Enzymatic preparation of functional polysaccharide hydrogels by phosphorylase catalysis

Preparation of polysaccharide hydrogels by phophorylase-catalyzed enzyamtic polymerization using immobilized primers

Polysaccharide primers having multiple maltooligosaccharide chains have been used for the phosphorylase-catalyzed enzymatic polymerization to produce hydrogels. The maltooligosaccharide primer chains have been introduced onto the main-chain polysaccharides by the following two types of the chemical reactions; one is reductive amination of maltooligosaccharides with basic polysaccharides having amino groups using reductants and the other one is condensation of amine-functionalized maltooligosaccharides with acidic polysaccharides having carboxylate groups using condensing agents (Fig. 2) [22], [28], [31], [32], [33], [34]. By means of the former reaction, followed by the phosphorylase-catalyzed enzymatic polymerization (chemoenzymatic approach), amylose-grafted chitosan and cellulose hydrogels have been obtained (Fig. 3) [35], [36], [37].

Fig. 3:

Amylose-grafted heteropolysaccharides from basic and acidic polysaccharides.

Chitosan is composed of β(1→4)-linked d-glucosamine (GlcN) repeating units, and is prepared by N-deacetylation of a natural polysaccharide, chitin [38]. The reducing end of maltoheptaose (G₇) was first reacted with amino groups at C-2 position in chitosan by the reductive amination using NaBH₃CN in a mixed solvent of aqueous acetic acid/methanol to give a maltooligosaccharide-grafted chitosan. The phosphorylase-catalyzed enzymatic polymerization of G-1-P from the maltooligosaccharide primers on the product was then carried out to obtain an amylose-grafted chitosan. When the reaction mixture was slowly dried in the vessel at 40–50°C, a hydrogel of the product was produced. The formation of double helix cross-linking points between the amylose graft chains on the chitosan probably induced the hydrogelation.

The amylose-grafted cellulose hydrogel was also fabricated by the chemoenzymatic method. To conduct reductive amination for the introduction of maltooligosaccharide primers, primary hydroxy groups at C-6 position in cellulose were first converted into amino groups by the following successive reactions; partial tosylation of hydroxy groups, displacement of the tosylates by azido groups, and reduction. Reductive amination of the produced amine-functionalized cellulose with G₇ was conducted to obtain a maltooligosaccharide-grafted cellulose. The phosphorylase-catalyzed enzymatic polymerization of G-1-P from the maltooligosaccharide primers on cellulose main-chain was subsequently carried out to produce an amylose-grafted cellulose. The reaction mixture totally turned into the gel form when it was left standing on a Petri dish at room temperature for several days. The resulting hydrogel was tougher than that formed from the amylose-grafted chitosan. A solid material was obtained by drying the hydrogel under the ambient atmosphere. The addition of water to the solid resulted in the hydrogelation again. Such conversion cycle was repeated by the wetting and drying process. Moreover, the reaction mixture of the enzymatic polymerization was spread thinly on a glass plate, and subsequently left standing at room temperature, resulting in a film.

Amylose-grafted chitin nanofibers were also fabricated by the similar chemoenzymatic approach [39]. Nanofibrillated materials from native chitin, so-called chitin nanofibers, have increasingly attracted much attention and are expected to find applications as new functional materials owing to their outstanding characteristics such as lightweight, high tensile strength, and low thermal expansion coefficient [40], [41], [42]. As native chitin sources are made up from assemblies of nanostructures, several methods for disentanglement of the assemblies have efficiently been developed to obtain nanofiber dispersions in water [43], [44], [45], [46]. It was also reported that re-dispersible amidinium chitin nanofibers were obtained from an amidinated chitin by CO₂ gas bubbling with ultrasonic treatment in water [47]. For the chemoenzymatic approach, malooligosaccharide primers were introduced by reductive amination with amino groups present on the nanofibers (Fig. 4). Grafting of amylose on the amidinium chitin nanofibers was then investigated by the phosphorylase-catalyzed enzymatic polymerization from the maltooligosaccharide graft chains to produce amylose-grafted chitin nanofiber materials. The reaction mixtures turned into hydrogels upon increasing the monomer/primer feed ratios. The formation of double helixes from a part of amylose graft chains closely preset among the nanofibers contributed to forming the smaller networks, resulting in hydrogelation. Lyophilization of the hydrogels gave rise to the controlled microstructures, which were changed from fibrous to porous morphologies depending on the molecular weights of the amylose graft chains. Most of the amylose graft chains with the higher molecular weights, which did not participate into double helixes, formed amorphous membranes in the nanofiber networks by lyophilization, to construct the porous structure.

Fig. 4:

Chemoenzymatic synthesis of amylose-grafted amidinium chitin nanofiber.

For the latter condensation reaction with acidic polysaccharides having carboxylate groups in the chemoenzymatic approach, functionalization of the reducing end of G₇ by an amino group was necessary because it can potentially react with the carboxylates by condensation to obtain maltooligosaccharide-grafted polysaccharides (Fig. 2). First, G₇ lactone was synthesized by successive I₂/KOH oxidation at the reducing end, cation exchange, and intramolecular dehydrative condensation [48]. The product was then reacted with 2-azidoethylamine, followed by reduction by NaBH₄, to give an amine-functionalized maltooligosaccharide at the reducing end. This substrate was reacted with xanthan gum and carboxymethyl cellulose sodium salt (NaCMC) by condensation. Then, the products were used for the phosphorylase-catalyzed enzymatic polymerization to obtain the corresponding hydrogels (Fig. 3) [49], [50], [51].

Xanthan gum, which is a water soluble polysaccharide produced by Xanthomonas campestris, has a cellulose-type main-chain (β(1→4)-glucan) with trisaccharide side-chains (mannose-β(1→4)-glucuronic acid-β(1→2)-mannose-α(1→3)-) attached to alternating glucose units in the main-chain [52]. C-6-Position in α-mannoside unit is acetylated and C-4 and -6 positions in β-mannoside unit are partially pyruvated. For the chemoenzymatic approach, condensation of an amine-functionalized maltooligosaccharide with carboxylates of xanthan gum was first conducted using water-soluble carbodiimide (WSC)/N-hydroxysuccinimide (NHS) as the condensing agent to produce a maltooligosaccharide-grafted xanthan gum. The phosphorylase-catalyzed enzymatic polymerization of G-1-P from the maltooligosaccharide primers on xanthan gum main-chain was then performed to obtain an amylose-grafted xanthan gum. An ion gel was formed by mixing and stirring the product with an ionic liquid, 1-butyl-3-methylimidazolium chloride, until the mixture became homogeneous. Exchange of dispersion media was conducted by immersing the ion gel in water to produce a hydrogel. Ionically cross-linking of carboxylates with trivalent metal cations in the hydrogel was additionally attempted by soaking it in aqueous FeCl₃. When the mechanical properties of the ionically cross-linked hydrogels with Fe³⁺ were evaluated by compressive testing, the fracture strain values increased with increasing functionalities and DPs of the amylose graft-chains. The formation of double helixes between the amylose graft chains probably contributed to the formation of looser network structures in the hydrogels, leading to the enhancement of elasticity.

An amylose-grafted NaCMC was also synthesized by the same chemoenzymatic approach to the amylose-grafted xanthan gum. NaCMC is one of the most widely used cellulose derivatives in practical application fields such as food industry [10]. A maltooligosaccharide-grafted NaCMC was first synthesized by condensation of an amine-functionalized maltooligosaccharide with carboxylates of NaCMC in the presence of WSC/NHS. The phosphorylase-catalyzed enzymatic polymerization of G-1-P using the maltooligosaccharide-grafted NaCMC was performed in sodium acetate buffer to produce a hydrogel. Lyophilization of the hydrogel was conducted to isolate the amylose-grafted NaCMC. A film was formed by drying a thinly spread alkaline solution of the isolated product in aqueous NaOH. The SEM image of the film showed the morphology of highly entangled nanofibers. NaOH present in the film was then removed by immersing in water. The SEM image of the resulting film showed that nanofibers were merged each other at interfacial area with remaining the fiber arrangements. The following self-assembling generative (bottom-up) process has been proposed for the formation of the nanofiber film. The introduction of the amylose graft chains on NaCMC prevented the construction of random-coil conformation and provided the rigid nature of the NaCMC chain. Some of these rigid materials regularly assembled in nano-scale with the slight double helix formation from the amylose graft chains on NaCMCs by drying the aqueous alkaline solution, but, the produced nano-assemblies do not aggregate further because of the prevention of the double helix formation from most of the amylose chains under the strong basic conditions. By washing out NaOH from the film, double helixes from amylose graft chains on the nanofibers are largely formed, resulting in merging on the surface of the fibers.

The above chemoenzymatic approach including condensation was employed for the synthesis of amylose-grafted polypeptide [53]. As the polypeptide main-chain, poly(γ-glutamic acid) (PGA) was selected because it has carboxylate groups and has been used for multifarious potential applications in foods, pharmaceuticals, healthcare, water treatment, and other fields (Fig. 5) [54], [55]. Maltooligosaccharide primers were first introduced on PGA main-chain by the condensation reaction using the WSC/NHS condensing agent in aqueous NaOH. The phosphorylase-catalyzed enzymatic polymerization was then performed from the primer chain ends of the product to obtain amylose-grafted PGAs, which formed hydrogels in reaction media depending on monomer/primer feed ratios. The amylose graft chains formed double helixes, which acted as cross-linking points for self-assembling hydrogelation. The SEM images of the cryogels, which were prepared by lyophilization of the hydrogels showed regularly controlled porous morphologies. Furthermore, pore sizes increased with increasing monomer/primer feed ratios, while the degrees of substitution of primer on the PGA main chain did not obviously affect pore sizes.

Fig. 5:

Chemoenzymatic synthesis of amylose-grafted PGA.

Preparation of polysaccharide hydrogels by phophorylase-catalyzed enzyamtic polymerization using natural polymeric primer, glycogen

When the phosphorylase-catalyzed enzymatic polymerization of G-1-P from glycogen as a polymeric primer was carried out, followed by standing the reaction mixture at room temperature for 24 h, a hydrogel was formed (Fig. 6) [56]. During the enzymatic polymerization, double helixes were formed from the elongated amylose chains among glycogen molecules, which acted as cross-linking points to give the hydrogel. The mechanical properties of the hydrogels produced by the different G-1-P/glycogen ratios were evaluated by compressive testing. The resulting stress-strain curves indicated that the properties were changed from elastic to hard and brittle with increasing the amounts of glycogen molecules. Because the number of elongated amylose chains increased with increasing amounts of glycogen, the hydrogel strength increased as more double helix cross-linking points were formed.

Fig. 6:

Preparation of (amphoteric) glycogen hydrogels by phosphorylase-catalyzed enzymatic reactions.

Lyophilziation of the hydrogels readily gave cryogels with porous morphology. Compressive testing of the cryogels showed that the mechanical properties were strengthened with increasing the amounts of glycogen. This is because of the formation of smaller networks owing to the higher cross-linking densities from the larger amounts of glycogen. A transparent film was produced by casting the alkaline solution of the cryogel on a glass plate and drying.

pH-Responsive amphoteric glycogen hydrogels were also fabricated by means of the similar approach through the phosphorylase-catalyzed enzymatic polymerization [57]. Based on the fact that phosphorylase catalyzes the enzymatic reactions using analog substrates of G-1-P such as α-d-glucuronic acid 1-phosphate (acidic substrate) and α-d-glucosamine 1-phosphate (basic substrate) [58], [59], amphoteric glycogens having both glucuronic acid and glucosamine units at the non-reducing ends have been synthesized by phosphorylase-catalyzed successive enzymatic reactions using the above analog substrates (Fig. 6). The functionalities of the acidic and basic units were controlled by the feed ratios. Furthermore, the products were found to exhibit the inherent isoelectric point (pI) values, which were reasonably changed in accordance with the acidic/basic unit ratios in the amphoteric products.

Accordingly, the amphoteric glycogen hydrogels were produced by the formation of double helix cross-linking points of the elongated amylose chains by the phosphorylase-catalyzed enzymatic polymerization of G-1-P from the nonfunctionalized, non-reducing ends in the amphoteric glycogens (Fig. 6). The amphoteric hydrogels produced exhibited pH responsive behavior. Under strong basic conditions by addition of NaOH aqueous solution, the gels were solubilized by the dissociation of double helix cross-linking points. By changing pH to weak basic by addition of acetic acid aqueous solution, reconstruction of double helix cross-linking points occurred, resulting in re-hydrogelation. The hydrogels exhibited pH-responsive shrinking/swelling behavior. The hydrogels are shrunk at pH=pI owing to the absence of intermolecular repulsive forces by neutral charges on the surfaces. At both weakly acidic and basic pHs, on the other hand, the surfaces of glycogens were charged, leading to swelling of the hydrogels owing to cationic/cationic or anionic/anionic electrostatic repulsion.