Chemoenzymatic synthesis of functional amylosic materials

Introduction

Amylose is a naturally occurring polysaccharide found in starch, which is composed of repeating glucose units linked through α-(1→4) glycosidic bonds [1]. Because the natural amylose product isolated from starch usually contains a small amount of amylopectin, which is the other component of starch, one of the efficient methods for the production of pure amylose is the following enzymatic polymerization approach, catalyzed by α-glucan phosphorylase (EC 2.4.1.1) [2–5].

α-Glucan phosphorylase is widely found in nature in animals, plants, and microorganisms, and it catalyzes the reversible phosphorolysis reactions at the nonreducing end of α-(1→4)-glucan, i.e., amylose, in the presence of inorganic phosphate to produce α-d-glucose 1-phosphate (G-1-P) [6–8]. It is well known that because of the reversibility of the reaction, α-glucan phosphorylase also catalyzes the enzymatic polymerization of G-1-P as a monomer to produce pure amylose with a well-defined structure (Fig. 1). To initiate the polymerization, maltooligosaccharides with degrees of polymerization (DPs) higher than that of the smallest maltooligosaccharide, which is recognized by α-glucan phosphorylase, should be present as a primer of the polymerization in the reaction system. The smallest substrates for phosphorolysis and polymerization by potato α-glucan phosphorylase catalysis are typically maltopentaose and maltotetraose, respectively. The polymerization proceeds in a chain-growth manner because the reaction is exactly initiated at the nonreducing end of the primer. Therefore, the α-glucan phosphorylase-catalyzed polymerization proceeds analogously to a living polymerization. Accordingly, the molecular weights of the produced amyloses can be controlled by the G-1-P/primer feed molar ratios, and their distributions are typically narrow (M_w/M_n < 1.2) [5, 9].

Fig. 1

α-Glucan phosphorylase-catalyzed enzymatic polymerization to produce amylose.

The α-glucan phosphorylase-catalyzed enzymatic polymerization can take place using immobilized maltooligosaccharides as the primer, whose reducing ends are covalently attached to another material such as a polymeric chain, because the reducing end of the primer does not participate in the polymerization [10]. The resulting immobilized product is typically multifunctional due to the presence of the plural nonreducing α-(1→4)-linked glucan ends. Using α-glucan phosphorylase-catalyzed enzymatic polymerization from the primer chains immobilized on synthetic polymers, various amylose-grafted polymeric materials have been synthesized (Fig. 2) [11–14], such as amylose-grafted polystyrene [15], polyacetylene [16, 17], poly(dimethylsiloxane) [18], poly(vinyl alcohol) [19], and poly(l-glutamic acid) [20].

Fig. 2

α-Glucan phosphorylase-catalyzed enzymatic polymerization using immobilized primer to produce amylose-grafted polymeric material.

Amylose has recently been recognized as a candidate for functional polymeric materials because it acts as a host molecule owing to its helical conformation and forms inclusion complexes with various guest molecules with a relatively low molecular weight [21]. The driving force for inclusion of the guest molecules in the cavity is the host-guest hydrophobic interaction, as the inside of the amylose helix is hydrophobic because the hydrophilic hydroxy groups of the glucose residues are situated on the outer part of the helix. Therefore, the guest molecules to be included by amylose are typically required to be hydrophobic [22–28]. However, little has been reported regarding the formation of inclusion complexes between amylose and polymeric guest molecules [29–31]. The principal difficulty for incorporating polymeric guest molecules into the amylose cavity is that the driving force for binding is caused only by weak hydrophobic interactions. It is therefore considered that amylose does not have a sufficient ability to directly include the long chains of polymeric guests into its cavity. An efficient method for the formation of amylose-polymer inclusion complexes has been found by means of α-glucan phosphorylase-catalyzed enzymatic polymerization. In the enzymatic polymerization system, which disperses appropriate hydrophobic guest polymers in an aqueous polymerization solvent, the propagation proceeds with the formation of inclusion complexes between the produced amylose and the guest polymers. The representation of this reaction system for the construction of such amylose-polymer inclusion complexes is similar to the way that vines of plants grow twining around a rod (Fig. 3) [14, 32–36]. Accordingly, it has been proposed that this polymerization method be named “vine-twining polymerization.” As the guest polymers for this polymerization system, hydrophobic polyethers (e.g., polytetrahydrofuran and polyoxetane) [37, 38], polyesters [e.g., poly(δ-valerolactone) and poly(ε-caprolactone)] [39, 40], and polycarbonates [e.g., poly(tetramethylene carbonate)] [41] have been used to form inclusion complexes with amylose.

Fig. 3

Image of “vine-twining polymerization” and representative guest polymers.

On the basis of the above background, in this article, the author reviews the synthesis of functional amylosic materials by means of the chemoenzymatic approach including the α-glucan phosphorylase-catalyzed enzymatic polymerization. The first topic is the chemoenzymatic synthesis of amylose-grafted heteropolysaccharide materials, which were efficiently achieved by the chemical immobilization of maltooligosaccharide primers on appropriate polysaccharide chains, followed by the α-glucan phosphorylase-catalyzed enzymatic polymerization. As a second topic, the vine-twining approach employing designed guest polymeric compounds successfully led to the production of amylosic supramolecular hydrogels and films with hierarchically controlled structures.

Chemoenzymatic synthesis of amylose-grafted heteropolysaccharide materials

In addition to linear polysaccharides such as cellulose, chitin/chitosan, and amylose, grafted structures have often been found in natural polysaccharides, where a polysaccharide of the main-chain is functionalized with different kinds of graft polysaccharide chains through glycosidic bonds [42]. Such a chemical structure probably contributes to their high-performance functions in nature. Accordingly, because the development of efficient methods for the preparation of the artificial grafted polysaccharides is a promising topic in polysaccharide material research fields, amylose-grafted heteropolysaccharide materials have been synthesized by the chemoenzymatic approach including the α-glucan phosphorylase-catalyzed enzymatic polymerization of G-1-P (Fig. 4) [11–14]. As aforementioned, the maltooligosaccharide primer has to be present in the system to initiate the polymerization. To produce the amylose-grafted heteropolysaccharides, therefore, maltooligosaccharides were first introduced onto the main-chain polysaccharides by appropriate chemical reactions, and then the α-glucan phosphorylase-catalyzed enzymatic polymerization from the immobilized primers was performed (chemoenzymatic method). Two types of chemical reactions have been employed to introduce maltooligosaccharides onto the main-chain polysaccharides: reductive amination of a maltooligosaccharide with cationic polysaccharides having amino groups using reductants and condensation of an amine-functionalized maltooligosaccharide with anionic polysaccharides having carboxylate groups using condensing agents.

Fig. 4

Chemoenzymatic synthesis of amylose-grafted heteropolysaccharide.

By means of the former approach using chitosan as a cationic polysaccharide with amino groups at the C-2 position in repeating glucosamine units, a maltooligosaccharide-grafted chitosan was prepared [degree of substitution (DS); 3.3 %]. This material was further converted into a maltooligosaccharide-grafted chitin by N-acetylation using acetic anhydride. Then, the α-glucan phosphorylase-catalyzed enzymatic polymerization of G-1-P from the maltooligosaccharide chains on the chitin and chitosan derivatives was performed to obtain amylose-grafted chitin/chitosan (Fig. 5) [43, 44]. A hydrogel of the amylose-grafted chitosan was formed by drying the polymerization mixture slowly at 40–50 °C.

Fig. 5

Structures of amylose-grafted chitin, chitosan, cellulose, alginate, xanthan gum, and carboxymethyl cellulose.

A similar approach was used to synthesize an amylose-grafted cellulose (Fig. 5) [45]. A partially aminated cellulose, which was successfully prepared by successive partial tosylation of the OH groups at the C-6 positions, displacement of the tosylates by azido groups, and reduction to amino groups, was first subjected to reductive amination with a maltooligosaccharide to give a maltooligosaccharide-grafted cellulose (DS; 3.1 %). Subsequently, the amylose-grafted cellulose was synthesized by the α-glucan phosphorylase-catalyzed enzymatic polymerization of G-1-P from the maltooligosaccharide chains on the cellulose derivative. The product has a very interesting and unique structure because it is made up of two representative polysaccharide chains, cellulose and amylose, which are composed of the same glucose units but linked through different glycosidic bonds [β-(1→4) and α-(1→4), respectively]. When the polymerization solution was left standing on a Petri dish at room temperature for several days, it was completely converted to the gel form. This hydrogel was tougher than that formed from the amylose-grafted chitosan and was further converted to the solid (or film) material by drying under ambient atmosphere. The addition of water to the solid gave the gel material again. The switching between the solid and gel could be repeated by the drying and wetting processes.

On the other hand, the latter condensation reaction was employed to introduce maltooligosaccharides to alginate [46], xanthan gum [47], and carboxymethyl cellulose (CMC) [48], which are anionic polysaccharides. A maltooligosaccharide with an amino group at the reducing end was first prepared by the reaction of a maltooligosaccharide lactone with 2-azidoethylamine, followed by a reduction using NaBH₄. Then, it was chemically introduced onto the anionic polysaccharides by condensation with the carboxylates of the polysaccharides using water-soluble carbodiimide (WSC)/N-hydroxysuccinimide (NHS) as the condensing agents to produce maltooligosaccharide-grafted alginate, xanthan gum, and CMC. Then, the α-glucan phosphorylase-catalyzed enzymatic polymerization of G-1-P from the maltooligosaccharide chains on the products was conducted to produce amylose-grafted alginate, xanthan gum, and CMC (Fig. 5).

A film of the amylose-grafted CMC was obtained by drying the thinly spread alkaline solution [0.040 g in 0.50 mol/L aqueous NaOH (1.50 mL)] (DS and DP of the amylose graft chain: 35.4 % and 214, respectively) [48]. The SEM image of the film showed highly entangled nanofibers (Fig. 6a). After removal of the residual alkali in the film by immersion in water, the SEM image showed that the nanofibers were merged at the interface, while the fiber arrangement was maintained (Fig. 6b). Conventional approaches to the production of cellulosic nanofibers are mainly top-down procedures that break down the starting bulk materials from native cellulose resources. The present method is a completely different approach to produce cellulosic nanofibers, a self-assembling generative (bottom-up) route that provides control over the morphology and self-assembly of heteropolysaccharides to produce new nanofibrillated materials. The stress-strain curve of the film after washing out the alkali by tensile testing showed superior mechanical properties to a CMC film (Fig. 7).

Fig. 6

SEM images of amylose-grafted CMC films before (a) and after (b) washing with water.

Fig. 7

Stress-strain curves of amylose-grafted CMC film (a) and CMC film (b) under tensile mode.

Preparation of amylosic supramolecular materials by a vine-twining polymerization approach

Recently, the aforementioned vine-twining approach was applied to the architecture of amylosic supramolecular materials [49, 50]. For example, the preparation of an amylosic supramolecular hydrogel was achieved through the formation of inclusion complexes by amylose during the progress of the α-glucan phosphorylase-catalyzed polymerization in the vine-twining polymerization system (Fig. 8) [49]. In this study, a graft copolymer as the guest compound was designed and chemically synthesized. This guest forms network structures by the formation of intermolecular inclusion complexes with amylose by vine-twining polymerization, giving rise to cross-linking points for the construction of hydrogels. Such a graft copolymer has to be dissolved in water at the initial stage of the polymerization to act as a component of the hydrogel; on the other hand, hydrophobicity is necessary so that the graft chain structure can be included by amylose between the intermolecular graft copolymers. Taking these two antagonistic properties required for the graft copolymer structure into account, poly(acrylic acid sodium salt-graft-δ-valerolactone) (PAA-Na-g-PVL) was designed, because PAA-Na has a strong hydrophilic nature to contribute to the water-solubility of the graft copolymer, and PVL has been reported to be included by amylose in the vine-twining polymerization [40]. When the vine-twining polymerization was conducted by the α-glucan phosphorylase-catalyzed enzymatic polymerization using G-1-P and a maltooligosaccharide in the presence of PAA-Na-g-PVL, the reaction mixture turned into a hydrogel. Because the produced amylose included the PVL graft chains in the intermolecular guest copolymers as the polymerization progressed, the formed inclusion complexes acted as the cross-linking points for the formation of the supramolecular hydrogel. Furthermore, enzymatic disruption and reproduction of the hydrogel were achieved by the combination of the β-amylase-catalyzed hydrolysis of the amylose component and the re-formation of amylose by the α-glucan phosphorylase-catalyzed enzymatic polymerization. Therefore, it should be noted that the present hydrogel exhibited enzymatically recyclable behavior by means of the two enzyme-catalyzed reactions.

Fig. 8

Preparation of amylosic supramolecular hydrogel through formation of inclusion complexes by vine-twining polymerization.

The author also investigated the preparation of an amylosic supramolecular film through hydrogelation by vine-twining polymerization using another designed graft copolymer (Fig. 9) [50]. To obtain the film form from the amylosic supramolecular hydrogel, CMC was selected as the main-chain structure in place of the PAA-Na used in the above study because of the film formability of CMC in addition to its water-soluble nature. Accordingly, CMC-g-poly(ε-caprolactone) (PCL) was chemically synthesized (PCL has also been found to be included by amylose in the vine-twining polymerization [39, 40]), and the hydrogel was prepared according to the vine-twining polymerization procedure using this graft copolymer. A film was obtained by adding water to a powdered sample obtained by lyophilization of the hydrogel, followed by drying. The mechanical properties of the resulting films composed of appropriate amounts of the amylose molecules with moderate molecular weights were superior to those of a CMC film. This result indicated that the supramolecular cross-linking structure in the films contributed to enhancing the mechanical properties.

Fig. 9

Preparation of amylosic supramolecular film through formation of hydrogel by vine-twining polymerization.

Conclusion and future outlook

In this review article, an overview of chemoenzymatic synthesis of functional amylosic materials by means of the α-glucan phosphorylase-catalyzed enzymatic polymerization of G-1-P, initiated from a maltooligosaccharide as the primer is presented. The amylose-grafted heteropolysaccharide materials with various abundant polysaccharide main chains were synthesized by a chemoenzymatic approach. The method included the introduction of the maltooligosaccharide primers on the main-chain polysaccharides by appropriate chemical reactions and the subsequent α-glucan phosphorylase-catalyzed enzymatic polymerization from the primer chain ends of the products. The resulting heteropolysaccharides exhibited original functions such as hydrogel- and film-forming properties. Furthermore, the amylosic supramolecular hydrogel and film were produced through the formation of inclusion complexes with amylose in the vine-twining polymerization approach using the designed graft copolymers, which were prepared by appropriate chemical reactions. The method was successful when performing the α-glucan phosphorylase-catalyzed enzymatic polymerization in the presence of the graft copolymers composed of hydrophilic main chains and hydrophobic guest graft chains. The resulting hydrogel and film showed enzymatic disruption-reproduction behavior and superior mechanical properties, respectively.

Because enzymatic reactions provides functional materials with well-defined structures in regio- and stereocontrolled fashions, the chemoenzymatic synthesis of polysaccharide materials with the wide variety of structures by the a-glucan phosphorylase catalysis, which are hardly produced by conventional chemical synthetic approaches, will be increasingly important and useful in material research field in the future. For example, the amylosic materials have potentials for the practical applications in the biomedical and environmentally benign fields of research because of the biodegradable, eco-friendly, and non-toxic properties of amylose.