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Rhodosorus marinus藻红蛋白的纯化、性质及稳定性研究
Thesis Advisor林惠民
Degree Grantor中国科学院水生生物研究所
Place of Conferral中国科学院水生生物研究所
Degree Discipline水生生物学
Abstract通过对四种红藻的培养及其光谱特性的比较,选出了具有代表性具尚未见报道的藻种 Rhodosorus marinus(R. m)进行藻红蛋白的研究,本实验改进了藻红蛋白的分离提纯方法,将R. m的水溶性精提物经硫酸铵沉淀后,用DEAE-52纤维素和Bio-gel-P300进行四次柱层析,得到三种藻红蛋白:B-PF_1,B-PE_2和b-PE,它们的纯度(A_(545)/A_(280))都达到4.0以上,而且在普通PAGE上鉴定纯度都达到了电泳纯。通过Sephadex G-200测定了三种藻红蛋白聚合体的分子量,它们分别是:b-PE为42KD;B-PE_1为330KD;B-PE_2为280KD。并通过SDS-PAGE测定了三种藻红蛋亚单位的分子量,由此推测它们的亚基组成如下:b- PE为(αβ)B-PE_1为( α'_2β')_6γ'和B-PE_2为(α'_2β')_6γ'。分别测定了纯化后的三种藻红蛋白(B-PE_1、B-PE_2和b-PE)的可见光谱和荧光光谱。两种P-PE的可见吸收峰比b-PE的多一个498nm的吸收峰,就是因为P-PE多一个亚单位的缘故。三种藻红蛋白的氨基酸组成以酸性氨基酸和疏水性氨基酸为主,所以经等电聚焦测定的三种藻红蛋白的等电点都是偏酸性的。研究了温度、PH、可见光和紫外光对R.m的藻红蛋白稳定性的影响。发现这种藻的藻红蛋白在PH4.5-8.0 范围内较稳定,而且可耐受60 ℃的高温,在可见光特别是紫外光下很不稳定。并通过加入一些还原剂保护藻红蛋白,减轻光氧化对藻红蛋白的存坏作用,提高了藻红蛋白的稳定性。能过酶解作用,得到了一种对乙醇有一定耐受力的藻红蛋白样品。
Other AbstractFor study of phycoerythrin(PE), We selected a strain algae (Rhodosorus marinus) which is significant and has not been reported before by means of culture of four strains of algae and comparison of their spectral character. The method for separating and purifying PE was improved. Three PE(b-PE, B-PE_1, B-PF_2) from R. m were purified with DEAD-52 cellulose column and Bio-gel P300 column by four times. The three PE were purified to electrophoresis purity. (A_(545nm)/A_(280nm) > 4.0). On a calibrated Sephadex G-200 column a molecular weight of 42KD was found for b-PE, a molecular weight of 330KD for P-PE_1 and a molecular weight of 280KD for B-PE_2. The SDS-PAGE indicated the subunit structure of the three PE was: (αβ) for b-PE; (α'_2β')_6γ' for B-PE_1 and (α"_2β")_6γ" for B-PE_2. The absorption spectra and the fluorescence spectra of the three PE were determined. The absorption spectra and the fluorescence spectra of the three PE were determined. The absence of presence of an absorption peak (or shoulder) at 498nm represents the major difference between the b-PE and B-PE. The difference is correlated with the present of one of subunit. The amino acid composition from the three PE are relatively rich in aliphatic and acidic amino acid. That is the reason why the isoelectric point of the three PE tends to acidity. The PE from R.m is stable from PH4.5-8.0, and thermostable up to 60 ℃ and it exhibits poor light stability especially to ultraviolet light. The PE were protect from the photo-oxydration by the means of mixing with some reductant. We obtained one PE which is resistant to alcohol.
Document Type学位论文
Recommended Citation
GB/T 7714
李邵蓉. Rhodosorus marinus藻红蛋白的纯化、性质及稳定性研究[D]. 中国科学院水生生物研究所. 中国科学院水生生物研究所,1992.
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