Cold Stress-induced Glucosyltransferase CsUGT78A15 is Involved in the Formation of Eugenol Glucoside in Camellia sinensis

Eugenol is a natural phenolic compound known for its health-promoting properties and its ability to add a floral scent to tea plants.Plant eugenol glycosides have been identified and shown to make important contributions to fruit floral quality.However,the details of their biosynthesis and metabolism in tea plants are still unknown.Here,eugenol glucoside was unambiguously identified as a native metabolite in the tea plant,and its biosynthesis was shown to be induced by low temperature treatment.Through the analysis of UGTs induced by low temperature,the glycosyltransferase CsUGT78A15 was identified in tea,and its encoded protein was shown to catalyze the glucosylation of eugenol.Vmax/Km ratios showed that eugenol was the most suitable substrate for CsUGT78A15.Sugar donor preference analysis showed that CsUGT78A15 had a higher selectivity for glucose,followed by galactose and glucuronic acid.The expression of CsUGT78A15was correlatedwith the accumulation of eugenol glucoside in different tissues and genotypes of tea.Down-regulation of CsUGT78A15 led to a decreased eugenol glucoside content under cold stress,indicating that CsUGT78A15 plays an important role in the biosynthesis of eugenol glucoside under cold stress.The identification of eugenol glucoside in the tea plant and the discovery of a cold stress-induced eugenol glucosyltransferase in tea provide the foundation for the improvement of tea flavor under cold stress and the biotechnological production of eugenol glucoside.

羧基和咪唑基团在柚柠檬苦素类化合物糖基转移酶催化反应中的作用(英文)

Limonoid bitterness is a serious problem in the citrus industry worldwide. Limonoid glucosyltransferase is an enzyme that catalyzes the conversion of bitter limonoid into non-bitter limonoid glucoside while retaining the health benefit of limonoids in the juice. The immobilization of this enzyme in a column can solve the juice bitterness problem. More information about the catalytic residues of the enzyme is needed in this immobilization process. Glutamate/aspartate,histidine,lysine,tryptophan,serine,and cysteine residues were chemi-cally modified to investigate their roles in the catalytic function of limonoid glucosyltransferase. Inactivation of the enzyme following modi-fication of carboxyl and imidazole moieties was a consequence of a loss in substrate binding and catalysis in the glucosyltransfer reaction. The modification of a single histidine residue completely destroyed the ability of limonoid glucosyltransferase to transfer the D-glucopyranosyl unit. Tryptophan seemed to have some role in maintaining the active conformation of the catalytic site. Lysine also seemed to have some direct or indirect role in this catalysis but the modification of serine and cysteine did not have any effect on catalysis. Therefore,we conclude that the carboxyl and imidazole groups containing amino acids are responsible for the catalytic action of the enzyme.

HCV NS5A and NS5B Enhance Expression of Human Ceramide Glucosyltransferase Gene

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-l-phenyl-2-decanoylamino-3- morpholino-l-propanol）. In agreement with these results, the expression level of GlcT-l（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

Biofilm Formation by <i>Streptococcus mutans</i>and Related Bacteria

Caries is a disease of human dentition characterized by the loss of mineralized surfaces of the tooth;it is an infectious disease of the oral cavity in which biofilms play a causative role. Control of biofilms has traditionally relied on non-specific removal of plaque by mechanical means such as brushing, although it is difficult to remove biofilms by this method. Caries is also a widespread infection in children. Streptococcus mutans and S. sobrinus are important causative agents of caries. They produce a homologous exocellular polysaccharide called glucan, which strongly adheres to the enamel surface. This is a review of oral microbial biofilm formation by S. mutans and other related bacteria.

Transcriptional Analysis of 9-Cis-Epoxycarotenoid Dioxygenase, Glucosyltransferase, 8＇-Hydroxylase and B-Glucosidase Genes that Regulate Abscisic Acid Homeostasis around the Onset of Grape Berry Ripening

The mechanisms for fine-tuning ABA level related to grape berry ripening remain elusive. Here, transcription analysis showed that the mRNA expression level of 9-cis-epoxycarotenoid dioxygenase gene （VvNCED1） increases first, rapidly in mesocarp before the onset of grape berry ripening. After VvNCED1 peaks its expression level, ABA content increases rapidly in mesocarp coupled with an increase in both soluble sugar content and pH value. On the onset of berry ripening, VvNCED1 transcripts decline rapidly to its lowest point, then increases slightly. Whereas, the mRNA expression level of B-glucosidase gene VvBGI, on the whole, increases constantly during grape berry ripening. During berry de-greening, ABA glucosyltransferase （VvGT） and ABA 8＇-hydroxylase （VvCYPI） equilibrate ABA level; during berry coloring-up, VvGT predominantly equilibrates ABA level, namely, the up-regulation of ABA level mainly leads from VvNCED1 and VvBG1 gene high expression; the down-regulation of ABA level leads mainly from VvCYP! transcript level both in ABA content- and developmental phase-dependence manner. In conclusion, our main results show that VvNCED1 and VvBG1 genes are closely related to grape berry ripening.

Preparation of high-quality resistant dextrin through pyrodextrin by a multienzyme complex

As a soluble food raw material with a low calorie content,resistant dextrin (RD) has broad application prospects in the food industry.Branching enzymes (BEs),as a key enzyme for RD preparation,can break the α-1,4 glycosidic bonds of donor chains and reconstruct the cleaved chains to acceptor chains through the α-1,6 glycosidic bonds.BEs with high transglucosidic activity toward amylopectin and short-chain substrates are urgently needed to increase the quality of RD.Herein,BE derived from Thermuobifida fusca (TfBE) was mined and characterized.The optimal temperature and pH of the TfBE were 40 ℃ and 6.5,respectively.A total of 1500 U/g substrate TfBE reacted with 20% (w/v) pyrodextrin for 12 h,the ratio of α-1,4 to α-1,6 glycosidic bonds was changed from 3.52:1 to 2.33:1,and the content of enzyme-resistant components notably increased from 44.0% to 53.8%.Furthermore,to make full use of receptor chains and small molecular sugars in the reaction system,a multienzyme complex of TfBE with T.fusca α-cyclodextrin glucosyltransferase (TfCGTase),TfBE with TfCGTase and Aspergillus nidulans α-glucosidase (AnGS) was used to further increase the enzyme resistance of RD from 44.0% to 65.3% and 70.6%,respectively.The developed multienzyme complex method could effectively contribute to improving the production quality and efficiency of RD preparation.

Crocin Synthesis Mechanism in Crocus sativus

Saffron （Crocus sativus） cells can synthesize crocin, crocetin digentiobiosyl ester, in suspension cultures. The crocin family biosynthesis mechanism was studied using high pressure liquid chromatography （HPLC） to determinate the glucosyltransferase activity and to develop a method for synthesizing medicine from saffron cells. Previous studies indicated that two glucosyltransferases might be involved in the formation of crocetin glucosyl- and gentiobiosyl-esters. GTasel formed an ester bond between crocetin carboxyl groups and glucose moieties while GTase2 catalyzed the formation of glucosidic bonds with glucosyl ester groups at both ends of the molecule. These enzymes can catalyze the formation of crocetin glucosides in vitro. GTasel activity is higher during the first four days of crocin glucosides biosynthesis, but decreases after four days. The formation and accumulation of crocin increase during the first six days and stabilized on the eighth day.

Molecular modification of Bacillus stearothermophilus NO_(2) cyclodextrin glucosyltransferase and preparation of α-cyclodextrin

Cyclodextrin(CD)is produced by the catalysis of starch or starch derivatives by cyclodextrin glucosyltransferase(CGTase),and its yield is mainly limited by the product and reaction specificity of CGTase.In this study,we use CGTase derived from Bacillus stearothermophilus NO_(2),exhibiting high expression levels and good stability for molecular modification.The N353A mutant effectively decreases the hydrolysis activity,and the ratio of the k_(cat) values(cyclization to hydrolysis activity)is 86.46,which is threefold that of the wild type.The E142P mutant effectively enhancesα-CD specificity,which increases the ratio of k_(cat) values(α-CD toβ-CD formation)from 2.18 of the wild-type to 2.42.The N353A/E142P mutant weakens the hydrolysis side reaction and enhancesα-CD specificity,and the proportion ofα-CD products is 53.67%,which is 15.62%higher than that of the wild-type.This research focuses on CGTase reaction and product specificities,which suggest a novel method for the industrial production ofα-CD.

Molecular cloning and functional characterization of an isoflavone glucosyltransferase from Pueraria thomsonii

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.

Synthesis of Camptothecin-lO-O-glucoside Using an Engineered Oleandomycin Glucosyltransferase

Oleandomycin glycosyltransferase variant P67T/S132F/A242V（ASP） was used to convert 10-hydroxycamptothecin into camptothecin-10-O-glucoside, which was confirmed by spectral analysis. Compared to the previously reported results, the present study reached the conversion rate up to 80% through the optimization of reaction conditions. In addition, compared with 10-hydroxycamptotheein（HCPT）, camptothecin-10-O-glucoside inhibited the proliferation of Huh7 cells in a concentration-dependent manner and showed stronger antineoplastic effect but lower toxicity. Furthermore, camptothecin-10-O-glucoside induced more apoptotic cells as compared with the parent compound.