Proteome analysis of recombinant xylose-fermenting Saccharomyces cerevisiae

Introduction of an active xylose utilization pathway into Saccharomyces cerevisiae, which does not naturally ferment pentose sugars, is likely to have a major impact on the overall cellular metabolism as the carbon introduced to the cells will now flow through the pentose phosphate pathway. The metabolic responses in the recombinant xylose-fermenting S. cerevisiae were studied at the proteome level by comparative two-dimensional gel electrophoresis of cellular proteins within a pH range of 3-10. Glucose-limited chemostat cultivations and corresponding chemostat cultivations performed in media containing xylose as the major carbon source were compared. The cultivations were studied in aerobic and anaerobic metabolic steady states and in addition at time points 5, 30 and 60 min after the switch-off of oxygen supply. We identified 22 proteins having a significant abundance difference on xylose compared to glucose, and 12 proteins that responded to change from aerobic to anaerobic conditions on both carbon sources. On xylose in all conditions studied, major changes were seen in the abundance of alcohol dehydrogenase 2 (Adh2p), acetaldehyde dehydrogenases 4 and 6 (Ald4p and Ald6p), and DL-glycerol 3-phosphatase (Gpp1p). Our results give indications of altered metabolic fluxes especially in the acetate and glycerol pathways in cells growing on xylose compared to glucose.     

Proteome analysis of the wort boiling process

A comprehensive proteome map was constructed of brewers wort. The map consisted of protein identification on two-dimensional gel electrophoresis (2DE) images and identified 63 out of 202 protein spots, which were categorized into 20 protein species. To analyze the modification of protein Z during wort boiling, protein Z spots on 2DE gel of the sweet wort, the boiled wort and the trub were analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF-MS) and then the spectra were compared. The analysis identified several specific signals detected only in the trub, suggesting that specific modification occurred in the precipitated protein Z during wort boiling. The protein Z spot on the 2DE gel of the precipitate was further analyzed by liquid chromatography mass spectrometry/mass spectrometry. The analysis identified low molecular weight fragment (1.3 kDa) derived from wound induced protein (barwin) in the protein Z spot of the trub. These results suggested that protein Z was precipitated by binding with comparatively small size specific fragment(s) derived from sweet wort protein, i.e., barwin during wort boiling. Our results and understandings have application for quality assurance and control in commercial brewing practice, and development of DNA markers for malting barley breeding.     

不同宰杀方式对鲫鱼肌肉质构和蛋白质组的影响

以异育银鲫为研究对象,利用质构仪和双向电泳技术研究2种不同宰杀方式对鲫鱼肌肉质构和蛋白质组的影响。结果表明,与直接打头致死组比较,氮气致死的宰杀方式可显著增加(P0.05)鲫鱼死后肌肉的硬度、咀嚼性和胶黏性(分别增加了62.24%、85.72%和75.47%)。2个处理的肌肉样品经双向电泳分析,均分离出1 000个以上的蛋白质点。与直接打头致死组比较,氮气致死组凝胶中小分子质量蛋白质点增多,相同蛋白质的相对含量下降,分布面积扩大。经PDQuest软件进行差异点分析和显著性丰度统计,得到12个有实际意义的差异蛋白质点。该结果揭示不同宰杀方式影响鲫鱼肌肉中蛋白质的含量与代谢,表现为肌肉的硬度、咀嚼性和胶黏性等质构指标发生变化。     

烟草叶片蛋白质组双向电泳实验体系的建立

选用干旱敏感型栽培烟草品种中烟100旺长后期的叶片和感黑胫病栽培烟草品种小黄金1025苗期的叶片作为实验材料,总蛋白质提取分别采用稍加改进的TCA/丙酮法和由蛋白质分级提取、酚抽提及丙酮洗涤相结合的方法。对中烟100旺长后期烟草叶片采用的总蛋白质TCA/丙酮提取方法进行了优化、对其使用的IPG胶条pH范围做了初步筛选,并在此研究基础上,对采用第二种提取方法获得的小黄金1025苗期烟草叶片总蛋白质样品进行了不同上样量对双向电泳结果影响的比较实验。初步建立起适合烟草叶片蛋白质组研究的双向电泳实验体系。     

猪肝脏蛋白质组双向电泳体系的建立及优化

建立猪肝脏蛋白质组的双向电泳体系,并从样品处理、电泳参数、染色方法等方面对该体系进行优化。样品处理采用超速离心法,Cleanup试剂盒处理,17cm pH5～8的双向电泳预制干胶条,蛋白上样量120μL,得出匹配率为76%,蛋白点有1412±8个。参照全盲法,对各种不同的猪肝脏样品进行准确性和重复性实验,样品匹配率均在70%以上,且图谱中点的相对迁移率都在实验室的控制标准之内,可以筛选出既具有统计学意义又具有量分析意义的蛋白点,也可用于区分不同的猪肝脏样品。该体系能满足不同猪肝脏样品的双向电泳分析。     

华根霉蛋白质组双向电泳体系的建立及优化

华根霉(Rhizopus chinensis CCTCC M201021)是从我国传统微生物载体——大曲中分离筛选得到的一株丝状真菌,具有重要的工业应用前景。为建立一种适于华根霉胞内蛋白质的双电泳体系以进行蛋白质组学研究,作者对华根霉胞内蛋白质的提取方法及双向电泳流程相关参数进行了考察和优化.确定采用液氮研磨与高速珠磨相结合的方法对华根霉进行细胞破碎,用TCA/丙酮对提取的蛋白质进行纯化,采用被动水化的上样方式上样,24 cm、pH 4～7的线性IPG胶条,上样量为1 200 μg蛋白质,考马斯亮蓝G-250股体染色法,最终得到了背景清晰、分辨率高的双向电泳图谱。     

非标记液质联用法测定市售鸡肉和猪肉的蛋白组

采用非标记液质联用法对市售鸡肉和猪肉样品进行蛋白组测定,比较两种肉在新鲜和熬煮后的小肽种类、量的相对变化。结果显示新鲜鸡肉的蛋白主要是大于100kD的蛋白,pI值范围为6～11;而鸡肉汤中蛋白主要是10～150kD,pI值范围为4～9;新鲜猪肉的蛋白主要是大于100kD的蛋白,pI值范围为6～11,与鸡肉相似;而猪肉汤中蛋白主要是10～100kD,pI值范围为3～9。显示了不同肉材质在相似加工中,其蛋白组的变化会有所不同,根据这些不同,提示由蛋白组测试提取出的指标可为肉质量的评价提供新的评价方式。     

桃果实总蛋白质双向电泳优化体系的建立

以"晖雨露"水蜜桃为试验材料,建立了一种适用于桃果实总蛋白质的双向电泳体系。最终试验确定采用酚抽法提取桃果实蛋白质,采用17 cm,pH 4~7的线性IPG胶条,800μg蛋白质上样,并用考马斯亮蓝G-250胶体考染,可以得到背景清晰、分辨率高的双向电泳图谱。对影响双向电泳图谱质量的因素进行了讨论分析。     

Proteome analysis of a temperature-inducible recombinant Escherichia coli for poly-beta-hydroxybutyrate production

A recombinant Escherichia coli strain harboring the lambdap(R)-p(L) promoter and heterologous poly-beta-hydroxybutyrate (PHB) biosynthesis genes was shown to accumulate PHB when the incubation temperature was changed from 34 degrees C to temperatures higher than 37 degrees C. In the present research, total gene expression patterns of the recombinant E. coli before and after induction were investigated by two-dimensional gel electrophoresis. Proteins encoded by serS, sucC, trpA, and alaS were found to be expressed before induction of phb genes at a culture temperature of 34 degrees C. On the other hand, proteins encoded by metG, rplI, and carA were found to be expressed after induction achieved by increasing the temperature to 40 degrees C. In the case of plasmid-free cells, all the selected genes have been shown to be expressed except metG, and ibpA and ibpB among the heat-shock proteins. The heat-shock proteins were found to be upregulated upon induction of phb genes, which may be due to the stress caused by the accumulation of PHB granules as well as by the temperature upshift. The changes in the expression of some of the metabolic pathway-related proteins before and after induction were interpreted in relation to the consumption of NADPH and acetyl-CoA for PHB synthesis.     

饮用水生产工艺的选择及进展

系统地介绍了饮用水生产的工艺进展，并就其关键工艺的选择，饮用水生产工艺的发展趋向进行了探讨。     

不同培养温度下腐败希瓦氏菌模式菌株DSM6067差异蛋白质组分析

腐败希瓦氏菌能够适应低温环境继续生长繁殖,是导致冷链流通过程中水产品腐败变质的优势腐败菌之一。运用Label-free非标记蛋白质组学技术对30、10?℃和4?℃培养温度下腐败希瓦氏菌模式菌株DSM6067所表达的蛋白质组差异进行分析,以期为探明腐败希瓦氏菌在低温胁迫下的分子调控机制提供理论依据。结果表明,腐败希瓦氏菌生长的环境温度越低,其所表达的蛋白质种类数越多。检测分析共得到2?165?条腐败希瓦氏菌蛋白质,其中30、10?℃和4?℃鉴定到的蛋白质条数分别为1?739、1?761和1?832。功能分析表明,这些差异蛋白涉及多种GO分类及KEGG通路,其中与能量代谢、催化活性、分子绑定功能相关的GO分类占较大比例。在这些差异蛋白中,与腐败希瓦氏菌模式菌株细胞膜脂肪酸合成相关的蛋白表达量相对较低,但4、10?℃与30?℃培养温度下相比差异较显著,且温度越低与细胞膜脂肪酸代谢相关的蛋白酶表达量越高。本研究为进一步探析腐败希瓦氏菌野生菌株适冷机制提供理论参考依据。     

Proteome analysis of the fungus Aspergillus carbonarius under ochratoxin A producing conditions

Aspergillus carbonarius is an important ochratoxin A producing fungus that is responsible for mycotoxin contamination of grapes and wine. In this study, the proteomes of highly (W04-40) and weakly (W04-46) OTA-producing A. carbonarius strains were compared to identify proteins that may be involved in OTA biosynthesis. Protein samples were extracted from two biological replicates and subjected to two dimensional gel electrophoresis analysis and mass spectrometry. Expression profile comparison (PDQuest software), revealed 21 differential spots that were statistically significant and showed a two-fold change in expression, or greater. Among these, nine protein spots were identified by MALDI-MS/MS and MASCOT database and twelve remain unidentified. Of the identified proteins, seven showed a higher expression in strain W04-40 (high OTA producer) and two in strain W04-46 (low OTA producer). Some of the identified amino acid sequences shared homology with proteins involved in regulation, amino acid metabolism, oxidative stress and sporulation. It is worth noting the presence of a protein with 126.5 fold higher abundance in strain W04-40 showing homology with protein CipC, a protein with unknown function related with pathogenesis and mycotoxin production by some authors. Variations in protein expression were also further investigated at the mRNA level by real-time PCR analysis. The mRNA expression levels from three identified proteins including CipC showed correlation with protein expression levels. This study represents the first proteomic analysis for a comparison of two A. carbonarius strains with different OTA production and will contribute to a better understanding of the molecular events involved in OTA biosynthesis.     

纳豆激酶的研究进展

纳豆激酶是一种由纳豆芽孢杆菌产生的具有强溶纤作用的碱性丝氨酸蛋白酶,具有安全性好、半衰期长、口服有效等优点。本文就纳豆激酶的基因结构、生化特性、生物学功能及酶活性测定等方面进行了综述。     

几种生物防腐剂的特性与研究进展

生物防腐剂是一类安全、高效、无毒的天然食品防腐剂.本文从特性、机理及应用等方面详细阐述了几种生物防腐剂,并对其做出了展望.     

外源CO对采后枣果实链格孢菌浸染过程中蛋白组的影响

为了阐明外源CO处理对链格孢菌(Aternaria alternata)浸染过程中的枣果实蛋白组影响,采用同位素标记蛋白组分析技术测定链格孢菌入侵枣果实过程中枣果实的蛋白质表达水平,鉴定差异蛋白,并对差异蛋白进行基因本体(gene oncology,GO)及基因通路(kyoto encyclopedia of gene...     

几种生物防腐剂的特性与研究进展

生物防腐剂是一类安全、高效、无毒的天然食品防腐剂.本文从特性、机理及应用等方面详细阐述了几种生物防腐剂,并对其做出了展望.     

Proteome changes during pork meat ageing following use of two different pre-slaughter handling procedures

The influence of postmortem storage time and pre-slaughter conditions (transport the day before slaughter or immediately before slaughter) on proteome changes of pork meat was investigated over a 72 h ageing period. Intensities of 37 spots varied significantly (p<0.05) with ageing time. Changes indicated proteolysis of troponin T, actin, -crystallin, myokinase, creatine kinase and mitochondrial ATPase, but also of proteins constitutive of the Z-lines, namely cypher proteins and myozenin. Other modifications were the intensity increase of a full-length protein of the sarcoplasmic reticulum, which may be linked to its increased extractibility after membrane disruption, and a gradual shift in pHi towards alkaline values of some forms of myosin light chains (MLC) 2 and 3. The pre-slaughter conditions affected significantly (p<0.05) 8 spots. Mitochondrial ATPase was over-expressed in the group transported immediately before slaughter, also characterised by a faster pH fall, and the shift in pHi of MLC 2 was more pronounced. The pre-slaughter conditions had no significant effect on the above proteolytic events.