肝素黄杆菌肝素酶性质探究.docVIP

肝素黄杆菌肝素酶性质探究摘要：目的研究肝素酶I的理化性质。方法测定肝素酶I的相对分子质量、等电点、紫外吸收光谱、N端序列，以及各种环境因素对酶活性和酶稳定性的影响。结果相对分子质量约为43×103，等电点为8.5。N端序列不能测到。酶的最适催化条件为：温度45℃，pH 6.4~7.0，离子强度150 mmol/L。酶在35℃以上极易失活，在pH 7~11之间基本稳定。该酶的最大紫外吸收位于280 nm。H2O2（1 mmol/L, 10 mmol/L），NaN3（10 mmol/L, 100 mmol/L），乙腈（1％，10％），1 mol/L盐酸胍和1 mol/L尿素对酶的活性无影响；1％SDS，4 mol/L盐酸胍和4 mol/L尿素则使酶完全失活；CaCl2和MgCl2是该酶的激活剂，FeCl2则对该酶的活性具有抑制作用。结论通过实验，测定了肝素酶I的主要理化性质。 关键词：肝素黄杆菌；肝素酶I；理化性质 中图分类号：R977.3文献标识码：A文章编号：1672-979X(2007)02-0014-04 Characterization of Flavobacteriun heparinum Heparinase I MA Xiao-lai，YUAN Qin-sheng （State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237，China） Abstract：Objective To study physico-chemical properties of heparinase I. Methods Properties of the enzyme including relative molecular mass, isoelectric point, N-terminal amino acid sequence, optimal temperature and pH, UV absorbance, and the influence of the environment factors on activity and stability of the enzyme were determined. Results The enzyme had a relative molecular mas of 43×103 and the pI was about 8.5. The N-terminal amino acid sequence could not be determined. It had an activity maximum at pH6.4~7.0 and 41℃. The enzyme with a maximum UV absorbance at 280 nm was very sensitive to thermal denaturation at temperature up to 35 ℃. H2O2(1 mmol/L, 10 mmol/L), NaN3(10 mmol/L, 100 mmol/L), acetonitrile（1％, 10％）, 1mol/L carbamidine hydrochloride and 1mol/L urea had no influence on the activity of the enzyme. The enzyme was sensitive to 1％ SDS，4mol/L carbamidine hydrocloride and 4mol/L urea. CaCl2 and MgCl2 were two activators of the enzyme, while FeCl2 was an inhibitor. Conclusion The main physico-chemical properties of heparinase I are clear by experiments. Key words：Flavobacteriun heparinum；heparinase I；physico-chemical characterization 肝素酶（heparinase）是指一类能够特异性地裂解肝素和类肝素主链糖苷键的酶，可以清除血液中的肝素，并常用于测定肝素的结构以及抗肿瘤等方面的研究[1-3]。肝素酶最初从肝素黄杆菌中发现并分离，后来发现其他一些微生物或动物组织中也存在[4]。近年，肝素酶用于低分子肝素生产和链上精确结构的