不同萃取体系对小球藻藻渣成分的影响

考察5种萃取体系（A：正己烷,B：正己烷/乙醇,C：正己烷/异丙醇,D：氯仿/甲醇,E：氯仿/乙醇）对小球藻（Chlorella phyrenoidosa）油脂的提取效果及藻渣成分的影响。实验结果表明：不同的萃取体系下,油脂得率为D（12.27%）、E（8.87%）、C（7.71%）、B（6.80%）、A（3.91%）,藻渣蛋白含量为A（52.60%）、E（46.23%）、B（40.19%）、C（39.52%）、D（32.52%）,藻渣碳水化合物含量为A（23.28%）、E（16.15%）、B（13.24%）、D（13.50%）、C（9.06%）;藻渣色素含量为A（1.75%）、E（1.29%）、B（1.14%）、C（0.96%）、D（0.58%）;藻渣灰分含量为D（3.63%）、E（2.94%）、C（2.23%）、B（2.25%）、A（1.48%）。综合考虑微藻生物柴油的生产及藻渣的可利用性,V（氯仿）/V（乙醇）=1是一种油脂萃取效果较好,藻渣营养成分损失较小的小球藻油脂萃取体系。     

小球藻高附加值生物活性物质“小球藻热水提取物”的研究现状与展望

小球藻是一种食用历史久、营养丰富的微藻功能食品,其中蛋白核小球藻已于2012年被我国批准为新资源食品,并成为国内外正在大力发展与培育的微藻能源及微藻固碳这一战略性新兴产业的主要藻种之一。在积累油脂的同时,小球藻自身还能合成高附加值生物活性物质,其合理利用可平衡微藻能源的高成本。小球藻热水提取物(CE),即商业上宣称的"小球藻生长因子(CGF)",是小球藻有别于其他微藻的主要生物活性物质,在促进生长、调节免疫等方面具有良好功效,且市场售价高。但迄今,有关CE的认识尚不清晰,尚未见CE方面的系统评述。本文对近年来CE的活性研究状况进行了系统的文献调查与梳理,综述了CE在增强免疫、抑制肿瘤、改善代谢综合征、清除自由基、抵御紫外损伤、螯合重金属以及保肝护肠等多个方面的功效,并分析了CE活性研究中存在的问题及CE的发展前景。     

Analytical and preparative isoelectric focusing of peptides in immobilized pH gradients

A new method for peptide analysis and purification is described, based on isoelectric focusing in immobilized pH gradients. On the analytical scale, the peptide zones can now be revealed by an stain for primary and secondary amino group (e.g. ninydrin, fluorescamine, dansyl chloride) since the buffering species, unlike conventional carrier ampholytes, contain only carboxyl and tertiary amino groups. For preparative purposes, conditions have been described to remove most contaminants (e.g. unreacted monomers, non-cross-linked, short polyacrylamide chains) from the gel matrix before the electrophoretic run. However, ca. 2% of the gel dry mass is still present as extractable material. The focused peptides can be recovered in higly yields (ca. 90%) with a fairly high degree of purity (75%), the contaminants being mostly components eluted from the polyacrylamide gel.     

Immunochemical characterization and localization of nitrate reductase in norflurazon-treated soybean cotyledons

Immunochemical procedures were used to characterize and localize NADH:nitrate reductase (NR; EC 1.6.6.1) in cotyledons of norflurazon-treated soybeans [ Glycine max (L.) Merr. cv. 'Hill']. Antiserum prepared to NR isolated from Chlorella strongly reacted against NR from norflurazon-treated cotyledons. This serum inhibited the NR activity in crude extracts of norflurazon-treated soybean cotyledons by 98% even at a 1:2000 dilution of crude serum. Pre-immune serum had no effect on the activity. These data indicate that there are similar antigenic determinants at the active site of both Chlorella and norflurazon-treated soybean NR. Whole cotyledons were homogenized in lithium dodecyl sulfate-containing buffer, electrophoretically separated and blotted to nitrocellulose. When the blots were reacted with the anti-NR serum only a single protein (M_r= 98 kdalton) was visualized. Immunofluorescence studies on fixed tissue sections revealed intense fluorescence in the cytoplasm. Weaker reactions were associated with organelles tentatively identified as plastids. Pre-immune serum controls were completely unstained using immunocytochemical procedures.     

Asymmetric ammonolysis of (R/S)-mandelic acid by immobilized lipases via direct amidation of mandelic acid in biphasic media

Abstract

We have investigated the direct enantioselective amidation of mandelic acid with ammonia, catalyzed by a variety of commercial lipases including those from Candida rugosa, Mucor miehei, Pseudomonas sp., Rhizomucor miehei, and Thermomyces lanuginosus covalently immobilized onto Florisil^® support via glutaraldehyde and polysuccinimide spacer arms. All the immobilized lipase preparations tested preferentially amidated the R isomer of mandelic acid. The highest amide yields were obtained for immobilized Pseudomonas sp. lipase preparations under the optimized reaction conditions. After 24 h of amidation, the reaction had proceeded with an excellent yield (50%) and enantiopurity (> 99%). The immobilized Pseudomonas sp. lipase preparations catalyzed the amidation reaction with the same yield and enantioselectivity. The enzyme immobilized via a glutaraldehyde spacer arm showed better reusability than that immobilized via a polysuccinimide spacer arm.

In view of these results, it is revealed that the direct amidation of mandelic acid catalyzed by the immobilized Pseudomonas sp. lipases is a facile and effective methodology for obtaining (S)-mandelic acid and (R)-mandelamide.     

Performance of an immobilized fuculose-1-phosphate aldolase for stereoselective synthesis

A derivative of fuculose-1-phosphate aldolase, immobilized with high loading on glyoxal–agarose gels, has been characterized and evaluated as a biocatalyst for an aldol addition reaction. The reaction of the solid biocatalyst was diffusion-controlled for conversion of its natural substrate. Nevertheless, when catalyzing the synthesis of a biologically active aminopolyol, the lower reaction rate with non-natural substrates led to a process controlled by the intrinsic enzyme kinetics. The resulting biocatalyst has high synthetic specific activity and has been successfully used in batch synthesis reactions with high conversion. In addition, the immobilized aldolase has been employed in fed-batch synthesis, increasing the selectivity of the reaction and obtaining high conversion (88%).     

Biodegradation of Phenol Using Immobilized Nocardia hydrocarbonoxydans in a Pulsed Plate Bioreactor: Effect of Packed Stages,Cell Carrier Loading,and Cell Acclimatization on Startup and Steady-State Behavior

The effect of the number of stages and cell carrier loading on the steady-state and startup performance of a continuous pulsed plate bioreactor with glass beads as the cell carrier material for biodegradation of phenol in wastewater using immobilized Nocardia hydrocarbonoxydans has been studied. It was found that the performance of the pulsed plate bioreactor during startup and at steady state can be improved by an increase in cell carrier loading, number of stages, total plate stack height, and with a decrease in plate spacing. The startup time for the continuous bioreactor can be decreased by increasing the number of preacclimatization steps for the cells. The attainment of steady effluent phenol concentration can be considered as an indication of steady state of the continuous bioreactor, as when phenol concentration attained a steady value, biofilm thickness, and the attached biomass dry weight also attained a constant value.     

A novel Aurora-A-mediated phosphorylation of p53 inhibits its interaction with MDM2

Purpose: Crosstalk between Aurora-A kinase and p53 has been proposed. While the genetic amplification of Aurora-A has been observed in many human cancers, how p53 is regulated by Aurora-A remains ambiguous. In this study, Aurora-A-mediated phosphorylation of p53 was analyzed by mass spectrometry in order to identify a new phosphorylation site. Subsequently, the functional consequences of such phosphorylation were examined. Experimental design: In vitro phosphorylation of p53 by Aurora-A was performed and the phosphorylated protein was then digested with trypsin and enriched for phosphopeptides by immobilized metal affinity chromatography. Subsequently, a combination of β-elimination and Michael addition was applied to the phosphopeptides in order to facilitate the identification of phosphorylation sites by MS. The functional consequences of the novel phosphorylation of p53 on the protein–protein interactions, protein stability and transactivation activity were then examined using co-immunoprecipitation, Western blotting and reporter assays. Results: Ser-106 of p53 was identified as a novel site phosphorylated by Aurora-A. A serine-to-alanine mutation at this site was found to attenuate Aurora-A-mediated phosphorylation in vitro. In addition, phosphate-sensitive Phos-tag SDS-PAGE was used to confirm that the Ser-106 of p53 is in vivo phosphorylated by Aurora-A. Finally, co-immunoprecipitation studies suggested that Ser-106 phosphorylation of p53 decreases its interaction with MDM2 and prolongs the half-life of p53. Conclusions: The inhibition of the interaction between p53 and MDM2 by a novel Aurora-A-mediated p53 phosphorylation was identified in this study and this provides important information for further investigations into the interaction between p53 and Aurora-A in terms of cancer biology.     

Proteome analysis of the plant-pathogenic bacterium Xanthomonas oryzae pv. oryzae

The plant-pathogenic bacterium Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of bacterial blight, which is one of the most serious diseases of rice. Xoo has been studied for over one century, and much has been learned about it, but proteomic investigation has been neglected. In this study, proteome reference maps of Xoo were constructed by two-dimensional gel electrophoresis, and 628 spots in the gels representing 469 different protein species were identified with MALDI-TOF/TOF MS. The identified spots were assigned to 15 functional categories according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the annotations from the National Center for Biotechnology Information (NCBI) database. The data set has been deposited in the World-2DPAGE database (Database ID: 0044). In addition, comparative proteomic analysis revealed that proteins related to the TonB-dependent transportation system and energy metabolism are involved in the phenazine-1-carboxylic acid resistance in Xoo. In conclusion, we have established a proteome database for Xoo and have used this database in a comparative proteomic analysis that identified proteins potentially contributing to phenazine-1-carboxylic acid resistance in Xoo.