壳聚糖螯合凝胶层析法纯化血液超氧化物歧化酶

Process of Purification on Superoxide Dismutase(SOD) from Blood by Chitosan Affinity Chromatography

DOI：10.3969/j.issn.1673-1689.2014.03.004

中文关键词: 相对分子质量 聚丙烯酸钠 血液 超氧化物歧化酶

英文关键词: chitosan IDA epichlorohydrin metal affinity chromatography

基金项目:国家自然科学基金项目(81273652)

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中文摘要:

首先利用不同相对分子质量聚丙烯酸钠从动物血液中分离纯化出超氧化物歧化酶(SOD),探索不同相对分子质量聚丙烯酸钠对SOD提取酶活性的影响。鉴于离子交换层析和凝胶过滤层析纯化时所用材料较昂贵,探索出一条适合工业化大规模生产的纯化方法。利用壳聚糖、亚氨基二乙酸(IDA)和环氧氯丙烷等材料合成一种新填料,根据亲和层析原理去除杂蛋白质,获得电泳纯级超氧化物歧化酶,使酶活性进一步提高,并和离子交换层析和凝胶过滤层析方法相对照,以期自制填料能代替Sephedex,DEAE-sepherose-fast flow等传统昂贵填料,以适合工业化生产。结果显示,低相对分子质量聚丙烯酸钠在血液SOD提纯工艺中更为适用,超滤后的粗酶液经离子交换层析、凝胶过滤层析和壳聚糖亲和层析纯化后经SDS-PAGE聚丙烯酰胺凝胶电泳后呈明显的单一条带,单亚基分子量16 kD,比活力达5 585 U/mg和6 148 U/mg,4 739 U/mg;产品得率分别为18.1%和13.7%,15.5%;纯化后活性回收率分别为61.2%,50.8%,42.6%。表明壳聚糖亲和层析方法在新工艺后续纯化工艺中有望代替Sephedex DEAEsepherose-fast flow为基础的传统纯化方法,适合工业化大规模生产。

英文摘要:

Making use of a definite molecular weight polyacylic acid sodium as dispersant to purify superoxide dismutase from blood,and to explore the effect of different molecular weight of PAAS on SOD activity of blood,in consideration of the purification materials of the ion exchange chromatography and gel filtration chromatography was expensive,so as to explore a suitable method of the SOD purification for the industrialized mass production.Making use of cheap materials such as:chitosan,IDA and epichlorohydrin to synthesis a new type of filler,according to the principle of chelating chromatography,raw protein was removed and electrophoresis pure level superoxide dismutase was obtained,the enzyme activity was further improved,the self-made materials can take place of the tradition materials such as Sephedex DEAE-sepherose-fast flow.The results of the SDS-PAGE of the crud enzyme after ultrafiltration by ion exchange chromatography showed a single band obviously,and reached the electrophoresis purity,the enzyme activity is up to 5 585 u/mg, 6 148 u/mg,4 739 u/mg,and the product yield is 18.1%,13.7%,15.5% respectively,active recovrery is 61.2%,50.8%,42.6% resectively after purification.The low molecular weight polyacrylic acid is more suitable in the Purification process of blood is suitable as a polymer electrolyte to purify the blood SOD,the medium and high molecular weight PAAS are not appropriate for industrialized purification application.

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