peptide synthesis. For example, the price per gram of γ-EC is more than 500 times higher than that of GSH in the Sigma-Aldrich product catalog. Several patents have revealed fermentation and enzymatic production procedures of γ-EC,^15–17) but the products have not been released in the market, probably due to high production costs. This supply limitation may delay further investigation of γ-EC functions and their commercial applications to medicine and functional additives to foods and cosmetics.

In our previous study, we reported that a phytochelatin synthase-like enzyme, NsPCS, derived from a cyanobacterium, Nostoc sp. mediated γ-EC synthesis from GSH.^18,19) This is the first reaction in phytochelatin ((γ-EC)_n-Gly) synthesis and is followed by the transfer of γ-EC to another GSH molecule to form (γ-EC)₂-Gly. Phytochelatin synthases that are derived from eukaryotic organisms, such as higher plants, mediate these multiple functional enzyme reactions only in the presence of heavy metals like cadmium,^20,21) whereas NsPCS mediates only the first reaction without requiring heavy metals. It means that NsPCS is a peptidase that hydrolyzes the Cys-Gly bond in GSH to cut out Gly as shown in Fig. 1. This reaction is a reversal of the GSH biosynthesis reaction catalyzed by GS and does not require ATP and any other cofactors. We revealed that NsPCS completely converted 100 mM GSH (30.7 g/L) to γ-EC (25.0 g/L) under optimized temperature, pH, and buffer conditions.²²⁾

Fig. 1. Reaction Equation for Hydrolysis of GSH to Produce γ-EC and Gly by NsPCS

In our current study, we investigated the use of NsPCS as an effective alternative for current γ-EC production procedures. We employed an enzyme immobilization technique to achieve a cost-effective and practical production procedure of γ-EC, since immobilized enzymes enable easy separation of enzyme from the reaction solution in the solid phase, which is applicable to both batch repeated and continuous productions. The property of NsPCS of requiring no cofactors is an advantage for immobilization because the production procedure can be easily regulated. Furthermore, the conversion ability of NsPCS for high concentration, 100 mM, of GSH can enable an effective production of γ-EC with high volumetric efficiency. However, we failed to obtain stable and sufficient activities of NsPCS immobilized by representative immobilization techniques such as encapsulation in calcium alginate, covalent binding to glass beads, and physical adsorption to various carriers. In this study, therefore, the availability of alternative technique, the covalent binding immobilization of NsPCS to cellulose carrier, was evaluated for stable and high-yielding production of γ-EC from GSH.

MATERIALS AND METHODS

Preparation of NsPCS

NsPCS was prepared as described previously.²²⁾ The plasmid pET25b-alr0975, previously constructed by,¹⁸⁾ was used to express NsPCS in Escherichia coli. Recombinant NsPCS was expressed in E. coli C43 (DE3) (Lucigen, WI, U.S.A.) transformed with pET25b-alr0975 at 20 °C in 2 L of Terrific Broth (TB) (Invitrogen, MA, U.S.A.). Protein expression was induced at the mid-log phase of the bacteria by the addition of isopropyl-β-D-thiogalactopyranoside to a final concentration of 0.5 mM, followed by expression for 20 h at 20 °C. Bacteria were harvested by centrifugation and disrupted by sonication in 200 mL of Buffer A (50 mM Tris–HCl buffer at pH 8.0, 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), and 1 mM dithiothreitol (DTT)). The supernatant obtained after centrifugation at 18000 × g for 30 min was filtered through a 0.22-µm membrane. The lysate was applied to a 5 mL HiTrap Q HP column (Cytiva, Japan) equilibrated with Buffer B (50 mM Tris–HCl buffer at pH 8.0, 10 mM NaCl, 1 mM EDTA, and 1 mM DTT). After washing twice with 10 mL Buffer B, elution was carried out with Buffer C (50 mM Tris–HCl buffer at pH 8.0, 1 M NaCl, 1 mM EDTA, and 1 mM DTT). The flow-through fraction was reapplied to the column four times. The flow-through and wash fractions were concentrated using Amicon Ultra-15 Centrifugal Filter Units (Merck Millipore, MA, U.S.A.) and dialyzed against 50 mM Tris–HCl buffer (pH 8.0) containing 100 mM NaCl, 5 mM tris(2-carboxyethyl)phosphine (TCEP), and 25% glycerol. The volume of the supernatant obtained after centrifugation (18000 × g for 60 min) was 22 mL. The homogeneity of the purified proteins was confirmed using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The darkest band corresponded to 27 kDa, which is the predicted molecular weight of NsPCS. The purity of the purified protein was 50%, as estimated by the intensity of the band using bovine serum albumin as a standard.

γ-EC Production by NsPCS Immobilized by Covalent Binding to the Cellulose Sheet

Cellulose sheet (filter paper quantitative Ashless No. 2, Advantec, Tokyo, Japan), cut into 5-mm diameter circles, was used as the immobilization carrier. Two times the amount of N,N′-disuccinimidyl carbonate (DSC) (TCI, Japan) as glucose and twice the amount of N,N-dimethyl-4-aminopyridine (DMAP) (FUJIFILM Wako Pure Chemical Corporation, Japan) as glucose were added to dehydrated acetonitrile and dissolved. After cellulose was added, the mixture was degassed under reduced pressure for 10 min and then stirred at 25–30 °C for 24 h. The mixture was then washed three times with dehydrated acetonitrile. Immediately before immobilization, the cellulose was dried under reduced pressure and washed with 20 mM potassium phosphate buffer (pH 8.0). The cellulose was immersed in a solution containing 220 µg/mL of NsPCS. After soaking for 30 min, the cellulose was washed three times with 20 mM potassium phosphate buffer (pH 8.0). The protein concentration of the remaining NsPCS and washed solutions was measured by Bradford method, following which the amount of immobilized enzyme was calculated.

The enzyme reaction was carried out in 100 µL of 200 mM potassium phosphate (pH 8.0) containing 1 mM TCEP, and 100 mM GSH with the prepared two cellulose sheet-immobilized NsPCS at 37 °C with shaking. The reaction was terminated by the addition of equal amounts of 3.6 N HCl. The substrate, GSH (γ-L-Glu-L-Cys-Gly), was purchased from Sigma-Aldrich.

To examine storage stability, immobilized NsPCS was soaked in 200 mM potassium phosphate buffer (pH 8.0) solution containing 25% glycerol and stored in liquid nitrogen or freeze-drying for 2 d. After storage, NsPCS was washed twice with 20 mM potassium phosphate buffer (pH 8.0) and examined as described above.

Repeated γ-EC Production by NsPCS Immobilized by Covalent Binding to the Cellulose Sheet

To examine γ-EC production after repeated use, 200 cellulose sheets covalently immobilized with NsPCS were used. The weight of 200 cellulose sheets was 73 mg and 61.8 µg NsPCS was remained on the sheets after immobilization. In this case, immobilized NsPCS on the sheets was 0.85 mg protein/g cellulose. The enzyme reaction was carried out by adding 10 mL reaction solution containing 200 mM potassium phosphate (pH 8.0), 1 mM TCEP, and 100 mM GSH at 37 °C with shaking. After confirming 100% conversion, the cellulose sheets were removed from the reaction solution, washed with 20 mM potassium phosphate buffer (pH 8.0), and then added to a new reaction solution. Conversion rate was examined after 2 h of solution exchange. The above-described tests were repeated five times. The reaction was terminated by the addition of equal amounts of 3.6 N HCl.

Continuous γ-EC Production by NsPCS Immobilized by Covalent Binding to Column-Shape Porous Cellulose Monolith

The cellulose monolith was synthesized according to a previous study.²³⁾ First, 0.20 g of cellulose acetate (CA) powder (Sigma-Aldrich, MO, U.S.A.) was completely dissolved in 1.0 mL of N,N-dimethylformide (TCI, Japan) at room temperature around 25 °C. 1-Hexanol (1.5 mL) was added dropwise into the solution with gentle stirring. The mixture was then heated at 70 °C until it became transparent. The temperature of the solution was then maintained at 20 °C for 24 h to complete phase separation. The solvent was replaced with ethanol three times and subsequently dried in vacuo to furnish the CA monolith. The CA monolith (50 mg) was first immersed in 2.0 mL of methanol. After degassing for 5 min, 0.15 mL of 2 M NaOH solution in methanol was added to initiate hydrolysis of the side chain alkyl group at room temperature. After 3 h, the solution was neutralized with 1 M HCl. The hydrolyzed cellulose monolith was successively rinsed with water and methanol and dried in vacuo.

A small column-shape porous cellulose monolith (5 mm in diameter and 15 mm in length) or a large cellulose monolith (13 mm in diameter and 17 mm in length) was used as the immobilization carrier. Immobilization was carried out as described for immobilization on a cellulose sheet by covalent binding. Four immobilized small column-shape porous cellulose monoliths with 234, 169, 151, and 129 µg of NsPCS were obtained. The weight of one of the small cellulose monolith used in this study was 75 mg. After washing, 234 µg NsPCS was remained on the monolith. In this case, immobilized NsPCS on the monolith was 3.52 mg protein/g cellulose. In case of large column-shaped porous cellulose monolith, the weight was 575 mg and remaining NsPCS in the monolith was 522 µg after immobilization. Then immobilized NsPCS was 0.91 mg protein/g cellulose.

A pump (MP-3000, EYELA, Japan) was connected to the prepared NsPCS-immobilized column-shaped porous cellulose monolith covered by a tubing (125610, EYELA) having an internal diameter of 1.15 mm and a heat-shrinkable tubing (HCT150-6.4-T, Denka Electron, Japan).

The reaction solution, 200 mM potassium phosphate (pH 8.0) containing 1 mM TCEP, and 10 mM GSH (for the small NsPCS-immobilized monolith) or 100 mM GSH (for the large NsPCS-immobilized monolith) was pumped at 35–37 °C. The reaction was terminated by the addition of 50 µL of 3.6 N HCl to 100 µL of the reaction solution.

Analysis of Enzyme Reaction Solution by HPLC

HPLC was conducted as described by Hirata et al.²⁴⁾ Reactants were centrifuged (15000 × g for 10 min at 4 °C), and 20 µL of the supernatant was diluted with water to a final volume of 200 µL. Each sample (100 µL) was injected onto a reverse-phase column (Inertsil ODS-3, 5 µm, 4.6 × 250 mm, GL Sciences, Japan) attached to a modified HPLC post-column system (Hitachi, Japan) and equilibrated with Buffer D (5 mM sodium 1-octane sulfonate solution containing 0.02% trifluoroacetic acid (TFA)). Thiols were eluted with a gradient of Buffer E (30% acetonitrile solution containing 0.02% TFA) at a flow rate of 1.5 mL/min at 40 °C. Buffer E was raised from 0% to 13% in 1 min, to 70% in the following 19 min, and then to 100% in 1 min and maintained for 7 min. Eluted thiols were mixed with 75.7 µM 5′-dithiobis (2-nitrobenzoic acid) in 50 mM potassium phosphate buffer (pH 8.0) containing 10% acetonitrile at 40 °C and quantified by absorbance at 412 nm (Hitachi). To examine for the presence of oxidized forms of GSH (GSSG) and γ-EC (Glu-Cys-Cys-Glu) in the reaction solution, the absorbance of these compounds at 215 nm were analyzed under the same HPLC conditions by using a Hitachi L7450 diode array detector. GSH (γ-L-Glu-L-Cys-Gly) and γ-EC (γ-L-Glu-L-Cys) were purchased from Sigma-Aldrich and used as standards for HPLC analysis. Oxidized form of GSH was purchased from Nacalai-tesque (Kyoto, Japan). Oxidized form of γ-EC was chemically synthesized by Scrum, Inc. (Tokyo, Japan). They were also used as standards for HPLC analysis.

RESULTS

To elucidate the availability of immobilization techniques for NsPCS-mediated stable and high-yielding conversion of GSH to γ-EC, we first examined representative immobilization techniques such as encapsulation, covalent binding, and physical adsorption. However, we found that none of these techniques were available to NsPCS immobilization because the enzyme activity decreased considerably (data not shown). To obtain stable immobilization that can endure repeated or continuous reactions without losing enzyme activity, we tried the covalently immobilization of NsPCS on cellulose carriers using DSC and DMAP²⁵⁾ as shown in Fig. 2.

Fig. 2. Reaction of Activation Used DSC in the Presence of DMAP Method of Cellulose and Immobilization of NsPCS on Activated Cellulose

A cellulose sheet was firstly used as the carrier for immobilization. As shown in Fig. 3, almost complete conversion of 100 mM GSH to γ-EC was achieved after 12.5 h (8 mM/h) with immobilized NsPCS, which is 38% of conversion rate of free enzyme at the same concentration. Product inhibition by the synthesized γ-EC was not observed. Since γ-EC is thought to be easily oxidized to its oxidized form (Glu-Cys-Cys-Glu) as same as GSH to GSSG, the presence of those oxidized forms was examined by HPLC analysis at 215 nm absorbance. The peaks of both oxidized forms were rarely detected at the retention times of their standards (data not shown). These results indicate that oxidation of the substrate, GSH, and the product, γ-EC, hardly occurred during conversion reaction with immobilized NsPCS.

Fig. 3. Conversion of GSH to γ-EC by Covalently Immobilized NsPCS on Cellulose Sheet; 100 µL of GSH (100 mM) Was Incubated with 2.6 µg of Immobilized NsPCS

Data are expressed as means ± standard deviation (S.D.) (n = 3).

Stable storage of enzymes is important for commercial level production. Storages in liquid nitrogen and at low temperatures after freeze-drying are mostly employed for industrially applicable enzymes. Free NsPCS can be stored using both the methods without decreasing its activity. When the immobilized NsPCS on cellulose sheet was stored in liquid nitrogen, the conversion rate reduced to 90%, but the reaction was completed without any change in γ-EC yield. On the other hand, the activity of immobilized NsPCS completely disappeared after freeze-drying. These results indicated that storage in liquid nitrogen was preferable for covalently immobilized NsPCS on cellulose.

To examine γ-EC production yield after repeated use, incubation of NsPCS immobilized on cellulose sheet with 100 mM GSH was repeated five times. In this experiment, the volume of reaction solution was enlarged to 10 mL by using 200 pieces of NsPCS-covalently immobilized cellulose sheet. As shown in Fig. 4, the conversion rate of 100 mM GSH did not reduce during five times repetitions whereas the time required for 100% conversion was extended gradually. These results indicated that the production of γ-EC from 100 mM GSH by covalently immobilized NsPCS on the cellulose sheet could be repeated without decreasing γ-EC yield.

Fig. 4. Conversion of GSH to γ-EC by Repeated Incubation with Covalently Immobilized NsPCS on Cellulose Sheet; 10 mL of 100 mM GSH Was Incubated with 61.8 µg of Immobilized NsPCS

The conversion rate was examined after 2h of solution exchange and yield of γ-EC was examined after the reaction.

To investigate the availability of the covalently immobilized NsPCS on cellulose for the continuous production of γ-EC, a column-shaped porous cellulose monolith was employed as an immobilization carrier instead of a cellulose sheet.^23,26) First, a small-scale column-shaped monolith (5 mm in diameter and 15 mm in length) was used for the continuous conversion of GSH to γ-EC. The concentration of GSH used was 10 mM, and the effect of flow rate on the yield of γ-EC was evaluated. Since the amount of immobilized NsPCS on the monolith fluctuated widely, the reaction was carried out using four different carriers of the same size prepared individually with the same immobilization procedure. As shown in Fig. 5, about 95% yields of γ-EC from GSH were obtained in the four different carriers at a flow rate of 0.25 mL/h, but the yields gradually decreased with wide fluctuation among the carriers at flow rates higher than 0.25 mL/h. When the conversion reaction was continued for 10 d at 0.25 mL/h, the yield was maintained without reduction (Fig. 6a), indicating that continuous production of γ-EC from 10 mM GSH was achieved using NsPCS immobilized on a column-shaped porous cellulose monolith.

Fig. 5. Effect of Flow Rate on the Yield of γ-EC from GSH (10 mM) with Covalently Immobilized NsPCS on Column-Shaped Cellulose Monolith (5 mm in Diameter and 15 mm in Length)

The conversion reactions were carried out using four different carriers—square, triangle, circle, and rhombus—with individually immobilized NsPCS on a cellulose monolith. The amounts of immobilized NsPCS were 234 µg for square, 151 µg for triangle, 169 µg for circle, and 129 µg for rhombus (individually).

Fig. 6. Continuous Production of γ-EC from GSH with Covalently Immobilized NsPCS on Column-Shaped Cellulose

The yield of γ-EC after 1, 4, 7, and 10 d of production are presented. (a) For a small-scale column-shaped monolith, 10 mM of GSH was added, and the flow rate was set at 0.25 mL/h ; (b) for a large-scale column-shaped monolith, 100 mM of GSH was added, and the flow rate was set at 0.1 mL/h. Data are expressed as means ± S.D. (n = 3).

To investigate the continuous conversion of 100 mM GSH to γ-EC, the volume of the column-shaped monolith was enlarged to 7.7 times (13 mm in diameter and 17 mm in length). At a flow rate of 0.2 mL/h, 99% of yield of γ-EC was maintained for the tested 3-d period. However, the yield decreased with increasing the flow rate from 0.2 mL/h (Fig. 7). Then the yield was 26% at the flow rate of 1.2 mL/h. When the conversion reaction was continued for 10 d at a flow rate of 0.1 mL/h, the yield was maintained without reduction (Fig. 6b). These results indicated that the covalently immobilized NsPCS on column-shaped porous cellulose monolith was applicable to the continuous production of γ-EC from 100 mM GSH.

Fig. 7. Effect of Flow Rate on Yield of γ-EC from GSH with NsPCS Covalently Immobilized on Column-Shaped Cellulose Monolith (13 mm in Diameter and 17 mm in Length)

The amount of immobilized NsPCS was 522 µg.

The molecular weight of γ-EC was 250.3. When 100 mM GSH was completely converted to γ-EC at a flow rate of 0.2 mL/h by immobilized NsPCS on column-shaped monolith, the yield of γ-EC obtained by continuous production for 10 d was about 1.2 g. In case of immobilized NsPCS on cellulose sheets, 10 mL of 100 mM GSH was completely converted to γ-EC in 26 h. When this batch production procedure was repeated five times with maintaining equivalent ability, 50 mL of 100 mM γ-EC (1.25 g) would be obtained for 139 h (26 h × 5). Although solution exchange is required, this simple repeated batch production procedure can be easily scaled up to any size without any additional operation.

DISCUSSION

Enzymes are widely utilized in various fields, such as food and pharmaceuticals for environmentally friendly chemical transformations.^27,28) However, free enzymes cannot achieve efficient production in industrial processes because they are unsuitable for repeated use and continuous performance in production procedures. This drawback of free enzymes can be solved by using immobilized enzymes, which can be easily separated from the reaction solution and be available for repeated and continuous use.²⁷⁾ In case of NsPCS, in addition, the two properties of NsPCS are preferable for the application of immobilization technique, in which this enzyme does not require any cofactors and can completely convert high concentration, 100 mM, of GSH to γ-EC.²²⁾

In our study, therefore, we tried to employ immobilization techniques to establish stable and high-yielding production procedure of γ-EC. The activity and stability of immobilized enzymes is affected by carriers and methods. In other words, they are dependent on the compatibility between enzymes and carriers and methods. In many cases, therefore, it is necessary to optimize immobilization conditions including carriers and methods to individual enzyme. We firstly examined the availability of representative immobilization techniques, encapsulation, covalent binding, and physical adsorption among the several immobilization carriers and methods. In cases of encapsulation in calcium-alginate gel and covalent binding to glass beads, the activities after immobilization were considerably lower than that of free NsPCS, and the reactions were not completed during the test period. In case of immobilization by physical adsorption on cellulose without any surface treatment or some modification, the first conversion reaction showed high conversion yield but we found the enzyme released from the carrier during reaction and could not be used repeatedly and continuously. It is required that considerably high and stable activity in immobilized enzyme for its application to industrial production. Since cellulose is non-active and stable materials for biological reactions with enzyme, we tried to apply covalent binding technique to bind enzyme to cellulose carrier more tightly and stably. The immobilization technique using DSC and DMAP was employed, because this technique has been already applied to horseradish peroxidase and other several enzymes useful for industrial application. The high and stable activity NsPCS sufficient for repeated and continuous production of γ-EC was obtained by this immobilization technique on cellulose sheets and column shape monolith.

Establishment of storage method of immobilized enzymes is very important for its industrial using. Freeze-drying and storage in liquid nitrogen are commonly available for free enzymes and functional proteins. These storages were also available to free NsPCS. Therefore, we examined the activity of immobilized NsPCS on cellulose sheet after storage in liquid nitrogen. Consequently, the storage in liquid nitrogen was preferable, but the activity was not maintained in immobilized NsPCS after freeze-drying. The reason why freeze-drying is not available to immobilized NsPCS would be irreversible denaturation of NsPCS caused by irregular or heterogenous dehydration around covalent binding site to cellulose or whole enzyme.

When NsPCS was immobilized on cellulose sheets and column-shaped cellulose monolith, repeated and continuous productions of γ-EC from 100 mM GSH were achieved without significant losing enzyme activity, respectively. Since 100 mM is close to the value of GSH solubility in water, both production procedures could be conducted with high volumetric efficiency. Impact of γ-EC on GSH status is promising for various potential health outcomes, in particular for prevention and improvement of Alzheimer’s diseases.^11–14) Therefore, more advanced and extensive investigation of γ-EC function and their industrial production for its commercial applications are expected to accelerate and progress. Both stable storage of immobilized NsPCS in liquid nitrogen as same as free enzyme, and stable repeated and continuous production of γ-EC with high yield and volumetric efficiency, which is unattainable in free enzyme, are thought to be advantageous for its application to industrial production. Further investigation of materials and reaction conditions should be done to establish scale-up production procedure applicable to industrial production. For example, the flow rate should be optimized in case of continuous production by using immobilized NsPCS on column shape cellulose monolith. The faster the flow rate, the fewer the number of molecular collisions of the substrate with the immobilized enzyme. In this study, therefore, at the flow rate higher than 0.25 mL/h, the frequency of collisions of GSH with NsPCS was not sufficient to complete conversion of GSH to γ-EC and GSH was remained in the flow out reaction solution.

Although further investigation as above is required, the covalently immobilized NsPCS on cellulose carrier is expected to solve an impasse of γ-EC production with high cost and low productivity and to break its supply limitation which delay further investigation and commercial applications.

Acknowledgments

This work was supported in part by a KAKENHI grant from the Japan Society for the Promotion of Science [Grant Nos. 16K14902 and 20K05923] and Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS]) from the Japan Agency for Medical Research and Development (AMED) [Grant No. JP21am0101084].

Conflict of Interest

The authors declare no conflict of interest.

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