沉淀方法对污泥提取蛋白质的选择性及结构的影响分析

Effects of precipitation methods on selectivity and structure of proteins extracted from sewage sludge

为提升污泥蛋白质的资源化利用,分别采用等电点沉淀法(Isoelectric Point Precipitation,IPP)、乙醇沉淀法(Ethanol Precipitation,EP)、硫酸铵沉淀法(Ammonium Sulfate precipitation,AS)以及联用沉淀法(Combined Precipitation,CP)分离蛋白质,用选择性因子α建立蛋白质、多糖、腐殖酸3者之间的数量关系,以考察不同沉淀方法中主导因素对蛋白质的选择性,并结合红外光谱分析其对蛋白质二级结构的影响以及蛋白质的应用潜能。结果表明:沉淀方法中的主导因素会对蛋白质的选择性及结构产生影响且所得蛋白质具有多重性。在主导因素pH=4.0、乙醇体积分数为40%、硫酸铵溶液饱和度为50%以及联用沉淀法时,蛋白质沉淀效率分别为84.76%、64.17%、65.84%和72.55%,选择性因子αPN/PS分别为3.11、4.22、3.80和6.51,均能显著分离蛋白质。在IPP、AS和CP分离得到的蛋白质中,污泥蛋白质以β-折叠结构为主。同时主导因素pH值与蛋白质中含醇羟基氨基酸的含量呈正相关;乙醇体积分数增加,蛋白质中含苯环氨基酸的含量减少,醇羟基氨基酸增加,且主要结构从β-折叠向α-螺旋转变;硫酸铵溶液饱和度的改变对蛋白质二级结构影响不显著。

To improve the resource utilization of sludge protein,a thermal-alkaline method was used to extract protein from sludge,and then protein was separated by isoelectric point precipitation(IPP),ethanol precipitation(EP),ammonium sulfate precipitation(AS),and combined precipitation(CP),respectively.Comparing the concentration changes of protein,polysaccharide and humic acid in the supernatant before and after precipitation,the quantitative relationship among protein,polysaccharide,and humic acid was constructed by selectivity factorα,and the selectivity of proteins by the dominant factor in different precipitation methods was investigated.The protein solids obtained by precipitation were dried at 50℃and then ground,which were used to analyze the effects of leading factors on the properties and secondary structure of a protein by Fourier translation infrared spectroscopy(FT-IR).Finally,the application potential of the protein was analyzed.The results show that while the protein is precipitated by different precipitation methods,polysaccharides and humic acid are also precipitated,and the protein can be significantly separated when the dominant factors are pH 4.0,ethanol concentration of 40%,ammonium sulfate saturation of 50%,and combined precipitation.The precipitation efficiency of protein at this time is 84.76%,64.17%,65.84%and 72.55%,the selectivity factorsαPN/PS are 3.11,4.22,3.80 and 6.51,respectively,and the selective order is protein,humic acid and polysaccharide in descending order.The protein obtained by the isoelectric precipitation method is mainly theβ-sheet structure.With the decrease of pH,the content of amino acid containing alcohol hydroxyl in protein decreases,and they are positively correlated.The main structure of protein obtained by the ethanol precipitation method will change fromβ-sheet toα-helix with the increase of ethanol concentration,the content of benzene-containing amino acid reduce,and the content of alcohol hydroxyl amino acids increase.The protein obtained by the ammonium sulfate precipitation method is mainlyβ-sheet structure,and the change of ammonium sulfate concentration has no significant effect on the secondary structure of the protein.Because of the safety,nutrition and plasticity,the obtained protein can be used as animal feed,material carriers,and gels.