Transformation of Tetracycline by Manganese Peroxidase from Phanerochaete chrysosporium.

The negative impacts on the ecosystem of antibiotic residues in the environment have become a global concern. However, little is known about the transformation mechanism of antibiotics by manganese peroxidase (MnP) from microorganisms. This work investigated the transformation characteristics, the antibacterial activity of byproducts, and the degradation mechanism of tetracycline (TC) by purified MnP from Phanerochaete chrysosporium. The results show that nitrogen-limited and high level of Mn2+ medium could obtain favorable MnP activity and inhibit the expression of lignin peroxidase by Phanerochaete chrysosporium. The purified MnP could transform 80% tetracycline in 3 h, and the threshold of reaction activator (H2O2) was about 0.045 mmol L-1. After the 3rd cyclic run, the transformation rate was almost identical at the low initial concentration of TC (77.05-88.47%), while it decreased when the initial concentration was higher (49.36-60.00%). The antimicrobial potency of the TC transformation products by MnP decreased throughout reaction time. We identified seven possible degradation products and then proposed a potential TC transformation pathway, which included demethylation, oxidation of the dimethyl amino, decarbonylation, hydroxylation, and oxidative dehydrogenation. These findings provide a novel comprehension of the role of MnP on the fate of antibiotics in nature and may develop a potential technology for tetracycline removal.

Simultaneously detecting ethyl carbamate and its precursors in rice wine based on a pH-responsive electrochemical impedance sensor.

Intensive efforts have been made to diminish ethyl carbamate in fermentation products. The presented research demonstrated an approach to simultaneously detecting ethyl carbamate and its precursors including urea, citrulline and arginine with nano Mn(Ⅱ)O modified composite working electrode via electrochemical impedance spectroscopy. Adjusting sample solutions at certain pH value leads the differentiated priority of protonation from nitrogen group in the ethyl carbamate (EC) and its precursors. Molecular recognition was achieved through attractive electrostatic interaction due to the negatively charged Mn(Ⅱ)O nanocrystal on the working electrode surface in aqueous sample solutions. Deconvolution and principle component analysis were applied to differentiate the specific scanning frequency for each analyte. The detection limits of EC, citrulline, urea and arginine are 0.8 ng L-1, 1.57 ng L-1, 0.54 ng L-1 and 1.56 ng L-1, respectively. The developed electrochemical sensor provides a sensitive and selective approach superior to the current reported label-free methodologies and offering a solution for ethyl carbamates in real time process control.

Enrichment-Free Rapid Detection of Phthalates in Chinese Liquor with Electrochemical Impedance Spectroscopy.

A non-invasive real-time detection technique for phthalates in Chinese liquor is proposed in this paper. This method is based on the measurement of Faradaic impedance in the presence of a redox probe, [Fe(CN)6]3-/4-, upon the absorption of phthalates to the graphene electrode surface. This absorption activity is according to the π-π stacking interactions between phthalates and the graphene working electrode which allows direct sampling and analyte preconcentration. The absorption of phthalates retards the interfacial electron-transfer kinetics and increases the charge-transfer resistance (Rct). Numerical values of Rct were extracted from a simulation of electrochemical impedance spectroscopy (EIS) spectra with the corresponding equivalent circuit. Cathodic polarization was employed prior to EIS measurements to effectively eliminate the metal ion interference. The results yielded a detection limit of 0.024 ng/L for diethyl phthalate (DEP) with a linear range from 2.22 ng to 1.11 µg. These results indicate a possibility of developing a household sensor for phthalate determination in Chinese liquor.

GUITAR-enhanced facile discrimination of aged Chinese Baijiu using electrochemical impedance spectroscopy.

The presented research demonstrated a method to evaluate aged Chinese Baijiu by measuring their electrochemical impedance spectrums (EIS) according to the distinctive colloidal impedance phenomena of verities' aged Baijiu. Graphite from the University of Idaho Thermolyzed Asphalt Reaction (GUITAR) electrode was employed for EIS data collection to achieve enhanced resolution. The obtained EIS Nyquist plots were simulated with the corresponding equivalent circuit to extract the numerical values of parameters which characterize the physicochemical properties of aged Baijiu. The curve fitting data were dimensionally reduced with principle components analysis (PCA). PCA score image originated from GUITAR electrode EIS measurement showed a more defined clustered profile in comparison to glassy carbon electrode. These results implicate GUITAR electrode based EIS is potentially available to develop a household device for aged Baijiu classification.

Highly efficient and regioselective synthesis of dihydromyricetin esters by immobilized lipase.

Dihydromyricetin is the principle component of the Chinese herbal tea Teng-cha and a promising ingredient for functional food and nutraceuticals, but its low solubility limits its application potentials. This study explored enzymatic acylation of dihydromyricetin to improve its solubility in lipid systems. Acylation was achieved with several lipases with the synthesis of a major (>86%) product and a minor product. Isolation and purification of the products by preparative HPLC followed by LC-MS, (13)C NMR, (1)H NMR and 2 D-HSQC NMR analyses showed that the major product was a dihydromyricetin monoester with the acylation site at the 3-OH group of C ring. Quantum chemical calculations revealed that the 3-OH had the lowest antioxidant activity, and therefore acylation at this site was expected to have minimum impact on the antioxidant activity. Several factors, including solvent, acyl donor, enzyme origin, molar ratio of substrates and reaction temperature and time, exhibited significant effects on the initial rate, conversion yield and regioselectivity of the reaction. Acylation occurred only with vinyl acetate as the acyl donor, and highest conversion yields were achieved with immobilized Penicillium expansum lipase and Novozyme 435 with DMSO and acetonitrile being the best solvents. In general, the acylation results were found to be superior to previous reports on acylation of aglycone flavonoids with respects to conversion yield and regioselectivity.

Design and synthesis of the novel DNA topoisomerase II inhibitors: esterification and amination substituted 4'-demethylepipodophyllotoxin derivates exhibiting anti-tumor activity by activating ATM/ATR signaling pathways.

According to the structure-activity relationship, drug combination principle and bioisosterism, a series of the novel esterification and amination 4'-demethylepipodophyllotoxin derivates were rationally designed in order to discover the potential antitumor prodrug. And then these compounds were tested by the drug-topoisomerase II docking models for virtual screening. Thus, twelve target compounds were screened out and synthesized. Most of compounds exhibited promising in vitro anti-tumor activity, particularly 4-N-tris(hydroxymethyl)metylaminomethane-4-deoxy-4'-demethylepipodophyllotoxin (Compound 1). The anti-tumor activity of Compound 1 against the tumor cell lines BGC-823 (i.e., the IC50 value of 5.35 ± 0.77 µM), HeLa (i.e., the IC50 value of 160.48 ± 14.50 µM), and A549 (i.e., the IC50 value of 13.95 ± 5.41 µM) was significantly improved by 706%, 31% and 900% than that of etoposide (i.e., the IC50 values of 43.74 ± 5.13, 209.90 ± 13.42, and 139.54 ± 7.05 µM), respectively. Moreover, the IC50 value of Compound 1 against the normal human cell line HK-2 (i.e., 16.3 ± 3.77 µM) was 78% lower than that of etoposide (i.e., 9.17 ± 1.58 µM). Compound 1 could diminish the relaxation reaction topoisomerase II DNA decatenation at a concentration of 10 µM and induce BGC-823 apoptosis by breaking DNA double-strand and activating ATM/ATR signaling pathways.

Actinobacillus succinogenes ATCC 55618 fermentation medium optimization for the production of succinic acid by response surface methodology.

As a potential intermediary feedstock, succinic acid takes an important place in bulk chemical productions. For the first time, a method combining Plackett-Burman design (PBD), steepest ascent method (SA), and Box-Behnken design (BBD) was developed to optimize Actinobacillus succinogenes ATCC 55618 fermentation medium. First, glucose, yeast extract, and MgCO(3) were identified to be key medium components by PBD. Second, preliminary optimization was run by SA method to access the optimal region of the key medium components. Finally, the responses, that is, the production of succinic acid, were optimized simultaneously by using BBD, and the optimal concentration was located to be 84.6 g L(-1) of glucose, 14.5 g L(-1) of yeast extract, and 64.7 g L(-1) of MgCO(3). Verification experiment indicated that the maximal succinic acid production of 52.7 ± 0.8 g L(-1) was obtained under the identified optimal conditions. The result agreed with the predicted value well. Compared with that of the basic medium, the production of succinic acid and yield of succinic acid against glucose were enhanced by 67.3% and 111.1%, respectively. The results obtained in this study may be useful for the industrial commercial production of succinic acid.

Manipulation of heterogeneity product in 4'-demethylepipodophyllotoxin biotransformation process by using yeast extract as nitrogen source.

Manipulation of product heterogeneity was attempted by using yeast extract as nitrogen source in Alternaria alternata S-f6 transformation process of 4'-demethylepipodophyllotoxin. When the nitrogen source of NaNO(3) was replaced by yeast extract, the heterogeneity of biotransformation products was significantly varied from a single product (i.e., 4'-demethylpodophyllotoxone) to four podophyllum derivates. According to the kinetics of 4'-demethylepipodophyllotoxin biotransformation process by A. alternata S-f6, the starting substrate of 4'-demethylepipodophyllotoxin was preferentially transformed to produce 4'-demethylpodophyllotoxone (1) with an oxidation reaction. By the further comparison of products configuration, 4ß-caprinoyl-4'-demethylepipodophyllotoxin (3) was produced from 4'-demethylpodophyllotoxone (1) instead of 4'-demethylisopicropodophyllone (2), which might be produced from 4'-demethylpodophyllotoxone (1) with the isomerization of lactone. Finally, 4'-demethylisopicropodophyllone (2) was hydrolyzed to produce 3α-hydroxymethyl-(6, 7)-dioxol-4-one-naphthalene (4). This work shows new information on the 4'-demethylepipodophyllotoxin biotransformation process by A. alternata S-f6 and provides a foundation for further studies on the structural diversification of a bioactive natural lead compound.

Comparison of lipid content and fatty acid composition between Tuber fermentation mycelia and natural fruiting bodies.

A comparison of lipid content and fatty acid (FA) composition between Tuber fermentation mycelia and natural fruiting bodies indicates that the lipid content in Tuber fermentation mycelia is higher than that in fruiting bodies. Unsaturated FAs (particularly linoleic acid and oleic acid) were the predominant constituents in total FAs in both Tuber fermentation mycelia and fruiting bodies. A total of 23 FAs, including arachidonic, eicosapentaenoic, docosahexaenoic, and γ-linolenic acids, were first identified in the Tuber species. A hierarchical clustering analysis showed that the FA profile of fermentation mycelia was quite similar, regardless of Tuber species. However, the FA profile of the fruiting bodies was significantly influenced by its species and habitat environments. Interestingly, the FA profile of the Tuber indicum and Tuber aestivum fruiting bodies was nearly identical to that of the Tuber fermentation mycelia, which partially confirms the similarity between the Tuber fermentation mycelia and the fruiting bodies.

Determination of the nucleosides and nucleobases in Tuber samples by dispersive solid-phase extraction combined with liquid chromatography-mass spectrometry.

A simple, fast and inexpensive method based on dispersive solid phase extraction (DSPE) combined with LC-MS was developed for simultaneous determination of 7 nucleosides and nucleobases (i.e., adenine, hypoxanthine, uridine, adenosine, guanine, guanosine, and inosine) in Tuber fruiting-bodies and fermentation mycelia. The DSPE procedure was firstly introduced to remove the protein interference from sample solutions, and D3520 macroporous resin was chosen as the DSPE sorbent because of its high removal capability on protein interferences, but low adsorption rate on analytes. Besides, key parameters on DSPE procedure (i.e., macroporous resin type, macroporous resin amount, methanol concentration, and vortex time) were optimized, and the protein removal efficacy could achieve about 95% after the process optimization. Though the method validation test, the DSPE-LC-MS method was confirmed to be precise, accurate and sensitive, and the column blinding problem was solved successfully. By using this established method, the total amount of nucleosides and nucleobases in the fermentation mycelia was determined to range from 4881.5 to 12,592.9µgg⁻¹, which was about 2-25 times higher than the fruiting-bodies (from 498.1 to 2274.1µgg⁻¹). The formulation of nucleosides and nucleobases in the fermentation mycelia maintained relatively constant, while the formulation in Tuber fruiting-bodies varied significantly with their species. Hierarchical cluster analysis (HCA) showed the formulation similarity of nucleosides and nucleobases between Tuber fermentation mycelia and the fruiting-bodies of Tuber indicum and Tuber himalayense. From the viewpoint of nucleosides and nucleobases, this work confirms the potentiality of Tuber fermentation mycelia as the alternative resource for its fruiting-bodies.