Production of succinate and polyhydroxyalkanoate from substrate mixture by metabolically engineered Escherichia coli

The strategic design of this study aimed at producing succinate and polyhydroxyalkanoate (PHA) from substrate mixture of glycerol/glucose and fatty acid in Escherichia coli. To accomplish this, an E. coli KNSP1 strain derived from E. coli LR1110 was constructed by deletions of ptsG, sdhA and pta genes and overexpression of phaC1 from Pseudomonas aeruginosa. Cultivation of E. coli KNSP1 showed that this strain was able to produce 21.07 g/L succinate and 0.54 g/L PHA (5.62 wt.% of cell dry weight) from glycerol and fatty acid mixture. The generated PHA composed of 58.7 mol% 3-hydroxyoctanoate (3HO) and 41.3 mol% 3-hydroxydecanoate (3HD). This strain would be useful for complete utilization of byproducts glycerol and fatty acid of biodiesel production process.     

Assay for glucose oxidase from Aspergillus niger and Penicillium amagasakiense by Fourier transform infrared spectroscopy

A simple and direct assay method for glucose oxidase (EC 1.1.3.4) from Aspergillus niger and Penicillium amagasakiense was investigated by Fourier transform infrared spectroscopy. This enzyme catalyzed the oxidation of d-glucose at carbon 1 into d-glucono-1,5-lactone and hydrogen peroxide in phosphate buffer in deuterium oxide ((2)H(2)O). The intensity of the d-glucono-1,5-lactone band maximum at 1212 cm(-1) due to CO stretching vibration was measured as a function of time to study the kinetics of d-glucose oxidation. The extinction coefficient epsilon of d-glucono-1,5-lactone was determined to be 1.28 mM(-1)cm(-1). The initial velocity is proportional to the enzyme concentration by using glucose oxidase from both A. niger and P. amagasakiense either as cell-free extracts or as purified enzyme preparations. The kinetic constants (V(max), K(m), k(cat), and k(cat)/K(m)) determined by Lineweaver-Burk plot were 433.78+/-59.87U mg(-1) protein, 10.07+/-1.75 mM, 1095.07+/-151.19s(-1), and 108.74 s(-1)mM(-1), respectively. These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on horseradish peroxidase in aqueous media: 470.36+/-42.83U mg(-1) protein, 6.47+/-0.85 mM, 1187.77+/-108.16s(-1), and 183.58 s(-1)mM(-1) for V(max), K(m), k(cat), and k(cat)/K(m), respectively. Therefore, this spectroscopic method is highly suited to assay for glucose oxidase activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of glucose oxidase activity.     

Whole-organism fingerprinting of the genus Carnobacterium using Fourier transform infrared spectroscopy (FT-IR)

Sixty seven strains of Carnobacterium, atypical Lactobacillus, Enterococcus durans, Lactobacillus maltaromicus and Vagacoccus salmoninarum were examined by Fourier transform infrared (FT-IR) spectroscopy. The effects of culture age and reproducibility over a six month period were also investigated. The results were analysed by multivariate statistics and compared with those from a previous numerical phenetic study, a pyrolysis mass spectrometry (PyMS) study and with investigations which used DNA-DNA and 16S rRNA sequencing homologies. Taxonomic correlations were observed between the FT-IR data and these studies. Culture age was observed to have little effect on the spectra obtained. The reproducibility study indicated that there was correlation between spectra produced on two occasions over the six month period. It was concluded that FTIR is a reliable method for investigating carnobacterial classification, and may have further potential as a rapid method for use in Carnobacterium identification.     

基于FTIR的三倍体罗汉果花粉直感效应研究

以四个品系的三倍体罗汉果雌株为材料,用五种不同的二倍体雄花分别对其授粉,利用傅里叶变换红外光谱法(FTIR)测定了其子代果实的红外光谱,并运用主成分分析和聚类分析研究了授粉雄花对子代果实化学成分的影响。结果表明:不同雄花授粉后,雌株F302、F323和F322各子代果实红外光谱中1 050 cm-1波数附近甜苷物质特征吸收峰的峰高有显著或极显著差异,雌株F311子代无籽果实的差异不显著;同时五种授粉雄花分别对雌株F302、F323、F322和F311子代果实在主成分二维投影图和聚类图中的排序也有明显的影响,但对不同品系雌株子代果实的排序影响不同,从而说明三倍体罗汉果的甜苷物质含量和整体成分含量均有较为明显的花粉直感效应,并且存在品种特异性。     

Discrimination of geographical origin and adulteration of radix astragali using fourier transform infrared spectroscopy and chemometric methods

Introduction – Radix Astragali, one of most widely used and important traditional Chinese medicines, is cultivated in different geographical regions. Because of varying growing conditions, the qualities of Radix Astragali vary, which can give rise to differences in clinical therapy. Detecting adulteration is a routine requirement in pharmaceutical practice. Objective – To develop a simple and accurate approach to discriminate the geographical origin and potential adulteration of Radix Astragali, derived from the root of Astragalus membranaceus (Fischer) Bunge var. mongholicus (Bunge) Hsiao, using Fourier transform infrared (FT‐IR) spectroscopy and chemometric methods. Methodology – To obtain characteristic IR spectra for accurate discrimination, a one‐solvent extraction method was utilised following a novel evaluation method for selecting appropriate solvents. Samples of Radix Astragali from different geographical origins were discriminated using FT‐IR spectroscopy and discriminant partial least squares (DPLS) methods. FT‐IR spectroscopy combined with Mahalanobis distance was employed to detect adulteration of Radix Astragali. Results – In comparison with other solvents, butanone was more effective at extracting samples. Radix Astragali samples were accurately assigned to their corresponding geographical origins by using FT‐IR spectroscopy and DPLS method. Most adulterated samples were detected accurately by application of FT‐IR spectroscopy combined with Mahalanobis distance. Conclusion – FT‐IR spectroscopy combined with chemometric method was developed and demonstrated to be a useful tool to discriminate geographical origin and adulteration of Radix Astragali. Copyright © 2010 John Wiley & Sons, Ltd.     

Application of Fourier transform infrared spectroscopy for monitoring hydrolysis and synthesis reactions catalyzed by a recombinant amidase

This study demonstrates the use of Fourier transform infrared (FTIR) spectroscopy for monitoring both synthesis and hydrolysis reactions catalyzed by a recombinant amidase (EC 3.5.1.4) from Pseudomonas aeruginosa. The kinetics of hydrolysis of acetamide, propionamide, butyramide, acrylamide, benzamide, phenylalaninamide, alaninamide, glycinamide, and leucinamide were determined. This revealed that very short-chain substrates displayed higher amidase activity than did branched side-chain or aromatic substrates. In addition, on reducing the polarity and increasing the substrates' bulkiness, a reduction of the amidase affinity for the substrates took place. Using FTIR spectroscopy it was possible to monitor and quantify the synthesis of several hydroxamic acid derivatives and ester hydrolysis products. These products may occur simultaneously in a reaction catalyzed by the amidase. The substrates used for the study of such reactions were ethyl acetate and glycine ethyl ester. Hydroxylamine was the nucleophile substrate used for the synthesis of acetohydroxamate compounds. Results presented in this article demonstrate the usefulness of FTIR spectroscopy as an important tool for understanding the enzyme structure-activity relationship because it provides a simple and rapid real-time assay for the detection and quantification of amidase hydrolysis and synthesis reactions in situ.     

Probing the Q-proton pathway of ba3-cytochrome c oxidase by time-resolved Fourier transform infrared spectroscopy

In cytochrome c oxidase, the terminal respiratory enzyme, electron transfers are strongly coupled to proton movements within the enzyme. Two proton pathways (K and D) containing water molecules and hydrophobic amino acids have been identified and suggested to be involved in the proton translocation from the mitochondrial matrix or the bacterial cytoplasm into the active site. In addition to the K and D proton pathways, a third proton pathway (Q) has been identified only in ba3-cytochrome c oxidase from Thermus thermophilus, and consists of residues that are highly conserved in all structurally known heme-copper oxidases. The Q pathway starts from the cytoplasmic side of the membrane and leads through the axial heme a3 ligand His-384 to the propionate of the heme a3 pyrrol ring A, and then via Asn-366 and Asp-372 to the water pool. We have applied FTIR and time-resolved step-scan Fourier transform infrared (TRS2-FTIR) spectroscopies to investigate the protonation/deprotonation events in the Q-proton pathway at ambient temperature. The photolysis of CO from heme a3 and its transient binding to CuB is dynamically linked to structural changes that can be tentatively attributed to ring A propionate of heme a3 (1695/1708 cm(-1)) and to deprotonation of Asp-372 (1726 cm(-1)). The implications of these results with respect to the role of the ring A propionate of heme a3-Asp372-H2O site as a proton carrier to the exit/output proton channel (H2O pool) that is conserved among all structurally known heme-copper oxidases, and is part of the Q-proton pathway in ba3-cytochrome c oxidase, are discussed.     

Rapid monitoring of recombinant antibody production by mammalian cell cultures using fourier transform infrared spectroscopy and chemometrics

Fourier transform infrared (FT‐IR) spectroscopy combined with multivariate statistical analyses was investigated as a physicochemical tool for monitoring secreted recombinant antibody production in cultures of Chinese hamster ovary (CHO) and murine myeloma non‐secreting 0 (NS0) cell lines. Medium samples were taken during culture of CHO and NS0 cells lines, which included both antibody‐producing and non‐producing cell lines, and analyzed by FT‐IR spectroscopy. Principal components analysis (PCA) alone, and combined with discriminant function analysis (PC‐DFA), were applied to normalized FT‐IR spectroscopy datasets and showed a linear trend with respect to recombinant protein production. Loadings plots of the most significant spectral components showed a decrease in the C–O stretch from polysaccharides and an increase in the amide I band during culture, respectively, indicating a decrease in sugar concentration and an increase in protein concentration in the medium. Partial least squares regression (PLSR) analysis was used to predict antibody titers, and these regression models were able to predict antibody titers accurately with low error when compared to ELISA data. PLSR was also able to predict glucose and lactate amounts in the medium samples accurately. This work demonstrates that FT‐IR spectroscopy has great potential as a tool for monitoring cell cultures for recombinant protein production and offers a starting point for the application of spectroscopic techniques for the on‐line measurement of antibody production in industrial scale bioreactors. Biotechnol. Bioeng. 2010; 106: 432–442. © 2010 Wiley Periodicals, Inc.     

Membrane permeability parameters for freezing of stallion sperm as determined by Fourier transform infrared spectroscopy

Cellular membranes are one of the primary sites of injury during freezing and thawing for cryopreservation of cells. Fourier transform infrared spectroscopy (FTIR) was used to monitor membrane phase behavior and ice formation during freezing of stallion sperm. At high subzero ice nucleation temperatures which result in cellular dehydration, membranes undergo a profound transition to a highly ordered gel phase. By contrast, low subzero nucleation temperatures, that are likely to result in intracellular ice formation, leave membrane lipids in a relatively hydrated fluid state. The extent of freezing-induced membrane dehydration was found to be dependent on the ice nucleation temperature, and showed Arrhenius behavior. The presence of glycerol did not prevent the freezing-induced membrane phase transition, but membrane dehydration occurred more gradual and over a wider temperature range. We describe a method to determine membrane hydraulic permeability parameters (E_Lp, Lpg) at subzero temperatures from membrane phase behavior data. In order to do this, it was assumed that the measured freezing-induced shift in wavenumber position of the symmetric CH₂ stretching band arising from the lipid acyl chains is proportional to cellular dehydration. Membrane permeability parameters were also determined by analyzing the H₂O-bending and -libration combination band, which yielded higher values for both E_Lp and Lpg as compared to lipid band analysis. These differences likely reflect differences between transport of free and membrane-bound water. FTIR allows for direct assessment of membrane properties at subzero temperatures in intact cells. The derived biophysical membrane parameters are dependent on intrinsic cell properties as well as freezing extender composition.     

红外光谱技术在滇龙胆栽培方式选择上的应用

该研究采用傅里叶变换红外光谱结合化学计量学,对条播、撒播、剪根后移栽、扦插和剪枝后移栽的滇龙胆进行了分析,以筛选滇龙胆的最佳栽培方式。结果表明:(1)不同栽培方式的滇龙胆原始谱图在峰形、峰位和峰强上有一定差异;用小波去噪法对光谱进行优化处理并进行偏最小二乘判别分析(Partial least squares discriminant analysis,PLS-DA),能较好地区分不同栽培方式的滇龙胆样品,PLS-DA二维得分图显示同一栽培方式的样品聚在一起,表明相同栽培方式的滇龙胆化学组成和含量差异较小;播种滇龙胆样品(条播和撒播)距离较近,移栽滇龙胆样品(剪根、扦插和剪枝)距离较近,而播种和移栽滇龙胆样品距离较远,表明栽培方式对滇龙胆化学成分的积累有影响。(2)滇龙胆四种主要成分总含量大小依次是剪枝剪根撒播条播扦插,除剪根后移栽,剪枝后移栽滇龙胆中四种主要成分总含量显著高于其他栽培方式下的滇龙胆(P0.05),剪枝后移栽滇龙胆质量最佳。(3)以液相数据为参考值,采用正交信号校正—偏最小二乘回归模型预测不同栽培模式滇龙胆中龙胆苦苷、马钱苷酸、獐牙菜苦苷和当药苷的含量。校正集和验证集的决定系数(R2)均大于0.90,校正均方根误差、交叉验证均方差和预测均方根误差均小于1.65,模型相关性和预测效果好,该方法对红外光谱分析在中药领域的推广应用提供了参考。     

Watching the components of photosynthetic bacterial membranes and their in situ organisation by atomic force microscopy

The atomic force microscope has developed into a powerful tool in structural biology allowing information to be acquired at submolecular resolution on the protruding structures of membrane proteins. It is now a complementary technique to X-ray crystallography and electron microscopy for structure determination of individual membrane proteins after extraction, purification and reconstitution into lipid bilayers. Moving on from the structures of individual components of biological membranes, atomic force microscopy has recently been demonstrated to be a unique tool to identify in situ the individual components of multi-protein assemblies and to study the supramolecular architecture of these components allowing the efficient performance of a complex biological function.Here, recent atomic force microscopy studies of native membranes of different photosynthetic bacteria with different polypeptide contents are reviewed. Technology, advantages, feasibilities, restrictions and limits of atomic force microscopy for the acquisition of highly resolved images of up to 10 Å lateral resolution under native conditions are discussed. From a biological point of view, the new insights contributed by the images are analysed and discussed in the context of the strongly debated organisation of the interconnected network of membrane-associated chlorophyll-protein complexes composing the photosynthetic apparatus in different species of purple bacteria.     

Properties of cells from inverted embryos ofXenopus laevis investigated by scanning electron microscopy

Summary Xenopus embryos held inverted from the one cell stage show a partial reversal of the pattern of cleavage: the blastocoel forms towards the new upper pole, and the non-pigmented cells forming the blastocoel roof are smaller than normal endoderm cells. Two properties of the cells from inverted embryos have been studied: their capacity to form cilia when cultured for 48 h, normally a property of ectoderm cells; and their scanning electron microscopical appearance when isolated and cultured for shorter periods, which differs for normal ectoderm and endoderm cells. Groups of the upper, non-pigmented cells from inverted embryos do not form cilia in a longerterm culture, whereas groups of the lower, pigmented cells do. In contrast, the scanning electron microscopical appearance of the upper, non-pigmented cells of inverted embryos is more like that of normal ectoderm cells; the appearance of lower, pigmented cells is more like that of normal endoderm. Thus the determination to form cilia is not reversed by inversion, whereas the control of cell morphology is.     

Gold-FISH: A new approach for the in situ detection of single microbial cells combining fluorescence and scanning electron microscopy

A novel fluorescence in situ hybridisation (FISH) method is presented that allows the combination of epifluorescence and scanning electron microscopy (SEM) to identify single microbial cells. First, the rRNA of whole cells is hybridised with horseradish peroxidase-labelled oligonucleotide probes and this is followed by catalysed reporter deposition (CARD) of biotinylated tyramides. This facilitates an amplification of binding sites for streptavidin conjugates covalently labelled with both fluorophores and nanogold particles. The deposition of Alexa Fluor 488 fluoro-nanogold–streptavidin conjugates was confirmed via epifluorescence microscopy and cells could be quantified in a similar way to standard CARD–FISH approaches. To detect cells by SEM, an autometallographic enhancement of the nanogold particles was essential, and allowed the in situ localisation of the target organisms at resolutions beyond light microscopy. Energy dispersive X-ray spectroscopy (EDS) was used to verify the effects of CARD and autometallography on gold deposition in target cells.     

Morphological characterization of the antennal sensilla of t he earwig Anisolabis maritima (Dermaptera: Carcinophoridae) with reference to their probable functions

The earwig, Anisolabis maritima (Dermaptera: Carcinophoridae), is one of the most significant insects in KSA because, it was recorded in Saudi Arabia as a beneficial predator on eggs and newly hatched larvae of the red palm weevil, Rhyncophorus ferrugineus. We examined the external morphology of the antennal sensilla of males and females of A. maritima using scanning electron microscopy (SEM). The filiform antennae of A. maritima were of the conventional type comprising a basal scape, pedicle and a long, thread-like flagellum, which was composed of 12 flagellomeres of males and 16 flagellomeres of females. Six morphologically unique sensillar types were found and described on the antennae of males and females of A. maritima. Of those identified, there were three types of porous trichoid sensilla (long, curved and arcuate), and two types of basiconic sensilla (short and curved), and one type of aporous trichoid sensilla. The shape, external morphology and array of sensilla on the antennae of males and females of A. maritima were similar.     

Localization of mannan at the surface of yeast protoplasts by scanning electron microscopy

The (13)glucanase of Basidiomycete QM 806 was used to prepare Saccharomyces cerevisiae and Candida utilis protoplasts. Plasma membranes isolated from S. cerevisiae contained a small amount of mannose and traces of glucose and ribose. Randomly distributed -mannan was detected by scanning electron microscopy at the surface of prefixed protoplasts using colloidal gold labelled with Concanavalin A as a marker. C. utilis protoplasts were also marked with anti-mannan antibodies. Again the distribution of mannan was random. This experiment indicated also that plasma membrane mannan has the same immunochemical determinants as cell wall mannan. It is hypothesized that mannan is mainly located in the outer layer of plasma membranes.     

Freezing and desiccation tolerance in the moss Physcomitrella patens: An in situ Fourier transform infrared spectroscopic study

In situ Fourier transform infrared spectroscopy (FTIR) was used in order to obtain more insights in the underlying protective mechanisms upon freezing and drying of ABA-treated tissues of the moss Physcomitrella patens. The effects of different treatments on the membrane phase behaviour, glassy state, and overall protein secondary structure were studied. We found that growth on ABA resulted in the accumulation of sucrose: up to 22% of the tissue on a dry weight basis, compared to only 3.7% in non-ABA-treated tissues. Sucrose functions as a protectant during freezing and drying, but accumulation of sucrose alone is not sufficient for survival. ABA-treated tissue survives a freeze–thaw cycle down to −80 °C only after addition of an additional cryoprotectant (DMSO). Survival correlates with preservation of membrane phase behaviour. We found that ABA-treated P. patens can survive slow but not rapid drying down to water contents as low as 0.02 g H₂O per g DW. Rapidly and slowly dried ABA-treated tissues were found to have similar sugar compositions and glass transition temperatures. The average strength of hydrogen bonding in the cytoplasmic glassy matrix, however, was found to be increased upon slow drying. In addition, slowly dried tissues were found to have a higher relative proportion of α-helical structures compared to rapidly dried tissues.     

Green synthesis and characterization of Carica papaya leaf extract coated silver nanoparticles through X-ray diffraction,electron microscopy and evaluation of bactericidal properties

The evolution of nanotechnology and the production of nanomedicine from various sources had proven to be of intense value in the field of biomedicine. The smaller size of nanoparticles is gaining importance in research for the treatment of various diseases. Moreover the production of nanoparticles is eco-friendly and cost effective. In the present study silver nanoparticles were synthesized from Carica papaya leaf extract (CPL) and characterized for their size and shape using scanning electron microscopy and transmission electron microscopy, respectively. Fourier transform infrared spectroscopy (FTIR), Energy dispersive X-ray spectroscopy (EDS/EDX) and X-ray diffraction spectroscopy (XRD) were conducted to determine the concentration of metal ions, the shape of molecules. The bactericidal activity was evaluated using Luria Bertani broth cultures and the minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) were estimated using turbidimetry. The data analysis showed size of 50–250 nm spherical shaped nanoparticles. The turbidimetry analysis showed MIC and MBC was >25 μg/mL against both Gram positive and Gram negative bacteria in Luria Bertani broth cultures. In summary the synthesized silver nanoparticles from CPL showed acceptable size and shape of nanoparticles and effective bactericidal activity.