Cloning and characterization of two rhamnose synthases from Sorbus aucuparia / 药学学报

Rhamnose synthase (RHM) is a key enzyme in the biosynthesis of uridine diphosphate rhamnose (UDP-Rha), reversibly converting uridine diphosphate-glucose (UDP-Glc) into UDP-Rha in the presence of NADH or NADPH. In this research, yeast extract (YE) was used to stimulate Sorbus aucuparia suspension cells. Based on a previous study of the transcriptome database of S. aucuparia suspension cells, two RHMs were cloned from S. aucuparia and named SaRHM1 (GenBank No.: MK213340) and SaRHM2 (GenBank No.: MK213341). The SaRHM1 gene contained a 2 007 bp open reading frame (ORF) encoding a polypeptide of 668 amino acids with a molecular weight of 75.25 kD, and a theoretical isoelectric point (pI) of 7.24. The SaRHM2 gene contained a 2 040 bp ORF encoding a polypeptide of 679 amino acids with a molecular weight of 76.26 kD and pI of 6.41. Bioinformatic analysis indicated that SaRHM1 and SaRHM2 contained two special sequences of GxxGxxG/A and YxxxK. Multiple sequence alignments and phylogenetic trees show that SaRHM1 and SaRHM2 have high sequence similarity with other plant species of RHMs. The results of enzyme activity assays in vitro revealed that both recombinant SaRHM1 and SaRHM2 are able to convert UDP-Glc into UDP-Rha. SaRHMs displayed maximum activity at 40 ℃ and a pH of 8 and 9, respectively. The Km values of SaRHM1 and SaRHM2 for UDP-Glc were 212.4 ± 56.70 and 361.0 ± 63.74 μmol·L-1, respectively, with Vmax values of 235.5 ± 18.98 and 516.5 ± 22.30 nmol·min-1·μg-1, respectively. This study reports the cloning and sequencing of RHMs from S. aucuparia and verifies their function, which likely provide rhamnose donors for the subsequent biosynthesis of rhamnosides.

Cloning and characterization of UDP-L-rhamnose synthase 1/2 from Fallopia multiflora / 药学学报

UDP-rhamnose is a rhamnose donor in a reaction catalyzed by UDP-rhamnose synthase (RHM), and plays an important role in the biosynthesis of rhamnoside compounds. The current literature suggests that there are only a few genes can encode the corresponding enzymes to participate in UDP-rhamnose biosynthesis in plants. In this study, two RHM genes (FmRHM1 & 2) were first cloned by using the transcriptomic data of Fallopia multiflora (Thunb) Harald and the multidimensional analysis, including bioinformatics, functional identification in vitro and tissue-specific expression analysis. The results showed that the open reading frame (ORF) of FmRHM1 & 2 genes both were 2 013 bp, encode proteins consisting of 670 amino acids with a calculated molecular mass of 75.6 kDa, and the theoretical isoelectric points of 6.20 and 7.19, respectively. Bioinformatic analysis also indicated that FmRHM1 & 2 contained 2 special sequences of GxxGxxG/A and YxxxK. The phylogenetic analysis showed that the FmRHM gene has a high homology with RHM of other species. The results of enzyme activity in vitro revealed that both recombinant FmRHM1 and FmRHM2 have catalytic activities for converting UDP-glucose into UDP-rhamnose. Measurements of tissue-specific expressions showed that the expression levels of FmRHM1 and FmRHM2 were lower in roots. On the contrary, the 2 genes showed significantly high expression in the stems and leaves. In conclusion, we have cloned and characterized the RHM gene function for the first time in F. multiflora. Here we have provided the preliminary data suggesting the need for further research on UDP-rhamnose biosynthesis by microorganisms.

Isolation and identification of chemical constituents from Disporum cantoniense / 中草药

Objective: To study the chemical constituents from Disporum cantoniense. Methods: Various column chromatographic techniques were used to separate and purify the chemical constituents and their structures were elucidated by spectral ananlysis. Results: Sixteen compounds were isolated and identified as 3-maleimide-5-oxime (1), 4-methylene-5-oxopyrrolidine-2- carboxylic acid (2), thymine (3), adenosine (4), 5'-deoxy-5'-methylamino-adenosine (5), ethyl-α-L-rhamnose (6), bergenin (7), 4-hydroxy- 2-methoxyphenyl-6-deoxy-α-L-talopyranoside (8), (-)-epicatechin (9), (6R,9R)-roseoside (10), 3-(4-hydroxy-3,5-dimethoxyphenyl) propane-1,2-diol (11), 3-hydroxy-5-(p-hydroxyphenyl) pentanoic acid (12), 1-ribityl-2,3-diketo-1,2,3,4-tetrahydro-6,7-dimethyl-quinoxaline (13), (6S,9R)-vomifoliol (14), 1-(3,4-dihydroxyphenyl)-2-hydroxye-thanone (15), and 1,2-dihydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)- ethane (16). Conclusion: Compounds 1, 2, 5-9, and 12-16 are isolated from genus Disporum Salisb. for the first time, and compounds 1, 2, 5-10, and 12-16 are isolated from this plant for the first time.

Monosaccharide composition analysis on polysaccharides in Polygonatum cyrtonema by high performance anion-exchange chromatography with pulsed amperometric detection / 中草药

Objective: To develop a method for determination of monosaccharide composition of Polygonatum cyrtonema polysaccharides (PCR) by high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC- PAD). Methods: The water-soluble crude polysaccharides were extracted from the P. cyrtonema Hua, then the polysaccharides were hydrolyzed with sulfuric acid, then separated by CarboPac PA1 column (250 mm × 4 mm, 10.0 μm) and with gradient elution, determination of monosaccharide composition in PCP by HPAEC-PAD. Results: The results showed that within the range of 1-100 mg/L it has good linearity (r > 0.999), The detection limitation was 0.015-0.025 mg/L, 1.35%-2.80% RSD, the recovery rates were 88.36%-111.3%. The results showed that the polysaccharides in P. cyrtonema Hua from different regions were composed of rhamnose, arabinose, galactose, glucose, mannose, and fructose. Conclusion: The proposed method has good separation effect and high sensitivity, which can be used for the study on monosaccharide composition and content determination of PCP.

Dynamic accumulations and distributions of eight chemical constituents of Hunan Belladonnae Herba in various parts and different growing periods / 中草药

Objective: To investigate the dynamic accumulations and distributions of eight chemical compounds of Belladonnae Herba in different growing periods and explore the distribution of each ingredient in various parts. Methods: The detection was performed by RP-HPLC with diode array detector (DAD). The mobile phase was acetonitrile-0.05% phosphonic acid in gradient elution. The HPLC method was established for the simultaneous determination of the eight compounds, such as scopolin, chlorogenic acid, quercetin-3-O-galactose (6→1) rhamnose-7-O-glucoside, quercetin-3-O-glucose (6→1) rhamnose-7-O-glucoside, kaempferol-3-O-galactose (6→1) rhamnose-7-O-glucoside, kaempferol-3-O-glucose-(6→1) rhamnose-7-O-glucoside, scopoletin, and rutin, and also for the the comparison on the changes of each ingredient in different growing periods and distribution in various parts. Results: The total amount of each ingredient in the whole plant increased along with its growth. However, each ingredient had different rate of increase, in addition, most compounds would reach to a balance of accumulation in the middle or the last third of June. The analysis results showed scopolin mainly distributed in the roots, while chlorogenic acid had a higher distribution in the leaves and flowers; Scopoletin distributed evenly among all parts in the plant, and the others possessed a higher distribution among the leaves and flowers, however, there was scare distribution in the roots. Conclusion: Each ingredient of Belladonnae Herba has different rate of increase and different distribution in each part, resulting in comparatively large difference of each compound. The experimental data of this research could provide the basis for the implantation, harvest and collection, and quality evaluation of Belladonnae Herba.

Effect of nutrients and fermentation conditions on the production of biosurfactants using rhizobacteria isolated from fique plants / Efecto de los nutrientes y las condiciones de fermentación en la producción de biosurfactantes con rizobacterias aisladas de fique / Efeito dos nutrientes e as condições de fermentação na produção de biossurfactantes com rizobacté rias isoladas da piteira

Objective. To isolate biosurfactant-producing microorganisms from the rhizosphere of fique and to select the best genus to evaluate the effect of nutritional and fermentation conditions on the production of rhamnolipids. Materials and methods. Rhizospheric soil was sampled in three areas of Cauca. The best genus was selected for the experimental designs (Plackett Burman and 2² factorial) and to find the production conditions for the growth kinetics at an Erlenmeyer flask scale. Results. Isolates from the rhizosphere of fique plants were from groups (or genera) of Bacillus, Pseudomonas and Actinomycetes, being Pseudomonas the more responsive in preliminary testing for emulsification. From the results of the experimental designs and the kinetics of production, we found that rhamnose synthesis associated with rhamnolipids (3.2 g/l) and emulsification (68% EC24) was significantly favored (p <0.0001) by cultivating an inoculum of 10% v/v of Pseudomonas fluorescens in a medium composed of: soybean oil 2% (v/v), K2HPO4 0.2% (w/v), yeast extract 0.4 g/l, NH4NO3 3.7 g/l, 1 ml trace elements (CoCl3 20 mg/l, H3BO(3)30 mg/l, ZnSO(4)10 mg/l, Cu2SO(4)1 mg/l, Na2MoO(4)3 mg/l, FeSO (4)10 mg/l MnSO(4)2,6 mg/l) and pH 7.2. Conclusion. Of all the microbial genera isolated from the rhizosphere of fique, Pseudomonas fluorescens had the greatest potential in the production of biosurfactants of the rhamnolipids family.

Objetivo. Aislar microorganismos de la rizosfera de fique capaces de producir biosurfactantes y seleccionar el mejor gé nero para evaluar el efecto de las condiciones nutricionales y de fermentación en la producción de ramnolípidos. Materiales y mé todos. Se realizaron muestreos de suelos rizosfé ricos en tres zonas del Cauca. El mejor gé nero fue seleccionado para realizar los diseños experimentales (Plackett Burman y factorial 2²) y establecer las condiciones de producción para las ciné ticas de crecimiento a escala de Erlemeyer. Resultados. Se aislaron bacterias del gé nero Bacillus, Pseudomonas y del grupo Actinomycetes, siendo Pseudomonas el grupo con mayor respuesta en las pruebas preliminares de emulsificación. A partir de los resultados obtenidos en los diseños experimentales y ciné ticas de producción, se estableció que la síntesis de ramnosa asociada con ramnolípidos (3,2 g/l) y la emulsificación (68% EC24) se favorecieron significativamente (p<0.0001) al cultivar un inoculo de 5% v/v de Pseudomonas fluorescens en un medio compuesto por: aceite de soya 2% (v/v), K2HPO4 0,2% (p/v), extracto de levadura 0,4 g/l, NH4NO(3)3,7 g/l, 1 ml de elementos traza (CoCl(3)20 mg/l, H3BO(3)30 mg/l, ZnSO(4)10 mg/l, Cu2SO4 1 mg/l, Na2MoO(4)3 mg/l, FeSO(4)10 mg/l MnSO(4)2,6 mg/l) y pH 7.2. Conclusión. Se aislaron 3 gé neros microbianos a partir de rizosfera de fique, siendo Pseudomonas fluorescens la bacteria con mayor potencial en la producción de biosurfactantes de la familia de los ramnolípidos.

Objetivo. Isolar microorganismos da rizosfera da piteira capazes de produzir biossurfactantes, selecionar o melhor gênero para avaliar o efeito das condições nutricionais e de fermentação na produção de rhamnolipídeos. Materiais e mé todos. Foram realizadas amostragem de solos rizosfé ricos em três á reas do Cauca. O melhor gênero foi selecionado para realizar desenhos experimentais (Plackett Burman e fatorial 2²) e definir as condições de produção para as ciné ticas em escala de erlenmeyer. Resultados. Foram isoladas bacté rias do gênero Bacillus, Pseudomonas e grupo de Actinomycetes. As Pseudomonas foram o grupo com maior resposta nos testes preliminares de emulsificação. A partir dos resultados obtidos nos desenhos experimentais e ciné ticas de produção foi estabelecido que a síntese de ramnose associados com rhamnolipídeos (3,2 g/l) e a emulsificação (68% EC24) foram favorecidos significativamente (p<0,0001) ao cultivar um inoculo de 5% v/ v de Pseudomonas fluorescens em um meio composto por: óleo de soja 2% (v/v), K2HPO(4)0,2% (p/v), extrato de levedura 0,4 g/l, NH4NO3 3,7 g/l, 1 ml de oligoelementos (CoCl(3)20 mg/l, H3BO(3)30 mg/l, ZnSO(4)10 mg/l, Cu2SO(4)1 mg/l, Na2MoO(4)3 mg/l, FeSO(4)10 mg/l MnSO4 2,6 mg/l) e pH 7,2. Conclusão. Três gêneros microbianos foram isolados da rizosfera da piteira, sendo Pseudomonas fluorescens a bacté ria com maior potencial na produção de biossurfactantes da família dos rhamnolipídeos.