Proteomic analysis of agar gel-entrapped Pseudomonas aeruginosa

We have compared the protein maps of agar-entrapped Pseudomonas aeruginosa cells to those of free counterparts grown in the presence or absence of the immobilized-cell gel support. Principal component analyses (PCAs) were used to interpret spot quantity variations observed on electropherograms obtained by two-dimensional gel electrophoresis. PCA of the data matrix (923 rows x 6 columns) in which spot density values were standardized horizontally extracted three principal components (PCs) with eigenvalues higher than 1, accounting together for 71.6% of the variability in the data. Principal component 1 (PC1) opposed free (F) and agar-entrapped (AE) cultures, with a low contribution of agar-released, free (ARF) cultures to PC1. Inversely, the contribution of ARF cultures to PC2 was high, opposing those of AE and F cultures. Component 3 was related to the duration of incubation. Only 10% of total proteins were upregulated in AE cells during the first 18 h of incubation, the number of underexpressed peptides balancing that of overexpressed ones. Downregulation clearly became the dominant tendency when the incubation time was extended to 48 h. These results demonstrate that AE and ARF bacteria are physiologically different from F organisms.     

The covalent-sorption reconstitution of steroid hydroxylases

The effect of covalent immobilization via free amino groups on the catalytic activity of individual components of the cholesterol side-chain cleavage and 11b-steroid hydroxylation systems (adrenodoxin reductase, adrenodoxin, cytochrome P-450scc and cytochrome P-450(11)b) as well as on that of co-immobilized protein complexes. The protein complex formation at different stages of the monooxygenase cycle (i.e., reduction, oxygenation) was followed by direct spectrophotometric monitoring of the functional state of the immobilized complexes. Cholesterol side-chain cleavage was carried out in minicolumns, using various combinations of immobilized and soluble proteins. Cytochromes P-450scc and P-450(11)b were found to retain their functional activities after immobilization via free SH-groups.     

Immobilized viable microbial cells: from the process to the proteome em leader or the cart before the horse

Biotechnological processes based on immobilized viable cells have developed rapidly over the last 30 years. For a long time, basic studies of the physiological behaviour of immobilized cells (IC) have remained in the shadow of the applications. Natural IC structures, i.e. biofilms, are being increasingly investigated at the cellular level owing to their definite importance for human health and in various areas of industrial and environmental relevance. This review illustrates this paradoxical development of research on ICs, starting from the initial rationale for IC emergence and main application fields of the technology—with particular emphasis on those that exploit the extraordinary resistance of ICs to antimicrobial compounds—to recent advances in the proteomic approach of IC physiology.     

Effect of PEG-mediated pore forming on Ca-alginate immobilization of nitrilase-producing bacteria Pseudomonas putida XY4

Effect of PEG-mediated pore forming on Ca-alginate immobilization of nitrilase-producing bacteria Pseudomonas putida XY4 was studied. Through using PEG as porogen, the environmental tolerance as well as the biocatalytic reaction efficiency of immobilized cells was greatly improved, i.e., Ca-alginate-PEG immobilized cells got better temperature and substrate concentration tolerance than Ca-alginate immobilized cells and showed similar efficiency with free cells, suggesting that the intrinsic mass transfer resistance of immobilization obviously decreased. It was also observed that the pore diameter and porosity of immobilization beads were related with the molecular weight of PEG. PEG400 was found to be a relatively suitable porogen for Ca-alginate-PEG immobilized cells catalyzed hydrolysis of glycinonitrile. It was noteworthy that the Ca-alginate-PEG immobilized cells could be reused more than 18 times with little loss of enzyme activity which had shown good operation ability and great application potential.     

中枢神经蛋白质组分析中双向电泳技术的建立

建立和优化了中枢神经组织蛋白质组分析所需的双向电泳及相关技术.由于中枢神经组织结构的特殊性,样品处理非常困难.对样品液组成、样品处理、上样方式、上样量、IPG胶条和SDS-聚丙烯酰胺凝胶电泳染色方法和保存等相关技术进行了比较研究和条件优化后,以固相pH梯度等电聚焦为第一向和SDS均一胶（T=12.5%）的水平电泳为第二向,成功地得到了神经组织双向电泳图谱.     

Hydrated conidia of Metarhizium anisopliae release a family of metalloproteases

Metarhizium anisopliae spores release isoforms of metalloprotease during hydration over a 4-day incubation period. The isoforms were identified and characterized by using one-dimensional native PAGE (1-DE nPAGE) and one-dimensional SDS non-dissociating (1-DE nSDS-PAGE) zymography. The ability of these isozymes to degrade gelatin varied as revealed by 2-D spot densitometry. 1-DE nPAGE zymography revealed five isoforms of gelatinase from Tween wash of conidia. Where as, one to three activities with different intensities appeared on gel from washing of conidia to incubation in water till day 4. The relative migrations of these activities on 1-DE nPAGE zymograms appeared as fast, medium and slow on gel. The 2-D spot densitometry of zymograms indicated isoforms have different proteolytic activity as quantified by pixel intensities. SDS-PAGE zymography indicated the release of two isozymes of Mr 103 and 12 kDa during Tween treatment of conidia. However, during the first washing step with water and incubation of spores at day 2 and 3, respectively, only 12 kDa protein was evident. Majority of these proteases were inhibited by EDTA, but stimulated by CaCl(2), and MgCl(2). The presence of isozymes in conidia and their release during hydration must have functional significance for fungi and in this case it should provide advantages to M. anisopliae in its saprobic or pathogenic modalities. To our knowledge this is the first report describing release of metalloprotease isozymes from conidia.     

Soil bioaugmentation by free and immobilized bacteria to reduce potentially phytoavailable cadmium

Soil bioaugmentation was performed in soil pots to reduce the cadmium potentially available for plants. A Bacillus sp. (isolate ZAN-044) and a Streptomyces sp. (isolate R25) were compared, just as the inoculation technique, i.e., inoculum size, free or immobilized cells. After 3 weeks of a batch incubation, the potentially phytoavailable Cd was reduced, at the maximum, to a factor 14.1 and 4.3 with Bacillus sp. ZAN-044 and Streptomyces sp. R25, respectively. The two bacteria survived and colonized the soil. The immobilization technique did not improve the cell survival in the bioaugmented soil. The potentially phytoavailable Cd was positively (r(2)=+0.73) or negatively correlated (r(2)=-0.78) to the cell concentration in the sterilized soil bioaugmented with Bacillus sp. ZAN-044 or Streptomyces sp. R25, respectively. The major effect upon the phytoavailable Cd was the microorganism used and, to a lesser extent, the inoculum size and the culture technique.     

Photonic activation of disulfide bridges achieves oriented protein immobilization on biosensor surfaces

Photonic induced immobilization is a novel technology that results in spatially oriented and spatially localized covalent coupling of biomolecules onto thiol-reactive surfaces. Immobilization using this technology has been achieved for a wide selection of proteins, such as hydrolytic enzymes (lipases/esterases, lysozyme), proteases (human plasminogen), alkaline phosphatase, immunoglobulins' Fab fragment (e.g., antibody against PSA [prostate specific antigen]), Major Histocompability Complex class I protein, pepsin, and trypsin. The reaction mechanism behind the reported new technology involves "photonic activation of disulfide bridges," i.e., light-induced breakage of disulfide bridges in proteins upon UV illumination of nearby aromatic amino acids, resulting in the formation of free, reactive thiol groups that will form covalent bonds with thiol-reactive surfaces (see Fig. 1). Interestingly, the spatial proximity of aromatic residues and disulfide bridges in proteins has been preserved throughout molecular evolution. The new photonic-induced method for immobilization of proteins preserves the native structural and functional properties of the immobilized protein, avoiding the use of one or more chemical/thermal steps. This technology allows for the creation of spatially oriented as well as spatially defined multiprotein/DNA high-density sensor arrays with spot size of 1 microm or less, and has clear potential for biomedical, bioelectronic, nanotechnology, and therapeutic applications.     

Characterization of wheat gliadin proteins by combined two-dimensional gel electrophoresis and tandem mass spectrometry

A proteomics-based approach was used for characterizing wheat gliadins from an Italian common wheat (Triticum aestivum) cultivar. A two-dimensional gel electrophoresis (2-DE) map of roughly 40 spots was obtained by submitting the 70% alcohol-soluble crude protein extract to isoelectric focusing on immobilized pH gradient strips across two pH gradient ranges, i.e., 3-10 or pH 6-11, and to sodium dodecyl sulfate-polyacrylamide electrophoresis in the second dimension. The chymotryptic digest of each spot was characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and nano electrospray ionization-tandem mass spectrometry (MS/MS) analysis, providing a "peptide map" for each digest. The measured masses were subsequently sought in databases for sequences. For accurate identification of the parent protein, it was necessary to determine de novo sequences by MS/MS experiments on the peptides. By partial mass fingerprinting, we identified protein molecules such as alpha/beta-, gamma-, omega-gliadin, and high molecular weight-glutenin. The single spots along the 2-DE map were discriminated on the basis of their amino acid sequence traits. alpha-Gliadin, the most represented wheat protein in databases, was highly conserved as the relative N-terminal sequence of the components from the 2-DE map contained only a few silent amino acid substitutions. The other closely related gliadins were identified by sequencing internal peptide chains. The results gave insight into the complex nature of gliadin heterogeneity. This approach has provided us with sound reference data for differentiating gliadins amongst wheat varieties.     

Hybrid nanoparticles based on organized protein immobilization on fullerenes

Nanoscale carbon materials (i.e., fullerenes and nanotubes) are an attractive platform for applications in biotransformations and biosensors. The interesting properties displayed by nanoparticles demand new strategies for the manipulation of these materials on the nanoscale. Controlled modification of their surface with biomolecules is required to fully realize their potential in bionanotechnology. In this work, immobilization of a fullerene derivative with a mutant subtilisin is demonstrated, and the effect of the fullerene on the protein activity is determined. The fullerene-conjugated enzyme had improved catalytic properties in comparison to subtilisin immobilized on nonporous silica. Further, the pH profile of free and fullerene-conjugated subtilisin were almost identical.     

Cr(VI) reduction by Pseudomonas aeruginosa immobilized in a polyvinyl alcohol/sodium alginate matrix containing multi-walled carbon nanotubes

Pseudomonas aeruginosa (P. aeruginosa) was immobilized with polyvinyl alcohol (PVA), sodium alginate and multiwalled carbon nanotubes (MCNTs). After immobilization, the beads were subjected to freeze-thawing to enhance mechanical strength. When exposed to 80 mg/L Cr(VI), the immobilized bacteria were able to reduce 50% of them in 84 h, however the free cells were deactivated at this concentration. The beads were used to reduce 50 mg/L Cr(VI) for nine times, with the reduction efficiency above 90% in the first five times and 65% in the end.     

Conformational flexibility of a model protein upon immobilization on self-assembled monolayers

The present study reports on the retention of conformational flexibility of a model allosteric protein upon immobilization on self-assembled monolayers (SAMs) on gold. Organothiolated SAMs of different compositions were utilized for adsorptive and covalent attachment of bovine liver glutamate dehydrogenase (GDH), a well-characterized allosteric enzyme. Sensitive fluorimetric assays were developed to determine immobilization capacity, specific activity, and allosteric properties of the immobilized preparations as well as the potential for repeated use and continuous catalytic transformations. The allosteric response of the free and immobilized forms towards ADP, L-leucine and high concentrations of NAD(+), some of the well-known activators for this enzyme, were determined and compared. The enzyme immobilized by adsorption or chemical binding responded similarly to the activators with a greater degree of activation, as compared to the free form. Also loss of activity involving the two immobilization procedures were similar, suggesting that residues essential for catalytic activity or allosteric properties of GDH remained unchanged in the course of chemical modification. A recently established method was used to predict GDH orientation upon immobilization, which was found to explain some of the experimental results presented. The general significance of these observations in connection with retention of native properties of protein structures upon immobilization on SAMs is discussed.     

Nature of antigens and antibodies in immune complexes isolated by staphylococcal protein A from plasma of melanoma patients

Immune complexes (IC) were isolated from plasma of melanoma patients by absorption to staphylococcal protein A and subsequent elution with MgCl2. The isolated ICs were purified by precipitation with polyethylene glycol and sucrose density gradient ultracentrifugation after radioiodination with 125I. The purified ICs were dissociated and radiolabeled antigen/antibody components were separated by ultracentrifugation at low pH (2.6). Under these conditions, about 72% radioactivity of the purified IC remained in the light-density region as a wide band. After neutralization, 26%-60% radioactivity in the region of 5S sedimentation bound to immobilized autologous immunoglobulins, as opposed to a maximum of 23% to immobilized immunoglobulins from human normal serum. Significant levels (73%-77%) of radioactivity in 7S region bound to rabbit anti-human IgG immunobeads. Immunoprecipitation of the antigen fraction by allogeneic anti-melanoma and rabbit anti-melanoma antibodies followed by SDS-polyacrylamide gel electrophoresis revealed the presence of a fetal antigen (FA) and a melanoma tumor-associated antigen (TAA). In addition, the presence of auto-antigen(s) was indicated by using autologous antibody in immunoprecipitation. Immunoglobulins (IgG) isolated from purified IC bound to cultured melanoma, sarcoma, and normal fibroblasts, although the binding to sarcoma and normal fibroblasts could be inhibited by preincubation of isolated IgG with soluble FA but not with soluble melanoma TAA. Thus, results of this investigation provide evidence that circulating IC in melanoma patients are composed of at least IgG and different antigens, and some of these antigens are produced by their tumor.     

Nitrate and phosphate removal by chitosan immobilized Scenedesmus

The effect of chitosan immobilization of Scenedesmus spp. cells on its viability, growth and nitrate and phosphate uptake was investigated. Scenedesmus sp. (strains 1 and 2) and Scenedesmus obliquus immobilized in chitosan beads showed high viability after the immobilization process. Immobilized Scenedesmus sp. strain 1 had a higher growth rate than its free living counterpart. Nitrate and phosphate uptake by immobilized cells of Scenedesmus sp. (strain 1), freely suspended cells and blank chitosan beads (without cells) were evaluated. Immobilized cells accomplished a 70% nitrate and 94% phosphate removal within 12h of incubation while free-living cells removed 20% nitrate and 30% phosphate within 36 h of treatment. Blank chitosan beads were responsible for up to 20% nitrate and 60% phosphate uptake at the end of the experiment. Chitosan is a suitable matrix for immobilization of microalgae, particularly Scenedesmus sp., but this system should be improved before its application for water quality control.     

Immobilization of pancreatic lipase on polyvinyl alcohol by cyanuric chloride

Porcine pancreatic lipase (EC 3.1.1.3) was covalently immobilized onto 2,4,6-trichloro-s-triazine (cyanuric chloride) activated polyvinyl alcohol (PVA). The influence of activating agent and enzyme concentration on the immobilization process were evaluated.Hydrolytic activities of free and immobilized enzyme were determined and the immobilization yield was estimated by measuring the quantity of protein, both in free enzyme solution and in washing solutions after immobilization. After the optimization of immobilization process, the physical and chemical characterization of immobilized enzyme was performed. Additionally, the thermal, pH, storage, and operational stability of the immobilized and free enzymes were tested. Obtained data showed that the immobilized enzyme seemed better and offered some advantages in comparison with free enzyme.     

Rotational dynamics of protein and boundary lipid in sarcoplasmic reticulum membrane

We have used spin labels and electron paramagnetic resonance (EPR) to study the correlation between the rotational dynamics of protein and lipid in sarcoplasmic reticulum (SR) membranes. A short-chain maleimide spin label was used to monitor the submillisecond rotational mobility of the Ca-ATPase enzyme (using saturation transfer EPR); a free fatty acid spin label was used to monitor the submicrosecond rotational mobility of the bulk lipid hydrocarbon chains (using conventional EPR); and a fatty acid spin label derivative (long-chain maleimide) attached to the enzyme was used to monitor the mobility of hydrocarbon chains adjacent to the protein (i.e., boundary lipid). In the native SR membranes, the protein was highly mobile (effective correlation time 50 microseconds). The spectra of the hydrocarbon probes both contained at least two components. For the unattached probe, the major component indicated nearly as much mobility as in the absence of protein (effective rotational correlation time 3 ns), while a minor component, corresponding to 25-30% of the total signal, indicated strong immobilization (effective correlation time greater than or equal to 10 ns). For the attached hydrocarbon probe, the major component (approximately 70% of the total) was strongly immobilized, and the mobile component was less mobile than that of the unattached probe. When the lipid-to-protein ratio was reduced 55% by treatment with deoxycholate, protein mobility decreased considerably, suggesting protein aggregation. A concomitant increase was observed in the fraction of immobilized spin labels for both the free and attached hydrocarbon probes. The observed hydrocarbon immobilization probably arises in part from immobilization at the protein-lipid boundary, but protein-protein interactions that trap hydrocarbon chains may also contribute. When protein aggregation was induced by glutaraldehyde crosslinking, submillisecond protein mobility was eliminated, but there was no effect on either hydrocarbon probe. Thus protein aggregation does not necessarily cause hydrocarbon chain immobilization.     

Cell immobilization technique for the enhanced production of alpha-galactosidase by Streptomyces griseoloalbus

Streptomyces griseoloalbus was immobilized in calcium alginate gel and the optimal immobilization parameters (concentrations of sodium alginate and calcium chloride, initial biomass and curing time) for the enhanced production of alpha-galactosidase were determined. The immobilization was most effective with 3% sodium alginate and 0.1M calcium chloride. The optimal initial biomass for immobilization was approximately 2.2g (wet wt.). The alginate-entrapped cells were advantageous because there was a twofold increase in the enzyme yield (55 U/ml) compared to the highest yield obtained with free cells (23.6 U/ml). Moreover, with immobilized cells the maximum yield was reached after 72 h of incubation in batch fermentation under optimal conditions, whereas in the case of free cells the maximum enzyme yield was obtained only after 96 h of incubation. The alginate beads had good stability and also retained 75% ability of enzyme production even after eight cycles of repeated batch fermentation. It is significant that this is the first report on whole-cell immobilization for alpha-galactosidase production.     

Protein hydrolysis by immobilized and stabilized trypsin

The preparation of novel immobilized and stabilized derivatives of trypsin is reported here. The new derivatives preserved 80% of the initial catalytic activity toward synthetic substrates [benzoyl-arginine p-nitroanilide (BAPNA)] and were 50,000-fold more thermally stable than the diluted soluble enzyme in the absence of autolysis. Trypsin was immobilized on highly activated glyoxyl-Sepharose following a two-step immobilization strategy: (a) first, a multipoint covalent immobilization at pH 8.5 that only involves low pK(a) amino groups (e.g., those derived from the activation of trypsin from trypsinogen) is performed and (b) next, an additional alkaline incubation at pH 10 is performed to favor an intense, additional multipoint immobilization between the high concentration of proximate aldehyde groups on the support surface and the high pK(a) amino groups at the enzyme surface region that participated in the first immobilization step. Interestingly, the new, highly stable trypsin derivatives were also much more active in the proteolysis of high molecular weight proteins when compared with a nonstabilized derivative prepared on CNBr-activated Sepharose. In fact, all the proteins contained a cheese whey extract had been completely proteolyzed after 6 h at pH 9 and 50°C, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Under these experimental conditions, the immobilized biocatalysts preserve more than 90% of their initial activity after 20 days. Analysis of the three-dimensional (3D) structure of the best immobilized trypsin derivative showed a surface region containing two amino terminal groups and five lysine (Lys) residues that may be responsible for this novel and interesting immobilization and stabilization. Moreover, this region is relatively far from the active site of the enzyme, which could explain the good results obtained for the hydrolysis of high-molecular weight proteins.     

Improved tolerance to bile salts of aggregated Bifidobacterium longum produced during continuous culture with immobilized cells

The effect of cell immobilization and continuous culture was studied on selected physiological and technological characteristics of Bifidobacterium longum NCC2705 cultivated for 20 days in a two stage continuous fermentation system. Continuous immobilized cell (IC) cultures with and without glucose limitation exhibited formation of macroscopic cell aggregates after 12 and 9 days, respectively. Auto-aggregation resulted in underestimation of viable cell counts by plate counts by more than 2 log units CFU/ml compared with qPCR method. Modifications of cell membrane composition might partially explain aggregate formation in IC cultures. Decreases in the ratio of unsaturated to saturated fatty acid content from 1.74 to 0.58 might also contribute to the enhanced tolerance of IC cells to porcine bile salts and aminoglycosidic antibiotics compared with free cells from batch cultures.The enhanced resistance against bile salts in combination with auto-aggregation may confer an advantage to probiotic bacteria produced by IC technology.     

玉米芯、竹炭及油枯吸附-海藻酸钠包埋施氏假单胞菌PFS-4对二氯喹啉酸的降解

采用玉米芯、竹炭及油枯吸附-海藻酸钠包埋对分离到的施氏假单胞菌PFS-4进行复合固定.采用正交试验对固定化条件进行优化,研究了固定化菌剂及游离菌体对二氯喹啉酸的降解效果.结果表明: 固定化菌剂制备的最佳条件为:海藻酸钠质量分数为4%、吸附载体比例(玉米芯∶竹炭∶油枯)为1∶2∶1、CaCl₂质量分数为3%、交联时间4 h.固定化菌剂在温度为30 ℃、初始pH=7的条件下,经6 d培养后,对浓度为800 mg·L^-1的二氯喹啉酸降解率为91.4%,而游离菌体的降解率为72.8%.将游离菌体和固定化菌剂用于实际污水及土壤处理时,固定化菌剂对水中及土壤中二氯喹啉酸去除率仍能分别达到84.2%和74.3%.研究结果表明,载体及其联结方式对土壤中二氯喹啉酸去除产生显著影响,翻动频率与土壤中二氯喹啉酸的去除率呈显著正相关.因此,玉米芯、竹炭及油枯吸附-海藻酸钠复合固定施氏假单胞菌PFS-4对不良环境具有较好的缓冲性能,对二氯喹啉酸污染水体及土壤原位生态修复具有潜力.