决明子营养价值分析及蛋白提取工艺研究

以大决明子为原材料,基于氨基酸比值系数法,对决明子的营养价值进行了分析;并以碱性缓冲液为提取剂,进行决明子蛋白提取工艺研究,探讨了缓冲液浓度及pH值、料液比、浸泡时间对蛋白提取率的影响,最后用正交试验确定大决明子蛋白的最佳提取工艺。结果表明决明子的营养价值高于大豆和紫花苜蓿,与南瓜接近略低于鸡蛋;提取工艺条件最佳为50 mmol/L、pH值=8.0的KH2PO4-NaOH缓冲液,料液比1 g：50 mL,浸泡提取时间12h。此条件下决明子蛋白的提取率为93.3%。     

黑米蛋白提取工艺的优化

以黑米为原料,研究黑米蛋白提取工艺的优化。采用"碱提酸沉"法,研究了提取液pH值、温度、搅拌速度、料液比、提取时间、及粒径大小等对黑米蛋白提取率的影响。在单因素试验的基础上,以L9(34)正交实验法优化黑米蛋白提取工艺。结果表明:各因素对黑米蛋白提取率的影响顺序依次为:pH值粒径料液比转速。本实验中,黑米蛋白提取的最佳工艺条件为:提取液p H 12.5、温度28℃、搅拌转速800 rpm、料液比1∶10 g/m L、提取时间为1 h、酸沉淀时间为1 h、粒径大小为能通过100目筛。优化后黑米水溶性蛋白提取率提高到96.77%,纯度为97.75%。     

大肠杆菌周质蛋白提取工艺的改进

介绍一种简捷高效的大肠杆菌周质蛋白提取工艺,即用含一定浓度溶菌酶的细胞裂解缓冲液一步提取周质蛋白,与传统的高渗和低渗两步提取法相比,不仅操作简单快捷,并且显著的提高了大肠杆菌周质蛋白的提取率.     

鹰嘴豆铁蛋白提取工艺优化研究

本研究以鹰嘴豆为原料,研究了缓冲液种类、缓冲液pH值、料液比、盐析盐种类、盐浓度等因素对鹰嘴豆铁蛋白提取率、总蛋白提取率及干重的影响。在单因素实验的基础上,以L9(34)正交实验方法优化鹰嘴豆铁蛋白提取工艺。结果表明:各因素对鹰嘴豆铁蛋白提取率的影响顺序依次为:料液比盐浓度温度pH。最优提取工艺为:料液比1∶4,浓度为70 mmol/L MgCl2盐析,温度50℃,KH2PO4-NaOH缓冲液pH 7.5,最优提取率为0.002643%。     

黄芪多糖的闪式提取技术研究

目的:建立黄芪多糖的闪式提取技术.方法:对黄芪多糖闪式提取技术中提取温度、提取时间、提取电机电压、pH值、料液比、乙醇浓度进行了单因素实验,以多糖得率为指标确定提取工艺,并与传统碱水提取法进行了比较,最后用该工艺对8种不同产地或级别的黄芪原料进行多糖提取和含量测定.结果:初步确定提取工艺为温度65℃、提取时间2 min、pH值为10、提取电机电压200V、料液比1:10、乙醇浓度为5%(V/V),提取所得黄芪多糖总量达到123.46mg/g,比碱提法多糖得率提高了近30%,该法检测不同黄芪原料多糖含量在87.44～187.74mg/g.结论:闪式提取技术能大大提高黄芪多糖提取率,不同产地或级别的黄芪原料在黄芪多糖含量上有较大差异.     

响应面法优化苦荞麸皮粉蛋白质提取工艺

目的：分析苦荞麸皮粉中蛋白含量及其蛋白组成。方法：采用碱法提取-等电点沉淀分离蛋白。在单因素试验基础上,选取料液比（w/v）、pH、时间等3个影响因素,采用响应面法 （Box-Behnken 中心组合）优化苦荞麸皮粉蛋白提取工艺。结果：料液比、pH、时间对麸皮蛋白提率有显著影响,影响顺序为料液比> pH>时间。响应面优化得到最佳提取条件为pH 10.5、料液比1∶35、时间3 h 40 min,此条件下苦荞麸皮蛋白提取率为（97.31±4.64）%。结论：本研究为有效开发利用苦荞麸皮粉的蛋白提供科学依据。     

响应面法优化低温豆粕大豆分离蛋白提取工艺

采用响应面分析法(RSM)对低温豆粕大豆分离蛋白(soybean protein isolated,SPI)的碱提工艺进行优化。在单因素实验的基础上,选取了影响SPI提取率的4个关键因素(pH、温度、时间和液料比)进行四因素五水平的中心组合旋转实验设计(CCRD)。通过RSM建立了响应值(SPI提取率)与各影响因素之间的回归方程,并获得了SPI的最优提取条件:pH 8.5,提取温度55℃,提取时间42.8 min,料水比1∶9.7(g/mL),此条件下SPI提取率预测值为37.12%,与实验值(36.69%)的误差为1.16%。将一次碱提后残渣进行二次碱提,二次提取率为10.16%。2次碱提上清液等电点沉淀的蛋白沉淀率分别为84.03%和85.84%。     

响应曲面法优化小牛胸腺肽提取工艺

目的:为了模拟并且优化小牛胸腺肽提取工艺,获得良好的活性肽提取效果.方法:在单因素试验的基础上,采用响应曲面法对影响胸腺肽提取率的四个因素即料液比、匀浆时间、匀浆液pH值和冻融次数进行优化.利用Design Expert7.0软件对胸腺肽提取率的二次多项数学模型分析.结果:在液料比1.94:1、匀浆时间3.38min、pH值3.23、冻融次数7次时,胸腺肽提取率达最大值,最佳提取率预测值为8.874mg/g,实测值为8.349mg/g.结论:采用响应曲面法能有效地优化小牛胸腺肽提取工艺,获得理想的提取效果.     

响应面法优化细果角茴香中总黄酮的超声提取条件

采用响应面法优化超声波辅助提取细果角茴香中总黄酮的工艺研究。在单因素试验的基础上,选择超声时间、乙醇浓度、料液比为自变量,以总黄酮的提取率为响应值,进行Box-Behnken中心组合实验设计,采用响应面法(RSM)评估了这些因素对总黄酮提取率的影响。结果表明:超声波法辅助提取细果角茴香中总黄酮的最佳工艺条件为液料比33 mL/g,乙醇浓度为58%,提取时间为34.5 min。在此条件下,总黄酮的提取率达1.525%。     

响应面法优化酶法提取枸杞多糖的研究

选用响应面法优化及正交实验法进行淀粉酶提取枸杞多糖实验设计及分析。通过单因素实验后,正交实验确定淀粉酶酶解提取枸杞多糖的最佳条件为:pH=5.0,温度50℃,时间80 min,加酶量为0.5%,枸杞多糖提取率12.1%;响应面分析确定淀粉酶酶解提取枸杞多糖的最佳条件为酶解温度49.56℃、酶解时间140 min、酶浓度0.3%,枸杞多糖提取率为13.25%。酶法提取枸杞多糖比传统热水浸提提高了枸杞多糖的提取率,反应条件温和,而且通过响应面法进行实验设计和优化比正交实验法能得到更高的枸杞多糖提取率。     

pH of the cytoplasm and periplasm of Escherichia coli: rapid measurement by green fluorescent protein fluorimetry

Cytoplasmic pH and periplasmic pH of Escherichia coli cells in suspension were observed with 4-s time resolution using fluorimetry of TorA-green fluorescent protein mutant 3* (TorA-GFPmut3*) and TetR-yellow fluorescent protein. Fluorescence intensity was correlated with pH using cell suspensions containing 20 mM benzoate, which equalizes the cytoplasmic pH with the external pH. When the external pH was lowered from pH 7.5 to 5.5, the cytoplasmic pH fell within 10 to 20 s to pH 5.6 to 6.5. Rapid recovery occurred until about 30 s after HCl addition and was followed by slower recovery over the next 5 min. As a control, KCl addition had no effect on fluorescence. In the presence of 5 to 10 mM acetate or benzoate, recovery from external acidification was diminished. Addition of benzoate at pH 7.0 resulted in cytoplasmic acidification with only slow recovery. Periplasmic pH was observed using TorA-GFPmut3* exported to the periplasm through the Tat system. The periplasmic location of the fusion protein was confirmed by the observation that osmotic shock greatly decreased the periplasmic fluorescence signal by loss of the protein but had no effect on the fluorescence of the cytoplasmic protein. Based on GFPmut3* fluorescence, the pH of the periplasm equaled the external pH under all conditions tested, including rapid acid shift. Benzoate addition had no effect on periplasmic pH. The cytoplasmic pH of E. coli was measured with 4-s time resolution using a method that can be applied to any strain construct, and the periplasmic pH was measured directly for the first time.     

Enhanced solubilization of immunoreactive proteins from Brugia malayi adult parasites using cetyltrimethylammonium bromide

To determine an optimal method for extracting immunoreactive proteins from filarial parasites, we have subjected Brugia malayi adult worms to a variety of solubilization regimens and compared the results. The parasites were extracted in one of seven detergents (including anionic, cationic, nonionic, and zwitterionic compounds) under varying conditions of pH, detergent concentrations, and incubation time. The individual antigen preparations were then compared both by one-dimensional SDS-PAGE and by immunoblotting analysis using a serum pool from individuals resident in an area endemic for lymphatic filariasis. The cationic detergent cetyltrimethylammonium bromide (CTAB) at 1.0% concentration, pH 7.2, consistently solubilized more proteins immunoreactive with the sera tested. Additionally, CTAB never failed to solubilize immunoreactive proteins solubilized by those other detergents or combinations of detergents studied.     

pH-dependent catabolic protein expression during anaerobic growth of Escherichia coli K-12

During aerobic growth of Escherichia coli, expression of catabolic enzymes and envelope and periplasmic proteins is regulated by pH. Additional modes of pH regulation were revealed under anaerobiosis. E. coli K-12 strain W3110 was cultured anaerobically in broth medium buffered at pH 5.5 or 8.5 for protein identification on proteomic two-dimensional gels. A total of 32 proteins from anaerobic cultures show pH-dependent expression, and only four of these proteins (DsbA, TnaA, GatY, and HdeA) showed pH regulation in aerated cultures. The levels of 19 proteins were elevated at the high pH; these proteins included metabolic enzymes (DhaKLM, GapA, TnaA, HisC, and HisD), periplasmic proteins (ProX, OppA, DegQ, MalB, and MglB), and stress proteins (DsbA, Tig, and UspA). High-pH induction of the glycolytic enzymes DhaKLM and GapA suggested that there was increased fermentation to acids, which helped neutralize alkalinity. Reporter lac fusion constructs showed base induction of sdaA encoding serine deaminase under anaerobiosis; in addition, the glutamate decarboxylase genes gadA and gadB were induced at the high pH anaerobically but not with aeration. This result is consistent with the hypothesis that there is a connection between the gad system and GabT metabolism of 4-aminobutanoate. On the other hand, 13 other proteins were induced by acid; these proteins included metabolic enzymes (GatY and AckA), periplasmic proteins (TolC, HdeA, and OmpA), and redox enzymes (GuaB, HmpA, and Lpd). The acid induction of NikA (nickel transporter) is of interest because E. coli requires nickel for anaerobic fermentation. The position of the NikA spot coincided with the position of a small unidentified spot whose induction in aerobic cultures was reported previously; thus, NikA appeared to be induced slightly by acid during aeration but showed stronger induction under anaerobic conditions. Overall, anaerobic growth revealed several more pH-regulated proteins; in particular, anaerobiosis enabled induction of several additional catabolic enzymes and sugar transporters at the high pH, at which production of fermentation acids may be advantageous for the cell.     

Proteomic characterization of acid stress response in Synechocystis sp. PCC 6803

A comparative proteomic analysis using 2-DE coupled with MALDI-MS and LC-MS/MS was performed in Synechocystis sp. PCC 6803 to identify protein candidates involved in acid stress response in cyanobacteria. Comparison of soluble proteins from the cytoplasmic fraction of cells grown on media set at pH 7.5 and 5.5 using 2-DE identified four proteins, which showed significant changes in the abundance. Surprisingly, several general stress proteins, either the heat shock family proteins or chaperonins, did not show perceptible fold changes in response to acidity. Compared to the cytoplasmic proteome, the periplasmic proteome showed remarkable changes as a function of external pH. Protein expression profiling at different external pH, i.e., 9.0, 7.5, 6.0 and 5.5, allowed classifying the periplasmic proteins depending on their preferential expression patterns towards acidity or alkalinity. Among the acid- and base-induced proteins, oxalate decarboxylase and carbonic anhydrase were already known for their role in pH homeostasis. Several unknown proteins from the periplasm, that showed significant changes in response to pH, provide ideal targets for further studies in understanding pH stress response in cyanobacteria. This study also identified 14 novel proteins, hitherto unknown from the periplasmic space of Synechocystis.     

Solubilization of protein aggregates by the acid stress chaperones HdeA and HdeB

The acid stress chaperones HdeA and HdeB of Escherichia coli prevent the aggregation of periplasmic proteins at acidic pH. We show in this report that they also form mixed aggregates with proteins that have failed to be solubilized at acidic pH and allow their subsequent solubilization at neutral pH. HdeA, HdeB, and HdeA and HdeB together display an increasing efficiency for the solubilization of protein aggregates at pH 3. They are less efficient for the solubilization of aggregates at pH 2, whereas HdeB is the most efficient. Increasing amounts of periplasmic proteins draw increasing amounts of chaperone into pellets, suggesting that chaperones co-aggregate with their substrate proteins. We observed a decrease in the size of protein aggregates in the presence of HdeA and HdeB, from very high molecular mass aggregates to 100-5000-kDa species. Moreover, a marked decrease in the exposed hydrophobicity of aggregated proteins in the presence of HdeA and HdeB was revealed by 1,1'-bis(4-anilino)naphtalene-5,5'-disulfonic acid binding experiments. In vivo, during the recovery at neutral pH of acid stressed bacterial cells, HdeA and HdeB allow the solubilization and renaturation of protein aggregates, including those formed by the maltose receptor MalE, the oligopeptide receptor OppA, and the histidine receptor HisJ. Thus, HdeA and HdeB not only help to maintain proteins in a soluble state during acid treatment, as previously reported, but also assist, both in vitro and in vivo, in the solubilization at neutral pH of mixed protein-chaperone aggregates formed at acidic pH, by decreasing the size of protein aggregates and the exposed hydrophobicity of aggregated proteins.     

Lateral diffusion of proteins in the periplasm of Escherichia coli.

We have introduced biologically active, fluorescently labeled maltose-binding protein into the periplasmic space of Escherichia coli and measured its lateral diffusion coefficient by the fluorescence photobleaching recovery method. Diffusion of this protein in the periplasm was found to be surprisingly low (lateral diffusion coefficient, 0.9 X 10(-10) cm2 s-1), about 1,000-fold lower than would be expected for diffusion in aqueous medium and almost 100-fold lower than for an equivalent-size protein in the cytoplasm. Galactose-binding protein, myoglobin, and cytochrome c were also introduced into the periplasm and had diffusion coefficients identical to that determined for the maltose-binding protein. For all proteins nearly 100% recovery of fluorescence was obtained after photobleaching, indicating that the periplasm is a single contiguous compartment surrounding the cell. These data have considerable implications for periplasmic structure and for the role of periplasmic proteins in transport and chemotaxis.     

A genetically incorporated crosslinker reveals chaperone cooperation in acid resistance

Acid chaperones are essential factors in preserving the protein homeostasis for enteric pathogens to survive in the extremely acidic mammalian stomach (pH 1-3). The client proteins of these chaperones remain largely unknown, primarily because of the exceeding difficulty of determining protein-protein interactions under low-pH conditions. We developed a genetically encoded, highly efficient protein photocrosslinking probe, which enabled us to profile the in vivo substrates of a major acid-protection chaperone, HdeA, in Escherichia coli periplasm. Among the identified HdeA client proteins, the periplasmic chaperones DegP and SurA were initially found to be protected by HdeA at a low pH, but they subsequently facilitated the HdeA-mediated acid recovery of other client proteins. This unique, ATP-independent chaperone cooperation in the ATP-deprived E. coli periplasm may support the acid resistance of enteric bacteria. The crosslinker would be valuable in unveiling the physiological interaction partners of any given protein and thus their functions under normal and stress conditions.     

Periplasmic proteins of Rhizobium: Variation with growth conditions and use in strain identification

Abstract The pattern of periplasmic proteins released by lysozyme-EDTA treatment of fast-growing rhizobia was influenced by the growth phase, the pH of the medium and the carbon source. The pattern for a particular strain of Rhizobium grown under defined conditions was characteristic of that strain, and we suggest that it can be used as an adjunct to existing methods of strain identification.