Isolation and characterisation of phytase from dormant Corylus avellana seeds

Phytase (myo-inositol-1,2,3,4,5,6-hexakisphosphate phosphohydrolase, EC 3.1.3.26), which catalyses the step-wise hydrolysis of phytic acid, was purified from cotyledons of dormant Corylus avellana L. seeds. The enzyme was separated from the major soluble acid phosphatase by successive (NH4)2SO4 precipitation, gel filtration and cation exchange chromatography resulting in a 300-fold purification and yield of 7.5%. The native enzyme positively interacted with Concanavalin A suggesting that it is putatively glycosylated. After size exclusion chromatography and SDS–PAGE it was found to be a monomeric protein with molecular mass 72±2.5 kDa. The hazel enzyme exhibited optimum activity for phytic acid hydrolysis at pH 5 and, like other phytases, had broad substrate specificity. It exhibited the lowest Km (162 μM) and highest specificity constant (Vmax/Km) for phytic acid, indicating that this is the preferred in vivo substrate. It required no metal ion as a co-factor, while inorganic phosphate and fluoride competitively inhibited enzymic activity (Ki=407 μM and Ki=205 μM, respectively).

Effect of biotic factors on the isolation of Lupinus albus L. protoplasts

Several tissue types of Lupinus albus L. were investigated as sources for the isolation of protoplasts. Cotyledons from in vitro seedlings were found to yield the highest number of protoplasts compared with leaves, hypocotyls and roots. A combination of the protoplast isolation enzymes, cellulase and Pectolyase Y23, was capable of releasing the highest number of protoplasts compared with a combination of cellulase and Macerase. Protoplast yield increased with increasing cotyledon age but was accompanied by a progressive decline in protoplast viability. The optimal combination of protoplast yield and viability occurred when the protoplasts were isolated from 14- to 18-day-old cotyledons. The ratio between the volume of enzyme solution and the tissue biomass did not affect the protoplast production significantly. This is the first report of the isolation of protoplasts from a lupin cotyledon and, following the procedure described in this paper, an average yield of 1.2 × 106 protoplasts per gram of fresh tissue was obtainable.

Enhanced phosphopeptide isolation by Fe(III)-IMAC using 1,1,1,3,3,3-hexafluoroisopropanol

IMAC can be used to selectively enrich phosphopeptides from complex peptide mixtures, but co-retention of acidic peptides together with the failure to retain some phosphopeptides restricts the general utility of the method. In this study Fe(III)-IMAC was qualitatively and quantitatively assessed using a panel of phosphopeptides, both synthetic and derived from proteolysis of known phosphoproteins, to identify the causes of success and failure in the application of this technique. Here we demonstrate that, as expected, peptides with a more acidic amino acid content are generally more efficiently purified and detected by MALDI-MS after Fe(III)-IMAC than those with a more basic content. Modulating the loading buffer used for Fe(III)-IMAC significantly affects phosphopeptide binding and suggests that conformational factors that lead to steric hindrance and reduced accessibility to the phosphate are important. The use of 1,1,1,3,3,3-hexa-fluoroisopropanol is shown here to significantly improve Fe(III)-IMAC enrichment and subsequent detection of phosphopeptides by MALDI-MS.

Liquid ultraviolet matrix-assisted laser desorption/ionization mass spectrometry for automated proteomic analysis

We have combined several key sample preparation steps for the use of a liquid matrix system to provide high analytical sensitivity in automated ultraviolet - matrix-assisted laser desorption/ ionisation - mass spectrometry (UV-MALDI-MS). This new sample preparation protocol employs a matrix-mixture which is based on the glycerol matrix-mixture described by Sze et al. U. Am. Soc. Mass Spectrom. 1998, 9, 166-174). The low-ferntomole sensitivity that is achievable with this new preparation protocol enables proteomic analysis of protein digests comparable to solid-state matrix systems. For automated data acquisition and analysis, the MALDI performance of this liquid matrix surpasses the conventional solid-state MALDI matrices. Besides the inherent general advantages of liquid samples for automated sample preparation and data acquisition the use of the presented liquid matrix significantly reduces the extent of unspecific ion signals in peptide mass fingerprints compared to typically used solid matrices, such as 2,5-dihydrox-ybenzoic acid (DHB) or alpha-cyano-hydroxycinnamic acid (CHCA). In particular, matrix and lowmass ion signals and ion signals resulting from cation adduct formation are dramatically reduced. Consequently, the confidence level of protein identification by peptide mass mapping of in-solution and in-gel digests is generally higher.

The gas-phase reactions Of SiCl4 and Si2Cl6 with CH3OH and C2H5OH: an investigation by mass spectrometry and matrix-isolation infrared spectroscopy

The gas phase reactions Of SiCl4 and Si2Cl6 With CH3OH and C2H5OH have been investigated using both mass spectrometry and matrix isolation techniques. SiCl4 reacts with both CH3OH and C2H5OH upon mixing of the vapours for times in excess of 3 h to generate the HCl-elimination products SiCl3OR (R = CH3 or C2H5). The identity of these products is confirmed by deuteration experiments and by ab initio calculations at the HF/6-31G(d) level. Further products are generated when the mixture is passed through a tube heated to 750degreesC. Si2Cl6 reacts with CH3OH and C2H5OH via a different mechanism in which the Si-Si bond is cleaved to yield SiCl3OR and HCl. Other products of the type SiCl4-n(OCH3)(n) are tentatively identified by a combination of mass spectrometric and matrix isolation measurements. These latter products indicate further replacement of Cl atoms by OR groups as a result of reaction of CH3OH or C2H5OH with the initial product.

Structure dependent rearrangement of the cyclopropylmethyl cation - isolation of a bicyclo[3.2.0]heptene

Access to 7-allyl substituted norbornene derivatives for tandem olefin metathesis via cationic rearrangement of cyclopropylmethanol substituted norbornenes is shown to be structure dependent. In some cases products that arise from cationic rearrangement of a cyclopropylmethyl cation are furnished. Thionyl chloride is shown to be superior to silica for inducing the desired rearrangement. (c) 2007 Elsevier Ltd. All rights reserved.

An in vitro assessment of the effects of broad-spectrum antibiotics on the human gut microflora and concomitant isolation of a Lactobacillus plantarum with anti-Candida activities

Chemostat culture was used to determine the effects of the antimicrobial agents tetracycline and nystatin on predominant components of the human gut microflora. Their addition to mixed culture systems caused a non-specific, and variable, decrease in microbial populations, although tetracycline allowed an increase in numbers of yeasts. Both had a profound inhibitory effect upon populations seen as important for gut health (bifidobacteria, lactobacilli). However, a tetracycline resistant Lactobacillus was enriched from the experiments. A combination of genotypic and phenotypic characterisations confirmed its identity as Lactobacillus plantarum. This strain exerted powerful inhibitory effects against Candida albicans. Because of its ability to resist the effects of tetracycline, this organism may be useful as a probiotic for the improved management of yeast related conditions such as thrush and irritable bowel syndrome. (C) 2004 Elsevier Ltd. All rights reserved.

Isolation of culturable yeasts from market wines and evaluation of the 5 center dot 8S-ITS rDNA sequence analysis for identification purposes

Aims: To test the possibility that wines available in the marketplace may contain culturable yeasts and to evaluate the 5.8S-ITS rDNA sequence analysis as adequate means for the identification of isolates. Methods and Results: As a case study, typical Greek wines were surveyed. Sequence analysis of the 5.8S-ITS rDNA was tested for its robustness in species or strain identification. Sixteen isolates could be assigned into the species Brettanomyces bruxellensis, Saccharomyces cerevisiae and Rhodotorula pinicola, whereas four isolates could not be safely identified. B. bruxellensis was the dominant species present in house wines, while non-Saccharomyces sp. were viable in aged wines of high alcohol content. Conclusions: Yeast population depends on postfermentation procedures or storage conditions. Although 5.8S-ITS rDNA sequence analysis is generally a rapid method to identify wine yeast isolates at the species level, or even below that, it may not be sufficient for some genera. Significance and Impact of the Study: This is the first report to show that commercial wines may possess diverse and potentially harmful yeast populations. The knowledge of yeasts able to reside in this niche environment is essential towards integrated quality assurance programmes. For selected species, the 5.8S-ITS rDNA sequence analysis is a rapid and accurate means.

Isolation of Corynebacterium falsenii and description of Corynebacterium aquilae sp. nov., from eagles

Biochemical, molecular chemical and molecular genetic studies were performed on seven unidentified Gram-positive, rod-shaped organisms recovered from eagles. The strains were provisionally identified as Corynebacterium jeikeium with the commercial API Coryne system, but they were able to grow under anaerobic conditions and were non-lipophilic. Comparative 16S rRNA gene sequencing studies demonstrated that the isolates belonged phylogenetically to the genus Corynebacterium. Three strains were identified genotypically as Corynebacterium falsenii; the remaining four strains corresponded to a hitherto unknown lineage within the genus Corynebacterium, associated with a small subcluster of species that included Corynebacterium diphtheriae and its close relatives. The unknown bacterial strains were readily distinguished from these and other species of the genus by biochemical tests. Based on both phenotypic and phylogenetic evidence, it is proposed that the unknown bacterial strains from eagles should be classified as Corynebacterium aquilae sp. nov. (type strain is S-613(T)=CECT 5993(T)=CCUG 46511(T)).

Isolation, fractionation and characterisation of proteins from Mucuna bean

The chemical composition and fractional distribution of protein isolates prepared from species of Mucuna bean were studied. Using six different extraction media, the yield of protein based on the Kjeldahl procedure varied from 8% to 34%, and the protein content varied from 75% to 95%. When the yields were high, the colour of the isolates generally tended to be dark and unsatisfactory. Hence, the use of chemical treatments and high pressure processing were explored. The solubility maxima for the protein isolates in water were found to occur at pH values of 2.0 and 11.0, while the pH corresponding to minimum solubility (i.e. isoelectric region) occurred at pH values of 4.0 and 5.0. The total essential amino acid in the isolates ranged from 495 to 557 mg g(-1) protein, which compares favourably with the recommended level for pre-school and school children. Methionine and cysteine were the limiting amino acids. A key nutritional attribute of the protein isolates was its high lysine content. The isolate can therefore complement cereal-based foods which are deficient in lysine. The proteins mainly consisted of albumins, glutelins and globulins. Prolamins were only present in trace concentration (< 0.3%). Gel filtration chromatograms of the isolates indicated the presence of major protein fractions with molecular weights of 40 and 15 kDa, while gel electrophoresis (SDS-PAGE) indicated a major broad zone with molecular weights of 36 +/- 7 and 17.3 +/- 13 kDa. (c) 2006 Elsevier Ltd. All rights reserved.

An automated calibration method for non-see-through head mounted displays

Accurate calibration of a head mounted display (HMD) is essential both for research on the visual system and for realistic interaction with virtual objects. Yet, existing calibration methods are time consuming and depend on human judgements, making them error prone. The methods are also limited to optical see-through HMDs. Building on our existing HMD calibration method [1], we show here how it is possible to calibrate a non-see-through HMD. A camera is placed inside an HMD displaying an image of a regular grid, which is captured by the camera. The HMD is then removed and the camera, which remains fixed in position, is used to capture images of a tracked calibration object in various positions. The locations of image features on the calibration object are then re-expressed in relation to the HMD grid. This allows established camera calibration techniques to be used to recover estimates of the display’s intrinsic parameters (width, height, focal length) and extrinsic parameters (optic centre and orientation of the principal ray). We calibrated a HMD in this manner in both see-through and in non-see-through modes and report the magnitude of the errors between real image features and reprojected features. Our calibration method produces low reprojection errors and involves no error-prone human measurements.

Hybrid fault detection and isolation in an heater bank of an HVAC system

In this work a hybrid technique that includes probabilistic and optimization based methods is presented. The method is applied, both in simulation and by means of real-time experiments, to the heating unit of a Heating, Ventilation Air Conditioning (HVAC) system. It is shown that the addition of the probabilistic approach improves the fault diagnosis accuracy.