文献类型：学位论文

中文题名：pH值调节诱导罗非鱼肌球蛋白结构和性质的变化

英文题名：PH-shifting Induced Changes in the Structure and Properties of Myosin from Tilapia

作者：王瑛[1];

机构：[1]广东海洋大学;

导师：洪鹏志;广东海洋大学

授予学位：硕士

语种：中文

中文关键词：罗非鱼;肌球蛋白;pH调节法;去折叠;重折叠;构象

外文关键词：tilapia;myosin;pH-shifting;unfolding;refolding;conformation

中文摘要：pH值调节法（pH-shifting，酸//碱溶解-等电点沉淀法）是一种新型的动物蛋白质提取方法，在低温偏离蛋白质等电点的极端酸性（pH≤3.0）或极端碱性（pH≥11.0）条件下使蛋白充分溶解，高速离心除去脂肪和不溶性杂质制备可溶性蛋白溶液，然后在等电点条件下使蛋白沉淀并回收，制备鱼分离蛋白（Fish Protein Isolates，FPI）。由于酸//碱法提取蛋白质回收率高、脂肪含量低、环境的污染小、凝胶强度好、色泽较好且具有较好的贮藏稳定性而受到广泛的关注，但提取过程中蛋白质结构和性质变化的研究相对较少，如不同阴阳离子的选择对pH值调节法的影响以及酸碱调节处理对肌球蛋白的结构-功能的影响。 为此，本研究以罗非鱼肉为原料，提取肌球蛋白，探讨pH值调节处理过程中，肌球蛋白分子酸//碱去折叠、等电点和中性调节重折叠过程中分子结构和性质的变化。其主要目的是进一步了解pH值调节处理过程中分子构象的动态变化，完善其基础理论体系，为低值海洋蛋白制备技术体系的建立和新型功能性分离鱼蛋白的开发提供依据。主要研究结果如下： 1.采用pH值调节（pH2.0-5.5、3.0-5.5、11.0-5.5、12.0-5.5）处理制备四种鱼分离蛋白，分别提取其水溶性组分和盐溶性组分，对其组成、溶解性、表面疏水性、总巯基和活性巯基含量、色氨酸荧光光谱、SDS-PAGE等进行检测和分析。结果表明：分离蛋白中盐溶性组分比例高于水溶性组分比例（p<0.05），且碱提蛋白中盐溶性蛋白的比例比酸提蛋白高（p<0.05）,表明碱法更易蛋白的提取；分离蛋白中水溶性组分和盐溶性组分的溶解性均受pH值的影响比较明显，酸提蛋白的溶解性较碱提蛋白的低；与新鲜鱼肉蛋白相比，四种分离蛋白中水溶性组分和盐溶性组分均发生了变性，体系的表面疏水性增加、巯基含量降低、色氨酸荧光强度下降，且酸性条件下蛋白质发生变性更严重；SDS-PAGE分析表明，肌球蛋白的变性最为明显。总体分析可得，碱调节处理更有利于蛋白的提取。 2.以罗非鱼肌球蛋白为原料，试验不同的pH值和酸//碱类型对肌球蛋白酸//碱溶解去折叠过程中分子结构和性质的变化。研究表明：在广泛pH值（pH2.0-12.0）范围内，肌球蛋白的溶解性受pH影响较明显；在极端酸性（pH≤3.0）和碱性（pH≥11.0）条件下，肌球蛋白的溶解度为60.6/%~76.65/%，而在pH4.0~6.0时溶解度较低；酸//碱诱导肌球蛋白去折叠过程中，在偏离pH7.0时，肌球蛋白的表面疏水性逐渐增大、巯基含量逐渐减小、色氨酸荧光强度减弱、Ca~/(2+/)-ATPase活性减小，这表明肌球蛋白疏水基团暴露、分子结构发生变化，且肌球蛋白在碱性条件变性程度较酸性条件下弱;在pH7.0条件下处理时，Ca~/(2+/)-ATPase活性为0.19μmol//mg//min，肌球蛋白变性程度较弱。 3.采用不同种类的酸诱导肌球蛋白去折叠，在HCl（pH2.0）处理条件下，表面疏水性最大、巯基含量最低、色氨酸荧光强大最小，肌球蛋白构象变性较严重，而在C/_6H/_8O/_7（pH3.0）处理条件下，肌球蛋白构象变化程度最弱；肌球蛋白在C/_6H/_8O/_7和H/_3PO/_4（pH2.0）处理条件下，-螺旋比例分别为20.5/%和9/%；不同种类的碱诱导肌球蛋白去折叠，NaOH（pH11.0）处理条件下的表面疏水性最小、巯基含量最高、色氨酸荧光强度最大、-螺旋比例最高，肌球蛋白构象变化程度最弱，此时肌球蛋白总巯基、活性巯基含量和-螺旋比例分别为4.054mol//10~5g、1.308mol//10~5g和15.3/%。总体分析可得，肌球蛋白在C/_6H/_8O/_7（pH3.0）、NaOH（pH11.0）诱导下变性程度最弱。 4.进一步研究酸//碱去折叠过程中NaCl的添加浓度和顺序对罗非鱼肌球蛋白结构和性质的影响，研究表明，添加0.5mol//L的NaCl时，肌球蛋白的溶解性最好；肌球蛋白在C/_6H/_8O/_7诱导去折叠过程中，NaCl的添加对疏水性影响不明显（p<0.05）；但用H/_3PO/_4、NaOH、KOH诱导去折叠时，在去折叠之前加NaCl时疏水性指数最小、巯基含量最高、荧光强大最大，且去折叠前加NaCl，肌球蛋白在C/_6H/_8O/_7、H/_3PO/_4（pH2.0）和NaOH、KOH（pH11.0）条件下，-螺旋比例分别为47.9/%、30.1/%、38.4/%和26.2/%；去折叠后加NaCl，-螺旋含量比例分别为31.2/%、8.2/%、13.1/%和10.1/%；这表明在用酸碱处理肌球蛋白溶液之前加NaCl能够抑制其构象的变化。总体分析可得，酸碱对肌球蛋白构象的影响程度为：弱酸（C/_6H/_8O/_7）＜强酸（H/_3PO/_4），NaOH＜KOH。 5.将酸//碱变性肌球蛋白调节到中性，考察重折叠过程中变性肌球蛋白结构和性质的变化。研究表明：未经酸//碱诱导重折叠的肌球蛋白溶解性明显高于经过酸//碱诱导重折叠的肌球蛋白溶解性，这表明经过酸//碱诱导处理后，肌球蛋白发生变性；碱性条件（尤其是pH11.0）下处理的肌球蛋白溶解性在广泛pH值（pH2.0-12.0）范围内最好；与酸//碱去折叠肌球蛋白相比，经过酸//碱诱导重折叠后的变性肌球蛋白表面疏水性降低、巯基含量和Ca~/(2+/)-ATPase活性增大、荧光强大增强，这表明酸//碱诱导部分变性的肌球蛋白发生了重折叠。 6.采用不同种类的酸和碱诱导变性肌球蛋白重折叠，研究表明，碱调节不同种类的酸变性肌球蛋白，对H/_3PO/_4（pH11.0）去折叠的变性肌球蛋白，经碱调节重折叠后-螺旋比例为26.4/%，肌球蛋白构象变性较轻，而在HCl（pH2.0）去折叠的变性肌球蛋白，-螺旋比例为14.9/%，肌球蛋白构象变化程度最严重；酸调节不同种类的碱变性肌球蛋白，NaOH（pH11.0）诱导去折叠的肌球蛋白构象变性较轻，而在KOH（pH12.0）诱导去折叠处理条件下，肌球蛋白构象变化程度最严重。与去折叠肌球蛋白相比，经pH值调节诱导重折叠后表面疏水性降低、巯基含量增大、荧光强大增强、-螺旋比例增大，这表明当把pH调回到中性时，部分变性的肌球蛋白发生重折叠。通过考查NaCl的添加顺序在酸//碱重折叠过程中对变性肌球蛋白构象的影响，研究表明，去折叠之前加NaCl时疏水性较小、巯基含量较高、-螺旋比例较高。 总体研究显示，酸//碱法提取蛋白质过程中肌球蛋白分子构象发生改变，采用C/_6H/_8O/_7和NaOH诱导肌球蛋白去折叠，肌球蛋白变性展开，且构象变化程度最小；随后调节pH到中性时，部分变性的肌球蛋白重折叠恢复到原有的结构；酸//碱提取蛋白质过程中，在去折叠之前添加NaCl，能够抑制蛋白质分子构象的变化。

外文摘要：The pH-shifting is a perfect approach for extracting protein. Fish protein isolates /(FPI/)were extracted at either acidic /(pH≤3.0/) or alkaline /(pH≥11.0/) at low temperature to obtainthe maximum solubility and remove fat and insoluble impurities via high-speedcentrifugation followed by the recovery of precipitated proteins at their isoelectric point.The pH-shifting has received widespread attention for high recovery rate, low fat content,few-environmental pollution, strongly gel intensity, good color and a better storage stability.The study on conformation and physicochemical property in the extraction process ofprotein, however, is rarely. Such as: the degree of influence by pH on the property andstructure of protein; the influence on pH-shifting by different ions; the influence onstructure and functions of myosin by pH-shifting. This paper taking tilapia as raw material, extract myosin and focus on the change ofconformation during unfolding and refolding of protein. The main purpose is to improvethe mechanism of pH-shifting and made it more mature, which will provide a fundamentalresearch for the widespread use of protein. This provides a bass for the processing system’sbuild of the low value marine proteins and development of new function of fish proteinisolates. The primary contents and results in this thesis as shown below: 1. Four protein isolates were prepared by pH-shifting /(pH2.0-5.5、3.0-5.5、11.0-5.5、12.0-5.5/) and extraction of water-soluble and salt soluble components from them. Thecomponent proportion, solubility, surface hydrophobicity, total sulfhydryl and activesulfhydryl content, the fluorescence of the Tyr residues and SDS-PAGE were detection andanalysis. The study showed that the proportion of salt-soluble protein was higher thanwater-soluble protein and the salt-solution extracted by alkali was higher than acid/(p<0.05/), which indicated alkali was easier to extract protein. pH has a significantinfluence on the solubility of water-soluble protein and salt-soluble protein of fish proteinisolate; Solubility of water-soluble protein and salt-soluble protein which were extracted byacid was lower than alkali; The water-soluble protein and salt-soluble protein extracted bypH-shifting from fish protein isolate were denatured compared with fresh fish. Surfacehydrophobicity was increased. Sulfhydryl content was decreased. The tryptophan fluorescence intensity was reduced. SDS-PAGE indicated the denaturation of myosin wasthe most serious. The overall analysis looked like that, alkali adjustment processing ismore advantageous to the extraction of proteins. 2. The changes of molecular structure and property of myosin during unfolding usingdifferent pH and types of acid base were studied. The results showed that the solubility ofmyosin was obvious influenced by pH between2.0to12.0, which was60.60-76.65/%under the extreme acidic /(pH≤3/) and alkaline /(pH≥11/) condition during unfolding andwhich was lower when pH4.0~6.0. There was a increased surface hydrophobicity,decreased sulfhydryl content, weaker tryptophan fluorescence intensity and decreasedactivity of Ca~/(2+/)-ATPase when unfolding, which indicated the hydrophobic group of myosinwas exposed and the changed of molecular structure, and a weaker degree of proteindenaturation in alkali-extracted process of myosin. The degeneration degree of myosin wasweaker when pH was7.0. The activity of Ca~/(2+/)-ATPase was0.19μmol//mg//min, which wasweaker in alkali than acid. 3. There was a higher degeneration of myosin during unfolding when pH was2.0adjust by strong acid /(hydrochloric acid, phosphoric acid/), while pH was3.0adjust bycitric acid is weakest. When the myosin was adjusted to pH2.0by citric acid andphosphoric acid, the-helix content were20.5/%and9.0/%respectively. When pH was11.0adjust by different alkali, the conformation change of myosin during unfolding wasvery small，especially by NaOH. The total sulfhydryl and active sulfhydryl content,proportion of-helix were4.054mol//10~5g、1.308mol//10~5g and15.3/%respectively. Theoverall analysis looked like that, the degeneration degree of myosin induced by citric acid/(pH3.0/) and NaOH /(pH11.0/) was weaker. 4. A further study have made in order to know the effect of add order andconcentrations of NaCl in acid//alkali unfolding process on structure and property ofmyosin from the tilapia.The results were showed when the concentration of NaCl was0.5M, there was a best solubility; There was no obvious difference in adjust by citric acidwhether add NaCl or not /(p<0.05/); Compared to absence of salt, myosin treated withphosphoric acid, NaOH and KOH with salt add before unfolding had minimumhydrophobic index, higher sulphur content, significantly higher fluorescence. When addNaCl before the myosin was adjusted to pH2.0by citric acid and phosphoric acid, the-helix content were47.9/%and30.1/%respectively. When add NaCl before the myosinwas adjusted to pH11.0by NaOH and KOH, the-helix content were38.4/%and26.2/%respectively; When add NaCl after unfolding, the-helix content were31.2/%,8.2/%,13.1/%and10.1/%respectively. This showed that when add salt before unfolding, it could inhibit myosin conformational changes. Total analysis showed: the conformation incidenceof myosin is weak acid /(citric acid/)

年份：2013