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.

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.

Optimization of maltooligosyl fructofuranosidesproduction by recombinant β-cyclodextrin glycosyltransferase / 生物工程学报

Coupling sugar is a kind of new sweetener which can substitute sucrose. It has a good application prospect in food, medicine and other fields because of its good coloration, water retention and anti caries. The purpose of this study was to find cheap and easily available donor and acceptor, and to optimize the preparation process of coupling sugar by using β-cyclodextrin glycosyltransferase from Bacilluscirculans 251. Using sucrose as acceptor, the factors of preparing coupling sugar was optimized, including enzyme dosage, starch types, temperature, pH, ratio of starch/sucrose, and cooperation of isoamylase and β-CGTase. When 105 g/L potato starch and 95 g/L sucrose was used as substrates, the yield of coupling sugar reached 88.4%, which was catalyzed by 13.5 U/g immobilized β-CGTase and 45.0 U/g isoamylase under the conditions of pH 5.5 and 40 °C for 21 h. In this study, isoamylase and β-CGTase were used to prepare coupling sugar innovatively. This method had obvious advantages in yield and cost, which laid both theoretical and experimental foundation for the industrial enzymatic preparation of coupling sugars.

Application of sucrose phosphorylase in glycosylation / 生物工程学报

Water solubility, stability, and bioavailability, can be substantially improved after glycosylation. Glycosylation of bioactive compounds catalyzed by glycoside hydrolases (GHs) and glycosyltransferases (GTs) has become a research hotspot. Thanks to their rich sources and use of cheap glycosyl donors, GHs are advantageous in terms of scaled catalysis compared to GTs. Among GHs, sucrose phosphorylase has attracted extensive attentions in chemical engineering due to its prominent glycosylation activity as well as its acceptor promiscuity. This paper reviews the structure, catalytic characteristics, and directional redesign of sucrose phosphorylase. Meanwhile, glycosylation of diverse chemicals with sucrose phosphorylase and its coupling applications with other biocatalysts are summarized. Future research directions were also discussed based on the current research progress combined with our working experience.

Function, structure and catalytic mechanism of sucrose phosphate synthase: a review / 生物工程学报

Sucrose is a natural product occurs widely in nature. In living organisms such as plants, sucrose phosphate synthase (SPS) is the key rate-limiting enzyme for sucrose synthesis. SPS catalyzes the synthesis of sucrose-6-phosphate, which is further hydrolyzed by sucrose phosphatase to form sucrose. Researches on SPS in recent decades have been focused on the determination of enzymatic activity of SPS, the identification of the inhibitors and activators of SPS, the covalent modification of SPS, the carbohydrate distribution in plants regulated by SPS, the mechanism for promoting plant growth by SPS, the sweetness of fruit controlled by SPS, and many others. A systematic review of these aspects as well as the crystal structure and catalytic mechanism of SPS are presented.

Molecular mechanisms of inhibiting glucosyltransferases for biofilm formation in Streptococcus mutans / 国际口腔科学杂志·英文版

Glucosyltransferases (Gtfs) play critical roles in the etiology and pathogenesis of Streptococcus mutans (S. mutans)- mediated dental caries including early childhood caries. Gtfs enhance the biofilm formation and promotes colonization of cariogenic bacteria by generating biofilm extracellular polysaccharides (EPSs), the key virulence property in the cariogenic process. Therefore, Gtfs have become an appealing target for effective therapeutic interventions that inhibit cariogenic biofilms. Importantly, targeting Gtfs selectively impairs the S. mutans virulence without affecting S. mutans existence or the existence of other species in the oral cavity. Over the past decade, numerous Gtfs inhibitory molecules have been identified, mainly including natural and synthetic compounds and their derivatives, antibodies, and metal ions. These therapeutic agents exert their inhibitory role in inhibiting the expression gtf genes and the activities and secretion of Gtfs enzymes with a wide range of sensitivity and effectiveness. Understanding molecular mechanisms of inhibiting Gtfs will contribute to instructing drug combination strategies, which is more effective for inhibiting Gtfs than one drug or class of drugs. This review highlights our current understanding of Gtfs activities and their potential utility, and discusses challenges and opportunities for future exploration of Gtfs as a therapeutic target.

Characterization and identification of Lactobacillus fermentum 4,6-α-glucosyltransferase and its products / 生物工程学报

4,6-α-glucosyltransferases (4,6-α-GTs), which converts amylose into α(1-6) bonds-containing α-glucan, possesses great application potential in enzymatic synthesis of dietary fiber. Primers were designed according to the conserved motifs existing in the amino acid sequence of 4,6-α-GTs, and used to amplify a putative GTFB-Like 4,6-α-GTs gene (named as gtf16) from the genomic DNA of Lactobacillus. The gtf16 gene was cloned into the plasmid pET15b, expressed in Escherichia coli BL21(DE3), followed by purification and characterization. The optimum pH and the optimum temperature of the purified enzyme were 5.0 and 40 °C, respectively. The biotransformation product of this enzyme was systematically characterized by thin-layer chromatography, NMR spectroscopy, and hydrolysis reaction. The Gtf16-catalyzed product shows a similar structure to that of the isomalto/malto-polysaccharide (IMMP), which is the amylose-derived product catalyzed by GtfB from Lactobacillus reuteri 121. Moreover, The Gtf16-catalyzed product contains up to 75% of α(1-6) bonds and has an average molecular weight of 23 793 Da. Furthermore, the content of the anti-digestive components was 88.22% upon hydrolysis with digestive enzymes.

Streptococcus Colonial Growth of Dental Plaque Inhibition Using Flavonoid Extract of Ants Nest (Myrmecodia pendans): An in Vitro Study

Objective: To know the activity of resistance of flavonoid content in ant nest plant in decreasing the number of colonies S. mutans oral cavity of children as a medic herbal material. Material and Methods: The subjects were plaque sample of 20 children aged 7-12 years. Research begins by making toothpaste from ant nest extract. Samples of children's dental plaque were inserted into BHIB media, after which incubated for 24 hours, 1/10 dilution with BHIB media three times, followed by TYC media planting and incubation of anaerob with temperature 370C for 48 hours. After that then count the number of colonies of S. mutans. Results: On ethyl acetate extract of ant nest incubated at room temperature with concentration 20%, 40%, 60%, 80%, 100% obtained a decrease from each treatment amount of Streptococcus mutans colony on TYC media with median value of each treatment was 89, 67, 64, 61, 59 and 51 for the ethyl acetate fraction, and 62, 61, 60, 59, 49 at the ethanol fraction. There was no significant difference between the six concentration groups (p>0.05). Conclusion: Flavonoids extract of ant nest plants have growth barrier on Streptococcus mutans bacteria, the greater the concentration given the greater the number of S. mutans colony.

Cloning,subcellular localization and spatio-temporal expression analysis of a flavonoid 3-O-glucosyltransferase gene( SmUF3GT) in Salvia miltiorrhiza / 中国中药杂志

The family of flavonoid 3-O-glucosyltransferase catalyzes the modification of anthocyanin from unstable-structure to stable-structure. In this study,based on homology cloning and transcriptome library,we isolated the full-length c DNA of UDP-glucose: flavonoid 3-O-glucosyltransferase( named SmUF3GT) from the flower tissues of S. miltiorrhiza. This gene was consisted of 1 353 bp open reading frames( ORF) encoding 450 amino acids. And the SmUF3GT protein was performed for the bioinformatic analysis. Our results showed that the protein was preliminary localized in the Golgi and peroxisome of cytosol,as well as plasma membrane and cell nuclear.QRT-PCR analyses indicated that SmUF3GT expressed differently in all tissues and organs but roots of S. miltiorrhiza and S. miltiorrhiza f.alba. During floral development,the expression of SmUF3GT showed a trend of rising fist and then down in purple-flower Danshen,whereas decreasing sharply fist and then slowly in white-flower Danshen. The present study provides basic information for further research on the network of synthesis and accumulation of flavonoids in S.miltiorrhiza.

Heterologous expression of Streptomyces coelicolor trehalose synthase and whole-cell biocatalyst production of trehalose in Escherichia coli / 生物工程学报

The trehalose synthase (ScTreS) gene from Streptomyces coelicolor was successfully cloned and heterologously expressed in Escherichia coli BL21(DE3). The protein purified by Ni-NTA affinity column showed an apparent molecular weight (MW) of 62.3 kDa analyzed by SDS-PAGE. The optimum temperature of the enzyme was 35 °C and the optimum pH was 7.0; the enzyme was sensitive to acidic conditions. By homologous modeling and sequence alignment, the enzyme was modified by site-directed mutagenesis. The relative activities of the mutant enzymes K246A and A165T were 1.43 and 1.39 times that of the wild type, an increased conversion rate of 14% and 10% respectively. To optimize the synthesis conditions of trehalose, the mutant strain K246A was cultivated in a 5-L fermentor and used for whole-cell transformation. The results showed that with the substrate maltose concentration of 300 g/L at 35 °C and pH 7.0, the highest conversion rate reached 71.3%, and the yield of trehalose was 213.93 g/L. However, when maltose concentration was increased to 700 g/L, the yield of trehalose can reach 465.98 g/L with a conversion rate of 66%.

Cloning and expression analysis in resurrection process of trehalose-6-phosphate synthase gene from Selaginella tamariscina / 中国中药杂志

Selaginella tamariscina is a typical resuscitation medicinal plant with extreme drought tolerance. Trehalose plays an important role in the resurrection process, and the trehalose-6-phosphate synthase(TPS) is the key enzyme to synthesize trehalose in plants. In this study, the sequence of TPS was obtained by splicing from the transcriptome data of S. tamariscina. After the synthesis of cDNA based on the template of total RNA, the sequence was cloned by RT-PCR for verification and then analyzed by bioinformatics methods. The results indicated that the full-length coding sequence of StTPS was 2 799 bp (GenBank accession no. MH155231), and the encoded protein contained 932 amino acids. StTPS could be located in the chloroplastid according to subcellular localization prediction. There were two conserved domains belonging to glycogen phosphorylase glycosyltransferase (GPGTF) family but no signal peptide or transmembrane domain in StTPS. The expression of StTPS was determined by qRT-PCR and the variation of trehalose content was measured by HPLC-ELSD during the resurrection process of S. tamariscina. Meanwhile, the correlation between them was analyzed. The results showed that both the expression level of StTPS and the trehalose content increased associated with the extension of dehydration time, and declined associated with the extension of rehydration time which proved a significant positive correlation between the StTPS expression level and the trehalose content. The results suggested that the StTPS probably plays a central role in recovery process in S. tamariscina.

Comparisons of IgA response in saliva and colostrum against oral streptococci species

Abstract The present study compared IgA specificity against oral streptococci in colostrum and saliva samples. Sixty-two mother-and-child pairs were included; samples of colostrum (C) and saliva (MS) were collected from the mothers and saliva samples were collected from babies (BS). The specificity of IgA against Streptococcus mutans and S. mitis were analyzed by western blot. Only 30% of babies’ samples presented IgA reactivity to S. mutans, while 74 and 80% of MS and C, respectively, presented this response. IgA reactivity to S. mutans virulence antigens (Ag I/II, Gtf and GbpB) in positive samples showed differences between samples for Gtf and especially for GbpB (p < 0.05), but responses to Ag I/II were similar (p > 0.05). The positive response of Gtf-reactive IgA was different between C (90%) and MS (58%) samples (p < 0.05), but did not differ from BS (p > 0.05). GbpB was the least detected, with 48 and 26% of C and MS, and only 5% of BS samples presenting reactivity (p > 0.05). Eight percent of MS and C samples presented identical bands to SM in the same time-point. In conclusion, the differences of IgA response found between C and MS can be due to the different ways of stimulation, proliferation and transportation of IgA in those secretions. The colostrum has high levels of IgA against S. mutans virulence antigens, which could affect the installation and accumulation process of S. mutans, mainly by supplying anti-GbpB IgA to the neonate.

A new alkalophilic isolate of Bacillus as a producer of cyclodextrin glycosyltransferase using cassava flour

Abstract Cyclodextrin glycosyltransferase (CGTase) catalyzes the conversion of starch into non-reducing cyclic sugars, cyclodextrins, which have several industrial applications. This study aimed to establish optimal culture conditions for β-CGTase production by Bacillus sp. SM-02, isolated from soil of cassava industries waste water lake. The optimization was performed by Central Composite Design (CCD) 2, using cassava flour and corn steep liquor as substrates. The maximum production of 1087.9 U mL−1 was obtained with 25.0 g L−1 of cassava flour and 3.5 g L−1 of corn steep after 72 h by submerged fermentation. The enzyme showed optimum activity at pH 5.0 and temperature 55 °C, and maintained thermal stability at 55 °C for 3 h. The enzymatic activity was stimulated in the presence of Mg+2, Ca+2, EDTA, K+, Ba+2 and Na+ and inhibited in the presence of Hg+2, Cu+2, Fe+2 and Zn+2. The results showed that Bacillus sp. SM-02 have good potential for β-CGTase production.

Immunogenicity and prediction of epitopic region of antigen Ag I/II and glucosyltransferase from Streptococcus mutans / 华中科技大学学报（医学）（英德文版）

The levels of Streptococcus (S.) mutans infections in saliva were evaluated and a comparison for specific antibody levels among children with different levels of S. mutans infection was made. The promising epitopic regions of antigen AgI/II (PAc) and glucosyltransferase (GTF) for potential vaccine targets related to S. mutans adherence were screened. A total of 94 children aged 3-4 years were randomly selected, including 53 caries-negative and 41 caries-positive children. The values of S. mutans and those of salivary total secretory immunoglobulin A (sIgA), anti-PAc and anti-Glucan binding domain (anti-GLU) were compared to determine the correlation among them. It was found the level of s-IgA against specific antigens did not increase with increasing severity of S. mutans infection, and the complete amino acid sequence of PAc and GTFB was analyzed using the DNAStar Protean system for developing specific anti-caries vaccines related to S. mutans adherence. A significantly positive correlation between the amount of S. mutans and children decayed, missing, and filled teeth index was observed. No significant difference was detected in specific sIgA against PAc or GLU between any two groups. No significant correlation was found between such specific sIgA and caries index. A total of 16 peptides from PAc as well as 13 peptides from GTFB were chosen for further investigation. S. mutans colonization contributed to early children caries as an important etiological factor. The level of sIgA against specific antigens did not increase with increasing severity of S. mutans infection in children. The epitopes of PAc and GTF have been screened to develop the peptide-based or protein-based anti-caries vaccines.

Effect of the Ethanol Extract of Propolis on Formation of Streptococcus mutans Biofilm

Streptococcus mutans (S. mutans) is one of the most important bacteria in the formation of dental plaque and dental caries. S. mutans adheres to an acquired pellicle formed on the tooth surface, and aggregates with many oral bacteria. It initiates plaque formation by synthesizing glucan from sucrose, which is catalyzed by glucosyltransferases. Propolis is a resinous mixture produced by honeybees, by mixing saliva and beeswax with secretions gathered from wood sap and flower pollen. Bees prevent pathogenic invasions by coating the propolis to the outer and inner surface of the honeycomb. Propolis has traditionally been used for the treatment of allergic rhinitis, asthma and dermatitis. We investigated the inhibitory effects of propolis ethanol extract on biofilm formation and gene expression of S. mutans. The biofilm formation of S. mutans was determined by scanning electron microscopy (SEM) and safranin staining. We observed that the extract of propolis had an inhibitory effect on the formation of S. mutans biofilms at concentrations higher than 0.2 mg/ml. Real-time PCR analysis showed that the gene expression of biofilm formation, such as gbpB, spaP, brpA, relA and vicR of S. mutans, was significantly decreased in a dose dependent manner. The ethanol extract of propolis showed concentration dependent growth inhibition of S. mutans, and significant inhibition of acid production at concentrations of 0.025, 0.05, 0.1 and 0.2 mg/ml, compared to the control group. These results suggest that the ethanol extract of propolis inhibits gene expression related to biofilm formation in S. mutans

Effects of ggpS over-expression on glycosylglycerol and glycerol biosynthesis of Synechocystis sp. PCC 6803 / 生物工程学报

To study the roles of glucosylglycerol phosphate synthase (Ggps) in glucosylglycerol (GG) and glycerol biosynthesis, we over-expressed Ggps from either Synechocystis sp. PCC 6803 or Synechococcus sp. PCC 7002 in a Synechocystis strain with a high GG titer, and determined the GG and glycerol accumulation in the resultant mutants grown under different NaCl-stress conditions. Ion chromatography results revealed that GG yield was not improved, but glycerol production was significantly enhanced by over-expression of Ggps from Synechocystis sp. PCC 6803 (6803ggpS). In addition, increasing the NaCl concentration of medium from 600 to 900 mmol/L led to a further 75% increase of glycerol accumulation in the mutant strain with 6803ggpS over-expression. These findings show the role of ggpS in driving the carbon flux to the glycerol biosynthesis pathway, and will be helpful for further improvement of GG and glycerol production in Synechocystis.

Expression and production optimization of sucrose isomerase from Pantoea dispersa in Escherichia coli / 生物工程学报

To improve the yield of sucrose isomerase from Pantoea dispersa UQ68J, we studied the effect of different signal peptides and fermentation conditions on sucrose isomerase expression in Escherichia coli. The gene of sucrose isomerase was optimized and expressed in E. coli BL21 (DE3) with native signal peptide which was named as ORI strain. The total and extracellular enzyme activity was 85 and 65 U/mL in the flask, respectively. The mature protein, which started from the 22th amino acid, was connected with the PelB and OmpA signal peptide to construct P22 and O22 strain, respectively. The total activity of P22 reached 138 U/mL, which was 1.6 times of ORI strain. The total activity of O22 strain was similar to that of ORI strain. Induced by 3.0 g/L lactose, the total activity of P22 strain increased to 168 U/mL. In 3 L fermentor, the effects of glycine concentration and induction time were studied. Induction when the DCW reached 18 g/L (OD₆₀₀=30), with 0.5% glycine, the extracellular enzyme activity reached 1 981 U/mL, and the total enzyme activity reached 2 640 U/mL, which is the highest activity of sucrose isomerase that was expressed in recombinant E. coli.

Overexpression of FKS1 to improve yeast autolysis-stress / 生物工程学报

With the development of high gravity brewing, yeast cells are exposed to multiple brewing-associated stresses, such as increased osmotic pressure, enhanced alcohol concentration and nutritional imbalance. These will speed up yeast autolysis, which seriously influence beer flavor and quality. To increase yeast anti-autolytic ability, FKS1 overexpression strain was constructed by 18S rDNA. The concentration of β-1,3-glucan of overexpression strain was 62% higher than that of wild type strain. Meantime, FKS1 overexpression strain increased anti-stress ability at 8% ethanol, 0.4 mol/L NaCl and starvation stress. Under simulated autolysis, FKS1 showed good anti-autolytic ability by slower autolysis. These results confirms the potential of FKS1 overexpression to tackle yeast autolysis in high-gravity brewing.

Construction of a recombinant Escherichia coli for high trehalose production / 生物工程学报

Trehalose, a compatible solute, is widely used in food, cosmetics, pharmaceutical products and organ transplantation. Nowadays, trehalose is mostly produced by enzymatic synthesis with many secondary products and lowpurity. In this study, high amount of trehalose was produced by recombinant E. ccli fermentation. First, a bifunctional trehalose gene TPSP was amplified from genome of C. hutchinscoii. Second, an expression vector pTac-HisA containing TPSP was constructed and transformed into the host E. coli. Expression of this bifunctional enzyme-TPSP converted glucose to trehalose. The result suggested that TPSP from C. hutchinsonji has been successfully expressed in E. ccoi. High amount of extracellular trehalose generated from glucose by whole-cell catalysis and After optimization, the production of trehalose in shake flasks was improved to 1.2 g/L and the relative conversion rate reached 21%. The production in bioreactor reached 13.3 g/L and the relative conversion rate reached 48.6%. It is the first time to realize the functional expression of the bifunctional enzyme-TPSP of C. hutchinsonii in E. coli and achieved the conversion form glucose to trehalose. This study laid a foundation for industrial large-scale production of trehalose.

Purified dextransucrase from Pediococcus pentosaceus CRAG3 as food additive.

The extracellular crude dextransucrase (0.67 U/mg) from P. pentosaceus CRAG3 (GenBank accession number JX679020) after PEG-1500 fractionation gave specific activity, 20.0 U/mg which by gel filtration resulted in 46.0 U/mg. The purified dextransucrase displayed molecular size of approximately, 224 kDa. The optimum assay conditions for dextransucrase activity were 5% sucrose in 20 mM sodium acetate buffer (pH 5.4) and 30 oC. The dextransucrase was stable up to 40 oC and at pH range of 5.4-7.0. The metal ions such as Co2+, Ca2+, Mg2+ and Zn2+ stimulated the dextransucrase activity by 56, 44, 14 and 12%, respectively. It was most stable at -20 oC with half-life of 307 days. Amongst various additives used, glycerol and Tween 80 provided significant stability to the enzyme with half-life 15.5 and 85.5 h, respectively as compared to control (6.9 h). The solidification of sucrose supplemented milk by purified dextransucrase due to dextran synthesis displayed its application as additive for improving the texture of dairy products.