Engineering the 182 site of cyclodextrin glucosyltransferase for glycosylated genistein synthesis / 生物工程学报

Genistein and its monoglucoside derivatives play important roles in food and pharmaceuticals fields, whereas their applications are limited by the low water solubility. Glycosylation is regarded as one of the effective approaches to improve water solubility. In this paper, the glycosylation of sophoricoside (genistein monoglucoside) was investigated using a cyclodextrin glucosyltransferase from Penibacillus macerans (PmCGTase). Saturation mutagenesis of D182 from PmCGTase was carried out. Compared with the wild-type (WT), the variant D182C showed a 13.42% higher conversion ratio. Moreover, the main products sophoricoside monoglucoside, sophoricoside diglucoside, and sophoricoside triglucoside of the variant D182C increased by 39.35%, 56.05% and 64.81% compared with that of the WT, respectively. Enzymatic characterization showed that the enzyme activities (cyclization, hydrolysis, disproportionation) of the variant D182C were higher than that of the WT, and the optimal pH and temperature of the variant D182C were 6 and 40℃, respectively. Kinetics analysis showed the variant D182C has a lower Km value and a higher kcat/Km value than that of the WT, indicating the variant D182C has enhanced affinity to substrate. Structure modeling and docking analysis demonstrated that the improved glycosylation efficiency of the variant D182C may be attributed to the increased interactions between residues and substrate.

Molecular cloning and functional characterization of an isoflavone glucosyltransferase from Pueraria thomsonii / 中国天然药物

Pueraria thomsonii has long been used in traditional Chinese medicine. Isoflavonoids are the principle pharmacologically active components, which are primarily observed as glycosyl-conjugates and accumulate in P. thomsonii roots. However, the molecular mechanisms underlying the glycosylation processes in (iso)flavonoid biosynthesis have not been thoroughly elucidated. In the current study, an O-glucosyltransferase (PtUGT8) was identified in the medicinal plant P. thomsonii from RNA-seq database. Biochemical assays of the recombinant PtUGT8 showed that it was able to glycosylate chalcone (isoliquiritigenin) at the 4-OH position and glycosylate isoflavones (daidzein, formononetin, and genistein) at the 7-OH or 4'-OH position, exhibiting no enzyme activity to flavonones (liquiritigenin and narigenin) in vitro. The identification of PtUGT8 may provide a useful enzyme catalyst for efficient biotransformation of isoflavones and other natural products for food or pharmacological applications.

Anti-caries activity of selected Sudanese medicinal plants with emphasis on Terminalia laxiflora

Abstract In Sudan, some medicinal plants, such as Acacia seyal, Calotropis procera and Balanites aegyptiaca have been used to prevent or treat oral health problems. The stem and stem bark of Terminalia laxiflora Engl., Combretaceae, are used as antiseptics for mouthwash to prevent gingivitis and thrush in Africa. Methanol and 50% hydroethanolic extracts of 25 plants that are used in traditional Sudanese medicine for several diseases and cavity disorders were screened for anti-cavity activities. T. laxiflora methanolic wood extracts, which exhibited such activity, were investigated. The crude extracts were assayed for their antimicrobial activities against Streptococcus sobrinus in terms of minimum inhibitory concentration and glucosyltransferase inhibition. The active extract of T. laxiflora wood was subsequently fractionated by different chromatographic techniques. Isolated compounds were identified by spectroscopic methods and assessed for S. sobrinus and glucosyltransferase inhibitory effects. Methanolic extracts of Terminalia brownii (bark), T. laxiflora (wood), A. seyal (bark), Persicaria glabra (leaves) and Tamarix nilotica (stem) showed good activities against both S. sobrinus and glucosyltransferase (MIC ≤ 1 mg/ml, IC50 values <50 µg/ml). Over all plant extracts, T. laxiflora demonstrated the good combined activities (MIC 0.5 mg/ml, glucosyltransferase, IC50 10.3 µg/ml); therefore, its methanolic wood extracts were selected for further phytochemical studies. Four constituents were isolated by chromatographic techniques and identified by spectroscopic techniques. Pharmacological evaluation of the obtained compounds showed that flavogallonic acid dilactone had comparatively good antibacterial activity. In the glucosyltransferase inhibitory test, terchebulin displayed potent activity with an IC50 of 7.5 µM. The screening presented in this study showed that methanol extracts of T. laxiflora wood possessed promising anti-cavity effects.

Using molecular markers to assess Streptococcus mutans variability and the biological risk for caries

AIM: To characterize the genetic variability of Streptococcus mutans isolates and to correlate this variability with different colonization profiles observed during dental caries in a sample of children. METHODS: S. mutans samples were isolated from the saliva of 30 children with varying histories of dental caries, and they were characterized according to morphological and biochemical markers and the sequences of their 16S-23S intergenic spacer region. The genetic variability of the isolates was first assessed using Random Amplified Polymorphic DNA (RAPD) markers. Next, the isolates were differentiated by sequencing a specific region of the gene encoding the enzyme glucosyltransferase B (gtfB). RESULTS: Characterization using RAPD markers uncovered significant genetic variability among the samples and indicated the existence of clusters, which allowed us to reconstruct both the origin and clinical history of the disease. By sequencing the 16S-23S intergenic region, it was found that all of the isolates belonged to the species S. mutans. Based on the genetic similarity of the isolates and pattern of amino acid variations identified by partial sequencing of the gtfB gene, base-pair changes were identified and correlated with different virulence patterns among the isolates. CONCLUSIONS: The partial sequencing of the gtfB gene can be a useful tool for elucidating the colonization patterns of S. mutans. As amino acid variations are likely to be correlated with differences in biological risk, molecular characterization, such as that described in this paper, could be the key for assessing the development of dental caries in children...

Effect of rubusoside on cariogenic potential of Streptococcus mutans / 中草药

Objective: To study the inhibitory effect of rubusoside on the growth of Streptococcus mutans. Methods: S. mutans was added to each group, cultured in anaerobic incubator with constant temperature, and the pH value was measured and compared. Microbial adhesion to hydrocarbons method was used to test the adhesion and cell surface hydrophobicity. Centrifugation was followed, Bradford method and Neson-samogyi method were used to measure the total contents of protein and reducing sugar and to calculate the viability of glucosyltransferase (GTF). Anihrone method was used to measure the contents of water-insoluble glucans (WIG). Results: There were highly significant differences among the sweet tea groups, the experiment groups, and control groups on the pH value, adhesion, cell surface hydrophobicity, GTF activity, and total content of WIG (P < 0.01). Conclusion: Rubusoside has the strong inhibition on the ability of acid production, adhesion, cell surface hydrophobicity, GTF activity, and the synthesis of WIG of S. mutans.

Genomic DNA cloning and analysis of uridine diphosphate-glucosyltransferase gene from Rhodiola crenulata / 中草药

Objective: To obtain the genomic DNA sequence of uridine diphosphate (UDP)-glucosyltransferase (GT) gene from Rhodiola crenulata and to analyze its DNA sequence at the level of bioinformatics. Methods: The optimized CTAB method was used to extract the genomic DNA from R. crenulata, and after three times genomic walking, the genomic DNA sequence of UDPGT in R. crenulata (RcUDPGT) was obtained by hiTAIR-PCR. The DNA sequence was analyzed by bioinformatics method. Results: The 2 977 bp DNA sequence of RcUDPGT was obtained, which contained the 1 499 bp gene sequence (including the 82 bp intron sequence) and 1 500 bp 5' upstream flanking sequence of coding region (including promoter sequence). The bioinformatic analysis showed that the RcUDPGT was hydrophilic, located in the cytoplasm, and had high homology with UDPGTs of other plants. The tertiary structure of RcUDPGT indicated that protein had UDPGT functional sites. Promoter analysis showed that it contained cis-acting elements responding to light, heat, pressure, temperature, anaerobic, anthocyanins biosynthesis sequences, etc. Conclusion The structure of RcUDPGT gene is integral and has functional sites. The research provides the reference for the molecular biology and metabolic engineering of R. crenulata.

HCV NS5A and NS5B Enhance Expression of Human Ceramide Glucosyltransferase Gene / 中国病毒学·英文版

Host genes involved in lipid metabolism are differentially affected during the early stages of hepatitis C virus (HCV) infection.Here we demonstrate that artificial up-regulation of fatty acid biosynthesis has a positive effect on the replication of the HCV full-length replicon when cells were treated with nystatin.Conversely,the HCV RNA replication was decreased when fatty acid biosynthesis was inhibited with 25-hydroxycholesterol and PDMP(D-threo-1-phenyl-2-decanoylamino-3- morpholino-1-propanol).In agreement with these results,the expression level of GlcT-1(ceramide glucosyltransferase),a host glucosyltransferase in the first step of GSL (glycosphingolipid) biosynthesis,was found to be closely associated with the expression and replication of HCV RNA.On the other hand,the viral RNA can also activate GlcT-1 in the early stage of viral RNA transfection in vitro.To identify viral factors that are responsible for GlcT-1 activation,we constructed ten stable Vero cell lines that express individual HCV proteins.Based on the analyses of these cell lines and transient transfection assay of the GlcT-1 promoter regions,we conclude that HCV proteins,especially NS5A and NS5B,have positive effects on the expression of GlcT-1.It is possible that NS5A and NS5B stimulate transcription factor(s) to activate the expression of GlcT-1 by increasing its transcription level.

Comparison of mRNA expression of gtf genes and adhesive ability of xylitol-sensitive and -resistant Streptococcus mutans by xylitol-treated concentrations / 대한구강보건학회지

OBJECTIVES: Xylitol is an effective anticarious natural sugar substitute, by inhibiting the virulence of Streptococcus mutans (S. mutans). However, long-term xylitol consumption leads to an emergence of the xylitol-resistant (XR) strains. This study aimed to confirm the general characteristics, mRNA expression of gtf genes, and adhesive ability of the xylitol-sensitive (XS) and XR S. mutans , by xylitol-treated concentrations. METHODS: S. mutans KCTC3065 was maintained in TYE medium, containing 0.4% glucose with 1% xylitol for 30 days at 37degrees C, 10% CO2 to form XR strain and the same procedures, without xylitol, were repeated for the formation of XS S. mutans. Both XS and XR were cultured by xylitol-treated concentrations (0%, 0.1% and 1%), then, general characteristics, such as growth and acid production, mRNA expression of gtf genes and adhesive ability were analyzed. RESULTS: Xylitol reduced the cell growth of XS S. mutans in a dose-dependent manner, but did not reduce the XR. Xylitol inhibited acid production of XS in a dose-dependent manner. However, it did not inhibit that of XR. Xylitol reduced the gtfB and gtfD mRNA expression of the XS S. mutans, which the genes synthesized soluble and insoluble extracellular polysaccharides, but not reduced that of the XR. By a microtiter plate assay, biofilm formation was more reduced in the XR strains, which means biofilm's adhesive ability of XR S. mutans was lower than that of the XS. CONCLUSIONS: These results indicate that a lower level of adhesive ability for XR S. mutans is related with mRNA expression level of gtf genes, which suggested that the XR strains may be less cariogenic than that of the XS.

Production of glucosyltransferase B and glucans by streptococcus mutans strains isolated from caries-free individuals

La glucosiltranferasa B es una enzima producida por Streptococcus mutans, que a partir de la sacarosa, cataliza la síntesis de glucanos insolubles los cuales dan soporte a la biopelícula, siendo uno de los principales factores de virulencia para la generación de la caries dental. Sin embargo, no se ha esclarecido su papel en los individuos libre de caries, portadores delmicroorganismo. El objetivo de este estudio fue determinar la producción de glucosiltransferasa B y la producción de glucanos por cepas de Streptococcus mutans aisladas de biopelícula de 30individuos libres de caries. Las cepas fueron cultivadas en caldo Todd Hewitt y las proteínas extracelulares fueron obtenidas por precipitación con sulfato de amonio las proteínas asociadas amembrana por extracción con urea. La presencia de GtfB fue determinada por peso molecular por SDS–PAGE, confirmada por Western Blot utilizando un anticuerpo específico y la producciónde polisacáridos por separación electroforética, incubación con sacarosa y coloración de Schiff. Los resultados muestran que el 96.7 por ciento de las cepas de Streptococcus mutans producen una banda a la altura del peso molecular correspondiente a las Gtf,de las cuales son reactivas por western blot el 63.4 por ciento El 93.3 por cientode las cepas producen polisacáridos. Conclusiones: la cepas de Streptococcus mutans aisladas de biopelícula de individuos sanos producen factores de virulencia asociados a la caries dental como glucosiltransferasa B y glucanos lo que indica que hay condiciones en la cavidad oral diferentes a estos factores que mantienen al individuo libre de caries dental, los cuales deben ser investigados en la búsqueda de estrategias para controlar la enfermedad.

Preparation and assay of C-glucosyltransferase from roots of Pueraria lobata.

C-glucosyltransferase (EC 2.4.1.X) is one of the key enzymes for the biosynthesis of puerarin. This paper describes the methodology in purification and assay of the enzyme for the first time in Puerarin lobata (Willd.) Ohwi. C-glucosyltransferase from roots of P. lobata was extracted and partially purified by (NH4)2SO4 saturation. The effects of pH, temperature, and substrate concentration on the activity of the enzyme were investigated. The properties of the puerarin produced by C-glucosyltransferase were studied by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The peak activity of C-glucosyltransferase was detected in fraction of by 80% saturation of (NH4)2SO4 and the optimal conditions for enzymatic reaction were 35.5 μmol l-1 of isoliquiritigenin and 560 μmol l-1 of UDP-G at pH 8.1, 28oC for 1 h. Mn2+ at 1 mmol l-1 and Al3+ at 1 mmol l-1 increased the enzyme activity, while Mg2+ inhibited its activity. The enzyme activity in Nicotiana tabacum and P. lobata were detected under the above assay conditions. Higher activity was found in roots than in leaves and stems of P. lobata, while no enzyme activity was detected in leaves of N. tabacum. It was the first time that activity of C-glucosyltransferase, which transforms isoliquiritigenin to puerarin, was detected in P. lobata.

Structure Elucidation of Product of β-1,3-Glucosyltransferase Encoded by wfgD Gene in Escherichia Coli O152 Using Mass Spectrometry / 分析化学

The O152 antigens of Escherichia coli contains a Glc-β-1,3-GlcNAc linkage within the repeating unit. The wfgD gene in E. coli O152 O antigen gene cluster had been demonstrated utilizing NMR technique to encode a glucosyltransferase which is responsible for the synthesis of Glc-β-1,3-GlcNAc linkage. In this study, a synthetic substrate analog of the natural acceptor substrate undecaprenol-pyrophosphate-lipid [GlcNAc-α-PO_3-PO_3-(CH_2)_(11))-O-phenyl]) was used as an acceptor and UDP-Glc as a donor substrate, and electrospray ionization tandem mass spectrometry(ESI-MS-MS) was used for the detailed structural characte-)rization) of the enzyme product. A systematic study was conducted on product to allow rationalization of the fragmentation) processes. The major fragments observed in the ESI-MS-MS spectra result from cleavage of glycosidic) bond and diphosphate moiety. The fragment originating from the nonreducing end of the product yields information on sequence). Cross-ring cleavages, which are very informative of the linkages of the monosaccharide residues constituting) the product, and "internal" cleavage ions which are derived from elimination of substituents from around) the pyranose ring, were also observed. This extensive fragmentation was shown that the expected Glc-β-1,3-GlcNAc linkage in the product, confirming that wfgD is in the form of UDP-Glc: GlcNAc-pyrophosphate-lipid β-1,3-glucosyltransferase.)

Glucosyltransferase production by Klebsiella sp. K18 and conversion of sucrose to palatinose using immobilized cells / Produção de glicosiltransferase por Klebsiella sp. K18 e conversão de sacarose em palatinose utilizando células imobilizadas

The strain Klebsiella sp. K18 produces the enzyme glucosyltransferase and catalyses the conversion of sucrose to palatinose, an alternative sugar that presents low cariogenicity. Response Surface Methodology was successfully employed to determine the optimal concentration of culture medium components. Maximum glucosyltransferase production (21.78 U mL-1) was achieved using the optimized medium composed by sugar cane molasses (80 g L-1), bacteriological peptone (7 g L-1) and yeast extract (20 g L-1), after 8 hours of fermentation at 28ºC. The conversion of sucrose to palatinose was studied utilizing immobilized cells in calcium alginate. The effects of the alginate concentration (2-4%), cell mass concentration (20-40%) and substrate concentration (25-45%) were evaluated and the yield of palatinose was approximately 62.5%.

A linhagem Klebsiella sp. K18 produz a enzima glicosiltransferase que catalisa a conversão de sacarose em palatinose, um açúcar alternativo que apresenta baixa cariogenicidade. Metodologia de Superfície de Resposta foi empregada com sucesso para determinar a concentração ótima dos componentes do meio de cultivo. A máxima produção deglicosiltransferase (21,78 U mL-1) foi obtida utilizando o meio de cultivo otimizado composto por melaço de cana de açúcar (80 g L-1), peptona bacteriológica (7 g L-1) e extrato de levedura (20 g L-1), após 8 horas de fermentação a 28ºC. A conversão desacarose em palatinose foi estudada utilizando células imobilizadas em alginato de cálcio. Os efeitos da concentração de alginato (2-4%), concentração de massa celular (20-40%) e concentração de substrato (25-45%) foram avaliados e a porcentagem de palatinose foi de aproximadamente 62,5%.

The roles of glycosphingolipids in the proliferation and neural differentiation of mouse embryonic stem cells

Glycosphingolipids including gangliosides play important regulatory roles in cell proliferation and differentiation. UDP-glucose:ceramide glucosyltransferase (Ugcg) catalyze the initial step in glycosphingolipids biosynthesis pathway. In this study, Ugcg expression was reduced to approximately 80% by short hairpin RNAs (shRNAs) to evaluate the roles of glycosphingolipids in proliferation and neural differentiation of mouse embryonic stem cells (mESCs). HPTLC/immunofluorescence analyses of shRNA-transfected mESCs revealed that treatment with Ugcg-shRNA decreased expression of major gangliosides, GM3 and GD3. Furthermore, MTT and Western blot/immunofluorescence analyses demonstrated that inhibition of the Ugcg expression in mESCs resulted in decrease of cell proliferation (P < 0.05) and decrease of activation of the ERK1/2 (P < 0.05), respectively. To further investigate the role of glycosphingolipids in neural differentiation, the embryoid bodies formed from Ugcg-shRNA transfected mESCs were differentiated into neural cells by treatment with retinoic acid. We found that inhibition of Ugcg expression did not affect embryoid body (EB) differentiation, as judged by morphological comparison and expression of early neural precursor cell marker, nestin, in differentiated EBs. However, RT-PCR/immunofluorescence analyses showed that expression of microtubule- associated protein 2 (MAP-2) for neurons and glial fibrillary acidic protein (GFAP) for glial cells was decreased in neural cells differentiated from the shRNA-transfected mESCs. These results suggest that glycosphingolipids are involved in the proliferation of mESCs through ERK1/2 activation, and that glycosphingolipids play roles in differentiation of neural precursor cells derived from mESCs.

Effect of propolis on cariogenesis of main cariogenic streptococci and their fluoride-resistant strains / 第三军医大学学报

Objective To evaluate the effects of propolis on the growth of S.mutans ATCC 25175(S.m),S.sobrinus 6715(S.s) and their fluoride-resistant strains(S.m-FR,S.s-FR),and the change of their glucosyltransferase(GTF).Methods S.m and S.s were induced with sodium fluoride by minimum inhibitory concentration(MIC) to induce fluoride-resistant strains(S.m-FR and S.s-FR).Fluid dilution method was used to observe the effects of different levels of propolis on growth of S.m,S.m-FR,S.s and S.s-FR.Chemistry enzyme analysis was used to detect the influence of propolis on the enzyme activity of GTF.Results MIC of propolis for S.m,S.m-FR,S.s and S.s-FR were respectively 0.39,0.78,0.20 and 0.39 g/L.MBC of propolis for them were respectively 0.78,1.56,1.56,and 1.56 g/L.GTF of S.m,S.m-FR,S.s,and S.s-FR was decreased gradually with the increase of concentration of propolis.There were significant differences among total sample groups,and each experimental group and positive control group as well(P