Thermostable ?-Glucan Phosphorylase-Catalyzed Enzymatic Copolymerization to Produce Partially 2-Deoxygenated Amyloses

: ?-Glucan phosphorylase catalyzes the enzymatic polymerization of ?-d-glucose 1-phosphate (Glc-1-P) monomers from a maltooligosaccharide primer to produce ?(1?4)-glucan—i.e., amylose. In this study, by exploiting the weak specificity for the substrate recognition of a thermostable ?-glucan phosphorylase (from Aquifex aeolicus VF5), we investigated the enzymatic copolymerization of 2-deoxy-?-d-glucose 1-phosphate (dGlc-1-P), which was produced in situ from d-glucal, with Glc-1-P to obtain non-natural heteropolysaccharides composed of ?(1?4)-linked dGlc/Glc units—i.e., partially 2-deoxygenated amylose. The reactions were carried out at different monomer feed ratios using a maltotriose primer at 40 °C for 24 h. The products were precipitated from the reaction medium, isolated by centrifugation, and subjected to 1H NMR spectroscopic and powder X-ray diffraction measurements to evaluate their chemical and crystalline structures, respectively. Owing to its amorphous nature, the partially 2-deoxygenated amylose with adapted unit ratios formed a film when subjected to a casting method. Abstract: α -Glucan phosphorylase catalyzes the enzymatic polymerization of α - d -glucose 1-phosphate (Glc-1-P) monomers from a maltooligosaccharide primer to produce α (1 → 4)-glucan—i.e., amylose. In this study, by exploiting the weak speciﬁcity for the substrate recognition of a thermostable α -glucan phosphorylase (from Aquifex aeolicus VF5), we investigated the enzymatic copolymerization of 2-deoxy- α - d -glucose 1-phosphate (dGlc-1-P), which was produced in situ from d -glucal, with Glc-1-P to obtain non-natural heteropolysaccharides composed of α (1 → 4)-linked dGlc / Glc units—i.e., partially 2-deoxygenated amylose. The reactions were carried out at di ﬀ erent monomer feed ratios using a maltotriose primer at 40 ◦ C for 24 h. The products were precipitated from the reaction medium, isolated by centrifugation, and subjected to 1 H NMR spectroscopic and powder X-ray di ﬀ raction measurements to evaluate their chemical and crystalline structures, respectively. Owing to its amorphous nature, the partially 2-deoxygenated amylose with adapted unit ratios formed a ﬁlm when subjected to a casting method.


Introduction
Polysaccharides are widely distributed and play important roles in nature as structural materials, suppliers of energy, and key materials for specific biological and vital functions [1]. Natural polysaccharides provide a great variety of chemical structures owing to the many types of monosaccharide units and different stereo-and regio-arrangements of the glycosidic linkages in the polymeric chains, which contributes to their many different in vivo functions [2]. In addition to homopolysaccharides composed of monosaccharide repeating units, such as cellulose, starch, and chitin, there are a variety of natural heteropolysaccharides which are composed of multiple types of monosaccharide units, such as glucomannans and glycosaminoglycans [3][4][5]. Accordingly, the synthesis of non-natural heteropolysaccharides with well-defined structures has attracted much attention, since they can be expected to exhibit new functions for possible applications as biofunctional materials in the fields of biomedical and tissue engineering.
The copolymerization approach catalyzed by enzymes (enzymatic copolymerization) has been identified as an efficient method for the synthesis of well-defined non-natural heteropolysaccharides [8][9][10]25]. For example, the hyaluronidase-catalyzed enzymatic copolymerization led to a hyaluronic acid-chondroitin hybrid polysaccharide [26]. In addition, we also revealed that the enzymatic copolymerization catalyzed by a thermostable α-glucan phosphorylase is a useful tool to synthesize non-natural heteropolysaccharides with a well-defined structure. The thermostable α-glucan phosphorylase-catalyzed enzymatic copolymerization of analog substrates, such as GlcN-1-P and α-d-mannose (Man) 1-phosphate, with the native substrate Glc-1-P, efficiently progressed under the above-mentioned conditions used for the removal of Pi to generate a non-natural glucosaminoglucan and mannoglucan composed of GlcN/Glc and Man/Glc units, respectively [27,28].
It was also reported that α-glucan phosphorylase isolated from several sources, such as potato, rabbit muscle, and Escherichia coli, could catalyze the enzymatic polymerization of 2-deoxy-α-d-glucose 1-phosphate (dGlc-1-P), which was produced in situ from 1,2-dideoxy-d-glucose (d-glucal) in the presence of Pi, using a maltooligosaccharide primer to afford α(1→4)-linked 2-deoxyglucose chains-i.e., 2-deoxyamylose [29][30][31]. The following mechanism was reported to allow for the generation of 2-deoxyamylose upon α-glucan phosphorylase-catalyzed enzymatic polymerization using d-glucal [29,32]. First, a dGlc unit is either chemically or enzymatically transferred from d-glucal to the primer via an addition reaction assisted by Pi. In the second step, a dGlc residue is released upon α-glucan phosphorylase-catalyzed enzymatic phosphorolysis to yield dGlc-1-P in situ, which acts as substrate of the enzymatic polymerization. It was found that when a thermostable α-glucan phosphorylase (from Aquifex aeolicus VF5) was used in the enzymatic polymerization using d-glucal, the mechanism of the in situ production of dGlc-1-P most likely differed from that observed using α-glucan phosphorylases from different sources. In particular, it can be assumed that a thermostable α-glucan phosphorylase catalyzes the enzymatic addition of a C-4 hydroxy group at the non-reducing end of the primer to d-glucal with the consequent formation of an α(1→4)-glycosidic linkage in the absence of Pi as the first step in the in situ production of dGlc-1-P, as it recognizes d-glucal as the only substrate. The in situ production of dGlc-1-P was confirmed by the direct measurement of the 1 H NMR spectra of the reaction mixtures in our experiment. Furthermore, we reported that the enzymatically synthesized 2-deoxyamylose spontaneously formed an antiparallel double-helical crystalline structure, which was completely different from the native parallel double helix built from amylose [33].
Based on the above background, in this study we investigated the thermostable α-glucan phosphorylase-catalyzed enzymatic copolymerization of dGlc-1-P, which was produced in situ from d-glucal, with Glc-1-P using maltotriose (Glc 3 ) as a primer to generate new heteropolysaccharides, and particularly partially 2-deoxygenated amyloses composed of dGlc/Glc units ( Figure 1). Furthermore, the crystalline structures of the products in accordance with the unit ratios of dGlc/Glc residues were evaluated. It was also found that the partially 2-deoxygenated amylose, which was composed of an adapted unit ratio, formed a film using a facile casting method. This study highlights the usefulness of the enzymatic copolymerization approach to obtain well-defined heteropolysaccharides. the reaction. The yield and Mn values by the enzymatic homopolymerization of D-glucal (entry 1) were lower than those by the present enzymatic copolymerization (entries 2-6). This is probably due to the low conversion of D-glucal to dGlc-1-P in situ at the non-reducing end of the 2-deoxyamylose homo-chain present in the progressed reaction system. The above results suggested that the native monomer, Glc-1-P, efficiently copolymerized with the analog monomer, dGlc-1-P, which was produced in situ, to obtain partially 2-deoxygenated amyloses.  Thermostable α-glucan phosphorylase-catalyzed enzymatic copolymerization of d-glucal with Glc-1-P from Glc 3 to produce partially 2-deoxygenated amyloses; m is the degree of polymerization of the produced polysaccharide, including both dGlc and Glc units.

Thermostable α-Glucan Phosphorylase-Catalyzed Enzymatic Copolymerization of d-Glucal with Glc-1-P
A typical experimental procedure was as follows (entry 4 in Table 1). A mixture of d-glucal (43.8 mg, 0.300 mmol), Glc-1-P (disodium salt, 91.2 mg, 0.300 mmol), and Glc 3 (2.5 mg, 5.00 µmol) in 20 mM of Tris-acetate buffer (pH 6.9, 1.0 mL) containing KH 2 PO 4 (0.70 mg, 5.00 µmol) was incubated in the presence of thermostable α-glucan phosphorylase (122 U) at 40 • C for 24 h. The precipitated product was isolated by centrifugation, followed by lyophilization to give a partially 2-deoxygenated amylose (50.6 mg) in a 53.3% yield based on the amounts of the total dGlc and Glc residues present in the reaction system. 1    The crystalline structures of the partially 2-deoxygenated amyloses were then investigated by powder XRD measurement. The XRD patterns of the products composed of the certainly higher unit ratios of dGlc to Glc (entries 2 and 3, Figure 3b,c) were identical with those of the homopolysaccharide, 2-deoxyamylose (entry 1, Figure 3a), as mainly detected at 10.5° and 19°, assigned to the arrangement of double helices; 13° and 19°, assigned to the distances between pyranose rings; 16.5°, assigned to the diameter of the double helix; and 21°, assigned to the helical radius. The crystalline structure evaluated by the XRD pattern was precisely reported in our previous study [33]. On the other hand, the XRD results of the products composed of the certainly higher unit ratios of Glc to dGlc (entries 5 and 6, Figure 3e,f) exhibited the same patterns as those of amylose (Figure 3g), as mainly observed at 17° and 22.5°. Such a majority rule in the crystalline structures of the partially 2-deoxygenated amyloses depending on the dGlc/Glc unit ratios is owing to the completely different double helical assembling fashions of the two homopolysaccharides-that is, 2deoxyamylose and amylose-as discussed in our previous publication [33].

Film Formation from Partially 2-Deoxygenated Amylose (Entry 4)
A solution of the partially 2-deoxygenated amylose (0.10 g) in DMSO (2.0 mL) was casted on a glass plate and dried under reduced pressure at 60 • C overnight to give a film.

Results and Discussion
The thermostable α-glucan phosphorylase-catalyzed enzymatic copolymerization of d-glucal with Glc-1-P was conducted at varying monomer feed ratios with Glc 3 as the primer, as shown in Table 1 (entries 2-6), in Tris-acetate buffer at 40 • C for 24 h (Figure 1). The feed ratio of the total comonomers to primer was adjusted to 120:1 to obtain the highest molecular weight products possible in the present reaction system, as it was the highest ratio in the enzymatic homopolymerization of d-glucal catalyzed by the same enzyme in our previous investigation [33]. An equimolar amount of KH 2 PO 4 with the primer as a Pi source was present in the buffer solvent because it was required for the in situ production of dGlc-1-P as the actual monomer. For comparison, the enzymatic homopolymerization of d-glucal catalyzed by thermostable α-glucan phosphorylase was performed by the same operation to produce 2-deoxyamylose (entry 1). As the products were precipitated from the reaction mixtures, they were isolated by centrifugation and dried by lyophilization. The 1 H NMR spectrum of the product in DMSO-d 6 + D 2 O (entry 4, Figure 2)) observed both the signals assignable to α(1→4)-linked dGlc units at δ 1.42-1.56 (H-2ax), 1.99-2.01 (H-2eq), and 5.30 (H-1) and α(1→4)-linked Glc units at δ 5.03 (H-1), supporting the structure of the partially 2-deoxygenated amylose; the signals' assignments accorded to the 1 H NMR analysis for the individual homopolysaccharides, amylose and 2-deoxyamylose, reported in our previous papers [33,36]. From the integrated ratios of the H-1 signals of these two units and the H-1 signals ascribable to the terminal reducing end at δ 4.33 (H-1β) and 4.95 (H-1α), the dGlc/Glc unit ratios and the number-average molecular weight (M n ) values of the produced polysaccharides including the primer chain were calculated as listed in Table 1. The unit ratios of dGlc to Glc were always lower than the feed ratios of d-glucal to Glc-1-P, owing to the lower reactivity of dGlc-1-P toward the native substrate, Glc-1-P. The presence of comparable amounts of d-glucal and Glc-1-P at feed gave a higher M n product (entry 4) than that produced by different feed ratios of comonomers. The copolymerization gave moderate yields in all cases, due to the precipitation of the products during the reaction. The yield and M n values by the enzymatic homopolymerization of d-glucal (entry 1) were lower than those by the present enzymatic copolymerization (entries 2-6). This is probably due to the low conversion of d-glucal to dGlc-1-P in situ at the non-reducing end of the 2-deoxyamylose homo-chain present in the progressed reaction system. The above results suggested that the native monomer, Glc-1-P, efficiently copolymerized with the analog monomer, dGlc-1-P, which was produced in situ, to obtain partially 2-deoxygenated amyloses.
The crystalline structures of the partially 2-deoxygenated amyloses were then investigated by powder XRD measurement. The XRD patterns of the products composed of the certainly higher unit ratios of dGlc to Glc (entries 2 and 3, Figure 3b,c) were identical with those of the homopolysaccharide, 2-deoxyamylose (entry 1, Figure 3a), as mainly detected at 10.5 • and 19 • , assigned to the arrangement of double helices; 13 • and 19 • , assigned to the distances between pyranose rings; 16.5 • , assigned to the diameter of the double helix; and 21 • , assigned to the helical radius. The crystalline structure evaluated by the XRD pattern was precisely reported in our previous study [33]. On the other hand, the XRD results of the products composed of the certainly higher unit ratios of Glc to dGlc (entries 5 and 6, Figure 3e,f) exhibited the same patterns as those of amylose (Figure 3g), as mainly observed at 17 • and 22.5 • . Such a majority rule in the crystalline structures of the partially 2-deoxygenated amyloses depending on the dGlc/Glc unit ratios is owing to the completely different double helical assembling fashions of the two homopolysaccharides-that is, 2-deoxyamylose and amylose-as discussed in our previous publication [33].
On the other hand, the XRD result of the partially 2-deoxygenated amylose composed of the dGlc/Glc unit ratio = 40/60 (entry 4, Figure 3d) showed a relatively broad pattern, indicating the amorphous nature of this product-specifically, which was probably owing to the random sequence of the two units. Additionally, the observation of such broad pattern, which did not clearly show the diffraction patterns from the individual crystalline structures of amylose and 2-deoxyamylose, supported the incorporation of both the units in the same polysaccharide chain by the copolymerization. Owing to the amorphous nature, the film was formed by casting a solution of this product in DMSO on a glass plate, followed by drying under reduced pressure. On the other hand, the other samples, which observed the diffraction patterns corresponding to either amylose or 2-deoxyamylose in their XRD results, did not exhibit a film formation property. The produced film showed a flexible nature and was bent as shown in Figure 4. Because only an enzymatically synthesized pure amylose specifically with a very high molecular weight (e.g., higher than ca. 5 × 10 5 ) exhibits an ability in film formation [37], the present copolymerization is a useful method to facilely obtain amylosic soft materials from α(1→4)-glucan substrates. Processes 2020, 8, x FOR PEER REVIEW 6 of 8 On the other hand, the XRD result of the partially 2-deoxygenated amylose composed of the dGlc/Glc unit ratio = 40/60 (entry 4, Figure 3d) showed a relatively broad pattern, indicating the amorphous nature of this product-specifically, which was probably owing to the random sequence of the two units. Additionally, the observation of such broad pattern, which did not clearly show the diffraction patterns from the individual crystalline structures of amylose and 2-deoxyamylose, supported the incorporation of both the units in the same polysaccharide chain by the copolymerization. Owing to the amorphous nature, the film was formed by casting a solution of this product in DMSO on a glass plate, followed by drying under reduced pressure. On the other hand, the other samples, which observed the diffraction patterns corresponding to either amylose or 2deoxyamylose in their XRD results, did not exhibit a film formation property. The produced film showed a flexible nature and was bent as shown in Figure 4. Because only an enzymatically synthesized pure amylose specifically with a very high molecular weight (e.g., higher than ca. 5 × 10 5 ) exhibits an ability in film formation [37], the present copolymerization is a useful method to facilely obtain amylosic soft materials from α(14)-glucan substrates.

Conclusions
Because non-natural heteropolysaccharides can be considered as new functional materials, in this study we investigated the thermostable α-glucan phosphorylase-catalyzed enzymatic copolymerization of D-glucal with Glc-1-P from the Glc3 primer via the in situ production of dGlc-1-P to obtain the partially 2-deoxygenated amyloses. The structures of the products were confirmed by  On the other hand, the XRD result of the partially 2-deoxygenated amylose composed of the dGlc/Glc unit ratio = 40/60 (entry 4, Figure 3d) showed a relatively broad pattern, indicating the amorphous nature of this product-specifically, which was probably owing to the random sequence of the two units. Additionally, the observation of such broad pattern, which did not clearly show the diffraction patterns from the individual crystalline structures of amylose and 2-deoxyamylose, supported the incorporation of both the units in the same polysaccharide chain by the copolymerization. Owing to the amorphous nature, the film was formed by casting a solution of this product in DMSO on a glass plate, followed by drying under reduced pressure. On the other hand, the other samples, which observed the diffraction patterns corresponding to either amylose or 2deoxyamylose in their XRD results, did not exhibit a film formation property. The produced film showed a flexible nature and was bent as shown in Figure 4. Because only an enzymatically synthesized pure amylose specifically with a very high molecular weight (e.g., higher than ca. 5 × 10 5 ) exhibits an ability in film formation [37], the present copolymerization is a useful method to facilely obtain amylosic soft materials from α(14)-glucan substrates.

Conclusions
Because non-natural heteropolysaccharides can be considered as new functional materials, in this study we investigated the thermostable α-glucan phosphorylase-catalyzed enzymatic copolymerization of D-glucal with Glc-1-P from the Glc3 primer via the in situ production of dGlc-1-P to obtain the partially 2-deoxygenated amyloses. The structures of the products were confirmed by

Conclusions
Because non-natural heteropolysaccharides can be considered as new functional materials, in this study we investigated the thermostable α-glucan phosphorylase-catalyzed enzymatic copolymerization of d-glucal with Glc-1-P from the Glc 3 primer via the in situ production of dGlc-1-P to obtain the partially 2-deoxygenated amyloses. The structures of the products were confirmed by the 1 H NMR spectra. The XRD results of the products indicated that the crystalline structures accorded to majority rule, in which the products with certainly higher ratios of dGlc units and Glc units to others formed the same crystalline structures as those of the corresponding homopolysaccharides-i.e., 2-deoxyamylose and amylose, respectively. The amorphous product, which was fabricated by a random sequence of the dGlc/Glc units, formed a flexible film. As the present heteropolysaccharides, the partially 2-deoxygenated amyloses, have a potential to exhibit new functions owing to different crystalline structures depending on the dGlc/Glc unit ratios, we are now working on further functions related to the structures of the present polysaccharides and their possible applications, such as in new practical biocompatible and environmentally benign materials.