重组羧肽酶B及其包涵体溶解性的研究

目的构建重组羧肽酶B(rCPB)的表达质粒和表达菌株,表达羧肽酶B(CPB),并对表达CPB包涵体的溶解性进行研究。方法构建CPB重组质粒pET-21a-CPB,将其导入表达菌株BL21(DE3)中,分别在25℃和37℃进行诱导表达;所得包涵体分别用不同浓度尿素添加不同还原剂溶解,以溶液中的蛋白质浓度判定其溶解性,利用非还原SDS-PAGE分析其溶解性提高的原因。结果诱导后生长温度不同对rCPB包涵体的纯度及后期处理均有影响;rCPB包涵体在10mol/L尿素溶液中的溶解度比在8mol/L尿素溶液中提高2￣3倍;添加0.75%β-巯基乙醇能显著改善rCPB包涵体的溶解效果。经非还原SDS-PAGE分析,添加β-巯基乙醇后,溶解rCPB聚体的含量减少。结论成功地表达了rCPB,并通过实验提高了rCPB包涵体的溶解度。     

重组阿片肽包涵体的溶解工艺

通过SDS PAGE凝胶电泳确定了目的重组阿片肽存在于包涵体中,系统研究了影响大肠杆菌表达的重组阿片肽包涵体溶解的因素,确定了重组阿片肽包涵体制备溶解的最佳工艺条件,即菌体超声破碎5min,间隔时间6s,破碎效果最好;包涵体洗涤温度为4℃,尿素洗涤液的浓度为2mol/L,洗涤时间7～8h,8mol/L的尿素溶液对包涵体进行溶解,蛋白质质量浓度约为600μg/mL,其效果最佳.     

诱导时机对羧肽酶原B包涵体质量和复性率影响的研究

目的研究诱导时机对羧肽酶原B包涵体质量和复性率的影响，探讨包涵体质量评价体系。方法在不同时间诱导产生重组羧肽酶原B包涵体，研究其变性和复性、对蛋白酶K的稳定性及在一定浓度盐酸胍溶液中的化学变性，分析诱导时间与包涵体复性率的相关性。结果诱导时机不同，所得重组羧肽酶原B包涵体复性率及对蛋白酶K的稳定性不同，在一定浓度盐酸胍溶液中的溶解性也不同。复性率越高的包涵体越不易被蛋白酶K酶解，对化学变性剂的稳定性也高。结论诱导时机影响重组羧肽酶原B包涵体的质量及复性率。     

诱导时机对羧肽酶原B包涵体质量和复性率影响的研究

目的研究诱导时机对羧肽酶原B包涵体质量和复性率的影响,探讨包涵体质量评价体系。方法在不同时间诱导产生重组羧肽酶原B包涵体,研究其变性和复性、对蛋白酶K的稳定性及在一定浓度盐酸胍溶液中的化学变性,分析诱导时间与包涵体复性率的相关性。结果诱导时机不同,所得重组羧肽酶原B包涵体复性率及对蛋白酶K的稳定性不同,在一定浓度盐酸胍溶液中的溶解性也不同。复性率越高的包涵体越不易被蛋白酶K酶解,对化学变性剂的稳定性也高。结论诱导时机影响重组羧肽酶原B包涵体的质量及复性率。     

诱导时机对羧肽酶原B包涵体质量和复性率影响的研究

目的 研究诱导时机对羧肽酶原B包涵体质量和复性率的影响,探讨包涵体质量评价体系.方法 在不同时间诱导产生重组羧肽酶原B包涵体,研究其变性和复性、对蛋白酶K的稳定性及在一定浓度盐酸胍溶液中的化学变性,分析诱导时间与包涵体复性率的相关性.结果 诱导时机不同,所得重组羧肽酶原B包涵体复性率及对蛋白酶K的稳定性不同,在一定浓度盐酸胍溶液中的溶解性也不同.复性率越高的包涵体越不易被蛋白酶K酶解,对化学变性剂的稳定性也高.结论 诱导时机影响重组羧肽酶原B包涵体的质量及复性率.     

植物乳杆菌重组亚油酸异构酶的表达与纯化

对转化有植物乳杆菌亚油酸异构酶基因的大肠杆菌进行诱导表达,表达出大量重组亚油酸异构酶蛋白,但大部分目的蛋白形成了包涵体,用8mol/L尿素将包涵体蛋白溶解变性后,用金属螯合层析对重组蛋白进行了纯化.当咪唑溶液浓度为50mmol/L时,重组蛋白开始被洗脱下来;当咪唑溶液浓度为200mmol/L时,蛋白洗脱效果最佳;浓度为400mmol/L时,重组蛋白完全被洗脱下来.纯化后重组蛋白的收率为84.71%.     

Streptoverticillium mobaraense谷氨酰胺转胺酶的表达、纯化和复性

以基因组DNA为模板 ,利用PCR技术从茂原轮链丝菌 (Streptoverticilliummobaraense)中扩增得到产成熟谷氨酰胺转胺酶MTG的结构基因mtg ,克隆于表达载体pQE 3 0T ,构建成lac启动子控制下的His6融合表达质粒pMTG ,将此重组质粒转化到受体菌E .coliM1 5中。对质粒稳定性的研究表明 ,E .coliM1 5在无选择压的情况下 ,于 3 7℃连续转接 5次 ,质粒丢失率仅有 2 4%,说明质粒基本稳定。重组菌经IPTG诱导 ,表达的重组谷氨酰胺转胺酶占菌体总蛋白的 1 8%,经SDS PAGE分析 ,表达的蛋白质的分子质量为 3 8ku ,与预期分子质量相符。表达产物主要以包涵体的形式存在。细胞经超声破碎 ,离心取包涵体溶于 8mol/L的尿素中 ,然后通过Ni NTA亲和柱分离纯化和稀释法复性。目的蛋白的纯度可达 95 %以上。比酶活为 1 0 3U/mg。     

草鱼潜在过敏原α-烯醇化酶原核表达条件的优化

目的 通过对草鱼α-烯醇化酶(α-Enolase)原核表达的条件进行优化,分别从表达的上清液与包涵体中纯化获得重组α-Enolase。方法 本研究基于前期克隆的草鱼α-Enolase基因序列设计表达引物,将草鱼α-Enolase基因克隆至pET-30a质粒中,得到pET-30a-α-Enolase重组质粒。然后,将重组质粒转入大肠杆菌Origami 2(DE3)感受态细胞中,并用异丙基-β-D-硫代半乳糖苷(Isopropyl-β-D-Thiogalactopyranoside, IPTG)进行体外诱导表达。采用SDS-PAGE电泳方法分析重组蛋白的可溶性,并对表达条件进行优化.。利用亲合层析法纯化目的蛋白。结果 在20℃诱导15 h得到的重组蛋白以二种形式存在：一部分为可溶性蛋白存在于上清液中,一部分以包涵体形式存在于沉淀中;在37℃诱导4 h得到的重组蛋白则只有包涵体形式。包涵体蛋白表达量最大的优化条件为：诱导温度为37℃,IPTG终浓度1.0 mmol/L,诱导时间4 h时。采用亲合层析法分别纯化上清液(20℃诱导15 h)和包涵体中(37℃诱导4 h)的重组蛋白,得率分别为3.5 mg /100 mL和4.9 mg/100 mL菌液。结论 利用原核表达从上清液与包涵体中均可纯化得到重组α-Enolase。但这二种α-Enolase在生物活性与免疫原性是否存在差异性,以及通过重组α-Enolase是否可以代替天然蛋白用于鱼类过敏原的研究,有待进一步研究。     

重组干扰素-tau在大肠杆菌中的高效表达

目的研究重组干扰素-tau的纯化和复性。方法将构建好的重组质粒pBV220转化E.coli BL21,发酵培养后迅速升温至42℃诱导干扰素-tau以包涵体的形式高效表达。裂菌后利用8mol/L尿素溶解目的蛋白质,经凝胶过滤色谱层析纯化后,再透析复性。结果干扰素-tau的表达量占菌体总蛋白质量的20%以上,纯化后纯度可达9 6%以上,活性得到有效恢复。结论建立了干扰素-tau纯化及复性的方法,获得了高表达、高纯度、有活性的干扰素-tau。     

类芽孢杆菌属β-葡萄糖苷酶在大肠杆菌中可溶性重组表达的优化

目的:β-葡萄糖苷酶在食品工业领域具有广泛的应用价值,但重组表达时容易形成无活性的包涵体。通过传统诱导条件优化无法完全解决包涵体积累问题,需探索新的策略。方法:从类芽孢杆菌属(Paenibacillus sp)基因组中克隆获得了β-葡萄糖苷酶基因bgl,构建到大肠杆菌表达载体p ET28a获得重组质粒p ET-bgl,转化大肠杆菌宿主细胞BL21(DE3)获得重组菌BL-ETbgl,并进行诱导条件优化。进一步通过复制起始位点替换,构建了新型大肠杆菌表达载体p ACYT-bgl,转化大肠杆菌宿主细胞BL21(DE3)后得到改进型重组菌BL-ATbgl。结果:BL-ETbgl经诱导表达后,所表达重组蛋白具有β-葡萄糖苷酶活性。经诱导条件优化后,仍有40%蛋白以包涵体存在。而BL-ATbgl经诱导表达后,可溶性的重组β-葡萄糖苷酶约占80%。自诱导培养基中β-葡萄糖苷酶产量可达2.31×106U/L。结论:通过降低质粒拷贝数、优化培养条件等手段,可以大幅度提高类芽孢杆菌β-葡萄糖苷酶在大肠杆菌中的可溶性表达水平。     

淡水鱼蛋白质冷冻变性的研究现状及前景

主要介绍了淡水鱼蛋白质冷冻变性的国内外研究现状,探讨了防止淡水鱼蛋白质冷冻变性的常用技术,并综述了淡水鱼蛋白质抗冷冻变性的研究方法和发展前景.     

Physicochemical properties of lactoferrin stabilized oil-in-water emulsions: Effects of pH,salt and heating

Lactoferrin is a globular protein from bovine milk with an unusually high isoelectric point (pI > 8), which may lead to novel functional properties in foods and other products because it is cationic across a wide pH range. In this study, we investigated the influence of pH (2–9), NaCl addition (0–200 mM), CaCl₂ addition (0–200 mM), and thermal processing (30–90 °C, 20 min) on the stability of lactoferrin (LF) stabilized oil-in-water emulsions. At ambient temperature, the emulsions were stable to droplet aggregation at low pH (pH ≤ 6), but exhibited some aggregation at pH ≈ pI (pH 7–9). The thermal stability of the emulsions depended on pH, holding temperature, and thermal history. When LF-coated droplets were heated in distilled water, and then their pH was adjusted in the range 2–9, they were highly unstable to aggregation at pH 7 and 8. On the other hand, when the pH was altered in the range 2–9 first, and then they were heated, the LF-coated droplets were highly unstable to aggregation at pH ≥ 5 when heated above 50 °C. The stability of the emulsions to salt addition depended on pH and salt type, which was attributed to counter-ion binding and electrostatic screening effects. For NaCl, emulsions were stable from 0 to 200 mM at pH 3 and 9, but aggregated at ≥100 mM at pH 6. For CaCl₂, emulsions were stable from 0 to 200 mM at pH 3, but aggregated with ≥150 mM CaCl₂ at pH 6 and 9. These results have important implications for the formulation and production of emulsion-based products using lactoferrin as an emulsifier.     

冷冻大豆分离蛋白凝胶的功能性分析

为明确冷冻后SPI凝胶的功能性变化,分析了SPI凝胶在冷冻前后的质构特性、可溶性蛋白含量及成分、扫描电镜的图像等指标。研究发现:SPI的凝胶在冷冻后硬度增加,弹力、持水率、可溶蛋白含量降低;SDS-PAGE电泳结果表明冷冻后凝胶可溶蛋白部分γ亚基消失、A亚基增多;扫描电镜分析结果显示,冷冻后水分聚集凝结成较大冰晶,凝胶由致密的结构变成多孔的网状结构。     

不同变性方式对Alcalase AF2.4L酶解玉米蛋白水解度的影响

玉米蛋白水溶性差一直是阻碍水解度提高的瓶颈 ,采用不同方式对玉米蛋白的变性进行了研究 ,以期摸索出提高酶解玉米蛋白水解度的途径。实验表明 ,采用“变性剂A”对玉米蛋白进行预处理后 ,能显著地提高玉米蛋白在时间和水解度上的酶解水平 ,使得AlcalaseAF -2 .4L酶解玉米蛋白的水解度经过 1h酶解可达 30 %以上。     

β-乳球蛋白的热变性聚集概述

介绍了天然β－乳球蛋白（β-Lg)的结构，叙述了β-Lg的热变性聚集机理，包括热变性聚集机理，热变性的结构变化及影响β-Lg热变性聚集的因素，最后介绍了β-Lg热变性聚集在乳品加工业中的应用。     

Myosins from red and white bovine muscles: Differences measured by turbidimetric,calorimetric and rheological techniques

The aim of this study was to elucidate the functional performance of the most abundant protein component in meat, ie myosin, which is recognised as important for binding in meat products. As several genetic variants of skeletal myosin exist, myosins from two bovine muscles of different fibre type composition, M masseter and M cutaneus trunci were compared with respect to filament forming properties and denaturation characteristics. The principal methods used were turbidimetric measurements, which were used to monitor filament formation, calorimetry and rheology. The myosin systems were examined at two different salt levels (0.2 and 0.6 M NaCl) and at pH 5.5–7.0. The method of preparing myosin suspensions/solutions was also examined. Differences in the filament-forming process for the two myosins were detected. Measurements of turbidity revealed that at conditions of low pH and low ionic strength white myosin had a higher ultimate turbidity compared with red myosin. Early in the transition from low to high turbidity, red myosin had a higher turbidity than white myosin corresponding to reduced solubility. The turbidity increased with time of storing the myosin suspensions/solutions. This change was attributed to formation of filaments and further association of filaments. White myosin had a smaller apparent enthalpy of denaturation than red myosin. The calorimetric measurements recorded in 0.2 M NaCl suggested that the head and the rod of white myosin were less stable than the corresponding parts of red myosin. However, exceptions to this rule were found at pH 6.0. In 0.6 M NaCl the identification of the transitions for red myosin was more difficult. The method of preparing myosin suspensions affected calorimetric and rheological measurements. In 0.6 M NaCl and pH 6.0 calorimetric thermograms of both myosins were affected by the preparation method. At pH 5.5 this change was interpreted as caused by denaturation promoted by the dilution/rapid titration technique compared with dialysing the systems to pH 5.5 from pH 7. Differences in the filaments formed might, however, also contribute to the variations seen in the calorimetric ther-mograms. The gelling properties of white myosin were most sensitive to the preparation method used. Systems prepared by dialysis gave stronger heat-induced gels than those prepared by ‘dilution’. White myosin always produced stronger gels than red myosin independent of the preparation technique. The rheological properties (at 80°C) of red myosin were less affected by the preparation method than were those of white myosin. At lower temperatures, however, there was more variation in the shapes of the rheological thermograms (? versus temperature) for red myosin than in the corresponding thermograms of white myosin.     

Effect of different denaturating methods on lipid-protein complex formation

To investigate the effect of denaturation of protein on lipid-protein complex formation in soybeans, different denaturing agents including urea, ethanol, autoclaving, and extreme pH treatment were used. Protein was denatured either in the presence of the oxidized crude soybean oil (MO_x OD), or before it was added to the crude oxidized oil (DMO_xO). Nitrogen solubility index and in vitro digestibility of the proteins were used to investigate the formation of the lipid-protein complexes. The meal resulting after the denaturation treatment was compared to untreated soybean meal. All treatments resulted in very highly significant differences (P>0.001) in the studied parameters when compared to untreated meal. Results proved that denaturation of protein in the presence of oxidized oil caused more lipid-protein complex formation than denaturation of the protein prior to the addition to the oxidized oil. Among the denaturing agents examined, urea caused the highest lipid-protein complex as indicated by the in vitro digestibility and nitrogen solubility of the protein.     

The physico-chemical properties and protein denaturation potential of surfactant mixtures

Protein denaturation was investigated to establish an in vitro evaluation method of surfactants in connection with their in vivo irritation potency to human skin. Eventually a new method with simplicity and high reproducibility was established by using quantitative analysis with gel-permeation chromatography (GPC). The protein denaturing potency of the commercially available surfactants was measured by using the developed method. Synergistic reduction in protein denaturation was observed in the mixed systems of anionic and amphoteric surfactants. The synergistic reduction was explained in terms of the physico-chemical properties of the mixed surfactants. A possible mechanism is the remarkable lowering of the total monomer concentration by the formation of hydrophobic complexes between the anionic and amphoteric surfactants.
Relation entre les propriétés physico-chimiques des mélanges de surfactifs et leur potentiel de dénaturation des protéines     

鱼肉蛋白质冷冻变性及抗冻剂的研究进展

本文简要分析了鱼肉蛋白冷冻变性的原因,主要综述了国内外鱼肉蛋白质冷冻变性的研究现状,并对糖类、盐类、乳蛋白、酶解产物等几种抗冻剂的应用作了详细介绍。展望了进一步研究鱼肉蛋白冷冻变性的新方向。