Biotransformation of 14-Deacetoxy-13-oxo sinenxan A by Ginkgo Cell Cultures

Deacetoxy-13-oxo sinenxan A (1) was converted to 9a-hydroxy-13-oxo-2a, 5a, 10b-triacetoxy-4(20),11-taxadiene (2) and 10b-hydroxy-13-oxo-2a,5a,9a-triacetoxy- 4(20), 11- taxadiene (3) by Ginkgo cell suspension cultures in 45% and 15% yields, respectively.

BIOTRANSFORMATION OF 2α，5α，10β，14β－TETRA－ACETOXY－4（20），11－TAXADIENE BY THE FUNGIJS CUNNINGHAMELLA ECHINULATA

?5α,10β,14β-Tetra-acctoxy-4(20).11-taxadicne (1) could be regioselectively transfonnedinto 6α 10β-dihydroxy-2α,5α,14β-triacetoxy-4(20). 11- taxadiene (2) by Cunninghamella echinnlata infair yield. 6βM.10β-Dihydroxy-2α,5α,14β-triacet 11-taxadienc (3). 10β-hydroxy-2α,5α,14β-triacetoxy-4(20).11-taxadiene (4), 10 β-hyboxy-4β,20-epoxy -2α,5α,14β-triacetoxy- tax - 11- ene (5)asminor prpducts were also isolated

Recent Progress on Biocatalysis and Biotransformations in Ionic Liquids

Ionic liquids have negligibly low vapor pressure, high stability and polarity. They are regarded as green solvents. Enzymes, especially lipases, as well as whole-cell of microbe, are catalytically active in ionic liquids or aqueous-ionic liquid biphasic systems. Up to date, there have been many reports on enzyme-exhibited features and enzyme-mediated reactions in ionic liquids. In many cases, remarkable results with respect to yield, catalytic activity, stability and (enantio-, regio-) selectivity were obtained in ionic liquids in comparison with those observed in conventional media. Accordingly, ionic liquids provide new possibilities for the application of new type of solvent in biocatalytic reactions.

Whole-cell recording of the robust nucleus of the arcopallium neurons in the adult zebra finch

BACKGROUND： Electrophysiological properties of the song nucleus have been revealed using conventional techniques, such as intracellular and extracellular recording. Research concerning the neuronal activation properties and regulations of the song system at the cellular and ion channel level may help reveal the neural mechanism of song learning. OBJECTIVE： To perform whole-cell recording of robust nucleus of the arcopallium （RA） neurons in brain slices from adult zebra finches （Taeniopygia guttata） and observe the action potential, sodium/potassium current and the spontaneous postsynaptic current of RA neurons. DESIGN, TIME AND SETTING： Self-controlled, neuroelectrophysiological experiment. The study was performed at the Neurophysiology Laboratory of South China Normal University from April to September 2008. MATERIALS： Flaming/Brown puller P-97 was purchased from Sutter Ins, USA; Axopatch 700B amplifier and Digidata 1332A converter were purchased from Axon Instrument, USA; pClamp software was provided by Axon Instrument, USA. METHODS： RA neurons were acutely isolated from 24 healthy male zebra finches. The action potential, voltage-gate sodium/potassium current and spontaneous postsynaptic current were recorded by whole-cell recording technology. Data were analyzed by pClamp software. MAIN OUTCOME MEASURES： The amplitude and frequency of the action potential, and the amplitude of the voltage-dependent and spontaneous postsynaptic currents, were measured. RESULTS： （1） Testing of action potential： Cells exhibited a stable current-voltage relationship following a series of hyperpolarization stepped currents, and an action potential was triggered by the spike threshold. All the recorded cells displayed repetitive firing following depolarizing current injection, with a frequency beyond 100 Hz. （2） Testing of voltage-gate currents： The inward and outward whole-cell currents were observed after a series of depolarizing voltage steps. The inward current disappeared following the application of tetrodotoxin and the outward current was significantly inhibited by application of 4-aminopyfidione and tetraethylammonium chloride. （3） Testing of spontaneous postsynaptic current： The majority of recorded cells exhibited an inward synaptic current when the membrane potential was maintained at -60 mV, with some cells exhibiting a robustly outward current when the membrane potential was maintained at -30 mV. Tetrodotoxin was unable to affect the spontaneous postsynaptic current. Following application of bicuculline [y-aminobutyric acid （A） receptor antagonist] and high concentration kynurenic acid （ionotropic glutamate receptor antagonist）, the inward and outward currents were completely inhibited. CONCLUSION： Under these experimental conditions, the action potential, sodium/potassium current and spontaneous postsynaptic current were recorded successfully in RA neurons. This indicates that the cells preserved relatively intact synaptic connections and normal physiological activity, which is required for investigating ion channels. The inward and outward whole-cell currents were sodium and potassium currents, respectively. The postsynaptic y-aminobutyric acid （A） receptors and ionotropic glutamate receptors contributed to the spontaneous postsynaptic current.

Biotransformation of aesculin by human gut bacteria and identification of its metabolites in rat urine

AIM:To observe the biotransformation process of a Chinese compound,aesculin,by human gut bacteria,and to identify its metabolites in rat urine.METHODS:Representative human gut bacteria were collected from 20 healthy volunteers,and then utilized in vitro to biotransform aesculin under anaerobic conditions.At 0,2,4,8,12,16,24,48 and 72 h post- incubation,10 mL of culture medium was collected.Metabolites of aesculin were extracted 3×from rat urine with methanol and analyzed by HPLC.For in vivo metabolite analysis,aesculetin(100 mg/kg)was administered to rats via stomach gavage,rat urine was collected from 6 to 48 h post-administration,and metabolite analysis was performed by LC/ESI-MS and MS/MS in the positive and negative modes.RESULTS:Human gut bacteria could completely convert aesculin into aesculetin in vitro.The biotransformation process occurred from 8 to 24 h post-incubation,with its highest activity was seen from 8 to 12 h.The in vitro process was much slower than the in vivo process.In contrast to the in vitro model,six aesculetin metabolites were identified in rat urine,including 6-hydroxy-7-gluco- coumarin(M1),6-hydroxy-7-sulf-coumarin(M2),6,7-di- gluco-coumarin(M3),6-glc-7-gluco-coumarin(M4),6-O-methyl-7-gluco-coumarin(M5)and 6-O-methyl-7- sulf-coumarin(M6).Of which,M2 and M6 were novel metabolites.CONCLUSION:Aesculin can be transferred into aesculetin by human gut bacteria and is further modified by the host in vivo.The diverse metabolites of aesculin may explain its pleiotropic pharmaceutical effects.

Study on Biotransformation of Natural Anti-tumor Drug Podophyllotoxin

[Objective] This study aimed to select stains for transforming podophyllotoxin, to isolate the transformation product and to identify the structure. [ Method~ Structure of podophyllotoxin was modified with microbial batch transformation method. Podophyllotoxin and its transformation products were detected by using TLC and HPLC methods to select strains with ability to transform podophyllotoxin. Scale-up fermentation of one podophyllotoxin-transforming strain was carried out and the fermented material was extracted with EtoAc, isolated with conventional silica gel column chromatography, structure of the transformation product was identified by using classic spectroscopy technology. [ Result ] Based on researches of pedophyllotoxin transformation, seven strains with ability to transform po- dophyllotoxin were selected, including BS. Str-1, BS. Str-2, BS. Str-3, BS. Str-7, BS. Str-18, BS. Str-21 and BS. Str-29. By scale up fermentation, the only trans- formation product of BS. Str-21 strain was extracted and isolated, which was identified as picropodophyllotoxin. [ Conclusion] This study demonstrates that podo- phyllotoxin derivatives can be obtained by biotransformation methods, which lays the foundation for acquisition of natural drugs with high anti-tumor activity.

Biotransformation of Carmoisine and Reactive Black 5 Dyes Using <i>Saccharomyces cerevisiae</i>

Saccharomyces cerevisiae (baker’s yeast) is the most important industrial microorganisms. This yeast is commonly used as a leavening agent in baking bread and bakery products, where it produces carbon dioxide from converting of the fermentable sugars present in the dough. Nowadays, industrial and chemical activities led to produce new compounds with new kinds of contamination in the environment. Discharge of untreated or partially treated industrial sewage has created the contamination problems of rivers and lakes such as drugs, oil, heavy metals, paints, pesticides and various chemical compounds in them. Hence, it is necessary to control and reduce the levels of these compounds in wastewater and bring them to permissible values. This study aims to study the bioconversion potential of commonly available Saccharomyces cerevisiae for the two textile dyes of Carmoisine and Reactive Black 5. Reaction mixtures for biotransformation of dyes included 50 mg/l Carmoisine or 25 mg/l Reactive Black 5 and 1% dried harvested cells of S. cerevisiae (bread’s yeast) were tested. Harvested dry and wet yeast were studied for this purpose. The results show that harvested cells of Saccharomyces cerevisiae are able to bioconvert Carmoisine and Reactive Black 5. Reactive Black 5, Carmoisine are degraded by biotransformation 85% and 53% within 24 hours in water at the room temperature.

Unbalanced biotransformation metabolism and oxidative stress status: implications for deficient fatty acid oxidation

The concept of accumulating xenobiotics within the human body as a health risk is well known. However, these compounds can also be endo-genous, as in the case of inborn errors of me-tabolism, and lead to some of the same symp-toms as seen in xenobiotic intoxication. Bio-transformation of both exogenous and endo-genous toxic compounds is an important function of the liver, and the critical balance between these systems is of fundamental importance for cellular health. We propose a novel model, to describe the critical balance between Phase I and Phase II biotransformation and how a disturbance in this balance will increase the oxidative stress status, with resulting pathological consequences. We further used deficient fatty acid oxidation to verify the proposed model, as deficient fatty acid oxidation is associated with the accumulation of characteristic metabolites. These accumulating metabolites undergo both Phase I and Phase II biotransformation reactions, with resulting depletion of biotransformation substrates and co-factors. Depletion of these important biomolecules is capable of disturbing the balance between Phase I and Phase II reactions, and disturbance of this balance will increase oxidative stress status. The value of the proposed model is illustrated by its application to a clinical case investigated in our laboratory. In this case the possibility of deficient fatty acid oxidation only became evident once the critical balance between Phase I and Phase II biotransformation was restored with oral replenishment of biotransformation substrates. In addition to bio-chemical improvement, there was also significant clinical improvement. The significance of this model lies within the treatment possibilities, as the assessment of biotransformation metabolism and oxidative stress status can lead to the development of nutritional treatment strategies to correct imbalances. This in turn may reduce the chances of, or delay the onset of certain disease states.

Production of Enzymes and Biotransformation of Orange Waste by Oyster Mushroom, <i>Pleurotus pulmonarius</i>(Fr.) Quél.

The wood-decay fungi are able to bioconvert a wide variety of lignocellulosic residues due to the secretion of extracellular enzymes. The use of agricultural wastes as substrate for mushroom cultivation or enzymes production can help to solve environmental problems caused by inadequate discharge in the nature. The production of hydrolytic and oxidative enzymes by Pleurotus pulmonarius developed in solid state system using orange waste as substrate was evaluated in this work. Among the hydrolytic enzymes, pectinase was the main enzyme produced by the fungus, presenting the highest enzymatic activity of 9.4 U/mL after 35 days of cultivation. Considering the oxidative enzymes, laccase was the main enzyme produced with maximal activity of 12.2 U/mL obtained after 20 days of cultivation. Low enzyme levels of manganese peroxidase, β-glucosidase and β-xy-losidase were detected with activity peaks at the end of the cultivation. The enzymatic levels of amylase, carboxymethyl cellulase and xylanase were similar and less than 1.5 U/mL. No aryl-alcohol oxidase activity was detected. NDF, ADF and cellulose values increased during 45 days of cultivation. There was no lignin degradation during the study period and the fungus culture in orange solid waste caused protein enrichment in the substrate. Our results demonstrate that P. pulmonarius was an efficient producer of two important industrial enzymes, pectinase and laccase in a cheap solid state system using orange waste as substrate.

Anaerobic Biotransformation of Nitro-Compounds to Amines by Bovine Rumen Fluid

Microorganims of the bovine rumen fluid biocatalyzed the reduction of nitro-compound substrates to yield the respective amines. This enzymatic process, using ruminal contents, has rarely been reported in associa- tion with the bioreduction of nitro groups. The biotransformation reactions catalyzed by this system were de- pendent of both the electronic characteristics and the area/volume of the nitro-substrates confirming the processes are enzymatic. The semi-preparative scale biotransformation went by in good yield showing the rumen fluid may be employed in the synthesis of amines under very mild conditions and, moreover, it may have application in the bioremediation of nitro-compounds.

Herba Epimedii′s biotransformation in vitro simulated gastrointestinal digestion and faecal fermentation systems

OBJECTIVE Epimedium is rich in a variety of beneficial active ingredients,and has been widely used in the ethnopharmacological practices,however,its biotransformation in gastrointestinal digestions remain unclear.This study aimed to investigate the dynamic changes of components and biological activity of Epimedium in the in vitro simulated digestion and subsequent human faecal fermentation.METHODS The models of in vitro simulated saliva,gastric and intestinal digestion,as well as colonic fermentation were constructed to simulate the digestion process of Epimedium.The dynamic changes of components of Epimedium during the simulated digestions in vitro and subsequent human faecal fermentation were investigated by UPLC-MS,HPLC-DAD combined with principal component analysis(PCA)and multi-ingredient quantitative analysis.RESULTS A variety of metabolites with high contents were produced after 0.5 h of intestinal digestion and colonic fermentation 0.5 h.Application of PCA to HPLC data showed the obvious separation of colonic fermentation 0.5 h stage samples from other colonic fermentation stages samples(24,48 and 72 h).Additionally,non-digestion and saliva digestion stage samples clustered together,and there was obvious separation between intestinal digestion samples and gastric digestion samples.The contents of epimedium C,icariin and baohuside I all increased significantly after intestinal digestion[58.70±7.08,47.15±5.68 and(12.78±0.55)mg·g^(-1)]compared with gastric digestion[29.00±5.65,17.40±4.55 and(2.77±0.19)mg·g^(-1)].There were significant differences between sample after 0.5 h of colonic fermentation[64.22±9.32,51.26±6.33 and(16.68±3.19)mg·g^(-1)]and other time points(24,48 and 72 h)in components and the contents of active ingredient,and the content of these components all decreased with the fermentation time.The ability of scavenging ABTS free radicals[IC50=(0.29±0.02)g·L^(-1)]increased significantly compared with gastric digestion[(1.57±0.02)g·L^(-1)],and after 0.5 h of colonic fermentation,the ability also increased significantly.CONCLUSION Gastrointestinal digestion had a significant impact on the contents of active components in Epimedium,and the metabolism of these components mainly occurred in the colon.The intestinal digestion and colonic fermentation significantly improved the anti-ABTS activity of epimedium.

Biotransformation of Flavor Compositions During Fermentation of Litchi (Litchi Chinensis Sonn.) Fruits into Wine

The aim was to examine the biotransformation of chemical compounds during the fermentation of litchi wine.S.bccyanus BV818 was inoculated to litchi juice(Heiye)to initiate the fermentation.Acetic acid decreased dramatically,succinic acid and DL-malic acid increased sharply.Saturated free fatty acid increased,especially the concentration of the free fatty acid with long carbon chain(more than 10 carbons)increased significantly.The unique flavor compounds of fresh litchi including linalool,α-terpineol,β-citronellol and other terpenoids remained in the litchi wine were transformed to other aroma constituents,by which the primary litchi flavor was retained.The wine had a fruity flavor and delicate bouquet and had harmonious sourness and sweetness.The litchi'Heiye'was suitable for being fermented into litchi wine.

Time-course monitoring of in vitro biotransformation reaction via solid-phase microextraction-ambient mass spectrometry approaches

The solid-phase microextraction technique quantifies analytes without considerably affecting the sample composition.Herein,a proof-of-concept study was conducted to demonstrate the use of coated probe electrospray ionization(coated-PESI)and coated blade spray(CBS)as ambient mass spectrometry approaches for monitoring drug biotransformation.The ability of these methods was investigated for monitoring the dephosphorylation of a prodrug,combretastatin A4 phosphate(CA4P),into its active form,combretastatin A4(CA4),in a cell culture medium supplemented with fetal bovine serum.The CBS spot analysis was modified to achieve the same extraction efficiency as protein precipitation and obtained results in 7 min.Because coated-PESI performs extraction without consuming any samples,it is the preferred technique in the case of a limited sample volume.Although coated-PESI only extracts small quantities of analytes,it uses the desorption solvent volume of 5-10 pL,resulting in high sensitivity,thus allowing the detection of compounds after only 1 min of extraction.The biotransformation of CA4P into CA4 via phosphatases occurs within the simple matrix,and the proposed sample preparation techniques are suitable for monitoring the biotransformation.

Fatty acid hydratase for value-added biotransformation:A review

The synthesis of hydroxy fatty acids(HFAs) from renewable oil feedstock by addition of water onto C_C bonds has attracted great attention in recent years. Given that selective asymmetric hydration of non-activated C_C bonds has been proven difficult to achieve with chemical catalysts, enzymatic catalysis by fatty acid hydratases(FAHs) presents an attractive alternative approach to produce value-added HFAs with high regio-, enantioand stereospecificity, as well as excellent atom economy. Even though FAHs have just been investigated as a potential biocatalyst for a decade, remarkable information about FAHs in different aspects is available;however, a comprehensive review has not been archived. Herein, we summarize the research progresses on biochemical characterization, structural and mechanistic determination, enzyme engineering, as well as biotechnological application of FAHs. The current challenges and opportunities for an efficient utilization of FAHs in organic synthesis and industrial applications are critically discussed.

Whole-cell recordings of voltage-gated Calcium,Potassium and Sodium currents in acutely isolated hippocampal pyramidal neurons

Objective：To record Calcium, Potassium and Sodium currents in acutely isolated hippocampal pyramidal neurons. Methods：Hippocampal CA3 neurons were freshly isolated by 1 mg protease/3 ml SES and mechanical trituration with polished pipettes of progressively smaller tip diameters. Patch clamp technique in whole-cell mode was employed to record voltage-gated channel currents. Results：The procedure dissociated hippocampal neurons, preserving apical dendrites and several basal dendrites, without impairing the electrical characteristics of the neurons. Whole-cell patch clamp configuration was successfully used to record voltage-gated Ca^2＋ currents, delayed rectifier K^＋ current and voltage-gated Na^＋ currents. Conclusion：Protease combined with mechanical trituration may be used for the dissociation of neurons from rat hippocampus. Voltage-gated channels currents could be recorded using a patch clamp technique.

Biotransformation of 6-deoxypseudoanisatin by Synechocystis sp. PCC6803

Objective:To explore the ability of Synechocystis sp.PCC6803 in transforming 6-deoxypseudoanisatin.Methods:The experiment was performed by incubating 6-deoxypseudoanisatin with the freshwater cyanobacterium Synechocystis sp.PCC6803 under continuous white light at 30C for 5 days.The crude converted product was detected using thin-layer chromatography(TLC)and further analyzed using high-performance liquid chromatography(HPLC)as well as HPLC with electron spray ionization mass spectrometry(HPLC-ESI-MS).Results:TLC results showed that 6-deoxypseudoanisatin was converted into a less polar product.HPLC and MS data indicated that the retention time of the converted product increased in comparison with the standard of 6-deoxypseudoanisatin.Conclusion:Thus,the study appears to demonstrate that Synechocystis sp.PCC6803 can transform 6-deoxypseudoanisatin.The polarity of the converted product is less than that of 6-deoxypseudoanisatin.

Study on Biotransformation of Traditional Chinese Herbal Medicines Complex

A newly designed enzymolysis-fermentation combined method to dramatically enhance the actives level and skin benefits of traditional Chinese herbs (TCHs) was developed, biotransformation process under optimal reaction conditions can significantly transform molecular structures and obtained fermentation extracts can deliver better skin benefits on anti-aging, hydration and whitening. Analytical results showed that the ginsenoside Rg3, total sugar, polyphenols and flavonoids in fermented extract were 2~4 times higher than unfermented extract. In-vitro tests including DPPH radical scavenging activity, tyrosinase inhibition, cells proliferation and Hyaluronan-CD44 activity, have showed significant enhancement of efficacy, inhibition rate on DPPH antioxidation as example achieved over 60% improvement, which makes traditional Chinese herbs application more feasible. It is inferred that this study potentially enables the herbs to deliver required bioefficacy in cosmetic application.

枯草芽孢杆菌LM 4-2发酵人参芦头生物转化人参皂苷Rd的研究

人参为我国传统的食药同源资源,但由于潜在的催吐等副作用,在人参炮制过程中需要做去芦处理,导致大量的人参芦头被丢弃,造成了严重的资源浪费,如何减少浪费、提高人参的综合应用需要进行相应研究。该研究建立了基于超高液相色谱法的人参皂苷检测方法,通过对13种人参皂苷单体进行测试,验证了该检测方法的准确性与可靠性。采用生物转化的方法,从6株候选菌株中筛选出转化人参皂苷Rd产量最高的菌株Bacillus subtilisLM 4-2,推测人参皂苷Rd经由Rc水解途径转化而来,进一步通过单因素试验与响应面试验设计,确定了菌株LM 4-2的最佳发酵条件为发酵时间20 d,温度30℃,料液比1∶4.3(g∶mL),接种量7.5%,该优化条件下Rd产量为(6.375±0.006)mg/g。同时,研究还对人参芦头发酵提取物的抗氧化性和抑菌效果进行了测定,抗氧化性试验发现DPPH自由基的清除率随着提取物浓度的增加而增加,提取物质量浓度为50 mg/mL时,DPPH自由基的清除率为82.55%;抑菌试验结果表明,在提取物质量浓度达到1.0 mg/mL时,对测试菌株金黄色葡萄球菌,枯草芽孢杆菌和大肠杆菌均能产生抑制作用,发酵液对3种测试菌株的最低抑制浓度分别为1.0、0.75和1.0 mg/mL。这些研究为通过菌株LM 4-2发酵人参芦头转化人参皂苷Rd、提高人参芦头的应用价值提供了参考。