Search Results (14)

In recent years, increased attention has been given to the effective use of chitin nanofibers (ChNFs). We have developed a method to fabricate thinner chitin nanomaterials, called scale-down chitin nanofibers (SD-ChNFs), by a bottom-up procedure at the nanoscale level, with subsequent disintegration by electrostatic repulsion. The surface modification of SD-ChNFs is anticipated to provide new properties and functions for their practical applications. Inspired by our previous reports, which found hydrophobicity in partially 2-deoxygenated (P2D-) amylose obtained by the glucan phosphorylase (GP)-catalyzed enzymatic copolymerization of α-d-glucose 1-phosphate/d-glucal as comonomers, this work investigated the hydrophobization of SD-ChNFs via an enzymatic approach. After the modification of maltooligosaccharide primers on SD-ChNFs was performed by a reductive alkylation toward ChNFs, the grafting of the P2D-amyloses was performed by GP-catalyzed enzymatic copolymerization. ¹H NMR analysis supported the production of P2D-amylose-grafted SD-ChNFs with different d-glucose/2-deoxy-d-glucose unit ratios on SD-ChNFs. The X-ray diffraction analysis of the products confirmed that the chain lengths and unit ratios of the grafted polysaccharides strongly affected the entire crystalline structures. Water contact angle measurements of the cast films of the products indicated that successful hydrophobization was achieved by the grafting of P2D-amylose chains with a sufficient chain length, a relatively high 2-deoxy-d-glucose unit ratio, and low crystallinity. Full article

The pseudotetrasaccharide acarbose, produced by Actinoplanes sp. SE50/110, is a relevant secondary metabolite used in diabetes type II medication. Although maltose plays a crucial role in acarbose biosynthesis, the understanding of the maltose/maltodextrin metabolism and its involvement in acarbose production is at an early stage. Here, we reconstructed the predicted maltose–maltodextrin pathway that involves four enzymes AmlE, MalZ, MalP, and MalQ. An investigation of enzyme activities was conducted through in vitro assays, leading to an expansion of previously postulated substrate spectra. The maltose-induced α-glucosidase AmlE is noteworthy for its high hydrolysis rate of linear α-1,4-glucans, and its capability to hydrolyze various glycosidic bonds. The predicted maltodextrin glucosidase MalZ showed slow hydrolysis activity on linear α-glucans, but it was resistant to acarbose and capable of releasing glucose from acarbose. AmlE compensates for the low activity of MalZ to ensure glucose supply. We determined the enzyme activity of MalP and its dual function as maltodextrin and glycogen phosphorylase. The 4-α-glucanotransferase MalQ plays a central role in the maltose/maltodextrin metabolism, alongside MalP. This study confirmed the simultaneous degradation and synthesis of long-chain α-glucans. The product distribution showed that with an increasing number of glycosidic bonds, less glucose is formed. We found that MalQ, like its sequence homolog AcbQ from the acarbose biosynthetic gene cluster, is involved in the formation of elongated acarviosyl metabolites. However, MalQ does not participate in the elongation of acarbose 7-phosphate, which is likely the more readily available acceptor molecule in vivo. Accordingly, MalQ is not involved in the formation of acarviosyl impurities in Actinoplanes sp. SE50/110. Full article

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. Full article

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. Full article

Honeybush (Cyclopia spp.) is a rich source of antioxidant properties and phenolic compounds. Water availability plays a crucial role in plant metabolic processes, and it contributes to overall quality. Thus, this study aimed to investigate changes in molecular functions, cellular components, and biological processes of Cyclopia subternata exposed to different water stress conditions, which include well-watered (as Control, T1), semi-water stressed (T2), and water-deprived (T3) potted plants. Samples were also collected from a well-watered commercial farm first cultivated in 2013 (T13) and then cultivated in 2017 (T17) and 2019 (T19). Differentially expressed proteins extracted from C. subternata leaves were identified using LC-MS/MS spectrometry. A total of 11 differentially expressed proteins (DEPs) were identified using Fisher’s exact test (p < 0.00100). Only α-glucan phosphorylase was found to be statistically common between T17 and T19 (p < 0.00100). Notably, α-glucan phosphorylase was upregulated in the older vegetation (T17) and downregulated in T19 by 1.41-fold. This result suggests that α-glucan phosphorylase was needed in T17 to support the metabolic pathway. In T19, five DEPs were upregulated, while the other six were downregulated. Based on gene ontology, the DEPs in the stressed plant were associated with cellular and metabolic processes, response to stimulus, binding, catalytic activity, and cellular anatomical entity. Differentially expressed proteins were clustered based on the Kyoto Encyclopedia of Genes and Genomes (KEGG), and sequences were linked to metabolic pathways via enzyme code and KEGG ortholog. Most proteins were involved in photosynthesis, phenylpropanoid biosynthesis, thiamine, and purine metabolism. This study revealed the presence of trans-cinnamate 4-monooxygenase, an intermediate for the biosynthesis of a large number of substances, such as phenylpropanoids and flavonoids. Full article

Understanding the physicochemical properties of starch during grain development and the mechanism for resistant starch (RS) accumulation will provide useful information for improving the RS content of wheat. The grains from wheat mutant lines with high RS contents and their corresponding wild-type control were analyzed to characterize the structural and physicochemical properties of wheat starch. A transcriptomic analysis was used to analyze the differentially expressed genes (DEGs) involved in RS accumulation. The results showed that the RS content increased with grain development, along with the total starch content, but a larger increase was observed in the middle and later stages of grain filling. The X-ray diffraction peak intensity and relative crystallinity of starch exhibited the lowest and highest values at 10 days after anthesis, respectively. Regarding the thermal properties of starch, the peak temperature and conclusion temperature generally decreased with grain development; however, the enthalpy values showed no apparent regularity. Compared to control cultivar ZM22, the RS639 and RS683 lines with high RS contents showed high amylose contents and high relative crystallinity and a large proportion of 2.0~9.8 µm starch granules. Furthermore, the transcriptomics analysis revealed that the average relative expression of the glucan-branching enzyme (GBE) α-1,4 glucan phosphorylase (Pho) and starch synthase (SS) in ZM22 was 2.47-, 2.70-, and 2.56-fold higher than that in RS639, respectively; which indicates that the downregulation of the expression of genes encoding GBE, Pho, and SS in wheat grain promotes the accumulation of RS. Full article

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. Full article

We have previously found that a partially 2-deoxygenated (P2D)-amylose, produced by glucan phosphorylase (GP)-catalyzed enzymatic copolymerization, shows hydrophobic nature. Based on this finding, the present study demonstrates hydrophobization of a strong hydrophilic polypeptide, i.e., poly(γ-glutamic acid) (PGA), by grafting of the P2D-amylose chains via GP-catalyzed enzymatic approach. After maltooligosaccharide primers for the enzymatic reaction were modified on the PGA chain, we performed GP-catalyzed copolymerization of d-glucan with α-d-glucose 1-phosphate as comonomers in different feed ratios from the primers to produce P2D-amylose-grafted PGAs. We analyzed the structures (chemical and crystalline) of the products, precipitated from reaction mixtures, by ¹H NMR and powder X-ray diffraction measurements, respectively. The values of the water contact angle of the cast films, prepared from DMSO solutions of the products with different 2-deoxyglucose/glucose unit ratios, were greater than 100°, indicating efficient hydrophobization of the hydrophilic polypeptide by the present approach. Full article

Resistant starch (RS) is a special group of starches which are slowly degraded and rarely digested in the gastrointestinal tract. It was recognized as a new type of dietary fiber that improved cardiovascular, cerebrovascular, and intestinal health. Breeding high-RS-content wheat is one of the most efficient and convenient approaches for providing an adequate amount of RS for a healthy diet. However, studies which aim to genetically illustrate RS content in wheat are still rare. In the present study, a panel of 207 wheat varieties were collected world-wide and planted under three locations. The RS content of each variety was measured, and 14 additive genetic loci were found to stably exist under more than two environments. Meanwhile, four genes were recognized as the putative candidates with annotated functions of β-amylase, α-1,4 glucan phosphorylase, sucrose transporter, and NAC domain protein. A kompetitive allele-specific PCR (KASP) marker was developed from the SNP AX-94546744, representing the genetic locus of β-amylase located. The AX-94546744-T allele can significantly increase the RS content compared to the AX-94546744-C allele. The genetic loci and KASP marker associated with RS content may be useful for wheat germplasm cultivation and variety breeding with a high RS content, further helping to improve the nutritional quality in wheat. Full article

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. Full article

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 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. Full article

Chestnuts are popular edible nuts that are rich in starch. In order to enhance the transcriptomic resources and further understand starch and sucrose metabolism in maturing chestnuts, a comparative transcriptomic study of Chinese chestnut kernels was conducted at three ripening stages (70, 82, and 94 DAF). At 82 and 94 days after flowering (DAF), starch continued to accumulate, and the amylopectin/amylose ratio increased. Transcriptomic profiling of kernels at 70 (stage I), 82 (stage II), and 94 DAF (stage III) indicated that soluble starch synthase and α-1,4-glucan branching enzyme genes are actively expressed at 82 and 94 DAF. The starch degradation enzymes amylase, phosphoglucan phosphatase DSP4, and maltose exporter did not show differential gene expression, while glycogen phosphorylase-encoding unigenes were significantly down-regulated at 94 DAF. In addition to starch and sucrose metabolism, RNA transport, RNA degradation, pyrimidine metabolism, purine metabolism, plant hormone signal transduction, plant–pathogen interactions, and glycerophospholipid metabolism were found to be significantly enriched in all comparisons included in the study. As Chinese chestnut matured, the unique enriched pathways switched from ribosomal biogenesis and RNA polymerase of eukaryotes to endocytosis and spliceosomes. These genomic resources and findings are valuable for further understanding starch and sucrose metabolism in the Chinese chestnut. Full article

As natural oligo- and polysaccharides are important biomass resources and exhibit vital biological functions, non-natural oligo- and polysaccharides with a well-defined structure can be expected to act as new functional materials with specific natures and properties. α-Glucan phosphorylase (GP) is one of the enzymes that have been used as catalysts for practical synthesis of oligo- and polysaccharides. By means of weak specificity for the recognition of substrates by GP, non-natural oligo- and polysaccharides has precisely been synthesized. GP-catalyzed enzymatic glycosylations using several analog substrates as glycosyl donors have been carried out to produce oligosaccharides having different monosaccharide residues at the non-reducing end. Glycogen, a highly branched natural polysaccharide, has been used as the polymeric glycosyl acceptor and primer for the GP-catalyzed glycosylation and polymerization to obtain glycogen-based non-natural polysaccharide materials. Under the conditions of removal of inorganic phosphate, thermostable GP-catalyzed enzymatic polymerization of analog monomers occurred to give amylose analog polysaccharides. Full article

The sequencing batch reactor (SBR) has been increasingly applied in the control of high organic wastewater. In this study, SBR with aerobic granular sludge was used for wastewater treatment in a noodle-manufacturing village in Vietnam. The results showed that after two months of operation, the chemical oxygen demand, total nitrogen and total phosphorous removal efficiency of aerobic granular SBR reached 92%, 83% and 75%, respectively. Bacterial diversity and bacterial community in wastewater treatment were examined using Illumina Miseq sequencing to amplify the V3-V4 regions of the 16S rRNA gene. A high diversity of bacteria was observed in the activated sludge, with more than 400 bacterial genera and 700 species. The predominant genus was Lactococcus (21.35%) mainly containing Lactococcus chungangensis species. Predicted functional analysis showed a high representation of genes involved in membrane transport (12.217%), amino acid metabolism (10.067%), and carbohydrate metabolism (9.597%). Genes responsible for starch and sucrose metabolism accounted for 0.57% of the total reads and the composition of starch hydrolytic enzymes including α-amylase, starch phosphorylase, glucoamylase, pullulanase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, and 1,4-α-glucan branching enzyme. The presence of these enzymes in the SBR system may improve the removal of starch pollutants in wastewater. Full article