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We prepared network polysaccharide nanoscopic hydrogels by crosslinking water-soluble chitosan (WSCS) with a carboxylate-terminated maltooligosaccharide crosslinker via condensation. In this study, the enzymatic elongation of amylose chains on chitosan-based network polysaccharides by glucan phosphorylase (GP) catalysis was performed to obtain assembly materials. Maltoheptaose (Glc7) primers for GP-catalyzed enzymatic polymerization were first introduced into WSCS by reductive amination. Crosslinking of the product with the above-mentioned crosslinker by condensation was then performed to produce Glc7-modified network polysaccharides. The GP-catalyzed enzymatic polymerization of the α-d-glucose 1-phosphate monomer from the Glc7 primers on the network polysaccharides was conducted, where the elongated amylose chains formed double helices. Enzymatic disintegration of the resulting network polysaccharide assembly successfully occurred by α-amylase-catalyzed hydrolysis of the double helical amyloses. The encapsulation and release of a fluorescent dye, Rhodamine B, using the CS-based network polysaccharides were also achieved by means of the above two enzymatic approaches. [ABSTRACT FROM AUTHOR]

This review article presents the biomimetic helical inclusion of amylose toward hydrophobic polyesters as guests through a vine-twining polymerization process, which has been performed in the glucan phosphorylase (GP)-catalyzed enzymatic polymerization field to fabricate supramolecules and other nanostructured materials. Amylose, which is a representative abundant glucose polymer (polysaccharide) with left-handed helical conformation, is well known to include a number of hydrophobic guest molecules with suitable geometry and size in its cavity to construct helical inclusion complexes. Pure amylose is prepared through enzymatic polymerization of α-d-glucose 1-phosphate as a monomer using a maltooligosaccharide as a primer, catalyzed by GP. It is reported that the elongated amylosic chain at the nonreducing end in enzymatic polymerization twines around guest polymers with suitable structures and moderate hydrophobicity, which is dispersed in aqueous polymerization media, to form amylosic nanostructured inclusion complexes. As the image of this system is similar to how vines of a plant grow around a support rod, this polymerization has been named 'vine-twining polymerization'. In particular, the helical inclusion behavior of the enzymatically produced amylose toward hydrophobic polyesters depending on their structures, e.g., chain lengths and substituents, has been systematically investigated in the vine-twining polymerization field. Furthermore, amylosic supramolecular network materials, such as hydrogels, are fabricated through vine-twining polymerization by using copolymers, where hydrophobic polyester guests or maltooligosaccharide primers are covalently modified on hydrophilic main-chain polymers. The vine-twining polymerization using such copolymers in the appropriate systems induces the formation of amylosic nanostructured inclusion complexes among them, which act as cross-linking points, giving rise to supramolecular networks at the nanoscale. The resulting materials form supramolecular hydrogels, films, and microparticles. [ABSTRACT FROM AUTHOR]

We prepared network polysaccharide nanoscopic hydrogels by crosslinking water-soluble chitosan (WSCS) with a carboxylate-terminated maltooligosaccharide crosslinker via condensation. In this study, the enzymatic elongation of amylose chains on chitosan-based network polysaccharides by glucan phosphorylase (GP) catalysis was performed to obtain assembly materials. Maltoheptaose (Glc7) primers for GP-catalyzed enzymatic polymerization were first introduced into WSCS by reductive amination. Crosslinking of the product with the above-mentioned crosslinker by condensation was then performed to produce Glc7-modified network polysaccharides. The GP-catalyzed enzymatic polymerization of the α-d-glucose 1-phosphate monomer from the Glc7 primers on the network polysaccharides was conducted, where the elongated amylose chains formed double helices. Enzymatic disintegration of the resulting network polysaccharide assembly successfully occurred by α-amylase-catalyzed hydrolysis of the double helical amyloses. The encapsulation and release of a fluorescent dye, Rhodamine B, using the CS-based network polysaccharides were also achieved by means of the above two enzymatic approaches.

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. [ABSTRACT FROM AUTHOR]

This review article presents the biomimetic helical inclusion of amylose toward hydrophobic polyesters as guests through a vine-twining polymerization process, which has been performed in the glucan phosphorylase (GP)-catalyzed enzymatic polymerization field to fabricate supramolecules and other nanostructured materials. Amylose, which is a representative abundant glucose polymer (polysaccharide) with left-handed helical conformation, is well known to include a number of hydrophobic guest molecules with suitable geometry and size in its cavity to construct helical inclusion complexes. Pure amylose is prepared through enzymatic polymerization of α-d-glucose 1-phosphate as a monomer using a maltooligosaccharide as a primer, catalyzed by GP. It is reported that the elongated amylosic chain at the nonreducing end in enzymatic polymerization twines around guest polymers with suitable structures and moderate hydrophobicity, which is dispersed in aqueous polymerization media, to form amylosic nanostructured inclusion complexes. As the image of this system is similar to how vines of a plant grow around a support rod, this polymerization has been named ‘vine-twining polymerization’. In particular, the helical inclusion behavior of the enzymatically produced amylose toward hydrophobic polyesters depending on their structures, e.g., chain lengths and substituents, has been systematically investigated in the vine-twining polymerization field. Furthermore, amylosic supramolecular network materials, such as hydrogels, are fabricated through vine-twining polymerization by using copolymers, where hydrophobic polyester guests or maltooligosaccharide primers are covalently modified on hydrophilic main-chain polymers. The vine-twining polymerization using such copolymers in the appropriate systems induces the formation of amylosic nanostructured inclusion complexes among them, which act as cross-linking points, giving rise to supramolecular networks at the nanoscale. The resulting materials form supramolecular hydrogels, films, and microparticles.

In this paper, we report that polysaccharide substrates bearing 2‐deoxygenated amylose chains are hydrophobic. A pure partially 2‐deoxygenated amylose was synthesized by the thermostable glucan phosphorylase‐catalyzed enzymatic copolymerization of d‐glucal, a non‐native monomer, with α‐d‐glucose 1‐phosphate, a native monomer from maltotriose, as a primer. A water contact angle on a cast film of the resulting polysaccharide confirms that it is hydrophobic. Enzymatic copolymerization using glycogen as a polymeric primer was used to investigate the possibility of engendering hydrophilic substrates with hydrophobicity by incorporating such polysaccharide chains; the resulting glycogen‐derived films with elongated partially 2‐deoxygenated amylose chains are strongly hydrophobic. [ABSTRACT FROM AUTHOR]

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.

In this study, we attempted to prepare an amylose-oligo[(R)-3-hydroxybutyrate] (ORHB) inclusion complex using a vine-twining polymerization approach. Our previous studies indicated that glucan phosphorylase (GP)-catalyzed enzymatic polymerization in the presence of appropriate hydrophobic guest polymers produces the corresponding amylose–polymer inclusion complexes, a process named vine-twining polymerization. When vine-twining polymerization was conducted in the presence of ORHB under general enzymatic polymerization conditions (45 °C), the enzymatically produced amylose did not undergo complexation with ORHB. However, using a maltotriose primer in the same polymerization system at 70 °C for 48 h to obtain water-soluble amylose, called single amylose, followed by cooling the system over 7 h to 45 °C, successfully induced the formation of the inclusion complex. Furthermore, enzymatic polymerization initiated from a longer primer under the same conditions induced the partial formation of the inclusion complex. The structures of the different products were analyzed by X-ray diffraction, 1H-NMR, and IR measurements. The mechanism of formation of the inclusion complexes discussed in the study is proposed based on the additional experimental results.

The β-glucans are structurally varied, naturally occurring components of the cell walls, and storage materials of a variety of plant and microbial species. In the human diet, mixed-linkage glucans [MLG - β-(1,3/4)-glucans] influence the gut microbiome and the host immune system. Although consumed daily, the molecular mechanism by which human gut Gram-positive bacteria utilize MLG largely remains unknown. In this study, we used Blautia producta ATCC 27340 as a model organism to develop an understanding of MLG utilization. B. producta encodes a gene locus comprising a multi-modular cell-anchored endo-glucanase (BpGH16 MLG ), an ABC transporter, and a glycoside phosphorylase (BpGH94 MLG ) for utilizing MLG, as evidenced by the upregulation of expression of the enzyme- and solute binding protein (SBP)-encoding genes in this cluster when the organism is grown on MLG. We determined that recombinant BpGH16 MLG cleaved various types of β-glucan, generating oligosaccharides suitable for cellular uptake by B. producta. Cytoplasmic digestion of these oligosaccharides is then performed by recombinant BpGH94 MLG and β-glucosidases (BpGH3-AR8 MLG and BpGH3-X62 MLG ). Using targeted deletion, we demonstrated BpSBP MLG is essential for B. producta growth on barley β-glucan. Furthermore, we revealed that beneficial bacteria, such as Roseburia faecis JCM 17581 T , Bifidobacterium pseudocatenulatum JCM 1200T, Bifidobacterium adolescentis JCM 1275T, and Bifidobacterium bifidum JCM 1254, can also utilize oligosaccharides resulting from the action of BpGH16 MLG . Disentangling the β-glucan utilizing the capability of B. producta provides a rational basis on which to consider the probiotic potential of this class of organism., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Crown Copyright © 2023. Published by Elsevier Inc. All rights reserved.)

Abstract: A glucan phosphorylase from the hyperthermoacidophilic crenarchaeaon Sulfolobus tokodaii strain 7 (stGP) was characterized. The enzyme displayed maximum activity at 75°C and pH 6.0 and was highly stable at 95°C. The enzyme showed distinct substrate specificity, with maltose being the minimum primer for glucan synthesis and maltotriose being the minimum substrate for degradation. Interestingly, the sequences of GPs from Crenarchaeota clustered all together and formed a distinct lineage. Compared with previously characterized GPs, stGP (56kDa) was much smaller. Structure modeling revealed that stGP was more compact than other GPs and it seemed that stGP was more primitive and adaptable to high temperature. Taken together, stGP and its homologues in Crenarchaeota probably constitute a novel group of GPs. [Copyright &y& Elsevier]

Abstract: The glgP gene in Thermoanaerobacter tengcongensis encodes a putative α-glucan phosphorylase (Tte-GlgP), which is much smaller than its homologs in most other bacteria and is grouped into a less-recognized family. To characterize this novel enzyme, it was overexpressed in Escherichia coli and purified to apparent homogeneity. This purified Tte-GlgP, with expected molecular size (∼64kDa) and isoelectric point (∼pI 6.2), exhibited a wide substrate spectrum in transformation of a variety of glucans such as soluble starch, maltodextrins and glycogen into glucose-1-phosphate. Western blot analysis indicated that the production of Tte-GlgP in T. tengcongensis was repressed by glucose but induced by maltose slightly, suggesting that it was involved in the carbohydrate metabolism in this thermophile. The Tte-GlgP was optimally active at 60°C, and was thermostable with 90% residual activity after treating at 60°C for 6h, thus the overexpressed Tte-GlgP might also be a good candidate for structure–function analysis in its family. [Copyright &y& Elsevier]

Amylose is a linear polymer of α-1,4-linked glucose and is expected to be used in various industries as a functional biomaterial. However, pure amylose is currently not available for industrial purposes, since the separation of natural amylose from amylopectin is difficult. It is known that amylose has been synthesized using various enzymes. Glucan phosphorylase, together with its substrate, glucose-1-phosphate, is the most suitable system for the production of amylose since the molecular size of amylose can be controlled precisely. However, the problem with this system is that glucose-1-phosphate is too expensive for industrial purposes. This review summarizes our work on the enzymatic synthesis of essentially linear amylose, together with recent progress in the production of synthetic amylose using sucrose or cellobiose through the combined actions of phosphorylases. [ABSTRACT FROM AUTHOR]

Glucan phosphorylase, branching enzyme, and 4-α-glucanotransferase were employed to produce glucose polymers with controlled molecular size and structures. Linear or branched glucan was produced from glucose-1-phosphate by glucan phosphorylase alone or together with branching enzyme, where the molecular weight of linear glucan was strictly controlled by the glucose-1-phosphate/primer molar ratio, and the branching pattern by the relative branching enzyme/glucan phosphorylase activity ratio. Cyclic glucans were produced by the cyclization reaction of 4-α-glucanotransferases and branching enzyme on amylose and amylopectin. Molecular size and structure of cyclic glucan was controlled by the type of enzyme and substrate chosen and by the reaction conditions. This in vitro approach can be used to manufacture novel glucose polymers with applicable value. [ABSTRACT FROM AUTHOR]

A procedure that allows the identification of polysaccharide binding polypeptides is described. The method can be applied to proteins whose enzymatic activity is either unknown or cannot be identified unambiguously by activity-staining procedures and it has been used for very complex protein mixtures, such as crude extracts of plant organs. The procedure consists of three steps. First, an affinity polyacrylamide gel electrophoresis using an inhomogeneous polyacrylamide slab gel composed of two triangular parts, an upper gel lacking the ligand and a lower triangular gel containing an immobilized ligand, is performed. Proteins that interact with the ligand form bands that deviate from those of nonbinding proteins and can be detected by protein staining (or, if possible, by activity staining). Second, the bands containing the interacting proteins are excised, denatured, and subjected to SDS–PAGE using a slab gel. In the resulting protein pattern the target proteins cover most of the length of the gel piece applied to the SDS gel, whereas contaminating proteins appear as spots or narrow bands. Suitable regions of the target protein bands are selected for tryptic digestion. Third, the resulting peptides are analyzed by matrix-assisted laser desorption ionization–mass spectrometry followed by database research. [Copyright &y& Elsevier]

Higher plants contain two types of phosphorylase (EC 2.4.1.1). One type is plastidic (Pho1) and the other resides in the cytosol (Pho2). For Solanum tuberosum L., two highly homologous Pho1-type sequences (designated as Pho1a and Pho1b, respectively) have been described that occur both in a homodimeric, (Pho1a)2, and a heterodimeric, Pho1a-Pho1b, state [U. Sonnewald et al. (1995) Plant Mol Biol 27:567–576; T. Albrecht et al. (1998) Eur J Biochem 251:981–991]. We present a spatial and temporal analysis of the expression patterns of the Pho1-type phosphorylases in S. tuberosum. Expression was analyzed at transcript, protein and activity levels. The specificity of both the probes and the antibodies used was carefully determined to ensure selectivity of detection. For both the Pho1a and Pho1b probes the degree of cross-hybridization was estimated. Peptide scanning identified the epitopes of the anti-Pho1a and anti-Pho1b antibodies. Expression of the two Pho1-type genes was analyzed in various organs of the potato plant. In all organs studied the Pho1a transcript levels exceeded those of Pho1b. Furthermore, leaves of a given developmental stage were sampled during the light period and were analyzed for transcript and protein levels and for various carbohydrate pools as well. The data show that in leaves the Pho1a gene expression closely corresponds to starch accumulation, suggesting that the enzyme fulfils a metabolic function within the process of starch biosynthesis. In tubers, Pho1a is constitutively expressed in the parenchyma cells whereas expression of the Pho1b gene is restricted to cells in close vicinity of the vascular tissue. [ABSTRACT FROM AUTHOR]

The occurrence, location, and biosynthesis of glucan-phosphorylase (EC 2.4.1.1) isoenzymes were studied in cotyledons of developing or germinating seeds of Pisum sativum L. Type-I and type-II isoenzymes were detected, and were also localized by indirect immunofluorescence using polyclonal anti-type-I or anti-type-II phosphorylase antibodies. Type-I isoenzyme was found in the cytosol of parenchyma cells whereas the type-II enzyme form is a plastid protein which resides either in amyloplasts (in developing seeds) or in proplastids (in germinating seeds). During seed development, type-II phosphorylase was the predominant isoenzyme and the type-I isoenzyme represented a very minor compound. During germination, the latter increased whilst type-II phosphorylase remained at a constant level. In in-vitro translation experiments, type-I isoenzyme was observed as a final-size product with an apparent molecular weight of approx. 90 kDa. In contrast, type-II phosphorylase was translated as a high-molecular-weight precursor (116 kDa) which, when incubated with a stromal fraction of isolated intact pea chloroplasts, was processed to the size of the mature protein (105 kDa). [ABSTRACT FROM AUTHOR]

The glucan specifity of the purified chloroplast and non-chloroplast forms of α-1,4-glucan phosphorylase (EC 2.4.1.1) from spinach leaves (Steup and E. Latzko (1979), Planta 145, 69-75) was investigated. Phosphorolysis by the two enzymes was studied using a series of linear maltodextrins (degree of polymerization ≦11), amylose, amylopectin, starch, and glycogen as substrates. For all unbranched glucans (amylose and maltodextrins G-G), the chloroplast phosphorylase had a 7-10-fold higher apparent affinity (determined by initial velocity measurements) than the non-chloroplast phosphorylase form. For both enzyme forms, the minimum chain length required for a significant rate of phosphorolysis was five glucose units. Likewise, phosphorolysis ceased when the maltodextrin was converted to maltotetraose. With the chloroplast phosphorylase, maltotetraose was a linear competitive inhibitor with respect to amylose or starch ( K-0.1 mmol 1); the inhibition by maltotetraose was less pronounced with the non-chloroplast enzyme. In contrast to unbranched glucans, the non-chloroplast phosphorylase exhibited a 40-, 50-, and 300-fold higher apparent affinity for amylopectin, starch, and glycogen, respectively, than the chloroplast enzyme. With respect to these kinetic properties the chloroplast phosphorylase resembled the type of 'maltodextrin phosphorylase'. [ABSTRACT FROM AUTHOR]

Plants are often challenged by an array of unfavorable environmental conditions. During cold exposure, many changes occur that include, for example, the stabilization of cell membranes, alterations in gene expression and enzyme activities, as well as the accumulation of metabolites. In the presented study, the carbohydrate metabolism was analyzed in the very early response of plants to a low temperature (2 °C) in the leaves of 5-week-old potato plants of the Russet Burbank cultivar during the first 12 h of cold treatment (2 h dark and 10 h light). First, some plant stress indicators were examined and it was shown that short-term cold exposure did not significantly affect the relative water content and chlorophyll content (only after 12 h), but caused an increase in malondialdehyde concentration and a decrease in the expression of NDA1, a homolog of the NADH dehydrogenase gene. In addition, it was shown that the content of transitory starch increased transiently in the very early phase of the plant response (3–6 h) to cold treatment, and then its decrease was observed after 12 h. In contrast, soluble sugars such as glucose and fructose were significantly increased only at the end of the light period, where a decrease in sucrose content was observed. The availability of the monosaccharides at constitutively high levels, regardless of the temperature, may delay the response to cold, involving amylolytic starch degradation in chloroplasts. The decrease in starch content, observed in leaves after 12 h of cold exposure, was preceded by a dramatic increase in the transcript levels of the key enzymes of starch degradation initiation, the α-glucan, water dikinase (GWD-EC 2.7.9.4) and the phosphoglucan, water dikinase (PWD-EC 2.7.9.5). The gene expression of both dikinases peaked at 9 h of cold exposure, as analyzed by real-time PCR. Moreover, enhanced activities of the acid invertase as well as of both glucan phosphorylases during exposure to a chilling temperature were observed. However, it was also noticed that during the light phase, there was a general increase in glucan phosphorylase activities for both control and cold-stressed plants irrespective of the temperature. In conclusion, a short-term cold treatment alters the carbohydrate metabolism in the leaves of potato, which leads to an increase in the content of soluble sugars. [ABSTRACT FROM AUTHOR]

In this study, we attempted to prepare an amylose-oligo[(R)-3-hydroxybutyrate] (ORHB) inclusion complex using a vine-twining polymerization approach. Our previous studies indicated that glucan phosphorylase (GP)-catalyzed enzymatic polymerization in the presence of appropriate hydrophobic guest polymers produces the corresponding amylose–polymer inclusion complexes, a process named vine-twining polymerization. When vine-twining polymerization was conducted in the presence of ORHB under general enzymatic polymerization conditions (45 °C), the enzymatically produced amylose did not undergo complexation with ORHB. However, using a maltotriose primer in the same polymerization system at 70 °C for 48 h to obtain water-soluble amylose, called single amylose, followed by cooling the system over 7 h to 45 °C, successfully induced the formation of the inclusion complex. Furthermore, enzymatic polymerization initiated from a longer primer under the same conditions induced the partial formation of the inclusion complex. The structures of the different products were analyzed by X-ray diffraction, 1H-NMR, and IR measurements. The mechanism of formation of the inclusion complexes discussed in the study is proposed based on the additional experimental results. [ABSTRACT FROM AUTHOR]