Development of a fed-batch process for a recombinant Pichia pastoris Δoch1 strain expressing a plant peroxidase.

Pichia pastoris is a prominent host for recombinant protein production, amongst other things due to its capability of glycosylation. However, N-linked glycans on recombinant proteins get hypermannosylated, causing problems in subsequent unit operations and medical applications. Hypermannosylation is triggered by an α-1,6-mannosyltransferase called OCH1. In a recent study, we knocked out OCH1 in a recombinant P. pastoris CBS7435 Mut(S) strain (Δoch1) expressing the biopharmaceutically relevant enzyme horseradish peroxidase. We characterized the strain in the controlled environment of a bioreactor in dynamic batch cultivations and identified the strain to be physiologically impaired. We faced cell cluster formation, cell lysis and uncontrollable foam formation.In the present study, we investigated the effects of the 3 process parameters temperature, pH and dissolved oxygen concentration on 1) cell physiology, 2) cell morphology, 3) cell lysis, 4) productivity and 5) product purity of the recombinant Δoch1 strain in a multivariate manner. Cultivation at 30°C resulted in low specific methanol uptake during adaptation and the risk of methanol accumulation during cultivation. Cell cluster formation was a function of the C-source rather than process parameters and went along with cell lysis. In terms of productivity and product purity a temperature of 20°C was highly beneficial. In summary, we determined cultivation conditions for a recombinant P. pastoris Δoch1 strain allowing high productivity and product purity.

Optimization and application of a fluorimetric assay for the identification of histone deacetylase inhibitors from plant origin.

CONTEXT: Histonedeacetylase inhibitors (HDACi) are the focus of enormous interest as a new class of anticancer drugs and discussed as novel treatment in cardiovascular diseases or memory enhancement. In the search for new active substances the structural diversity of secondary plant metabolites provides an indispensable resource. Several molecules from the plant kingdom have gained importance as anticancer drugs. Thus, a search for new therapeutic agents inhibiting histonedeacetylases (HDACs) is an important topic. To accelerate the isolation of potential candidates from plants bioassays well suited for screenings of extracts are an indispensable prerequisite. OBJECTIVE: In the presented study an enzymatic assay was modified, optimized and validated for the search for HDACi from plant origin. MATERIALS AND METHODS: A fluorimetric assay was validated with respect to parameters such as temperature, incubation times, reproducibility, applicability of different enzyme sources and HDAC substrate. For the determination of the HDAC inhibitory potential of extracts the detailed study of the influence of different classes of primary and secondary metabolites probably interfering with the assay was most important. RESULTS: In the experimental design autofluorescent coumarins and tannins were identified to disrupt the assay. Possibilities to avoid disturbances are demonstrated and the applicability of the method in the bioactivity-guided search for new HDACi was proven on the example Leonuri herba (Leonurus cardiaca L.; Lamiaceae). CONCLUSION: The optimization of the assay led to a highly efficient fluorimetric method to study plant extracts and fractions of medium/high polarity for HDAC inhibition. In the bioactivity-guided fractionation of extracts from Leonuri herba the applicability for the aimed purpose was clearly demonstrated.

Identification and quantification of flavonoids and ellagic acid derivatives in therapeutically important Drosera species by LC-DAD, LC-NMR, NMR, and LC-MS.

Droserae herba is a drug commonly used for treatment of convulsive or whooping cough since the seventeenth century. Because of the contribution of flavonoids and ellagic acid derivatives to the therapeutic activity of Droserae herba, an LC-DAD method has been developed for quantification of these analytes in four Drosera species used in medicine (Drosera anglica, D. intermedia, D. madagascariensis, and D. rotundifolia). During elaboration of the method 13 compounds, including three substances not previously described for Drosera species, were detected and unambiguously identified by means of extensive LC-MS and LC-NMR experiments and by off-line heteronuclear 2D NMR after targeted isolation. The most prominent component of D. rotundifolia and D. anglica, 2″-O-galloylhyperoside, with myricetin-3-O-ß-glucopyranoside and kaempferol-3-O-(2″-O-galloyl)-ß-galactopyranoside, were identified for the very first time in this genus. The LC-DAD method for quantification was thoroughly validated, and enables, for the first time, separation and precise analysis of these analytes in Droserae herba. Simple sample preparation and use of a narrow-bore column guarantee low cost and simplicity of the suggested system, which is excellently suited to quality control of the drug or herbal medicinal products containing this drug.

Apparently no sedative benzoflavone moiety in passiflorae herba.

Due to the fact that an Indian group had reported a benzoflavone moiety (BZF) as an active principle in the herb of Passiflora incarnata L. (Passifloraceae), this study was performed to isolate the compound for analytical purposes. In Passiflorae herba from three different origins (cultivations in India, Italy and France) a compound with the published TLC characteristics was detected in trace amounts only in the Italian material. No traces of the substance were found in the drugs from India and France. In a commercial extract two compounds with the respective TLC characteristics were detected. One was identified as a phytol isomer. Due to the very small amounts of the second compound its structure elucidation was not successful. The amount of extract for the isolation corresponded to approximately the 10-fold amount of the drug, from which the isolation of 332 mg "BZF" had been reported. The detection of only trace amounts of a BZF-like compound in one of three commercial samples of Passiflorae herba and in an extract suggests for the first time that BZF is not the active principle in this drug and should not serve as an active marker.

In vitro anti-leukemic activity of the ethno-pharmacological plant Scutellaria orientalis ssp. carica endemic to western Turkey.

AIM OF THIS STUDY: Within the genus Scutellaria various species are used in different folk medicines throughout Asia. Traditional Chinese Medicine (TCM) uses S. baicalensis (Labiatae) to treat various inflammatory conditions. The root shows strong anticancer properties in vitro and was suggested for clinical trials against multiple myeloma. Further, S. barbata was successfully tested against metastatic breast cancer in a phase I/II trial. Therefore, we investigated the anti-cancer properties of S. orientalis L. ssp. carica Edmondson, an endemic subspecies from the traditional medicinal plant S. orientalis L. in Turkey, which is used to promote wound healing and to stop haemorrhage. MATERIALS AND METHODS: Freeze-dried plant material was extracted with petroleum ether, dichloromethane, ethyl acetate, and methanol and the bioactivity of these extracts was analysed by proliferation assay, cell death determination, and by investigating protein expression profiles specific for cell cycle arrest and apoptosis. RESULTS: The strongest anti-leukemic activity was shown by the methanol extract, which contained apigenin, baicalein, chrysin, luteolin and wogonin, with an IpC50 of 43 microg/ml (corresponding to 1.3mg/ml of dried plant material) which correlated with cyclin D1- and Cdc25A suppression and p21 induction. At 132 microg/ml (=4 mg/ml of the drug) this extract caused genotoxic stress indicated by substantial phosphorylation of the core histone H2AX (gamma-H2AX) followed by activation of caspase 3 and signature-type cleavage of PARP resulting in a 55% apoptosis rate after 48 hours of treatment. CONCLUSIONS: Here, we report for the first time that S. orientalis L. ssp. carica Edmondson exhibited potent anti-leukaemic properties likely through the anti-proliferative effect of baicalein and the genotoxic property of wogonin.

In vitro anti-neoplastic activity of the ethno-pharmaceutical plant Hypericum adenotrichum Spach endemic to Western Turkey.

Hypericum perforatum (St. John's wort) is well-established for its antidepressant activity throughout the world and also various other species within this genus are used in different folk medicines. Hyperforin of St. John's wort inhibited growth of cancer cell lines and the use of hypericin (another compound of H. perforatum) in cancer photodynamic therapy is proposed. Therefore, we investigated the anti-cancer properties of H. adenotrichum Spach (Guttiferae), an endemic species in Turkey called 'kantaron', which is used for wound healing and antiseptic effects. Freeze-dried plant was extracted with petroleum ether, dichloromethane, ethyl acetate, and methanol and the bioactivity of these extracts was analysed by proliferation assay, cell death determination, by investigating protein expression profiles specific for cell cycle arrest and apoptosis as well as composition by HPLC. The strongest anti-proliferative activity was determined for the petroleum ether extract with an IpC50 of approximately 5.8 microg/ml medium (referring to 1 mg dried plant) which correlated with cyclin D1 suppression and p21 induction. This extract also induced phosphorylation of H2AX, and activated caspase-3 followed by signature-type cleavage of PARP resulting in approximately 50% apoptosis at 23.2 microg/ml after 24 h of treatment. Neither hyperforin, hypericin, or amentoflavone contributed to these properties. To the best of our knowledge, we report for the first time that the endemic plant H. adenotrichum Spach exhibits potent p53-independent anti-neoplastic properties due to yet unexplored Hypericum constituents.

Characterization and molecular cloning of a heterodimeric beta-galactosidase from the probiotic strain Lactobacillus acidophilus R22.

Beta-galactosidase from the probiotic strain Lactobacillus acidophilus R22 was purified to apparent homogeneity by ammonium sulphate fractionation, hydrophobic interaction, and affinity chromatography. The enzyme is a heterodimer consisting of two subunits of 35 and 72 kDa, as determined by gel electrophoresis. The optimum temperature of beta-galactosidase activity was 55 degrees C (10-min assay) and the range of pH 6.5-8, respectively, for both o-nitrophenyl-beta-D-galactopyranoside (oNPG) and lactose hydrolysis. The Km and Vmax values for lactose and oNPG were 4.04+/-0.26 mM, 28.8+/-0.2 micromol D-glucose released per min per mg protein, and 0.73+/-0.07 mM, 361+/-12 micromol o-nitrophenol released per min per mg protein, respectively. The enzyme was inhibited by high concentrations of oNPG with Ki,s=31.7+/-3.5 mM. The enzyme showed no specific requirements for metal ions, with the exception of Mg2+, which enhanced both activity and stability. The genes encoding this heterodimeric enzyme, lacL and lacM, were cloned, and compared with other beta-galactosidases from lactobacilli. Beta-galactosidase from L. acidophilus was used for the synthesis of prebiotic galacto-oligosaccharides (GOS) from lactose, with the maximum GOS yield of 38.5% of total sugars at about 75% lactose conversion.