从牛眼玻璃体中分离纯化透明质酸的新方法  

A New Method of Purification of Hyaluronic Acid from Bovine Vitreous Body

作　　者：郭育涛[1] 陈斌[1] 江元汝[1] 李东亮[1] 

机构地区：[1]西安建筑科技大学化学系,陕西西安710055

出　　处：《精细化工》2008年第3期246-250,259,共6页Fine Chemicals

基　　金：陕西省教育厅自然科学专项基金(2001JK210)

摘　　要：为了得到一种简便的从动物组织中分离纯化透明质酸(HA)的新方法,用牛眼玻璃体为原料,对HA的分离纯化进行了研究。在ρ(NaHCO3)=7.56 g/L和ρ(Na2CO3)=1.06 g/L的碱性缓冲水溶液中,用胰蛋白酶水解牛眼HA提取物中的蛋白质,经氯苯除杂后,用乙醇析出并分离得到HA初级沉淀物,将其溶解于ρ(NaC l)=11.6 g/L,ρ(NaH2PO4.12H2O)=0.45 g/L,ρ(Na2HPO4.2H2O)=0.06 g/L的含盐磷酸盐水溶液中,再用3倍体积的乙醇析出并分离得到HA的首次纯化沉淀物,此纯化阶段分离系数(α)=19.03,蛋白质和HA中w(HA)=63.9%,HA回收率91.84%。其次,用含盐磷酸盐水溶液溶解HA的首次纯化沉淀物,再加入ρ(活性炭)=50 g/L及ρ(高岭土)=100 g/L进行吸附纯化,此纯化阶段分离系数(α)=27.69,HA回收率94.0%,w(DNA)=0%,蛋白质和HA中w(HA)=98.0%。最后,经50 000 Da透析袋用去离子水透析4 h,用乙醇析出并分离得到纯化了的HA沉淀,此纯化阶段分离系数(α)=17.64,HA回收率93.5%。最终HA沉淀经冷冻干燥后获白色粉末状HA纯化品,无水HA纯化品中w(HA)=99.77%,HA相对分子质量Mη=6.3×105。该方法牛眼玻璃体质量扩大至2 000 mL的扩试结果与小试结果相近。该文报道工作的新颖性,已为2007年4月18日由陕西省科学技术信息研究所查新中心出具的第2007360-3号《科技查新报告》所证实。In order to get a simplified method to purify hyaluronic acid （HA） from tissues of animals, procedure of purification of HA from bovine vitreous body was studied. The protein in the extracts including HA from bovine vitreous body was hydrolyzed by trypsin in the basic buffer water solution [ρ（NaHCO3） = 7.56 g/L and ρ（Na2CO3 ） = 1.06 g/L ]. After the non-aqueous impurities were removed by chlorobenzene, preliminary precipitates containing HA were got by using ethyl alcohol, and after the precipitates were dissolved in a phosphate buffer water solution with sodium chloride （ρ（NaCl） = 11.6 g/L,ρ（ NaH2PO4·12H2O ） = 0. 45 g/L and ρ （ Na2HPO4·2H2O ） = 0.06 g/L） initial purified HA precipitates[w（HA） = 63.9% according to two components of HA and polypetid] were obtained by addition of 3 time the volume of 95% ethyl alcohol. In this main stage of purification, separation factor （α） = 19. 03 and recovery ratio of HA was 91.84%. Then, after the initial precipitates was dissolved in the phosphate buffer water solution, and ρ （ charcoal ） = 50 g/L and p （ kaolin ） = 100 g/L were added, HA solution was obtained by centrifugalization and MF with 0.22μm membrane. In this auxiliary stage of purification, the separation factor （ α ） = 27.69, w （ HA ） = 98.0% , w （ DNA ） = 0% , and recovery ratio of HA was 94.0%. Finally, after the HA solution was dialyzed by dialysis membrane of MWCO of 50 000 in deionized water for 4 h,the purified HA precipitate was isolated by alcohol. In this subsidiary stage of purification, the separation factor （α） = 17.64, and recovery ratio of HA was 93.5%. The purified HA precipitate was dried by vacuum freeze drying,and a white powder of HA was got [ w （HA） =99.77% and Mη = 6. 3 × 10^5]. Results of bench-scale test（ bovine vitreous body 2 000 mL） approached that of the research. The novelty and originality of this work was proved by No. 2007360 -3 Report of Consultation on Science and Technology, which was issued by Consu

关 键 词：透明质酸 纯化 胰蛋白酶 酒精-盐溶 多肽 牛眼玻璃体 活性炭 透析 

分 类 号：Q503[生物学—生物化学] Q539.7