Designing starch derivatives with desired structures and functional properties via rearrangements of glycosidic linkages by starch-active transglycosylases.

Modification of starch by transglycosylases from glycoside hydrolase families has attracted much attention recently; these enzymes can produce starch derivatives with novel properties, i.e. processability and functionality, employing highly efficient and safe methods. Starch-active transglycosylases cleave starches and transfer linear fragments to acceptors introducing α-1,4 and/or linear/branched α-1,6 glucosidic linkages, resulting in starch derivatives with excellent properties such as complexing and resistance to digestion characteristics, and also may be endowed with new properties such as thermo-reversible gel formation. This review summarizes the effects of variations in glycosidic linkage composition on structure and properties of modified starches. Starch-active transglycosylases are classified into 4 groups that form compounds: (1) in cyclic with α-1,4 glucosidic linkages, (2) with linear chains of α-1,4 glucosidic linkages, (3) with branched α-1,6 glucosidic linkages, and (4) with linear chains of α-1,6 glucosidic linkages. We discuss potential processability and functionality of starch derivatives with different linkage combinations and structures. The changes in properties caused by rearrangements of glycosidic linkages provide guidance for design of starch derivatives with desired structures and properties, which promotes the development of new starch products and starch processing for the food industry.

miR-101-5p suppresses trophoblast cell migration and invasion via modulating the DUSP6-ERK1/2 axis in preeclampsia.

PURPOSE: Dysregulated behaviors of trophoblast cells leading to defective placentation are considered the main cause of preeclampsia (PE). Abnormal miRNA expression profiles have been observed in PE placental tissue, indicating the significant role of miRNAs in PE development. This study aimed to investigate the expression of miR-101-5p in PE placental tissue and its biological functions. METHODS: The expression of miR-101-5p in placental tissue was detected by quantitative real-time PCR (qRT-PCR). The localization of miR-101-5p in term placental tissue and decidual tissue was determined by the fluorescence in situ hybridization (FISH)-immunofluorescence (IF) double labeling assay. The effect of miR-101-5p on the migration, invasion, proliferation, and apoptosis of the HTR8/SVneo trophoblast cells was investigated. Online databases combined with transcriptomics were used to identify potential target genes and related pathways of miR-101-5p. Finally, the interaction between miR-101-5p and the target gene was verified by qRT-PCT, WB, dual-luciferase reporter assay, and rescue experiments. RESULTS: The study found that miR-101-5p was upregulated in PE placental tissue compared to normal controls and was mainly located in various trophoblast cell subtypes in placental and decidual tissues. Overexpression of miR-101-5p impaired the migration and invasion of HTR8/SVneo cells. DUSP6 was identified as a potential downstream target of miR-101-5p. The expression of miR-101-5p was negatively correlated with DUSP6 expression in HTR8/SVneo cells, and miR-101-5p directly bound to the 3' UTR region of DUSP6. DUSP6 upregulation rescued the migratory and invasive abilities of HTR8/SVneo cells in the presence of miR-101-5p overexpression. Additionally, miR-101-5p downregulated DUSP6, resulting in enhanced ERK1/2 phosphorylation. CONCLUSION: This study revealed that miR-101-5p inhibits the migration and invasion of HTR8/SVneo cells by regulating the DUSP6-ERK1/2 axis, providing a new molecular mechanism for the pathogenesis of PE.

Increasing Protein Content of Rice Flour with Maintained Processability by Using Granular Starch Hydrolyzing Enzyme.

Rice flour (RF) has become a promising food material. In the present study, RF with higher protein content was prepared using a granular starch hydrolyzing enzyme (GSHE). Particle size, morphology, crystallinity, and molecular structures of RF and rice starch (RS) were characterized to establish a hydrolytic mechanism; thermal, pasting, and rheological properties were determined to evaluate processability using differential scanning calorimetry (DSC), rapid viscosity analysis (RVA), and rheometer, respectively. The GSHE treatment resulted in pinholes, pits, and surface erosion through sequential hydrolysis of crystalline and amorphous areas on the starch granule surface. The amylose content decreased with hydrolysis time, while the very short chains (DP < 6) increased rapidly at 3 h but decreased slightly later. After hydrolysis for 24 h, the protein content in RF increased from 8.52% to 13.17%. However, the processability of RF was properly maintained. Specifically, the data from DSC showed that the conclusion temperature and endothermic enthalpy of RS barely changed. The result of rapid RVA and rheological measurement indicated that RF paste viscosity and viscoelastic properties dropped rapidly after 1 h hydrolysis and thereafter recovered slightly. This study provided a new RF raw material useful for improving and developing RF-based foods.

Impact of Starch Binding Domain Fusion on Activities and Starch Product Structure of 4-α-Glucanotransferase.

A broad range of enzymes are used to modify starch for various applications. Here, a thermophilic 4-α-glucanotransferase from Thermoproteus uzoniensis (TuαGT) is engineered by N-terminal fusion of the starch binding domains (SBDs) of carbohydrate binding module family 20 (CBM20) to enhance its affinity for granular starch. The SBDs are N-terminal tandem domains (SBDSt1 and SBDSt2) from Solanum tuberosum disproportionating enzyme 2 (StDPE2) and the C-terminal domain (SBDGA) of glucoamylase from Aspergillus niger (AnGA). In silico analysis of CBM20s revealed that SBDGA and copies one and two of GH77 DPE2s belong to well separated clusters in the evolutionary tree; the second copies being more closely related to non-CAZyme CBM20s. The activity of SBD-TuαGT fusions increased 1.2-2.4-fold on amylose and decreased 3-9 fold on maltotriose compared with TuαGT. The fusions showed similar disproportionation activity on gelatinised normal maize starch (NMS). Notably, hydrolytic activity was 1.3-1.7-fold elevated for the fusions leading to a reduced molecule weight and higher α-1,6/α-1,4-linkage ratio of the modified starch. Notably, SBDGA-TuαGT and-SBDSt2-TuαGT showed Kd of 0.7 and 1.5 mg/mL for waxy maize starch (WMS) granules, whereas TuαGT and SBDSt1-TuαGT had 3-5-fold lower affinity. SBDSt2 contributed more than SBDSt1 to activity, substrate binding, and the stability of TuαGT fusions.

Enzymatic preparation and potential applications of agar oligosaccharides: a review.

Oligosaccharides derived from agar, that is, agarooligosaccharides and neoagarooligosaccharides, have demonstrated various kinds of bioactivities which have been utilized in a variety of fields. Enzymatic hydrolysis is a feasible approach that principally allows for obtaining specific agar oligosaccharides in a sustainable way at an industrial scale. This review summarizes recent technologies employed to improve the properties of agarase. Additionally, the relationship between the degree of polymerization, bioactivities, and potential applications of agar-derived oligosaccharides for pharmaceutical, food, cosmetic, and agricultural industries are discussed. Engineered agarase exhibited general improvement of enzymatic performance, which is mostly achieved by truncation. Rational and semi-rational design assisted by computational methods present the latest strategy for agarase improvement with greatest potential to satisfy future industrial needs. Agarase immobilized on magnetic Fe3O4 nanoparticles via covalent bond formation showed characteristics well suited for industry. Additionally, albeit with the relationship between the degree of polymerization and versatile bioactivities like anti-oxidants, anti-inflammatory, anti-microbial agents, prebiotics and in skin care of agar-derived oligosaccharides are discussed here, further researches are still needed to unravel the complicated relationship between bioactivity and structure of the different oligosaccharides.

Malto-oligosaccharides as critical functional ingredient: a review of their properties, preparation, and versatile applications.

Malto-oligosaccharides (MOS) are α-1,4 glycosidic linked linear oligosaccharides of glucose, which have a diverse range of functional applications in the food, pharmaceutical, and other industries. They can be used to modify the physicochemical properties of foods thereby improving their quality attributes, or they can be included as prebiotics to improve their nutritional attributes. The degree of polymerization of MOS can be controlled by using specific enzymes, which means their functionality can be tuned for specific applications. In this article, we review the chemical structure, physicochemical properties, preparation, and functional applications of MOS in the food, health care, and other industries. Besides, we offer an overview for this saccharide from the perspective of prospect functional ingredient, which we feel lacks in the current literature. MOS could be expected to provide a novel promising substitute for functional oligosaccharides.

Potential risks of nicotine on the germination, growth, and nutritional properties of broad bean.

This study evaluated the allelopathy, uptake and accumulation, and potential agricultural and food safety risks of nicotine in broad bean (Vicia faba L.) during seed germination and seedling growth. Nicotine stress has an allelopathic inhibitory effect on seeds and a hormesis effect on germinated seeds and seedlings, which has an enhancement effect (<50 mg kg-1) and an inhibition effect (>100 mg kg-1) on the germinated seeds and an enhancement effect (<100 mg kg-1) and an inhibition effect (>200 mg kg-1) on the seedlings. Exogenous nicotine can be absorbed by broad bean roots from nicotine-contaminated soil and accumulated in the main organs of the seedlings, especially the leaves, which exceeded the maximum residue level (0.03 mg kg-1 DW) at 50 mg kg-1. Moreover, nicotine resulted in a bitter taste in the edible broad bean leaves, disrupting the balance of basic nutritional properties, decreasing sucrose, and increasing bitter substances such as choline and procyanidin. These results demonstrated that residual nicotine in the soil not only poses potential risks to sustainable agricultural development but also a food safety risk for consumers. The present study provides insight into the potential risks of nicotine in agroecosystems.

Phosphorylation of Yes-associated protein impairs trophoblast invasion and migration: implications for the pathogenesis of fetal growth restriction†.

Fetal growth restriction (FGR) is a condition in which a newborn fails to achieve his or her prospective hereditary growth potential. This condition is associated with high newborn mortality, second only to that associated with premature birth. FGR is associated with maternal, fetal, and placental abnormalities. Although the placenta is considered to be an important organ for supplying nutrition for fetal growth, research on FGR is limited, and treatment through the placenta remains challenging, as neither proper uterine intervention nor its pathogenesis have been fully elucidated. Yes-associated protein (YAP), as the effector of the Hippo pathway, is widely known to regulate organ growth and cancer development. Therefore, the correlation of the placenta and YAP was investigated to elucidate the pathogenic mechanism of FGR. Placental samples from humans and mice were collected for histological and biomechanical analysis. After investigating the location and role of YAP in the placenta by immunohistochemistry, we observed that YAP and cytokeratin 7 have corresponding locations in human and mouse placentas. Moreover, phosphorylated YAP (p-YAP) was upregulated in FGR and gradually increased as gestational age increased during pregnancy. Cell function experiments and mRNA-Seq demonstrated impaired YAP activity mediated by extracellular signal-regulated kinase inhibition. Established FGR-like mice also recapitulated a number of the features of human FGR. The results of this study may help to elucidate the association of FGR development with YAP and provide an intrauterine target that may be helpful in alleviating placental dysfunction.

Trophoblastic proliferation and invasion regulated by ACTN4 is impaired in early onset preeclampsia.

Successful pregnancy requires normal placentation, which largely depends on the tight regulation of proliferation, invasion, and migration of trophoblast cells. Abnormal functioning of trophoblast cells may cause failure of uterine spiral artery remodeling, which may be related to pregnancy-related disorders, such as preeclampsia. Here, we reported that an actin-binding protein, α-actinin (ACTN)4, was dysregulated in placentas from early onset preeclampsia. Moreover, knockdown of ACTN4 markedly inhibited trophoblast cell proliferation by reducing AKT membrane translocation. Furthermore, E-cadherin regulated ACTN4 and ß-catenin colocalization on trophoblast cell podosomes, and ACTN4 down-regulation suppressed the E-cadherin-induced cell invasion increase via depolymerizing actin filaments. Moreover, loss of ACTN4 recapitulated a number of the features of human preeclampsia. Therefore, our data indicate that ACNT4 plays a role in trophoblast function and is required for normal placental development.-Peng, W., Tong, C., Li, L., Huang, C., Ran, Y., Chen, X., Bai, Y., Liu, Y., Zhao, J., Tan, B., Luo, X., Wang, H., Wen, L., Zhang, C., Zhang, H., Ding, Y., Qi, H., Baker, P. N. Trophoblastic proliferation and invasion regulated by ACTN4 is impaired in early onset preeclampsia.

A Cyclodextrin-Based Controlled Release System in the Simulation of In Vitro Small Intestine.

A novel cyclodextrin (CD)-based controlled release system was developed in the small intestine to control the rate of drug release, on the premise of enteric-coated tablets. The system was designed based on the enzymes exogenous ß-cyclodextrin glycosyltransferase (ß-CGTase) and endogenous maltase-glucoamylase (MG), wherein MG is secreted in the small intestine and substituted by a congenerous amyloglucosidase (AG). The vanillin-/curcumin-ß-CD complexes were prepared and detected by Fourier transform infrared (FT-IR), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC), and host CD degradation was measured based on the glucose yield. The combination of ß-CGTase and AG was also functional in the CD complex system. The variations in the concentrations of added ß-CGTase, with AG constantly in excess, could effectively alter the rate of host CD degradation and guest release by monitoring glucose production and color disappearance, thus, demonstrating that guest release in the CD complex system could be precisely controlled by changing the amount of ß-CGTase used. Thus, the in vitro simulation of the system indicated that a novel controlled release system, based on endogenous MG, could be established in the small intestine. The CD-based controlled release system can be potentially applied in drug delivery and absorption in the small intestine.

Profiles of circular RNAs in human placenta and their potential roles related to preeclampsia.

To identify the profiles of circular RNAs (circRNAs) in human placental tissues and to explore the potential roles of dysregulated circRNAs in the pathological genesis of preeclampsia, expression profiles of circRNAs in human placentas were performed in this study. Utilizing high-throughput technology, based on fold changes and P values, 300 circRNAs that are differentially expressed between preeclampsia and normal placental tissues were identified. Among them, hg38_circ_0014736 and hsa_circ_0015382 were validated as significantly upregulated by real-time quantitative PCR with divergent primers. At the same time, hsa_circ_0007121 was significantly downregulated. GO analysis revealed that the three altered circRNAs had a relationship with transcription regulation, proliferation, protein binding, and response to hypoxia. KEGG analysis yielded that apoptosis, Wnt signaling, and HIF-1 pathways were significantly enriched. Interestingly, hsa_circ_0007121 was found to be expressed differently in plasma between preeclampsia and normal pregnancy and this difference could be detected before 20 gestational weeks. Besides, addition receiver operating characteristic (ROC) curve analysis showed that the area under the ROC curve of hsa_circ_0007121 reached 0.72 ([0.59-0.85], P = 0.004) with a sensitivity of 0.77 and specificity of 0.70. Collectively, this study demonstrates the existence of dysregulated circRNAs in the placenta of preeclampsia patients and annotates their potential roles in the pathogenesis of the disease. Encouragingly, hsa_circ_0007121 was found to be a potential noninvasive biomarker for the prediction of preeclampsia.

SATB1 downregulation induced by oxidative stress participates in trophoblast invasion by regulating ß-catenin.

Preeclampsia (PE) is characterized by abnormal placentation in the early stages of pregnancy. Adequate migration and invasion of trophoblasts into the uterine wall and spiral arteries to form a functional maternal-fetal interface are pivotal for normal placentation, but the exact mechanism remains unclear. Growing evidence has revealed that special AT-rich sequence binding protein 1 (binds to nuclear matrix/scaffold-associating DNA) (SATB1) is a tumor promoter that participates in cancer cell migration and invasion. However, the expression and function of SATB1 in trophoblasts is unknown. Here, we characterize the stimulatory effect of SATB1 on the migration and invasion of trophoblasts and identify the regulatory events and downstream signaling components. Downregulated SATB1 was detected in PE placentae and villous explants cultured under hypoxia/reoxygenation (H/R) conditions. H/R-treated trophoblasts with lower SATB1 levels exhibited weaker invasive and growth capacities, whereas upregulation of the SATB1 level with recombinant SATB1 restored these impairments. This restoration was especially apparent with the sumoylation-deficient SATB1 variant, which contained a mutated site that blocked sumoylation. Moreover, the elevated concentration of SATB1 also increased the expression of ß-catenin, which is involved in human placental trophoblast invasion and differentiation is downregulated in PE. However, a specific activator, namely, lithium chloride (LiCl), increased ß-catenin expression but had no evident influence on SATB1 expression. Furthermore, upregulated SATB1 failed to restore trophoblast function when Wnt/ß-catenin was suppressed by dickkopf (Xenopus laevis) homolog 1, dickkopf 1 homolog (Xenopus laevis) (DKK1). Together, these data show that SATB1expression in the human placenta is affected by oxidative stress and might regulate the migration and invasion of trophoblasts via ß-catenin signaling.

A Novel Cyclodextrin-Functionalized Hybrid Silicon Wastewater Nano-Adsorbent Material and Its Adsorption Properties.

A novel cyclodextrin-functionalized hybrid silicon nano-adsorbent material (6-EA-ß-CD-Si) was synthesized via the nucleophilic substitution method. The structure was detected by Fourier transform infrared (FT-IR), X-ray, thermogravimetric analysis, and Brunauer-Emmett-Teller (BET) analysis. Results reveal that the BET surface area of 6-EA-ß-CD-Si is 240 m²/g and the average pore size is 4.16 nm. The adsorption properties of 6-EA-ß-CD-Si onto methylene blue (MB) were studied and fitted with adsorption kinetic models. Both the Freundlich adsorption isotherm model and pseudo-second-order model were fitted with well shows that the multi-layer adsorption with chemisorption and physisorption co-existing in the system. The maximum adsorption capacities are 39.37, 39.21, 36.90, and 36.36 mg/g at temperatures 303, 313, 323, and 333 K, respectively. The maximum removal rate of MB could reach 99.5%, indicating the potential application value of 6-EA-ß-CD-Si in wastewater treatment. The adsorption mechanisms of 6-EA-ß-CD-Si showed that the hydrophobic cave of ß-CD plays an important role on the adsorption of MB.

Structure-function relationships of family GH70 glucansucrase and 4,6-α-glucanotransferase enzymes, and their evolutionary relationships with family GH13 enzymes.

Lactic acid bacteria (LAB) are known to produce large amounts of α-glucan exopolysaccharides. Family GH70 glucansucrase (GS) enzymes catalyze the synthesis of these α-glucans from sucrose. The elucidation of the crystal structures of representative GS enzymes has advanced our understanding of their reaction mechanism, especially structural features determining their linkage specificity. In addition, with the increase of genome sequencing, more and more GS enzymes are identified and characterized. Together, such knowledge may promote the synthesis of α-glucans with desired structures and properties from sucrose. In the meantime, two new GH70 subfamilies (GTFB- and GTFC-like) have been identified as 4,6-α-glucanotransferases (4,6-α-GTs) that represent novel evolutionary intermediates between the family GH13 and "classical GH70 enzymes". These enzymes are not active on sucrose; instead, they use (α1 â†’ 4) glucans (i.e. malto-oligosaccharides and starch) as substrates to synthesize novel α-glucans by introducing linear chains of (α1 â†’ 6) linkages. All these GH70 enzymes are very interesting biocatalysts and hold strong potential for applications in the food, medicine and cosmetic industries. In this review, we summarize the microbiological distribution and the structure-function relationships of family GH70 enzymes, introduce the two newly identified GH70 subfamilies, and discuss evolutionary relationships between family GH70 and GH13 enzymes.

Acrylated Composite Hydrogel Preparation and Adsorption Kinetics of Methylene Blue.

By using cyclodextrin (α-CD) self-assembly into a hydrogel with the triblock copolymer Pluronic F127, nanomicrocrystalline cellulose was introduced into a gel system to form a composite CNC-ß-CD/α-CD/Pluronic F127 hydrogel (CCH). CCH was modified further by grafting acrylic acid to form a novel acrylated composite hydrogel (ACH). The swelling degree of ACH was 156 g/g. Adsorption isotherms show that the adsorption process for methylene blue proximity fitted the Freundlich model. The adsorption kinetics showed that ACH followed a quasi-second-order kinetic model. Methylene blue desorption showed that ACH was a temperature- and pH-dependent gel. Repeated adsorption and desorption experiments were carried out three times, and the removal rate of methylene blue at 75 mg/L was still 70.1%.

C/EBP ß Mediates Endoplasmic Reticulum Stress Regulated Inflammatory Response and Extracellular Matrix Degradation in LPS-Stimulated Human Periodontal Ligament Cells.

Periodontitis is an oral inflammatory disease that not only affects the integrity of local tooth-supporting tissues but also impacts systemic health. A compositional shift in oral microbiota has been considered as the main cause of periodontitis; however, the potential mechanism has not been fully defined. Herein, we investigated the role of CCAAT/enhancer-binding protein ß (C/EBP ß), a member of the C/EBP family of transcription factors, in human periodontal ligament cells (hPDLCs) exposed to Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS). RT-PCR and Western blotting analysis showed that the expression of C/EBP ß was significantly increased in hPDLCs stimulated with LPS stimuli. Overexpression of C/EBP ß by the recombinant adenoviral vector pAd/C/EBP ß markedly increased the expression of the pro-inflammatory cytokines IL-6 and IL-8, and matrix metalloproteinases (MMP)-8 and -9 in hPDLCs in response to LPS. Furthermore, the activation of endoplasmic reticulum (ER) stress was confirmed in LPS-stimulated hPDLCs by measuring the expression of the ER stress marker molecules protein kinase-like ER kinase (PERK), eIF2α, GRP78/Bip, and C/EBP homologous protein (CHOP). The ER stress inhibitor salubrinal repressed, but inducer tunicamycin enhanced, the production of IL-6, IL-8, MMP-8, and MMP-9 in hPDLCs. Additionally, ER stress inducer tunicamycin significantly increased the expression level of C/EBP ß in hPDLCs. Blocking of C/EBP ß by siRNA resulted in a significant decrease in the secretion of IL-6 and IL-8 and expression of MMP-8 and MMP-9 induced by tunicamycin treatment in hPDLCs. Taken together, ER stress appears to play a regulatory role in the inflammatory response and extracellular matrix (ECM) degradation in hPDLCs in response to LPS stimuli by activating C/EBP ß expression. This enhances our understanding of human periodontitis pathology.

Characterization of the 4,6-α-glucanotransferase GTFB enzyme of Lactobacillus reuteri 121 isolated from inclusion bodies.

BACKGROUND: The GTFB enzyme of the probiotic bacterium Lactobacillus reuteri 121 is a 4,6-α-glucanotransferase of glycoside hydrolase family 70 (GH70; http://www.cazy.org ). Contrary to the glucansucrases in GH70, GTFB is unable to use sucrose as substrate, but instead converts malto-oligosaccharides and starch into isomalto-/malto- polymers that may find application as prebiotics and dietary fibers. The GTFB enzyme expresses well in Escherichia coli BL21 Star (DE3), but mostly accumulates in inclusion bodies (IBs) which generally contain wrongly folded protein and inactive enzyme. METHODS: Denaturation followed by refolding, as well as ncIB preparation were used for isolation of active GTFB protein from inclusion bodies. Soluble, refolded and ncIB GTFB were compared using activity assays, secondary structure analysis by FT-IR, and product analyses by NMR, HPAEC and SEC. RESULTS: Expression of GTFB in E. coli yielded > 100 mg/l relatively pure and active but mostly insoluble GTFB protein in IBs, regardless of the expression conditions used. Following denaturing, refolding of GTFB protein was most efficient in double distilled H2O. Also, GTFB ncIBs were active, with approx. 10 % of hydrolysis activity compared to the soluble protein. When expressed as units of activity obtained per liter E. coli culture, the total amount of ncIB GTFB expressed possessed around 180 % hydrolysis activity and 100 % transferase activity compared to the amount of soluble GTFB enzyme obtained from one liter culture. The product profiles obtained for the three GTFB enzyme preparations were similar when analyzed by HPAEC and NMR. SEC investigation also showed that these 3 enzyme preparations yielded products with similar size distributions. FT-IR analysis revealed extended ß-sheet formation in ncIB GTFB providing an explanation at the molecular level for reduced GTFB activity in ncIBs. The thermostability of ncIB GTFB was relatively high compared to the soluble and refolded GTFB. CONCLUSION: In view of their relatively high yield, activity and high thermostability, both refolded and ncIB GTFB derived from IBs in E. coli may find industrial application in the synthesis of modified starches.

Biochemical Characterization of the Lactobacillus reuteri Glycoside Hydrolase Family 70 GTFB Type of 4,6-α-Glucanotransferase Enzymes That Synthesize Soluble Dietary Starch Fibers.

4,6-α-Glucanotransferase (4,6-α-GTase) enzymes, such as GTFB and GTFW of Lactobacillus reuteri strains, constitute a new reaction specificity in glycoside hydrolase family 70 (GH70) and are novel enzymes that convert starch or starch hydrolysates into isomalto/maltopolysaccharides (IMMPs). These IMMPs still have linear chains with some α1→4 linkages but mostly (relatively long) linear chains with α1→6 linkages and are soluble dietary starch fibers. 4,6-α-GTase enzymes and their products have significant potential for industrial applications. Here we report that an N-terminal truncation (amino acids 1 to 733) strongly enhances the soluble expression level of fully active GTFB-ΔN (approximately 75-fold compared to full-length wild type GTFB) in Escherichia coli. In addition, quantitative assays based on amylose V as the substrate are described; these assays allow accurate determination of both hydrolysis (minor) activity (glucose release, reducing power) and total activity (iodine staining) and calculation of the transferase (major) activity of these 4,6-α-GTase enzymes. The data show that GTFB-ΔN is clearly less hydrolytic than GTFW, which is also supported by nuclear magnetic resonance (NMR) analysis of their final products. From these assays, the biochemical properties of GTFB-ΔN were characterized in detail, including determination of kinetic parameters and acceptor substrate specificity. The GTFB enzyme displayed high conversion yields at relatively high substrate concentrations, a promising feature for industrial application.

The effect of electronic medical record application on the length of stay in a Chinese general hospital: a department- and disease-focused interrupted time-series study.

A key purpose of electronic medical records (EMR) introduced in medical institutions is to improve work efficiency. The average length of stay (LOS) is just an important indicator to evaluate work efficiency of medical care in hospitals. Recently, there have been reports about effects of EMR application on LOS in medical institutions, but they have been mostly based on the overall analysis of a region or a hospital and not of specific clinical departments and diseases or based on longer time periods. Therefore, in this study, we selected four clinical departments and four diseases with the largest number of inpatients from January 2004 to December 2012 in a Chinese 3A general hospital and used an interrupted time-series method by the departments and diseases to analyze the relationship of EMR application and LOS. Through our analyses, we concluded that, under unadjusted condition, LOS were all reduced (P < 0.001) after EMR application in four departments and for four diseases. After adjustment by gender, age or admission condition, LOS still all decreased after EMR application (P < 0.05) regardless of departments or diseases. The trend changes in LOS reversed from increasing to decreasing in the orthopedics department (coefficient: 0.016 to -0.079), the cardiovascular surgery department (coefficient: 0.007 to -0.126) and all departments overall (coefficient: 0.004 to -0.070), as well as for the intervertebral disc disorders (coefficient: 0.026 to -0.068). Furthermore, the decreasing trend gained a larger slope in the cardiology department (coefficient: -0.017 to -0.023), the neurology department (coefficient: -0.012 to -0.043) and for the coronary heart disease (coefficient: -0.010 to -0.018), the ventricular septal defect (coefficient: -0.024 to -0.059), and the cerebral infarction (coefficient: -0.031 to -0.040). Together, these findings indicate that EMR application coincided with a decrease in LOS and may have a contribution to the decrease.

Progress in cyclodextrins as important molecules regulating catalytic processes of glycoside hydrolases.

Cyclodextrins (CDs) are important starch derivatives and commonly comprise α-, ß-, and γ-CDs. Their hydrophilic surface and hydrophobic inner cavity enable regulation of enzyme catalysis through direct or indirect interactions. Clarifying interactions between CDs and enzyme is of great value for enzyme screening, mechanism exploration, regulation of catalysis, and applications. We summarize the interactions between CDs and glycoside hydrolases (GHs) according to two aspects: 1) CD as products, substrates, inhibitors and activators of enzymes, directly affecting the reaction process; 2) CDs indirectly affecting the enzymatic reaction by solubilizing substrates, relieving substrate/product inhibition, increasing recombinant enzyme production and storage stability, isolating and purifying enzymes, and serving as ligands in crystal structure to identify functional amino acid residues. Additionally, CD enzyme mimetics are developed and used as catalysts in traditional artificial enzymes as well as nanozymes, making the application of CDs no longer limited to GHs. This review concerns the regulation of GHs catalysis by CDs, and gives insights into research on interactions between enzymes and ligands.