Arthrobacter simplex 156 is a microorganism that is used for steroid drug biotransformation of cortisone acetate (CA) to prednisone acetate (PA). The enzyme 3-ketosteroid-△(1)-dehydrogenase encoded by the ksdD gene plays an important role in the bioconversion process. To further improve the biotransformation efficiencies of the industrial strain, a genetic manipulation system for A. simplex 156 was developed. Additional copies of the ksdD gene under the control of the cat promoter (from pXMJ19) were transferred into the strain A. simplex 156 and integrated into the 16S rDNA sites, yielding a series of recombinant strains. One of these recombinant strains, designated A. simplex M158, exhibited superior properties for CA biotransformation. At the substrate concentration of 83.6 g/l, the highest PA production of the recombinant strain reached 66.7 g/l, which is approximately 32.9 % higher than that of wild-type strains, and the incubation time for CA to PA bioconversion was reduced by 20 h. Southern blotting analysis of the recombinant strain indicated two copies of deregulated ksdD genes were integrated into the 16S rDNA sites, which means two of five 16S rRNA operons were insertionally disrupted in the recombinant strain. However, the disruption of the two 16S rRNA operons did not affect the growth rate of the recombinant strain, which survived and thrived under desired conditions. In addition, the new strain was genetically stable for more than 100 generations without the use of antibiotics for selection. These superior characteristics make the new strain more suitable than the wild-type strain for PA production.

An aptamer-based electrochemical biosensor was successfully developed and applied in the rapid detection of pathogenic Escherichia coli (E. coli) in licorice extract. The thiolated capture probes were firstly immobilized on a gold electrode, and then the biotinylated aptamer probes for E. coli were introduced by hybridization with the capture probes. Due to the stronger interaction between the aptamer and the E. coli, a part of the biotinylated aptamers will dissociate from the capture probes in the presence of E. coli. The residual biotinylated aptamer probes can quantitatively bind with streptavidin-alkaline phosphatase. Subsequently, α-naphthyl phosphate substrate was catalytically hydrolyzed to generate electrochemical response, which could be recorded by a differential pulse voltammetry. The dependence of the peak current on the logarithm of E. coli concentration in the range from 5.0 × 10 2 colony forming units (CFU)/mL to 5.0 × 10 7 CFU/mL exhibited a linear trend with a detection limit of 80 CFU/mL. The relative standard deviation of 5 successive scans was 5.3%, 4.5% and 1.1% for 5.0 × 10 2 , 5.0 × 10 5 and 5.0 × 10 7 CFU/mL E. coli, respectively. In the detection of the licorice extract samples, the results obtained from the proposed strategy and traditional culture counting method were close to each other, but the time consumption was only~1/30 compared with the traditional method. These results demonstrate that the designed biosensor can be potentially utilized for rapid microbial examination in traditional Chinese medicine and relevant fields. Appl. Sci. 2019, 9, 295 2 of 15 bacterial colony morphology, color change, and biochemical reactions for quantitative detection. This detection method is economical and relatively simple to operate, but the detection time is generally 48-72 h or even longer, which cannot meet the needs for rapid detection. As an effective molecular biological detection technology, polymerase chain reaction (PCR) and the derived technology of PCR has been widely used in detecting E. coli [4,7]. PCR is based on the principle that DNA templates can denature at high temperatures, reactivate and extend (one cycle) at low temperatures by using appropriate DNA polymerase and primers. As the template DNA amplification product increases exponentially, increasing the number of cycles can magnify the specified DNA sequence by several million-fold in a short time, which greatly improves detection sensitivity [8]. However, PCR technology is costly and prone to produce false negatives [9], which limits the extensive application of PCR. In addition, enzyme-linked immunosorbent assay (ELISA), as a common immunological detection technology, has also been used in bacterial test [5,10]. ELISA technology undergoes a color change by the catalysis of the enzyme in the specific recognition process. Owing to the immune activity between antigen and antibody, ELISA method has superior specificity, high sensitivity and short detection time, and has been used in detection of different subtypes of E. col...

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Gynostemma pentaphyllum (thumb.) Makino is a functional herbal tea commonly used in Asian countries and regions to reduce blood lipid levels. G. pentaphyllum saponin is the main component, but there are still a large number of components with lipid-lowering activity that have not been found. In this study, 10 novel dammarane-type saponins, (1–10) and a known one (11) were isolated from G. pentaphyllum. Ten new compounds were identified and named as yunnangypenosides A-J (1–10), and another known one (11) was also obtained. Their chemical structures were determined by MS, NMR spectroscopic analyses. Moreover, the cytotoxicities on human HepG-2 hepatocellular carcinoma cells of these isolates were evaluated, and the results showed that compounds 1–11 had no obvious cytotoxicity. Finally, all these compounds were evaluated for their lipid-lowering effect by means of the oil red O staining method. Ten compounds could significantly reduce lipid levels except of 2, especially 8 exhibite the strongest hypolipidemia activity.

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