Phosphorylase-catalyzed Enzymatic Polymerization Research Articles

Hydrogelation from Self-Assembled and Scaled-Down Chitin Nanofibers by the Modification of Highly Polar Substituents.

Chitin nanofibers (ChNFs) with a bundle structure were fabricated via regenerative self-assembly at the nanoscale from a chitin ion gel with an ionic liquid using methanol. Furthermore, the bundles were disentangled by partial deacetylation under alkaline conditions, followed by cationization and electrostatic repulsion in aqueous acetic acid to obtain thinner nanofibers called scaled-down ChNFs. This review presents a method for hydrogelation from self-assembled and scaled-down ChNFs by modifying the highly polar substituents on ChNFs. The modification was carried out by the reaction of amino groups on ChNFs, which were generated by partial deacetylation, with reactive substituent candidates such as poly(2-oxazoline)s with electrophilic living propagating ends and mono- and oligosaccharides with hemiacetallic reducing ends. The substituents contributed to the formation of network structures from ChNFs in highly polar dispersed media, such as water, to produce hydrogels. Moreover, after the modification of the maltooligosaccharide primers on ChNFs, glucan phosphorylase-catalyzed enzymatic polymerization was performed from the primer chain ends to elongate the amylosic graft chains on ChNFs. The amylosic graft chains formed double helices between ChNFs, which acted as physical crosslinking points to construct network structures, giving rise to hydrogels.

Vine-Twining Inclusion Behavior of Amylose towards Hydrophobic Polyester, Poly(β-propiolactone), in Glucan Phosphorylase-Catalyzed Enzymatic Polymerization.

This study investigates inclusion behavior of amylose towards, poly(β-propiolactone) (PPL), that is a hydrophobic polyester, via the vine-twining process in glucan phosphorylase (GP, isolated from thermophilic bacteria, Aquifex aeolicus VF5)-catalyzed enzymatic polymerization. As a result of poor dispersibility of PPL in sodium acetate buffer, the enzymatically produced amylose by GP catalysis incompletely included PPL in the buffer media under the general vine-twining polymerization conditions. Alternatively, we employed an ethyl acetate-sodium acetate buffer emulsion system with dispersing PPL as the media for vine-twining polymerization. Accordingly, the GP (from thermophilic bacteria)-catalyzed enzymatic polymerization of an α-d-glucose 1-phosphate monomer from a maltoheptaose primer was performed at 50 °C for 48 h in the prepared emulsion to efficiently form the inclusion complex. The powder X-ray diffraction profile of the precipitated product suggested that the amylose-PPL inclusion complex was mostly produced in the above system. The 1H NMR spectrum of the product also supported the inclusion complex structure, where a calculation based on an integrated ratio of signals indicated an almost perfect inclusion of PPL in the amylosic cavity. The prevention of crystallization of PPL in the product was suggested by IR analysis, because it was surrounded by the amylosic chains due to the inclusion complex structure.

Preparation of Supramolecular Material from Amylosic Inclusion Complex with Thermoresponsive Guest Polymer Obtained by Vine-Twining Polymerization

Amylose constructs supramolecular inclusion complexes with polymeric guests in phosphorylase-catalyzed enzymatic polymerization field, called ‘vine-twining polymerization’. However, such inclusion complexes have not exhibited specific property and processability as functional supramolecular materials. In a recent study, n amphiphilic triblock guest copolymer (poly(2-methyl-2-oxazoline-block-tetrahydrofuran-block-2-methyl-2-oxazoline, (P(MeOx-block-THF-block-MeOx)) was employed for the vine-twining polymerization to form soft materials from an amylosic inclusion complex. In this study, we investigated the vine-twining polymerization using a thermoresponsive triblock guest copolymer, that is, poly(2-isopropyl-2-oxazoline-block-tetrahydrofuran-block-2-isopropyl-2-oxazoline (P(iPrOx-block-THF-block-iPrOx)). The vine-twining polymerization at the temperature below the lower critical solution temperature (LCST) of PiPrOx gave the inclusion complex, while no inclusion by an enzymatically produced amylose took place at the temperature above the LCST of PiPrOx due to aggregation of P(iPrOx-block-THF-block-iPrOx) in aqueous buffer (polymerization solvent). The product's characterization results suggested that the cavity of the enzymatically elongated amylose chain included the PTHF block in the triblock copolymer by hydrophobic interaction. Accordingly, the outer PiPrOx blocks constructed spaces among the inclusion complex segments. Such higher-order structure formed supramolecular networks, leading to the formation of a hydrogel. The product showed thermoresponsive property depending upon the temperatures below and above the LCST of PiPrOx.

Evaluation of artificial crystalline structure from amylose analog polysaccharide without hydroxy groups at C-2 position

In this study, we found that a new artificial crystalline structure was fabricated from an amylose analog polysaccharide without hydroxy groups at the C-2 position, i.e., 2-deoxyamylose. The polysaccharide with a well-defined structure was synthesized by facile thermostable α-glucan phosphorylase-catalyzed enzymatic polymerization. Powder X-ray diffraction (XRD) analysis of the product indicated the formation of a specific crystalline structure that was completely different from the well-known double helix of the natural polysaccharide, amylose. Molecular dynamics simulations showed that the isolated chains of 2-deoxyamylose spontaneously assembled to a novel double helix based on building blocks with controlled hydrophobicity arising from pyranose ring stacking. The simulation results corresponded with the XRD patterns.

Synthesis of Amylosic Supramolecular Materials by Glucan Phosphorylase-Catalyzed Enzymatic Polymerization According to the Vine-Twining Approach

This article overviews the synthesis of amylosic supramolecular materials through inclusion complexation in glucan phosphorylase (GP)-catalyzed enzymatic polymerization. Amylose is a polysaccharide that is known to form inclusion complexes with a number of hydrophobic small guest molecules. A pure amylose can be synthesized by the enzymatic polymerization of α-d-glucose 1-phosphate monomer with a maltooligosaccharide primer catalyzed by GP. The author has reported that the propagating amylosic chain in the enzymatic polymerization twines around hydrophobic polymers present in aqueous reaction media to form supramolecular inclusion complexes. As it is similar to the way that vines of a plant grow around a rod, this polymerization is termed ‘vine-twining polymerization’. Amylosic supramolecular network materials have been obtained through the vine-twining polymerization by using copolymers, where hydrophobic guest polymers are covalently grafted on hydrophilic main-chain polymers. The enzymatically produced amylosic chains form complexes with the guest polymers among graft copolymers, which act as cross-linking points to form supramolecular networks, resulting in the formation of soft materials, such as gels and films. Vine-twining polymerization using appropriately designed guest polymers has also been performed, which leads to supramolecular products that exhibit new functionality.1 Introduction2 Vine-Twining Polymerization to Form Supramolecular Inclusion Complexes3 Selective Complexation of Amylose toward Guest Polymers in Vine-Twining Polymerization4 Hierarchical Architecture of Amylosic Supramolecular Network Materials by Vine-Twining Polymerization Approach5 Hierarchical Fabrication of Amylosic Supramolecular Materials by Vine-Twining Polymerization Using Designed Guest Polymers6 Conclusions

Formation of Supramolecular Soft Materials from Amylosic Inclusion Complexes with Designed Guest Polymers Obtained by Vine-Twining Polymerization.

Amylose forms supramolecular inclusion complexes with polymeric guests in the phosphorylase-catalyzed enzymatic polymerization field, so-called “vine-twining polymerization”. However, such inclusion complexes have not exhibited specific properties and processability as functional supramolecular materials. In this study, we found that amylosic inclusion complexes, which were obtained by vine-twining polymerization using a designed guest polymer, that is, an amphiphilic triblock copolymer poly(2-methyl-2-oxazoline-block-tetrahydrofuran-block-2-methyl-2-oxazoline), exhibited gel and film formation properties. The characterization results of the products suggested that enzymatically elongated amylose chains complexed with the polytetrahydrofuran block in the triblock copolymer. Accordingly, the outer poly(2-methyl-2-oxazoline) blocks constructed hydrophilic spaces among the inclusion complex segments. Furthermore, the presence of such outer blocks affected the lower regularity of crystalline alignment among the inclusion complex segments in the products. Such higher-order structures probably induced the formation of supramolecular soft materials, such as gels and films.

Preparation of Amylose-Carboxymethyl Cellulose Conjugated Supramolecular Networks by Phosphorylase-Catalyzed Enzymatic Polymerization

Enzymatic polymerization has been noted as a powerful method to precisely synthesize polymers with complicated structures, such as polysaccharides, which are not commonly prepared by conventional polymerization. Phosphorylase is one of the enzymes which have been used to practically synthesize well-defined polysaccharides. The phosphorylase-catalyzed enzymatic polymerization is conducted using α-d-glucose 1-phosphate as a monomer, and maltooligosaccharide as a primer, respectively, to obtain amylose. Amylose is known to form supramolecules owing to its helical conformation, that is, inclusion complex and double helix, in which the formation is depended on whether a guest molecule is present or not. In this paper, we would like to report the preparation of amylose-carboxymethyl cellulose (CMC) conjugated supramolecular networks, by the phosphorylase-catalyzed enzymatic polymerization, using maltoheptaose primer-grafted CMC. When the enzymatic polymerization was carried out using the graft copolymer, either in the presence or in the absence of a guest polymer poly (ε-caprolactone) (PCL), the enzymatically elongated amylose chains from the primers on the CMC main-chain formed double helixes or inclusion complexes, depending on the amounts of PCL, which acted as cross-linking points for the construction of network structures. Accordingly, the reaction mixtures totally turned into hydrogels, regardless of the structures of supramolecular cross-linking points.

Enzymatic Preparation of Supramolecular Networks Composed of Amylosic Inclusion Complexes with Grafted Guest Polymers

In this study, we attempted to prepare supramolecular networks composed of amylosic inclusion complexes with grafted guest polymers by means of phosphorylase-catalyzed enzymatic polymerization of α-d-glucose 1-phosphate as a monomer from a maltooligosaccharide primer. When the enzymatic polymerization was examined in the presence of poly(γ-glutamic acid-graft-tetrahydrofuran) (PGA-g-PTHF), the reaction mixture totally turned into a hydrogel form. The powder X-ray diffraction measurement of a lyophilized sample of the hydrogel suggested that enzymatically elongated amyloses formed inclusion complexes with the PTHF graft chains among the PGA main-chains according to vine-twining polymerization manner to construct the supramolecular network structure. Therefore, the product formed the hydrogel with inclusion complex cross-linking as the enzymatic polymerization progressed. The vine-twining polymerization is the method for the formation of amylose-polymer inclusion complexes in the phosphorylase-catalyzed enzymatic polymerization field, which has been previously developed by us. On the other hand, the enzymatic polymerization mixture in the presence of poly(γ-glutamic acid-graft-l-lactic acid) (PGA-g-PLLA) did not induce the hydrogel formation upon the same operation. In this system, enzymatically elongated amyloses did not form inclusion complexes with the PLLA graft chains due to their bulkiness, but sorely constructed well-known double helical assemblies, resulting in their aggregation in the reaction mixture.

Formation of microparticles from amylose-grafted poly(γ-glutamic acid) networks obtained by thermostable phosphorylase-catalyzed enzymatic polymerization.

Amylose is a natural polysaccharide with helical conformation, which spontaneously forms water-insoluble assemblies, such as double helixes and inclusion complexes, at ambient temperatures in aqueous media, whereas it is synthesized as a water-soluble single chain by thermostable phosphorylase-catalyzed enzymatic polymerization at elevated temperatures in aqueous buffer solvents. In this study, we investigated the enzymatic polymerization at 80 °C using a primer-grafted poly(γ-glutamic acid) (PGA) in the presence or absence of poly(l-lactic acid) (PLLA) as a guest polymer for inclusion by amylose. Consequently, the produced amylose-grafted PGAs formed microparticles by cooling the mixtures at room temperature after the enzymatic polymerization in either the presence or the absence of PLLA. The particle sizes, which were evaluated by SEM measurement, were dependent on the feed ratios of PLLA. Based on the characterization results by the powder X-ray diffraction, IR, and dynamic light scattering measurements, a mechanism for the formation of the microparticles in the present system is proposed.

Difference in Macroscopic Morphologies of Amylosic Supramolecular Networks Depending on Guest Polymers in Vine-Twining Polymerization.

Amylose, a natural polysaccharide, acts as a host molecule to form supramolecular inclusion complexes in its enzymatically formation process, that is, phosphorylase-catalyzed enzymatic polymerization using the α-d-glucose 1-phosphate monomer and the maltooligosaccharide primer, in the presence of appropriate guest polymers (vine-twining polymerization). Furthermore, in the vine-twining polymerization using maltooligosaccharide primer-grafted polymers, such as maltoheptaose (G7)-grafted poly(γ-glutamic acid) (PGA), in the presence of poly(ε-caprolactone) (PCL), the enzymatically elongated amylose graft chains have formed inclusion complexes with PCL among the PGA main-chains to construct supramolecular networks. Either hydrogelation or aggregation as a macroscopic morphology from the products was observed in accordance with PCL/primer feed ratios. In this study, we evaluated macroscopic morphologies from such amylosic supramolecular networks with different guest polymers in the vine-twining polymerization using G7-grafted PGA in the presence of polytetrahydrofuran (PTHF), PCL, and poly(l-lactide) (PLLA). Consequently, we found that the reaction mixture using PTHF totally turned into a hydrogel form, whereas the products using PCL and PLLA were aggregated in the reaction mixtures. The produced networks were characterized by powder X-ray diffraction and scanning electron microscopic measurements. The difference in the macroscopic morphologies was reasonably explained by stabilities of the complexes depending on the guest polymers.

(Invited) Enzymatic Preparation of Supramolecular Hydrogels by Means of Amylosic Helical Assemblies

Amylose, which is a helical polysaccharide and well-known as a component of starch, forms controlled assemblies, such as a double helix and inclusion complexes depending on whether guest compounds are present or not. Therefore, amylose has been employed as a functional polymeric material. However, little has been reported regarding the formation of inclusion complexes between amylose and polymeric compounds. The main difficulty in incorporating polymeric compounds into the amylose cavity is that the driving force for the binding is only caused by hydrophobic interactions. Amylose, therefore, does not have sufficient ability to include the long polymeric chains. The author has developed a new method of the polymerization system called “vine-twining polymerization” for the formation of well-defined polysaccharide supramolecules, that is, amylose–polymer inclusion complexes. The method was achieved by phosphorylase-catalyzed enzymatic polymerization of α-D-glucose 1-phosphate (G-1-P) monomer from malooligosaccharide primer to produce amylose, in the presence of hydrophobic synthetic guest polymers. The image of this polymerization system is the similar as the way that the vines of a plant grow twining around a rod. By means of this method using designed guest polymers, the author has prepared hierarchically controlled amylosic supramolecular network materials, which formed hydrogels in aqueous media. For example, the author performed the preparation of supramolecular network hydrogels by the vine-twining polymerization using graft copolymers composed of hydrophobic graft guest polymers, such poly(γ-glutamic acid-graft-ε-caprolactone) (PGA-g-PCL). A supramolecular network hydrogel was obtained with the progress of the vine-twining polymerization, which showed a self-standing property. Macroscopic interfacial healing was achieved by the formation of inclusion complexes at interface between two hydrogel pieces through the enzymatic polymerization. A porous cryogel based on supramolecular network structure was obtained by lyophilization of a hydrogel. The XRD result of the lyophilized sample indicated the presence of inclusion complexes of amylose with the PCL graft-chains between intermolecular (PGA-g-PCL)s, which acted as supramolecular cross-linking points for the hydrogelation. Furthermore, ion gels were fabricated by soaking the hydrogels in an ionic liquid, 1-butyl-3-methylimidazolium chloride.

Gelation Behaviors of Amylosic Host-guest Supramolecular Networks Depending on Guest Polymers

Amylose, which is a helical polysaccharide, is known to form inclusion complexes with relatively low molecular weight guest compounds by hydrophobic interaction. However, little has been reported regarding the formation of inclusion complexes between amylose and polymeric compounds. The main difficulty in incorporating polymeric compounds into the amylose cavity is that the driving force for the binding is only caused by hydrophobic interactions. Amylose, therefore, does not have sufficient ability to include the long polymeric chains. The author has developed a new method of the polymerization system called “vine-twining polymerization” for the formation of well-defined polysaccharide supramolecules, that is, amylose–polymer inclusion complexes. The method was achieved by phosphorylase-catalyzed enzymatic polymerization of α-D-glucose 1-phosphate (G-1-P) monomer from malooligosaccharide primer to produce amylose, in the presence of hydrophobic synthetic guest polymers. The image of this polymerization system is the similar as the way that the vines of a plant grow twining around a rod. In this study, we attempted to prepare supramolecular hydrogels composed of amylosic host-guest networks from different guest polymers by the vine-twining polymerization using maltooligosaccharide primer-grafted poly(g-glutamic acid) in the presence of the following guest polymers, polytetrahydrofuran (PTHF), poly(ε-caprolactone) (PCL), and poly(L-lactide) (PLLA). Consequently, we found that the reaction mixture using PTHF totally turned into hydrogel form, whereas the products using PCL and PLLA were aggregated in the reaction mixtures. It was speculated that the different behaviors were owing to stabilities of inclusion complexes as cross-linking points in the networks, depending on the guest polymers.

Enzymatic preparation of functional polysaccharide hydrogels by phosphorylase catalysis

Abstract This article reviews enzymatic preparation of functional polysaccharide hydrogels by means of phosphorylase-catalyzed enzymatic polymerization. A first topic of this review deals with the synthesis of amylose-grafted polymeric materials and their formation of hydrogels, composed of abundant natural polymeric main-chains, such as chitosan, cellulose, xantham gum, carboxymethyl cellulose, and poly(γ-glutamic acid). Such synthesis was achieved by combining the phosphorylase-catalyzed enzymatic polymerization forming amylose with the appropriate chemical reaction (chemoenzymatic method). An amylose-grafted chitin nanofiber hyrogel was also prepared by the chemoenzymatic approach. As a second topic, the preparation of glycogen hydrogels by the phosphorylase-catalyzed enzymatic reactions was described. When the phosphorylase-catalyzed enzymatic polymerization from glycogen as a polymeric primer was carried out, followed by standing the reaction mixture at room temperature, a hydrogel was obtained. pH-Responsive amphoteric glycogen hydrogels were also fabricated by means of the successive phosphorylase-catalyzed enzymatic reactions.

Preparation of supramolecular network materials by means of amylose helical assemblies

Amylose, a natural polysaccharides, is a well-known functional material, because it forms double helix and inclusion complex assemblies depending on whether guest compounds are present or not, owing to its left-handed helical conformation. Amylose is precisely synthesized by phosphorylase-catalyzed enzymatic polymerization. In this study, we investigated the phosphorylase-catalyzed enzymatic polymerization initiated from maltoheptalose (primer for the polymerization)-grafted poly(γ-glutamic acid) in the presence of different feed ratios of a guest polymer, poly(ε-caprolactone) (PCL). In the absence of PCL or presence of less amount of PCL, the reaction mixtures totally turned into hydrogel form, predominantly composed of amylose double helixes. On the other hand, aggregates, which were largely composed of amylose inclusion complexes, were formed in the reaction mixtures in the presence of larger amount of PCL. The analytical results indicated that double helix cross-linking points participated into the formation of larger network structure, whereas smaller network structure was fabricated from inclusion complex cross-linking points. These structures on molecular level hierarchically constructed different macroscopic network sizes, leading to difference in the material forms.

Preparation and Material Application of Amylose-Polymer Inclusion Complexes by Enzymatic Polymerization Approach.

This review presents our researches on the preparation and material application of inclusion complexes that comprises an amylose host and polymeric guests through phosphorylase-catalyzed enzymatic polymerization. Amylose is a well-known polysaccharide and forms inclusion complexes with various hydrophobic small molecules. Pure amylose is produced by enzymatic polymerization by using α-d-glucose 1-phosphate as a monomer and maltooligosaccharide as a primer catalyzed by phosphorylase. We determined that a propagating chain of amylose during enzymatic polymerization wraps around hydrophobic polymers present in the reaction system to form inclusion complexes. We termed this polymerization “vine-twining polymerization” because it is similar to the way vines of a plant grow around a rod. Hierarchical structured amylosic materials, such as hydrogels and films, were fabricated by inclusion complexation through vine-twining polymerization by using copolymers covalently grafted with hydrophobic guest polymers. The enzymatically produced amyloses induced complexation with the guest polymers in the intermolecular graft copolymers, which acted as cross-linking points to form supramolecular hydrogels. By including a film-formable main-chain in the graft copolymer, a supramolecular film was obtained through hydrogelation. Supramolecular polymeric materials were successfully fabricated through vine-twining polymerization by using primer-guest conjugates. The products of vine-twining polymerization form polymeric continuums of inclusion complexes, where the enzymatically produced amylose chains elongate from the conjugates included in the guest segments of the other conjugates.

Saltiness enhancement of reduced sodium pork breakfast sausages

In this study, we investigated chmemoenzymatic synthesis of amylose-grafted poly(γ-glutamic acid) (PGA) as a new artificial saccharide-peptide conjugate composed of two biological macromolecules. Maltooligosaccharide as a primer of enzymatic polymerization by phosphorylase catalysis was first introduced on the PGA main chain by the condensation reaction using the condensing agent in NaOH aq. Thermostable phosphorylase-catalyzed enzymatic polymerization of α-d-glucose 1-phosphate (G-1-P) as a monomer was then performed from the primer chain ends of the product to obtain amylose-grafted PGAs, which formed hydrogels in reaction media depending on the G-1-P/primer feed ratios. The powder X-ray diffraction patterns of lyophilized samples (cryogels) from the hydrogels suggested that the amylose graft chains formed double helixes, which acted as cross-inking points for self-assembling hydrogelation. The scanning electron microscopic images of the cryogels showed regularly controlled porous morphologies. Moreover, pore sizes of the cryogels increased with increasing the G-1-P/primer feed ratios, whereas the degrees of substitution of primer on the PGA main chain did not obviously affect pore sizes.

α-Glucan Phosphorylase: A Useful Catalyst for Precision Enzymatic Synthesis of Oligo- and Polysaccharides

Oligo- and polysaccharides have complicated structures because of the structurally different monosaccharide units and differences in stereo- and regioarrangements of glycosidic bonds. As enzymatic reactions proceed in highly regio- and stereo-controlled manner, these are well accepted as a powerful tool for the precise synthesis of well-defined oligo- and polysaccharides. For example, α-glucan phosphorylase has increasingly been identified as the crucial and useful catalyst to provide oligo- and polysaccharide substrates with various structures, which are difficult to synthesize by conventional chemical synthetic approaches. This enzyme catalyzes the polymerization of α-D-glucose 1-phosphate (Glc-1-P) from a maltooligosaccharide primer to produce pure amylose. After the discussion on the principle reaction manner and specificity by α-glucan phosphorylase catalysis, the present review discloses the synthesis of various amylose diblock copolymers by α-glucan phosphorylase-catalyzed enzymatic polymerization using polymeric primers with a maltooligosaccharide moiety at the chain end. The following chemoenzymatic approach including α-glucan phosphorylase-catalyzed enzymatic polymerization is also disclosed. When bio-related polymeric primers with multiple maltooligosaccharide chains are used for enzymatic polymerization, amylose-grafted bio-related polymers, e.g., amylose-grafted polysaccharides and polypeptides, are produced. Based on the fact that α-glucan phosphorylase exhibits weak specificity for substrate recognition, the next section deals with the synthesis of non-natural oligo- and polysaccharides by α-glucan phosphorylase-catalyzed glycosylation using analog substrates of Glc-1-P, which have monosaccharide residues different from Glc. The last section describes the preparation of amylose inclusion supramolecules with polymeric guests from α-glucan phosphorylase- catalyzed enzymatic polymerization in the presence of guest polymers. This polymerization system has been referred to as “vine-twining polymerization.” Keywords: Amylose, analog substrate, enzymatic glycosylation and polymerization, α-glucan phosphorylase, maltooligosaccharide, nonnatural oligo- and polysaccharides, supramolecules.

Preparation and characterizations of all-biodegradable supramolecular hydrogels through formation of inclusion complexes of amylose

In this study, we found that the hydrogel containing cross-linking points of amylose/PCL graft chain inclusion complexes was obtained by vine-twining polymerization using a water-soluble chitosan-graft-poly(e-caprolactone) (chitosan-g-PCL). When phosphorylase-catalyzed enzymatic polymerization was performed in the presence of chitosan-g-PCL, the reaction mixture turned into a gel form. The IR spectrum of the sample obtained by lyophilization of the hydrogel indicated that amylose included the PCL graft chains in the intermolecular (chitosan-g-PCL)s to produce cross-linking points. The evaluation of the hydrogels obtained under various conditions was conducted by the shear-viscosity measurement. Because amylose, chitosan, and PCL are known to be enzymatically hydrolyzed, we have investigated enzymatic disruption behaviors of the hydrogels by hydrolysis of these components catalyzed by the corresponding enzymes, i.e., β-amylase, chitosanase, and lipase, respectively. In all cases, the hydrogels were transformed into solution or suspension states, indicating the occurrence of enzymatic disruption of hydrogels. Furthermore, the hydrogel was reproduced when the vine-twining polymerization was carried out in the presence of phosphorylase in the resulting solution by the β-amylase treatment.

Chemoenzyamtic synthesis and self-assembling gelation behavior of amylose-grafted poly(γ-glutamic acid)

In this study, we investigated chmemoenzymatic synthesis of amylose-grafted poly(γ-glutamic acid) (PGA) as a new artificial saccharide-peptide conjugate composed of two biological macromolecules. Maltooligosaccharide as a primer of enzymatic polymerization by phosphorylase catalysis was first introduced on the PGA main chain by the condensation reaction using the condensing agent in NaOH aq. Thermostable phosphorylase-catalyzed enzymatic polymerization of α-d-glucose 1-phosphate (G-1-P) as a monomer was then performed from the primer chain ends of the product to obtain amylose-grafted PGAs, which formed hydrogels in reaction media depending on the G-1-P/primer feed ratios. The powder X-ray diffraction patterns of lyophilized samples (cryogels) from the hydrogels suggested that the amylose graft chains formed double helixes, which acted as cross-inking points for self-assembling hydrogelation. The scanning electron microscopic images of the cryogels showed regularly controlled porous morphologies. Moreover, pore sizes of the cryogels increased with increasing the G-1-P/primer feed ratios, whereas the degrees of substitution of primer on the PGA main chain did not obviously affect pore sizes.

Precision Synthesis of Functional Polysaccharide Materials by Phosphorylase-Catalyzed Enzymatic Reactions.

In this review article, the precise synthesis of functional polysaccharide materials using phosphorylase-catalyzed enzymatic reactions is presented. This particular enzymatic approach has been identified as a powerful tool in preparing well-defined polysaccharide materials. Phosphorylase is an enzyme that has been employed in the synthesis of pure amylose with a precisely controlled structure. Similarly, using a phosphorylase-catalyzed enzymatic polymerization, the chemoenzymatic synthesis of amylose-grafted heteropolysaccharides containing different main-chain polysaccharide structures (e.g., chitin/chitosan, cellulose, alginate, xanthan gum, and carboxymethyl cellulose) was achieved. Amylose-based block, star, and branched polymeric materials have also been prepared using this enzymatic polymerization. Since phosphorylase shows a loose specificity for the recognition of substrates, different sugar residues have been introduced to the non-reducing ends of maltooligosaccharides by phosphorylase-catalyzed glycosylations using analog substrates such as α-d-glucuronic acid and α-d-glucosamine 1-phosphates. By means of such reactions, an amphoteric glycogen and its corresponding hydrogel were successfully prepared. Thermostable phosphorylase was able to tolerate a greater variance in the substrate structures with respect to recognition than potato phosphorylase, and as a result, the enzymatic polymerization of α-d-glucosamine 1-phosphate to produce a chitosan stereoisomer was carried out using this enzyme catalyst, which was then subsequently converted to the chitin stereoisomer by N-acetylation. Amylose supramolecular inclusion complexes with polymeric guests were obtained when the phosphorylase-catalyzed enzymatic polymerization was conducted in the presence of the guest polymers. Since the structure of this polymeric system is similar to the way that a plant vine twines around a rod, this polymerization system has been named “vine-twining polymerization”. Through this approach, amylose supramolecular network materials were fabricated using designed graft copolymers. Furthermore, supramolecular inclusion polymers were formed by vine-twining polymerization using primer–guest conjugates.