肠球菌中α-l-鼠李糖苷酶的分离纯化及酶学性质分析-purification and characterization of α - l - rhamnosidase from enterococcus.docxVIP

ABSTRACTABSTRACTIIIIrange of sodium chloride was controlled between 0.2 and 0.6 mol/L. Below 0.2 mol/L or above 0.6 mol/L, there was almost no protein eluted out. The velocity eluate is 0.6 ml/min, and collect each tube every 15 min. The NO.12 tube displayed the highest activity among all tubes, and the first protein peak has no activity. When the sodium chloride concentration was between 0.3 mol/L and 0.5 mol/L, the α-L-rhamnosidase can be eluted completely.④ The unrefined α-L-rhamnosidase was applied to the column of gel filtrationchrmatography on Sephadex G-100. The velocity was 0.4 mL/min, and collect each tube every 15 min. Three protein peaks were eluted out, the third one is target peak.The homogeneity of the enzyme was verified by using SDSafter salting out and Gel-filtration chromatography. The concentration of separate glue is 10% and the concentrate glue is 4%. The result showed that after the ammonium suphate fractional precipitation it contained some proteins with different molecular weights. However, after gel-filtration chromatography on Sephadex G-100, the α-L-rhamnosidase was purified completely and it showed homogeneity by SDS. The molecular weight was about 90 K Da.Enzymatic properties study indicate that this enzyme has high acid tolerance and thermal stability. The enzyme activity synergistically increased by the addition of metal cations such as K+ ,and Mg2+, meanwhile, Ca2+ and Fe2+ can significantly inhibit its activity. Some kinds of organic solvents can inhibit its activity as well, especially acetone. Kinetic studies showed that Michaelis constant Km is 4.58 mmol/L, Vmax is 16.13 U/L for the enzyme, Pnpr was used as substrate.Keywords: α-L-rhamnosidase; Enterococcus durans; condition optimization; isolation and山东轻工业学院硕士学位论文山东轻工业学院硕士学位论文IIIIIIpurification; enzyme characteristics学位论文独创性声明本人声明，所呈交的学位论文系在导师指导下本人独立完成的研究成果。文中引用 他人的成果，均已做出明确标注或得到许可。论文内容未包含法律意义上已属于他人的 任何形式的研究成果，也不包含本人已用于其他学位申请的论文或成果，与我一同工作 的同志对本研究所做的任何贡献均已在论文中作了明确的说明并表示